Identification of Novel Kaposi's Sarcoma-Associated Herpesvirus Orf50 Transcripts: Discovery of New RTA Isoforms with Variable Transactivation Potential

PubMed Central

Wakeman, Brian S.; Izumiya, Yoshihiro

2016-01-01

ABSTRACT Kaposi's sarcoma-associated herpesvirus (KSHV) is a gammaherpesvirus that has been associated with primary effusion lymphoma and multicentric Castleman's disease, as well as its namesake Kaposi's sarcoma. As a gammaherpesvirus, KSHV is able to acutely replicate, enter latency, and reactivate from this latent state. A key protein involved in both acute replication and reactivation from latency is the replication and transcriptional activator (RTA) encoded by the gene Orf50. RTA is a known transactivator of multiple viral genes, allowing it to control the switch between latency and virus replication. We report here the identification of six alternatively spliced Orf50 transcripts that are generated from four distinct promoters. These newly identified promoters are shown to be transcriptionally active in 293T (embryonic kidney), Vero (African-green monkey kidney epithelial), 3T12 (mouse fibroblast), and RAW 264.7 (mouse macrophage) cell lines. Notably, the newly identified Orf50 transcripts are predicted to encode four different isoforms of the RTA which differ by 6 to 10 residues at the amino terminus of the protein. We show the global viral transactivation potential of all four RTA isoforms and demonstrate that all isoforms can transcriptionally activate an array of KSHV promoters to various levels. The pattern of transcriptional activation appears to support a transcriptional interference model within the Orf50 region, where silencing of previously expressed isoforms by transcription initiation from upstream Orf50 promoters has the potential to modulate the pattern of viral gene activation. IMPORTANCE Gammaherpesviruses are associated with the development of lymphomas and lymphoproliferative diseases, as well as several other types of cancer. The human gammaherpesvirus, Kaposi's sarcoma-associated herpesvirus (KSHV), is tightly associated with the development of Kaposi's sarcoma and multicentric Castleman's disease, as well as a rare form of B cell lymphoma (primary effusion lymphoma) primarily observed in HIV-infected individuals. RTA is an essential viral gene product involved in the initiation of gammaherpesvirus replication and is conserved among all known gammaherpesviruses. We show here for KSHV that transcription of the gene encoding RTA is complex and leads to the expression of several isoforms of RTA with distinct functions. This observed complexity in KSHV RTA expression and function likely plays a critical role in the regulation of downstream viral and cellular gene expression, leading to the efficient production of mature virions. PMID:27795414

Novel Nine-Exon AR Transcripts (Exon 1/Exon 1b/Exons 2–8) in Normal and Cancerous Breast and Prostate Cells

PubMed Central

Hu, Dong Gui; McKinnon, Ross A.; Hulin, Julie-Ann; Mackenzie, Peter I.; Meech, Robyn

2016-01-01

Nearly 20 different transcripts of the human androgen receptor (AR) are reported with two currently listed as Refseq isoforms in the NCBI database. Isoform 1 encodes wild-type AR (type 1 AR) and isoform 2 encodes the variant AR45 (type 2 AR). Both variants contain eight exons: they share common exons 2–8 but differ in exon 1 with the canonical exon 1 in isoform 1 and the variant exon 1b in isoform 2. Splicing of exon 1 or exon 1b is reported to be mutually exclusive. In this study, we identified a novel exon 1b (1b/TAG) that contains an additional TAG trinucleotide upstream of exon 1b. Moreover, we identified AR transcripts in both normal and cancerous breast and prostate cells that contained either exon 1b or 1b/TAG spliced between the canonical exon 1 and exon 2, generating nine-exon AR transcripts that we have named isoforms 3a and 3b. The proteins encoded by these new AR variants could regulate androgen-responsive reporters in breast and prostate cancer cells under androgen-depleted conditions. Analysis of type 3 AR-GFP fusion proteins showed partial nuclear localization in PC3 cells under androgen-depleted conditions, supporting androgen-independent activation of the AR. Type 3 AR proteins inhibited androgen-induced growth of LNCaP cells. Microarray analysis identified a small set of type 3a AR target genes in LNCaP cells, including genes known to modulate growth and proliferation of prostate cancer (PCGEM1, PEG3, EPHA3, and EFNB2) or other types of human cancers (TOX3, ST8SIA4, and SLITRK3), and genes that are diagnostic/prognostic biomarkers of prostate cancer (GRINA3, and BCHE). PMID:28035996

Identification of Novel Androgen-Regulated Pathways and mRNA Isoforms through Genome-Wide Exon-Specific Profiling of the LNCaP Transcriptome

PubMed Central

Carling, Phillippa J.; Buist, Thomas; Zhang, Chaolin; Grellscheid, Sushma N.; Armstrong, Kelly; Stockley, Jacqueline; Simillion, Cedric; Gaughan, Luke; Kalna, Gabriela; Zhang, Michael Q.; Robson, Craig N.; Leung, Hing Y.; Elliott, David J.

2011-01-01

Androgens drive the onset and progression of prostate cancer (PCa) by modulating androgen receptor (AR) transcriptional activity. Although several microarray-based studies have identified androgen-regulated genes, here we identify in-parallel global androgen-dependent changes in both gene and alternative mRNA isoform expression by exon-level analyses of the LNCaP transcriptome. While genome-wide gene expression changes correlated well with previously-published studies, we additionally uncovered a subset of 226 novel androgen-regulated genes. Gene expression pathway analysis of this subset revealed gene clusters associated with, and including the tyrosine kinase LYN, as well as components of the mTOR (mammalian target of rapamycin) pathway, which is commonly dysregulated in cancer. We also identified 1279 putative androgen-regulated alternative events, of which 325 (∼25%) mapped to known alternative splicing events or alternative first/last exons. We selected 30 androgen-dependent alternative events for RT-PCR validation, including mRNAs derived from genes encoding tumour suppressors and cell cycle regulators. Of seven positively-validating events (∼23%), five events involved transcripts derived from alternative promoters of known AR gene targets. In particular, we found a novel androgen-dependent mRNA isoform derived from an alternative internal promoter within the TSC2 tumour suppressor gene, which is predicted to encode a protein lacking an interaction domain required for mTOR inhibition. We confirmed that expression of this alternative TSC2 mRNA isoform was directly regulated by androgens, and chromatin immunoprecipitation indicated recruitment of AR to the alternative promoter region at early timepoints following androgen stimulation, which correlated with expression of alternative transcripts. Together, our data suggest that alternative mRNA isoform expression might mediate the cellular response to androgens, and may have roles in clinical PCa. PMID:22194994

Another face of the Treacher Collins syndrome (TCOF1) gene: identification of additional exons.

PubMed

So, Rolando B; Gonzales, Bianca; Henning, Dale; Dixon, Jill; Dixon, Michael J; Valdez, Benigno C

2004-03-17

Treacher Collins syndrome (TCS) is characterized by an abnormality in craniofacial development during early embryogenesis. TCS is caused by mutations in the gene TCOF1, which encodes the nucleolar phosphoprotein treacle. Genetic and proteomic characterizations of TCS/treacle are based on the previously reported 26 exons of TCOF1. Here, we report the identification of 231-nucleotide (nt) exon 6A (between exons 6 and 7) and 108-nt exon 16A (between exons 16 and 17). Isoforms with exon 6A are up to 3.7-fold more abundant than alternatively spliced variants without exon 6A, but only minor isoforms contain exon 16A. Exon 6A encodes a peptide sequence containing basic and acidic domains similar to 10 other exons of TCOF1. Unlike the other exons, exon 6A encodes a nuclear localization signal (NLS) which does not, however, alter the nucleolar localization of full-length treacle. The discovery of exons 6A and 16A is relevant to mutational analysis of the TCOF1 gene in TCS patients, and to functional analysis of its gene product.

Merlin Isoforms 1 and 2 Both Act as Tumour Suppressors and Are Required for Optimal Sperm Maturation

PubMed Central

Zoch, Ansgar; Mayerl, Steffen; Schulz, Alexander; Greither, Thomas; Frappart, Lucien; Rübsam, Juliane; Heuer, Heike; Giovannini, Marco; Morrison, Helen

2015-01-01

The tumour suppressor Merlin, encoded by the gene NF2, is frequently mutated in the autosomal dominant disorder neurofibromatosis type II, characterised primarily by the development of schwannoma and other glial cell tumours. However, NF2 is expressed in virtually all analysed human and rodent organs, and its deletion in mice causes early embryonic lethality. Additionally, NF2 encodes for two major isoforms of Merlin of unknown functionality. Specifically, the tumour suppressor potential of isoform 2 remains controversial. In this study, we used Nf2 isoform-specific knockout mouse models to analyse the function of each isoform during development and organ homeostasis. We found that both isoforms carry full tumour suppressor functionality and can completely compensate the loss of the other isoform during development and in most adult organs. Surprisingly, we discovered that spermatogenesis is strictly dependent on the presence of both isoforms. While the testis primarily expresses isoform 1, we noticed an enrichment of isoform 2 in spermatogonial stem cells. Deletion of either isoform was found to cause decreased sperm quality as observed by maturation defects and head/midpiece abnormalities. These defects led to impaired sperm functionality as assessed by decreased sperm capacitation. Thus, we describe spermatogenesis as a new Nf2-dependent process. Additionally, we provide for the first time in vivo evidence for equal tumour suppressor potentials of Merlin isoform 1 and isoform 2. PMID:26258444

Proteomic Analysis of Parkin Isoforms Expression in Different Rat Brain Areas.

PubMed

D'Amico, Agata Grazia; Maugeri, Grazia; Reitano, Rita; Cavallaro, Sebastiano; D'Agata, Velia

2016-10-01

PARK2 gene's mutations are related to the familial form of juvenile Parkinsonism, also known as the autosomic recessive juvenile Parkinsonism. This gene encodes for parkin, a 465-amino acid protein. To date, a large number of parkin isoforms, generated by an alternative splicing mechanism, have been described. Currently, Gene Bank lists 27 rat PARK2 transcripts, which matches to 20 exclusive parkin alternative splice variants. Despite the existence of these isoforms, most of the studies carried out so far, have been focused only on the originally cloned parkin. In this work we have analyzed the expression profile of parkin isoforms in some rat brain areas including prefrontal cortex, hippocampus, substantia nigra and cerebellum. To discriminate among these isoforms, we detected their localization through the use of two antibodies that are able to identify different domains of the parkin canonical sequence. Our analysis has revealed that at least fourteen parkin isoforms are expressed in rat brain with a various distribution in the regions analyzed. Our study might help to elucidate the pathophysiological role of these proteins in the central nervous system.

Function of Protein Phosphatase 2A in Control of Proliferation: Isolation and Analysis of Dominant-Defective Mutants

DTIC Science & Technology

1999-06-01

subunits are expressed ubiquitously and appear to be encoded by small and quite homogeneous gene families. In plants , however, A and C subunit gene...1996). In both plants and animals, different B subunit isoforms are encoded by two or more unrelated gene families, some of which are expressed in a...PP2A functions in whole plants and in mammalian tissue culture cells. This genetic system may also prove useful for analyzing interactions between

Loss of GATA-1 Full Length as a Cause of Diamond–Blackfan Anemia Phenotype

PubMed Central

Parrella, Sara; Aspesi, Anna; Quarello, Paola; Garelli, Emanuela; Pavesi, Elisa; Carando, Adriana; Nardi, Margherita; Ellis, Steven R.; Ramenghi, Ugo; Dianzani, Irma

2015-01-01

Mutations in the hematopoietic transcription factor GATA-1 alter the proliferation/differentiation of hemopoietic progenitors. Mutations in exon 2 interfere with the synthesis of the full-length isoform of GATA-1 and lead to the production of a shortened isoform, GATA-1s. These mutations have been found in patients with Diamond–Blackfan anemia (DBA), a congenital erythroid aplasia typically caused by mutations in genes encoding ribosomal proteins. We sequenced GATA-1 in 23 patients that were negative for mutations in the most frequently mutated DBA genes. One patient showed a c.2T > C mutation in the initiation codon leading to the loss of the full-length GATA-1 isoform. PMID:24453067

The Dynein Gene Family in Chlamydomonas Reinhardtii

PubMed Central

Porter, M. E.; Knott, J. A.; Myster, S. H.; Farlow, S. J.

1996-01-01

To correlate dynein heavy chain (Dhc) genes with flagellar mutations and gain insight into the function of specific dynein isoforms, we placed eight members of the Dhc gene family on the genetic map of Chlamydomonas. Using a PCR-based strategy, we cloned 11 Dhc genes from Chlamydomonas. Comparisons with other Dhc genes indicate that two clones correspond to genes encoding the alpha and beta heavy chains of the outer dynein arm. Alignment of the predicted amino acid sequences spanning the nucleotide binding site indicates that the remaining nine clones can be subdivided into three groups that are likely to include representatives of the inner-arm Dhc isoforms. Gene-specific probes reveal that each clone represents a single-copy gene that is expressed as a transcript of the appropriate size (>13 kb) sufficient to encode a high molecular weight Dhc polypeptide. The expression of all nine genes is upregulated in response to deflagellation, suggesting a role in axoneme assembly or motility. Restriction fragment length polymorphisms between divergent C. reinhardtii strains have been used to place each Dhc gene on the genetic map of Chlamydomonas. These studies lay the groundwork for correlating defects in different Dhc genes with specific flagellar mutations. PMID:8889521

Identification of Novel Kaposi's Sarcoma-Associated Herpesvirus Orf50 Transcripts: Discovery of New RTA Isoforms with Variable Transactivation Potential.

PubMed

Wakeman, Brian S; Izumiya, Yoshihiro; Speck, Samuel H

2017-01-01

Kaposi's sarcoma-associated herpesvirus (KSHV) is a gammaherpesvirus that has been associated with primary effusion lymphoma and multicentric Castleman's disease, as well as its namesake Kaposi's sarcoma. As a gammaherpesvirus, KSHV is able to acutely replicate, enter latency, and reactivate from this latent state. A key protein involved in both acute replication and reactivation from latency is the replication and transcriptional activator (RTA) encoded by the gene Orf50 RTA is a known transactivator of multiple viral genes, allowing it to control the switch between latency and virus replication. We report here the identification of six alternatively spliced Orf50 transcripts that are generated from four distinct promoters. These newly identified promoters are shown to be transcriptionally active in 293T (embryonic kidney), Vero (African-green monkey kidney epithelial), 3T12 (mouse fibroblast), and RAW 264.7 (mouse macrophage) cell lines. Notably, the newly identified Orf50 transcripts are predicted to encode four different isoforms of the RTA which differ by 6 to 10 residues at the amino terminus of the protein. We show the global viral transactivation potential of all four RTA isoforms and demonstrate that all isoforms can transcriptionally activate an array of KSHV promoters to various levels. The pattern of transcriptional activation appears to support a transcriptional interference model within the Orf50 region, where silencing of previously expressed isoforms by transcription initiation from upstream Orf50 promoters has the potential to modulate the pattern of viral gene activation. Gammaherpesviruses are associated with the development of lymphomas and lymphoproliferative diseases, as well as several other types of cancer. The human gammaherpesvirus, Kaposi's sarcoma-associated herpesvirus (KSHV), is tightly associated with the development of Kaposi's sarcoma and multicentric Castleman's disease, as well as a rare form of B cell lymphoma (primary effusion lymphoma) primarily observed in HIV-infected individuals. RTA is an essential viral gene product involved in the initiation of gammaherpesvirus replication and is conserved among all known gammaherpesviruses. We show here for KSHV that transcription of the gene encoding RTA is complex and leads to the expression of several isoforms of RTA with distinct functions. This observed complexity in KSHV RTA expression and function likely plays a critical role in the regulation of downstream viral and cellular gene expression, leading to the efficient production of mature virions. Copyright © 2016 American Society for Microbiology.

MAISTAS: a tool for automatic structural evaluation of alternative splicing products.

PubMed

Floris, Matteo; Raimondo, Domenico; Leoni, Guido; Orsini, Massimiliano; Marcatili, Paolo; Tramontano, Anna

2011-06-15

Analysis of the human genome revealed that the amount of transcribed sequence is an order of magnitude greater than the number of predicted and well-characterized genes. A sizeable fraction of these transcripts is related to alternatively spliced forms of known protein coding genes. Inspection of the alternatively spliced transcripts identified in the pilot phase of the ENCODE project has clearly shown that often their structure might substantially differ from that of other isoforms of the same gene, and therefore that they might perform unrelated functions, or that they might even not correspond to a functional protein. Identifying these cases is obviously relevant for the functional assignment of gene products and for the interpretation of the effect of variations in the corresponding proteins. Here we describe a publicly available tool that, given a gene or a protein, retrieves and analyses all its annotated isoforms, provides users with three-dimensional models of the isoform(s) of his/her interest whenever possible and automatically assesses whether homology derived structural models correspond to plausible structures. This information is clearly relevant. When the homology model of some isoforms of a gene does not seem structurally plausible, the implications are that either they assume a structure unrelated to that of the other isoforms of the same gene with presumably significant functional differences, or do not correspond to functional products. We provide indications that the second hypothesis is likely to be true for a substantial fraction of the cases. http://maistas.bioinformatica.crs4.it/.

Genetic disruption of the sh3pxd2a gene reveals an essential role in mouse development and the existence of a novel isoform of tks5.

PubMed

Cejudo-Martin, Pilar; Yuen, Angela; Vlahovich, Nicole; Lock, Peter; Courtneidge, Sara A; Díaz, Begoña

2014-01-01

Tks5 is a scaffold protein and Src substrate involved in cell migration and matrix degradation through its essential role in invadosome formation and function. We have previously described that Tks5 is fundamental for zebrafish neural crest cell migration in vivo. In the present study, we sought to investigate the function of Tks5 in mammalian development by analyzing mice mutant for sh3pxd2a, the gene encoding Tks5. Homozygous disruption of the sh3pxd2a gene by gene-trapping in mouse resulted in neonatal death and the presence of a complete cleft of the secondary palate. Interestingly, embryonic fibroblasts from homozygous gene-trap sh3pxd2a mice lacked only the highest molecular weight band of the characteristic Tks5 triplet observed in protein extracts, leaving the lower molecular weight bands unaffected. This finding, together with the existence of two human Expressed Sequence Tags lacking the first 5 exons of SH3PXD2A, made us hypothesize about the presence of a second alternative transcription start site located in intron V. We performed 5'RACE on mouse fibroblasts and isolated a new transcript of the sh3pxd2a gene encoding a novel Tks5 isoform, that we named Tks5β. This novel isoform diverges from the long form of Tks5 in that it lacks the PX-domain, which confers affinity for phosphatidylinositol-3,4-bisphosphate. Instead, Tks5β has a short unique amino terminal sequence encoded by the newly discovered exon 6β; this exon includes a start codon located 29 bp from the 5'-end of exon 6. Tks5β mRNA is expressed in MEFs and all mouse adult tissues analyzed. Tks5β is a substrate for the Src tyrosine kinase and its expression is regulated through the proteasome degradation pathway. Together, these findings indicate the essentiality of the larger Tks5 isoform for correct mammalian development and the transcriptional complexity of the sh3pxd2a gene.

Genetic Disruption of the Sh3pxd2a Gene Reveals an Essential Role in Mouse Development and the Existence of a Novel Isoform of Tks5

PubMed Central

Cejudo-Martin, Pilar; Yuen, Angela; Vlahovich, Nicole; Lock, Peter; Courtneidge, Sara A.; Díaz, Begoña

2014-01-01

Tks5 is a scaffold protein and Src substrate involved in cell migration and matrix degradation through its essential role in invadosome formation and function. We have previously described that Tks5 is fundamental for zebrafish neural crest cell migration in vivo. In the present study, we sought to investigate the function of Tks5 in mammalian development by analyzing mice mutant for sh3pxd2a, the gene encoding Tks5. Homozygous disruption of the sh3pxd2a gene by gene-trapping in mouse resulted in neonatal death and the presence of a complete cleft of the secondary palate. Interestingly, embryonic fibroblasts from homozygous gene-trap sh3pxd2a mice lacked only the highest molecular weight band of the characteristic Tks5 triplet observed in protein extracts, leaving the lower molecular weight bands unaffected. This finding, together with the existence of two human Expressed Sequence Tags lacking the first 5 exons of SH3PXD2A, made us hypothesize about the presence of a second alternative transcription start site located in intron V. We performed 5′RACE on mouse fibroblasts and isolated a new transcript of the sh3pxd2a gene encoding a novel Tks5 isoform, that we named Tks5β. This novel isoform diverges from the long form of Tks5 in that it lacks the PX-domain, which confers affinity for phosphatidylinositol-3,4-bisphosphate. Instead, Tks5β has a short unique amino terminal sequence encoded by the newly discovered exon 6β; this exon includes a start codon located 29 bp from the 5'-end of exon 6. Tks5β mRNA is expressed in MEFs and all mouse adult tissues analyzed. Tks5β is a substrate for the Src tyrosine kinase and its expression is regulated through the proteasome degradation pathway. Together, these findings indicate the essentiality of the larger Tks5 isoform for correct mammalian development and the transcriptional complexity of the sh3pxd2a gene. PMID:25259869

Subcellular distribution of serine acetyltransferase from Pisum sativum and characterization of an Arabidopsis thaliana putative cytosolic isoform.

PubMed

Ruffet, M L; Lebrun, M; Droux, M; Douce, R

1995-01-15

The intracellular compartmentation of serine acetyltransferase, a key enzyme in the L-cysteine biosynthesis pathway, has been investigated in pea (Pisum sativum) leaves, by isolation of organelles and fractionation of protoplasts. Enzyme activity was mainly located in mitochondria (approximately 76% of total cellular activity). Significant activity was also identified in both the cytosol (14% of total activity) and chloroplasts (10% of total activity). Three enzyme forms were separated by anion-exchange chromatography, and each form was found to be specific for a given intracellular compartment. To obtain cDNA encoding the isoforms, functional complementation experiments were performed using an Arabidopsis thaliana expression library and an Escherichia coli mutant devoid of serine acetyltransferase activity. This strategy allowed isolation of three distinct cDNAs encoding serine acetyltransferase isoforms, as confirmed by enzyme activity measurements, genomic hybridizations, and nucleotide sequencing. The cDNA and related gene for one of the three isoforms have been characterized. The predicted amino acid sequence shows that it encodes a polypeptide of M(r) 34,330 exhibiting 41% amino acid identity with the E. coli serine acetyltransferase. Since none of the general features of transit peptides could be observed in the N-terminal region of this isoform, we assume that it is a cytosolic form.

The Guinea Pig as a Model for Sporadic Alzheimer’s Disease (AD): The Impact of Cholesterol Intake on Expression of AD-Related Genes

PubMed Central

Ong, Daniel; Wijaya, Linda; Laws, Simon M.; Taddei, Kevin; Newman, Morgan; Lardelli, Michael; Martins, Ralph N.; Verdile, Giuseppe

2013-01-01

We investigated the guinea pig, Cavia porcellus, as a model for Alzheimer’s disease (AD), both in terms of the conservation of genes involved in AD and the regulatory responses of these to a known AD risk factor - high cholesterol intake. Unlike rats and mice, guinea pigs possess an Aβ peptide sequence identical to human Aβ. Consistent with the commonality between cardiovascular and AD risk factors in humans, we saw that a high cholesterol diet leads to up-regulation of BACE1 (β-secretase) transcription and down-regulation of ADAM10 (α-secretase) transcription which should increase release of Aβ from APP. Significantly, guinea pigs possess isoforms of AD-related genes found in humans but not present in mice or rats. For example, we discovered that the truncated PS2V isoform of human PSEN2, that is found at raised levels in AD brains and that increases γ-secretase activity and Aβ synthesis, is not uniquely human or aberrant as previously believed. We show that PS2V formation is up-regulated by hypoxia and a high-cholesterol diet while, consistent with observations in humans, Aβ concentrations are raised in some brain regions but not others. Also like humans, but unlike mice, the guinea pig gene encoding tau, MAPT, encodes isoforms with both three and four microtubule binding domains, and cholesterol alters the ratio of these isoforms. We conclude that AD-related genes are highly conserved and more similar to human than the rat or mouse. Guinea pigs represent a superior rodent model for analysis of the impact of dietary factors such as cholesterol on the regulation of AD-related genes. PMID:23805206

Localization and expression of clarin-1, the Clrn1 gene product, in auditory hair cells and photoreceptors

PubMed Central

Zallocchi, Marisa; Meehan, Daniel T.; Delimont, Duane; Askew, Charles; Garrige, Suneetha; Gratton, Michael Anne; Rothermund-Franklin, Christie A.; Cosgrove, Dominic

2009-01-01

The Usher syndrome 3A (CLRN1) gene encodes clarin-1, which is a member of the tetraspanin family of transmembrane proteins. Although identified more than 6 years ago, little is known about its localization or function in the eye and ear. We developed a polyclonal antibody that react with all clarin-1 isoforms and used it to characterize protein expression in cochlea and retina. In the cochlea, we observe clarin-1expression in the stereocilia of P0 mice, and in synaptic terminals present at the base of the auditory hair cells from E18 to P6. In the retina, clarin-1 localizes to the connecting cilia, inner segment of photoreceptors and to the ribbon synapses. RT-PCR from P0 cochlea and P28 retina show mRNAs encoding only isoforms 2 and 3. Western-blots show that only isoform 2 is present in protein extracts from these same tissues. We examined clarin-1 expression in the immortomouse-derived hair cell line UB/OC-1. Only isoform 2 is expressed in UB/OC-1 at both mRNA and protein levels, suggesting this isoform is biologically relevant to hair cell function. The protein co-localizes with microtubules and post-transgolgi vesicles. The sub-cellular localization of clarin-1 in hair cells and photoreceptors suggests it functions at both the basal and apical poles of neurosensoriepithelia. PMID:19539019

The Isoforms of the p53 Protein

PubMed Central

Khoury, Marie P.; Bourdon, Jean-Christophe

2010-01-01

p53 is a transcription factor with a key role in the maintenance of genetic stability and therefore preventing cancer formation. It belongs to a family of genes composed of p53, p63, and p73. The p63 and p73 genes have a dual gene structure with an internal promoter in intron-3 and together with alternative splicing, can express 6 and 29 mRNA variants, respectively. Such a complex expression pattern had not been previously described for the p53 gene, which was not consistent with our understanding of the evolution of the p53 gene family. Consequently, we revisited the human p53 gene structure and established that it encodes nine different p53 protein isoforms because of alternative splicing, alternative promoter usage, and alternative initiation sites of translation. Therefore, the human p53 gene family (p53, p63, and p73) has a dual gene structure. We determined that the dual gene structure is conserved in Drosophila and in zebrafish p53 genes. The conservation through evolution of the dual gene structure suggests that the p53 isoforms play an important role in p53 tumor-suppressor activity. We and others have established that the p53 isoforms can regulate cell-fate outcome in response to stress, by modulating p53 transcriptional activity in a promoter and stress-dependent manner. We have also shown that the p53 isoforms are abnormally expressed in several types of human cancers, suggesting that they play an important role in cancer formation. The determination of p53 isoforms' expression may help to link clinical outcome to p53 status and to improve cancer patient treatment. PMID:20300206

Pleiotropic biological activities of alternatively spliced TMPRSS2/ERG fusion gene transcripts

PubMed Central

Wang, Jianghua; Cai, Yi; Yu, Wendong; Ren, Chengxi; Spencer, David M.; Ittmann, Michael

2008-01-01

TMPRSS2/ERG gene fusions are found in the majority of prostate cancers; however, there is significant heterogeneity in the 5′ region of the alternatively spliced fusion gene transcripts. We have found that there is also significant heterogeneity within the coding exons as well. There is variable inclusion of a 72-bp exon and other novel alternatively spliced isoforms. To assess the biological significance of these alternatively spliced transcripts, we expressed various transcripts in primary prostatic epithelial cells and in an immortalized prostatic epithelial cell line, PNT1a. The fusion gene transcripts promoted proliferation, invasion and motility with variable activities that depended on the structure of the 5′ region encoding the TMPRSS2/ERG fusion and the presence of the 72-bp exon. Cotransfection of different isoforms further enhanced biological activity, mimicking the situation in vivo, in which multiple isoforms are expressed. Finally, knockdown of the fusion gene in VCaP cells resulted in inhibition of proliferation in vitro and tumor progression in an in vivo orthotopic mice model. Our results indicate that TMPRSS2/ERG fusion isoforms have variable biological activities promoting tumor initiation and progression and are consistent with our previous clinical observations indicating that certain TMPRSS2/ERG fusion isoforms are significantly correlated with more aggressive disease. PMID:18922926

Characterization of the Drosophila Group Ortholog to the Amino-Terminus of the Alpha-Thalassemia and Mental Retardation X-Linked (ATRX) Vertebrate Protein

PubMed Central

Hernández-Rodríguez, Benjamín; Campos, Adam; Montero, Daniel; Rudiño, Enrique; Vázquez, Martha; Zurita, Mario; Valadez-Graham, Viviana

2014-01-01

The human ATRX gene encodes hATRX, a chromatin-remodeling protein harboring an helicase/ATPase and ADD domains. The ADD domain has two zinc fingers that bind to histone tails and mediate hATRX binding to chromatin. dAtrx, the putative ATRX homolog in Drosophila melanogaster, has a conserved helicase/ATPase domain but lacks the ADD domain. A bioinformatic search of the Drosophila genome using the human ADD sequence allowed us to identify the CG8290 annotated gene, which encodes three ADD harboring- isoforms generated by alternative splicing. This Drosophila ADD domain is highly similar in structure and in the amino acids which mediate the histone tail contacts to the ADD domain of hATRX as shown by 3D modeling. Very recently the CG8290 annotated gene has been named dadd1. We show through pull-down and CoIP assays that the products of the dadd1 gene interact physically with dAtrxL and HP1a and all of them mainly co-localize in the chromocenter, although euchromatic localization can also be observed through the chromosome arms. We confirm through ChIP analyses that these proteins are present in vivo in the same heterochromatic regions. The three isoforms are expressed throughout development. Flies carrying transheterozygous combinations of the dadd1 and atrx alleles are semi-viable and have different phenotypes including the appearance of melanotic masses. Interestingly, the dAdd1-b and c isoforms have extra domains, such as MADF, which suggest newly acquired functions of these proteins. These results strongly support that, in Drosophila, the atrx gene diverged and that the dadd1-encoded proteins participate with dAtrx in some cellular functions such as heterochromatin maintenance. PMID:25437195

Molecular cloning and characterization of a novel salt-inducible gene encoding an acidic isoform of PR-5 protein in soybean (Glycine max [L.] Merr.).

PubMed

Onishi, M; Tachi, H; Kojima, T; Shiraiwa, M; Takahara, H

2006-10-01

We identified a novel salt-inducible soybean gene encoding an acidic-isoform of pathogenesis-related protein group 5 (PR-5 protein). The soybean PR-5-homologous gene, designated as Glycine max osmotin-like protein, acidic isoform (GmOLPa)), encodes a putative polypeptide having an N-terminal signal peptide. The mature GmOLPa protein without the signal peptide has a calculated molecular mass of 21.5 kDa and a pI value of 4.4, and was distinguishable from a known PR-5-homologous gene of soybean (namely P21 protein) through examination of the structural features. A comparison with two intracellular salt-inducible PR-5 proteins, tobacco osmotin and tomato NP24, revealed that GmOLPa did not have a C-terminal extension sequence functioning as a vacuole-targeting motif. The GmOLPa gene was transcribed constitutively in the soybean root and was induced almost exclusively in the root during 24 h of high-salt stress (300 mM NaCl). Interestingly, GmOLPa gene expression in the stem and leaf, not observed until 24 h, was markedly induced at 48 and 72 h after commencement of the high-salt stress. Abscisic acid (ABA) and dehydration also induced expression of the GmOLPa gene in the root; additionally, dehydration slightly induced expression in the stem and leaf. In fact, the 5'-upstream sequence of the GmOLPa gene contained several putative cis-elements known to be involved in responsiveness to ABA and dehydration, e.g. ABA-responsive element (ABRE), MYB/MYC, and low temperature-responsive element (LTRE). These results suggested that GmOLPa may function as a protective PR-5 protein in the extracellular space of the soybean root in response to high-salt stress and dehydration.

Identification and differential induction of the expression of aquaporins by salinity in broccoli plants.

PubMed

Muries, Beatriz; Faize, Mohamed; Carvajal, Micaela; Martínez-Ballesta, María Del Carmen

2011-04-01

Plant aquaporins belong to a large superfamily of conserved proteins called the major intrinsic proteins (MIPs). There is limited information about the diversity of MIPs and their water transport capacity in broccoli (Brassica oleracea) plants. In this study, the cDNAs of isoforms of Plasma Membrane Intrinsic Proteins (PIPs), a class of aquaporins, from broccoli roots have been partially sequenced. Thus, sequencing experiments led to the identification of eight PIP1 and three PIP2 genes encoding PIPs in B. oleracea plants. The occurrence of different gene products encoding PIPs suggests that they may play different roles in plants. The screening of their expression as well as the expression of two specific PIP2 isoforms (BoPIP2;2 and BoPIP2;3), in different organs and under different salt-stress conditions in two varieties, has helped to unravel the function and the regulation of PIPs in plants. Thus, a high degree of BoPIP2;3 expression in mature leaves suggests that this BoPIP2;3 isoform plays important roles, not only in root water relations but also in the physiology and development of leaves. In addition, differences between gene and protein patterns led us to consider that mRNA synthesis is inhibited by the accumulation of the corresponding encoded protein. Therefore, transcript levels, protein abundance determination and the integrated hydraulic architecture of the roots must be considered in order to interpret the response of broccoli to salinity.

The significance of alternative transcripts for Caenorhabditis elegans transcription factor genes, based on expression pattern analysis

PubMed Central

2013-01-01

Background Sequence-specific DNA-binding proteins, with their paramount importance in the regulation of expression of the genetic material, are encoded by approximately 5% of the genes in an animal’s genome. But it is unclear to what extent alternative transcripts from these genes may further increase the complexity of the transcription factor complement. Results Of the 938 potential C. elegans transcription factor genes, 197 were annotated in WormBase as encoding at least two distinct isoforms. Evaluation of prior evidence identified, with different levels of confidence, 50 genes with alternative transcript starts, 23 with alternative transcript ends, 35 with alternative splicing and 34 with alternative transcripts generated by a combination of mechanisms, leaving 55 that were discounted. Expression patterns were determined for transcripts for a sample of 29 transcription factor genes, concentrating on those with alternative transcript starts for which the evidence was strongest. Seamless fosmid recombineering was used to generate reporter gene fusions with minimal modification to assay expression of specific transcripts while maintaining the broad genomic DNA context and alternative transcript production. Alternative transcription factor gene transcripts were typically expressed with identical or substantially overlapping distributions rather than in distinct domains. Conclusions Increasingly sensitive sequencing technologies will reveal rare transcripts but many of these are clearly non-productive. The majority of the transcription factor gene alternative transcripts that are productive may represent tolerable noise rather than encoding functionally distinct isoforms. PMID:23586691

Northwestern’s Kelleher Laboratory Develops Top-Down KRAS Isoform Assay to Detect Protein Mutations and Modifications | Office of Cancer Clinical Proteomics Research

Cancer.gov

Mutations in the RAS genes — KRAS, HRAS, and NRAS — have been identified in approximately 30% of all human cancers. While RAS gene family members encode proteins that are pivotal for cytoplasmic cell signaling, RAS oncogenes

The structural role of high molecular weight tropomyosins in dipteran indirect flight muscle and the effect of phosphorylation.

PubMed

Mateos, Jesús; Herranz, Raúl; Domingo, Alberto; Sparrow, John; Marco, Roberto

2006-01-01

In Drosophila melanogaster two high molecular weight tropomyosin isoforms, historically named heavy troponins (TnH-33 and TnH-34), are encoded by the Tm1 tropomyosin gene. They are specifically expressed in the indirect flight muscles (IFM). Their N-termini are conventional and complete tropomyosin sequences, but their C-termini consist of different IFM-specific domains that are rich in proline, alanine, glycine and glutamate. The evidence indicates that in Diptera these IFM-specific isoforms are conserved and are not troponins, but heavy tropomyosins (TmH). We report here that they are post-translationally modified by several phosphorylations in their C-termini in mature flies, but not in recently emerged flies that are incapable of flight. From stoichiometric measurements of thin filament proteins and interactions of the TmH isoforms with the standard Drosophila IFM tropomyosin isoform (protein 129), we propose that the TmH N-termini are integrated into the thin filament structural unit as tropomyosin dimers. The phosphorylated C-termini remain unlocated and may be important in IFM stretch-activation. Comparison of the Tm1 and Tm2 gene sequences shows a complete conservation of gene organisation in other Drosophilidae, such as Drosophila pseudoobscura, while in Anopheles gambiae only one exon encodes a single C-terminal domain, though overall gene organization is maintained. Interestingly, in Apis mellifera (hymenopteran), while most of the Tm1 and Tm2 gene features are conserved, the gene lacks any C-terminal exons. Instead these sequences are found at the 3' end of the troponin I gene. In this insect order, as in Lethocerus (hemipteran), the original designation of troponin H (TnH) should be retained. We discuss whether the insertion of the IFM-specific pro-ala-gly-glu-rich domain into the tropomyosin or troponin I genes in different insect orders may be related to proposals that the IFM stretch activation mechanism has evolved independently several times in higher insects.

The rat alpha-tropomyosin gene generates a minimum of six different mRNAs coding for striated, smooth, and nonmuscle isoforms by alternative splicing.

PubMed Central

Wieczorek, D F; Smith, C W; Nadal-Ginard, B

1988-01-01

Tropomyosin (TM), a ubiquitous protein, is a component of the contractile apparatus of all cells. In nonmuscle cells, it is found in stress fibers, while in sarcomeric and nonsarcomeric muscle, it is a component of the thin filament. Several different TM isoforms specific for nonmuscle cells and different types of muscle cell have been described. As for other contractile proteins, it was assumed that smooth, striated, and nonmuscle isoforms were each encoded by different sets of genes. Through the use of S1 nuclease mapping, RNA blots, and 5' extension analyses, we showed that the rat alpha-TM gene, whose expression was until now considered to be restricted to muscle cells, generates many different tissue-specific isoforms. The promoter of the gene appears to be very similar to other housekeeping promoters in both its pattern of utilization, being active in most cell types, and its lack of any canonical sequence elements. The rat alpha-TM gene is split into at least 13 exons, 7 of which are alternatively spliced in a tissue-specific manner. This gene arrangement, which also includes two different 3' ends, generates a minimum of six different mRNAs each with the capacity to code for a different protein. These distinct TM isoforms are expressed specifically in nonmuscle and smooth and striated (cardiac and skeletal) muscle cells. The tissue-specific expression and developmental regulation of these isoforms is, therefore, produced by alternative mRNA processing. Moreover, structural and sequence comparisons among TM genes from different phyla suggest that alternative splicing is evolutionarily a very old event that played an important role in gene evolution and might have appeared concomitantly with or even before constitutive splicing. Images PMID:3352602

Pectin engineering to modify product quality in potato.

PubMed

Ross, Heather A; Morris, Wayne L; Ducreux, Laurence J M; Hancock, Robert D; Verrall, Susan R; Morris, Jenny A; Tucker, Gregory A; Stewart, Derek; Hedley, Pete E; McDougall, Gordon J; Taylor, Mark A

2011-10-01

Although processed potato tuber texture is an important trait that influences consumer preference, a detailed understanding of tuber textural properties at the molecular level is lacking. Previous work has identified tuber pectin methyl esterase (PME) activity as a potential factor impacting on textural properties, and the expression of a gene encoding an isoform of PME (PEST1) was associated with cooked tuber textural properties. In this study, a transgenic approach was undertaken to investigate further the impact of the PEST1 gene. Antisense and over-expressing potato lines were generated. In over-expressing lines, tuber PME activity was enhanced by up to 2.3-fold; whereas in antisense lines, PME activity was decreased by up to 62%. PME isoform analysis indicated that the PEST1 gene encoded one isoform of PME. Analysis of cell walls from tubers from the over-expressing lines indicated that the changes in PME activity resulted in a decrease in pectin methylation. Analysis of processed tuber texture demonstrated that the reduced level of pectin methylation in the over-expressing transgenic lines was associated with a firmer processed texture. Thus, there is a clear link between PME activity, pectin methylation and processed tuber textural properties. © 2011 The Authors. Plant Biotechnology Journal © 2011 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd.

Differential accumulation of β-carotene and tissue specific expression of phytoene synthase (MaPsy) gene in banana (Musa sp) cultivars.

PubMed

Dhandapani, R; Singh, V P; Arora, A; Bhattacharya, R C; Rajendran, Ambika

2017-12-01

An experiment was conducted with twelve major Indian banana cultivars to investigate the molecular relationship between the differential accumulation of β-carotene in peel and pulp of the banana fruit and carotenoid biosynthetic pathway genes. The high performance liquid chromatography showed that all banana cultivars accumulated two-three fold more β-carotene in non-edible portion of the banana fruit. However, Nendran , a famous orange fleshed cultivar of South India, had high β-carotene content (1362 µg/100 g) in edible pulp. The gene encoding Musa accuminata phytoene synthase ( MaPsy ) was successfully amplified using a pair of degenerate primers designed from Oncidium orchid. The deduced amino acid sequences shared a high level of identity to phytoene synthase gene from other plants. Gene expression analysis confirmed the presence of two isoforms ( MaPsy1 and MaPsy2 ) of MaPsy gene in banana fruits. Presence of two isoforms of MaPsy gene in peel and one in pulp confirmed the differential accumulation of β-carotene in banana fruits. However, Nendran accumulated more β-carotene in edible pulp due to presence of both the isoforms of MaPsy gene. Thus, carotenoid accumulation is a tissue specific process strongly dependent on differential expression pattern of two isoforms of MaPsy gene in banana.

Cell-type specific features of circular RNA expression.

PubMed

Salzman, Julia; Chen, Raymond E; Olsen, Mari N; Wang, Peter L; Brown, Patrick O

2013-01-01

Thousands of loci in the human and mouse genomes give rise to circular RNA transcripts; at many of these loci, the predominant RNA isoform is a circle. Using an improved computational approach for circular RNA identification, we found widespread circular RNA expression in Drosophila melanogaster and estimate that in humans, circular RNA may account for 1% as many molecules as poly(A) RNA. Analysis of data from the ENCODE consortium revealed that the repertoire of genes expressing circular RNA, the ratio of circular to linear transcripts for each gene, and even the pattern of splice isoforms of circular RNAs from each gene were cell-type specific. These results suggest that biogenesis of circular RNA is an integral, conserved, and regulated feature of the gene expression program.

Loss of functional K+ channels encoded by ether-à-go-go-related genes in mouse myometrium prior to labour onset

PubMed Central

Greenwood, I A; Yeung, S Y; Tribe, R M; Ohya, S

2009-01-01

There is a growing appreciation that ion channels encoded by the ether-à-go-go-related gene family have a functional impact in smooth muscle in addition to their accepted role in cardiac myocytes and neurones. This study aimed to assess the expression of ERG1–3 (KCNH1–3) genes in the murine myometrium (smooth muscle layer of the uterus) and determine the functional impact of the ion channels encoded by these genes in pregnant and non-pregnant animals. Quantitative RT-PCR did not detect message for ERG2 and 3 in whole myometrial tissue extracts. In contrast, message for two isoforms of mERG1 were readily detected with mERG1a more abundant than mERG1b. In isometric tension studies of non-pregnant myometrium, the ERG channel blockers dofetilide (1 μm), E4031 (1 μm) and Be-KM1 (100 nm) increased spontaneous contractility and ERG activators (PD118057 and NS1643) inhibited spontaneous contractility. In contrast, neither ERG blockade nor activation had any effect on the inherent contractility in myometrium from late pregnant (19 days gestation) animals. Moreover, dofetilide-sensitive K+ currents with distinctive ‘hooked’ kinetics were considerably smaller in uterine myocytes from late pregnant compared to non-pregnant animals. Expression of mERG1 isoforms did not alter throughout gestation or upon delivery, but the expression of genes encoding auxillary subunits (KCNE) were up-regulated considerably. This study provides the first evidence for a regulation of ERG-encoded K+ channels as a precursor to late pregnancy physiological activity. PMID:19332483

NHS-A isoform of the NHS gene is a novel interactor of ZO-1.

PubMed

Sharma, Shiwani; Koh, Katrina S Y; Collin, Caitlin; Dave, Alpana; McMellon, Amy; Sugiyama, Yuki; McAvoy, John W; Voss, Anne K; Gécz, Jozef; Craig, Jamie E

2009-08-15

Mutations in the NHS (Nance-Horan Syndrome) gene lead to severe congenital cataracts, dental defects and sometimes mental retardation. NHS encodes two protein isoforms, NHS-A and -1A that display cell-type dependent differential expression and localization. Here we demonstrate that of these two isoforms, the NHS-A isoform associates with the cell membrane in the presence of intercellular contacts and it immunoprecipitates with the tight junction protein ZO-1 in MDCK (Madin Darby Canine Kidney) epithelial cells and in neonatal rat lens. The NHS-1A isoform however is a cytoplasmic protein. Both Nhs isoforms are expressed during mouse development. Immunolabelling of developing mouse with the anti-NHS antibody that detects both isoforms revealed the protein in the developing head including the eye and brain. It was primarily expressed in epithelium including neural epithelium and certain vascular endothelium but only weakly expressed in mesenchymal cells. In the epithelium and vascular endothelium the protein associated with the cell membrane and co-localized with ZO-1, which indirectly indicates expression of the Nhs-A isoform in these structures. Membrane localization of the protein in the lens vesicle similarly supports Nhs-A expression. In conclusion, the NHS-A isoform of NHS is a novel interactor of ZO-1 and may have a role at tight junctions. This isoform is important in mammalian development especially of the organs in the head.

The combined expression patterns of Ikaros isoforms characterize different hematological tumor subtypes.

PubMed

Orozco, Carlos A; Acevedo, Andrés; Cortina, Lazaro; Cuellar, Gina E; Duarte, Mónica; Martín, Liliana; Mesa, Néstor M; Muñoz, Javier; Portilla, Carlos A; Quijano, Sandra M; Quintero, Guillermo; Rodriguez, Miriam; Saavedra, Carlos E; Groot, Helena; Torres, María M; López-Segura, Valeriano

2013-01-01

A variety of genetic alterations are considered hallmarks of cancer development and progression. The Ikaros gene family, encoding for key transcription factors in hematopoietic development, provides several examples as genetic defects in these genes are associated with the development of different types of leukemia. However, the complex patterns of expression of isoforms in Ikaros family genes has prevented their use as clinical markers. In this study, we propose the use of the expression profiles of the Ikaros isoforms to classify various hematological tumor diseases. We have standardized a quantitative PCR protocol to estimate the expression levels of the Ikaros gene exons. Our analysis reveals that these levels are associated with specific types of leukemia and we have found differences in the levels of expression relative to five interexonic Ikaros regions for all diseases studied. In conclusion, our method has allowed us to precisely discriminate between B-ALL, CLL and MM cases. Differences between the groups of lymphoid and myeloid pathologies were also identified in the same way.

Mitrocomin from the jellyfish Mitrocoma cellularia with deleted C-terminal tyrosine reveals a higher bioluminescence activity compared to wild type photoprotein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Burakova, Ludmila P.; Natashin, Pavel V.; Markova, Svetlana V.

The full-length cDNA genes encoding five new isoforms of Ca2 +-regulated photoprotein mitrocomin from a small tissue sample of the outer bell margin containing photocytes of only one specimen of the luminous jellyfish Mitrocoma cellularia were cloned, sequenced, and characterized after their expression in Escherichia coli and subsequent purification. The analysis of cDNA nucleotide sequences encoding mitrocomin isoforms allowed suggestion that two isoforms might be the products of two allelic genes differing in one amino acid residue (64R/Q) whereas other isotypes appear as a result of transcriptional mutations. In addition, the crystal structure of mitrocomin was determined at 1.30 Åmore » resolution which expectedly revealed a high similarity with the structures of other hydromedusan photoproteins. Although mitrocomin isoforms reveal a high degree of identity of amino acid sequences, they vary in specific bioluminescence activities. At that, all isotypes displayed the identical bioluminescence spectra (473–474 nm with no shoulder at 400 nm). Fluorescence spectra of Ca2 +-discharged mitrocomins were almost identical to their light emission spectra similar to the case of Ca2 +-discharged aequorin, but different from Ca2 +-discharged obelins and clytin which fluorescence is red-shifted by 25–30 nm from bioluminescence spectra. The main distinction of mitrocomin from other hydromedusan photoproteins is an additional Tyr at the C-terminus. Using site-directed mutagenesis, we showed that this Tyr is not important for bioluminescence because its deletion even increases specific activity and efficiency of apo-mitrocomin conversion into active photoprotein, in contrast to C-terminal Pro of other photoproteins. Since genes in a population generally exist as different isoforms, it makes us anticipate the cloning of even more isoforms of mitrocomin and other hydromedusan photoproteins with different bioluminescence properties.« less

The presence of both negative and positive elements in the 5'-flanking sequence of the rat Na,K-ATPase alpha 3 subunit gene are required for brain expression in transgenic mice.

PubMed Central

Pathak, B G; Neumann, J C; Croyle, M L; Lingrel, J B

1994-01-01

The Na,K-ATPase is an integral plasma membrane protein consisting of alpha and beta subunits, each of which has discrete isoforms expressed in a tissue-specific manner. Of the three functional alpha isoform genes, the one encoding the alpha 3 isoform is the most tissue-restricted in its expression, being found primarily in the brain. To identify regions of the alpha 3 isoform gene that are involved in directing expression in the brain, a 1.6 kb 5'-flanking sequence was attached to a reporter gene, chloramphenicol acetyltransferase (CAT). The alpha 3-CAT chimeric gene construct was microinjected into fertilized mouse eggs, and transgenic mice were produced. Analysis of adult transgenic mice from different lines revealed that the transgene is expressed primarily in the brain. To further delineate regions that are needed for conferring expression in this tissue, systematic deletions of the 5'-flanking sequence of the alpha 3-CAT fusion constructs were made and analyzed, again using transgenic mice. The results from these analyses indicate that DNA sequences required for mediating brain-specific expression of the alpha 3 isoform gene are present within 210 bp upstream of the transcription initiation site. alpha 3-CAT promoter constructs containing scanning mutations in this region were also assayed in transgenic mice. These studies have identified both a functional neural-restrictive silencer element as well as a positively acting cis element. Images PMID:7984427

Expression of the tachykinin receptor mRNAs in healthy human colon.

PubMed

Jaafari, Nadia; Hua, Guoqiang; Adélaïde, José; Julé, Yvon; Imbert, Jean

2008-12-03

Tachykinins are a family of neuropeptides, involved in a variety of physiological and pathological processes occurring in the gastrointestinal tract. They act via three distinct types of receptors, tachykinin NK(1), NK(2), and NK(3) receptors, which belong to the family of G protein-coupled receptors. The aim of the present study was to characterize, for the first time in the healthy human colon, the TACR(1), TACR(2) and TACR(3) mRNAs encoding the three different tachykinin receptors and to measure their relative expression by quantitative reverse transcription-PCR assay. Our results confirm the broad distribution of the tachykinin receptors but evidenced significant differences in the expression level of their respective mRNAs. A higher expression level of the TACR2 mRNA alpha isoform, the gene encoding the functional tachykinin NK(2) receptor, was observed in comparison to TACR1 and TACR3 mRNAs genes encoding for NK(1) and NK(3) receptors respectively. The prevalence of the TACR2 mRNA alpha isoform strongly suggests a major involvement of tachykinin NK(2) receptor in the regulation of human colonic functions.

Effects of PTCs on nonsense-mediated mRNA decay are dependent on PTC location.

PubMed

Moon, Heegyum; Zheng, Xuexiu; Loh, Tiing Jen; Jang, Ha Na; Liu, Yongchao; Jung, Da-Woon; Williams, Darren R; Shen, Haihong

2017-03-01

The récepteur d'origine nantais (RON) gene is a proto-oncogene that is responsible for encoding the human macrophage-stimulating protein (MSP) 1 receptor. MSP activation induces RON-mediated cell dissociation, migration and matrix invasion. Isoforms of RON that exclude exons 5 and 6 encode the RONΔ160 protein, which promotes cell transformation in vitro and tumor metastasis in vivo . Premature termination codons (PTCs) in exons activate the nonsense-mediated mRNA decay (NMD) signaling pathway. The present study demonstrated that PTCs at various locations in the alternative exons 5 and 6 could induce NMD of the majority of the spliced, or partially spliced, isoforms. However, the isoforms that excluded exon 6 or exons 5 and 6 were markedly increased when produced from mutated minigenes with inserted PTCs. Furthermore, the unspliced isoform of intron 5 was not observed to be decreased by the presence of PTCs. Notably, these effects may be dependent on the location of the PTCs. The current study demonstrated a novel mechanism underlying the regulation of NMD in alternative splicing.

Subcellular distribution of human RDM1 protein isoforms and their nucleolar accumulation in response to heat shock and proteotoxic stress.

PubMed

Messaoudi, Lydia; Yang, Yun-Gui; Kinomura, Aiko; Stavreva, Diana A; Yan, Gonghong; Bortolin-Cavaillé, Marie-Line; Arakawa, Hiroshi; Buerstedde, Jean-Marie; Hainaut, Pierre; Cavaillé, Jérome; Takata, Minoru; Van Dyck, Eric

2007-01-01

The RDM1 gene encodes a RNA recognition motif (RRM)-containing protein involved in the cellular response to the anti-cancer drug cisplatin in vertebrates. We previously reported a cDNA encoding the full-length human RDM1 protein. Here, we describe the identification of 11 human cDNAs encoding RDM1 protein isoforms. This repertoire is generated by alternative pre-mRNA splicing and differential usage of two translational start sites, resulting in proteins with long or short N-terminus and a great diversity in the exonic composition of their C-terminus. By using tagged proteins and fluorescent microscopy, we examined the subcellular distribution of full-length RDM1 (renamed RDM1alpha), and other RDM1 isoforms. We show that RDM1alpha undergoes subcellular redistribution and nucleolar accumulation in response to proteotoxic stress and mild heat shock. In unstressed cells, the long N-terminal isoforms displayed distinct subcellular distribution patterns, ranging from a predominantly cytoplasmic to almost exclusive nuclear localization, suggesting functional differences among the RDM1 proteins. However, all isoforms underwent stress-induced nucleolar accumulation. We identified nuclear and nucleolar localization determinants as well as domains conferring cytoplasmic retention to the RDM1 proteins. Finally, RDM1 null chicken DT40 cells displayed an increased sensitivity to heat shock, compared to wild-type (wt) cells, suggesting a function for RDM1 in the heat-shock response.

Splice-mediated Variants of Proteins (SpliVaP) - data and characterization of changes in signatures among protein isoforms due to alternative splicing.

PubMed

Floris, Matteo; Orsini, Massimiliano; Thanaraj, Thangavel Alphonse

2008-10-02

It is often the case that mammalian genes are alternatively spliced; the resulting alternate transcripts often encode protein isoforms that differ in amino acid sequences. Changes among the protein isoforms can alter the cellular properties of proteins. The effect can range from a subtle modulation to a complete loss of function. (i) We examined human splice-mediated protein isoforms (as extracted from a manually curated data set, and from a computationally predicted data set) for differences in the annotation for protein signatures (Pfam domains and PRINTS fingerprints) and we characterized the differences & their effects on protein functionalities. An important question addressed relates to the extent of protein isoforms that may lack any known function in the cell. (ii) We present a database that reports differences in protein signatures among human splice-mediated protein isoform sequences. (i) Characterization: The work points to distinct sets of alternatively spliced genes with varying degrees of annotation for the splice-mediated protein isoforms. Protein molecular functions seen to be often affected are those that relate to: binding, catalytic, transcription regulation, structural molecule, transporter, motor, and antioxidant; and the processes that are often affected are nucleic acid binding, signal transduction, and protein-protein interactions. Signatures are often included/excluded and truncated in length among protein isoforms; truncation is seen as the predominant type of change. Analysis points to the following novel aspects: (a) Analysis using data from the manually curated Vega indicates that one in 8.9 genes can lead to a protein isoform of no "known" function; and one in 18 expressed protein isoforms can be such an "orphan" isoform; the corresponding numbers as seen with computationally predicted ASD data set are: one in 4.9 genes and one in 9.8 isoforms. (b) When swapping of signatures occurs, it is often between those of same functional classifications. (c) Pfam domains can occur in varying lengths, and PRINTS fingerprints can occur with varying number of constituent motifs among isoforms - since such a variation is seen in large number of genes, it could be a general mechanism to modulate protein function. (ii) The reported resource (at http://www.bioinformatica.crs4.org/tools/dbs/splivap/) provides the community ability to access data on splice-mediated protein isoforms (with value-added annotation such as association with diseases) through changes in protein signatures.

Cell-Type Specific Features of Circular RNA Expression

PubMed Central

Salzman, Julia; Chen, Raymond E.; Olsen, Mari N.; Wang, Peter L.; Brown, Patrick O.

2013-01-01

Thousands of loci in the human and mouse genomes give rise to circular RNA transcripts; at many of these loci, the predominant RNA isoform is a circle. Using an improved computational approach for circular RNA identification, we found widespread circular RNA expression in Drosophila melanogaster and estimate that in humans, circular RNA may account for 1% as many molecules as poly(A) RNA. Analysis of data from the ENCODE consortium revealed that the repertoire of genes expressing circular RNA, the ratio of circular to linear transcripts for each gene, and even the pattern of splice isoforms of circular RNAs from each gene were cell-type specific. These results suggest that biogenesis of circular RNA is an integral, conserved, and regulated feature of the gene expression program. PMID:24039610

Forced expression of the Ikaros 6 isoform in human placental blood CD34(+) cells impairs their ability to differentiate toward the B-lymphoid lineage.

PubMed

Tonnelle, C; Bardin, F; Maroc, C; Imbert, A M; Campa, F; Dalloul, A; Schmitt, C; Chabannon, C

2001-11-01

Studies in mice suggest that the Ikaros (Ik) gene encodes several isoforms and is a critical regulator of hematolymphoid differentiation. Little is known on the role of Ikaros in human stem cell differentiation. Herein, the biological consequences of the forced expression of Ikaros 6 (Ik6) in human placental blood CD34(+) progenitors are evaluated. Ik6 is one of the isoforms produced from the Ikaros premessenger RNA by alternative splicing and is thought to behave as a dominant negative isoform of the gene product because it lacks the DNA binding domain present in transcriptionally active isoforms. The results demonstrate that human cord blood CD34(+) cells that express high levels of Ik6 as a result of retrovirally mediated gene transfer have a reduced capacity to produce lymphoid B cells in 2 independent assays: (1) in vitro reinitiation of human hematopoiesis during coculture with the MS-5 murine stromal cell line and (2) xenotransplantation in nonobese diabetic-severe combined immunodeficient mice. These results suggest that Ikaros plays an important role in stem cell commitment in humans and that the balance between the different isoforms is a key element of this regulatory system; they support the hypothesis that posttranscriptional events can participate in the control of human hematopoietic differentiation.

Nance-Horan syndrome protein, NHS, associates with epithelial cell junctions.

PubMed

Sharma, Shiwani; Ang, Sharyn L; Shaw, Marie; Mackey, David A; Gécz, Jozef; McAvoy, John W; Craig, Jamie E

2006-06-15

Nance-Horan syndrome, characterized by congenital cataracts, craniofacial, dental abnormalities and mental disturbances, is an X-linked disorder with significant phenotypic heterogeneity. Affected individuals have mutations in the NHS (Nance-Horan syndrome) gene typically resulting in premature truncation of the protein. This report underlines the complexity of the regulation of the NHS gene that transcribes several isoforms. We demonstrate the differential expression of the two NHS isoforms, NHS-A and NHS-1A, and differences in the subcellular localization of the proteins encoded by these isoforms. This may in part explain the pleiotropic features of the syndrome. We show that the endogenous and exogenous NHS-A isoform localizes to the cell membrane of mammalian cells in a cell-type-dependent manner and that it co-localizes with the tight junction (TJ) protein ZO-1 in the apical aspect of cell membrane in epithelial cells. We also show that the NHS-1A isoform is a cytoplasmic protein. In the developing mammalian lens, we found continuous expression of NHS that became restricted to the lens epithelium in pre- and postnatal lens. Consistent with the in vitro findings, the NHS-A isoform associates with the apical cell membrane in the lens epithelium. This study suggests that disturbances in intercellular contacts underlie cataractogenesis in the Nance-Horan syndrome. NHS is the first gene localized at TJs that has been implicated in congenital cataracts.

ΔN-P63α and TA-P63α exhibit intrinsic differences in transactivation specificities that depend on distinct features of DNA target sites

PubMed Central

Foggetti, Giorgia; Raimondi, Ivan; Campomenosi, Paola; Menichini, Paola

2014-01-01

TP63 is a member of the TP53 gene family that encodes for up to ten different TA and ΔN isoforms through alternative promoter usage and alternative splicing. Besides being a master regulator of gene expression for squamous epithelial proliferation, differentiation and maintenance, P63, through differential expression of its isoforms, plays important roles in tumorigenesis. All P63 isoforms share an immunoglobulin-like folded DNA binding domain responsible for binding to sequence-specific response elements (REs), whose overall consensus sequence is similar to that of the canonical p53 RE. Using a defined assay in yeast, where P63 isoforms and RE sequences are the only variables, and gene expression assays in human cell lines, we demonstrated that human TA- and ΔN-P63α proteins exhibited differences in transactivation specificity not observed with the corresponding P73 or P53 protein isoforms. These differences 1) were dependent on specific features of the RE sequence, 2) could be related to intrinsic differences in their oligomeric state and cooperative DNA binding, and 3) appeared to be conserved in evolution. Since genotoxic stress can change relative ratio of TA- and ΔN-P63α protein levels, the different transactivation specificity of each P63 isoform could potentially influence cellular responses to specific stresses. PMID:24926492

The intranuclear PEX domain of MMP involves proliferation, migration, and metastasis of aggressive adenocarcinoma cells.

PubMed

Okusha, Yuka; Eguchi, Takanori; Sogawa, Chiharu; Okui, Tatsuo; Nakano, Keisuke; Okamoto, Kuniaki; Kozaki, Ken-Ichi

2018-05-15

Members of matrix metalloproteinase (MMP) family promote cancer cell migration, invasion, and metastasis through alteration of the tumor milieu, intracellular signaling pathways, and transcription. We examined gene expression signatures of colon adenocarcinoma cell lines with different metastatic potentials and found that rapidly metastatic cells powerfully expressed genes encoding MMP3 and MMP9. The non-proteolytic PEX isoform and proteolytic isoforms of MMPs were significantly expressed in the metastatic cells in vitro. Knockdown of MMP3 attenuated cancer cell migration and invasion in vitro and lung metastasis in vivo. Profound nuclear localization of MMP3/PEX was found in tumor-stroma marginal area. In contrast, MMP9 was localized in central area of subcutaneous tumors. Overexpression of the PEX isoform of MMP3 promoted proliferation and migration of the rapidly metastatic cells in vitro. Taken together, the non-proteolytic PEX isoform of MMPs locating in cell nuclei involves proliferation, migration, and subsequent metastasis of aggressive adenocarcinoma cells. © 2018 Wiley Periodicals, Inc.

Genome-wide identification and evolutionary analysis of algal LPAT genes involved in TAG biosynthesis using bioinformatic approaches.

PubMed

Misra, Namrata; Panda, Prasanna Kumar; Parida, Bikram Kumar

2014-12-01

Lysophosphatidyl acyltransferase (LPAT) is one of the major triacylglycerol synthesis enzymes, controlling the metabolic flow of lysophosphatidic acid to phosphatidic acid. Experimental studies in Arabidopsis have shown that LPAT activity is exhibited primarily by three distinct isoforms, namely the plastid-located LPAT1, the endoplasmic reticulum-located LPAT2, and the soluble isoform of LPAT (solLPAT). In this study, 24 putative genes representing all LPAT isoforms were identified from the analysis of 11 complete genomes including green algae, red algae, diatoms and higher plants. We observed LPAT1 and solLPAT genes to be ubiquitously present in nearly all genomes examined, whereas LPAT2 genes to have evolved more recently in the plant lineage. Phylogenetic analysis indicated that LPAT1, LPAT2 and solLPAT have convergently evolved through separate evolutionary paths and belong to three different gene families, which was further evidenced by their wide divergence at gene structure and sequence level. The genome distribution supports the hypothesis that each gene encoding a LPAT is not duplicated. Mapping of exon-intron structure of LPAT genes to the domain structure of proteins across different algal and plant species indicates that exon shuffling plays no role in the evolution of LPAT genes. Besides the previously defined motifs, several conserved consensus sequences were discovered which could be useful to distinguish different LPAT isoforms. Taken together, this study will enable the generation of experimental approximations to better understand the functional role of algal LPAT in lipid accumulation.

DNA polymerase ι: The long and the short of it!

PubMed

Frank, Ekaterina G; McLenigan, Mary P; McDonald, John P; Huston, Donald; Mead, Samantha; Woodgate, Roger

2017-10-01

The cDNA encoding human DNA polymerase ι (POLI) was cloned in 1999. At that time, it was believed that the POLI gene encoded a protein of 715 amino acids. Advances in DNA sequencing technologies led to the realization that there is an upstream, in-frame initiation codon that would encode a DNA polymerase ι (polι) protein of 740 amino acids. The extra 25 amino acid region is rich in acidic residues (11/25) and is reasonably conserved in eukaryotes ranging from fish to humans. As a consequence, the curated Reference Sequence (RefSeq) database identified polι as a 740 amino acid protein. However, the existence of the 740 amino acid polι has never been shown experimentally. Using highly specific antibodies to the 25 N-terminal amino acids of polι, we were unable to detect the longer 740 amino acid (ι-long) isoform in western blots. However, trace amounts of the ι-long isoform were detected after enrichment by immunoprecipitation. One might argue that the longer isoform may have a distinct biological function, if it exhibits significant differences in its enzymatic properties from the shorter, well-characterized 715 amino acid polι. We therefore purified and characterized recombinant full-length (740 amino acid) polι-long and compared it to full-length (715 amino acid) polι-short in vitro. The metal ion requirements for optimal catalytic activity differ slightly between ι-long and ι-short, but under optimal conditions, both isoforms exhibit indistinguishable enzymatic properties in vitro. We also report that like ι-short, the ι-long isoform can be monoubiquitinated and polyubiuquitinated in vivo, as well as form damage induced foci in vivo. We conclude that the predominant isoform of DNA polι in human cells is the shorter 715 amino acid protein and that if, or when, expressed, the longer 740 amino acid isoform has identical properties to the considerably more abundant shorter isoform. Published by Elsevier B.V.

Differential Splicing of Oncogenes and Tumor Suppressor Genes in African- and Caucasian-American Populations: Contributing Factor in Prostate Cancer Disparities

DTIC Science & Technology

2015-10-01

signaling protein as defined by in vitro assays and mouse xenograft studies, ii) is associated with worse prognosis in patients, and iii) is resistant to...available. Specific Aim 2. To characterize oncogenic differences of splice variant pairs in vivo using xenograft animal models. Task 1. Validate...idelalisib as defined by in vitro assays and mouse xenograft models. In contrast, the corresponding EA isoform (PI3Kδ-L) encodes a less aggressive isoform

Selective ablation of Ppp1cc gene in testicular germ cells causes oligo-teratozoospermia and infertility in mice.

PubMed

Sinha, Nilam; Puri, Pawan; Nairn, Angus C; Vijayaraghavan, Srinivasan

2013-11-01

The four isoforms of serine/threonine phosphoprotein phosphatase 1 (PP1), derived from three genes, are among the most conserved proteins known. The Ppp1cc gene encodes two alternatively spliced variants, PP1 gamma1 (PPP1CC1) and PP1 gamma2 (PPP1CC2). Global deletion of the Ppp1cc gene, which causes loss of both isoforms, results in male infertility due to impaired spermatogenesis. This phenotype was assumed to be due to the loss of PPP1CC2, which is abundant in testis. While PPP1CC2 is predominant, other PP1 isoforms are also expressed in testis. Given the significant homology between the four PP1 isoforms, the lack of compensation by the other PP1 isoforms for loss of one, only in testis, is surprising. Here we document, for the first time, expression patterns of the PP1 isoforms in postnatal developing and adult mouse testis. The timing and sites of testis expression of PPP1CC1 and PPP1CC2 in testis are nonoverlapping. PPP1CC2 is the only one of the four PP1 isoforms not detected in sertoli cells and spermatogonia. Conversely, PPP1CC2 may be the only PP1 isoform expressed in postmeiotic germ cells. Deletion of the Ppp1cc gene in germ cells at the differentiated spermatogonia stage of development and beyond in Stra8 promoter-driven Cre transgenic mice results in oligo-terato-asthenozoospermia and male infertility, thus phenocopying global Ppp1cc null (-/-) mice. Taken together, these results confirm that spermatogenic defects observed in the global Ppp1cc knockout mice and in mice expressing low levels of PPP1CC2 in testis are due to compromised functions of PPP1CC2 in meiotic and postmeiotic germ cells.

Comparative genomic analysis reveals a novel mitochondrial isoform of human rTS protein and unusual phylogenetic distribution of the rTS gene

PubMed Central

Liang, Ping; Nair, Jayakumar R; Song, Lei; McGuire, John J; Dolnick, Bruce J

2005-01-01

Background The rTS gene (ENOSF1), first identified in Homo sapiens as a gene complementary to the thymidylate synthase (TYMS) mRNA, is known to encode two protein isoforms, rTSα and rTSβ. The rTSβ isoform appears to be an enzyme responsible for the synthesis of signaling molecules involved in the down-regulation of thymidylate synthase, but the exact cellular functions of rTS genes are largely unknown. Results Through comparative genomic sequence analysis, we predicted the existence of a novel protein isoform, rTS, which has a 27 residue longer N-terminus by virtue of utilizing an alternative start codon located upstream of the start codon in rTSβ. We observed that a similar extended N-terminus could be predicted in all rTS genes for which genomic sequences are available and the extended regions are conserved from bacteria to human. Therefore, we reasoned that the protein with the extended N-terminus might represent an ancestral form of the rTS protein. Sequence analysis strongly predicts a mitochondrial signal sequence in the extended N-terminal of human rTSγ, which is absent in rTSβ. We confirmed the existence of rTS in human mitochondria experimentally by demonstrating the presence of both rTSγ and rTSβ proteins in mitochondria isolated by subcellular fractionation. In addition, our comprehensive analysis of rTS orthologous sequences reveals an unusual phylogenetic distribution of this gene, which suggests the occurrence of one or more horizontal gene transfer events. Conclusion The presence of two rTS isoforms in mitochondria suggests that the rTS signaling pathway may be active within mitochondria. Our report also presents an example of identifying novel protein isoforms and for improving gene annotation through comparative genomic analysis. PMID:16162288

OCA-B regulation of B-cell development and function.

PubMed

Teitell, Michael A

2003-10-01

The transcriptional co-activator OCA-B [for Oct co-activator from B cells, also known as OBF-1 (OCT-binding factor-1) and Bob1] is not required for B-cell genesis but does regulate subsequent B-cell development and function. OCA-B deficient mice show strain-specific, partial blocks at multiple stages of B-cell maturation and a complete disruption of germinal center formation in all strains, causing humoral immune deficiency and susceptibility to infection. OCA-B probably exerts its effects through the regulation of octamer-motif controlled gene expression. The OCA-B gene encodes two proteins of distinct molecular weight, designated p34 and p35. The p34 isoform localizes in the nucleus, whereas the p35 isoform is myristoylated and is bound to the cytoplasmic membrane. p35 can traffic to the nucleus and probably activates octamer-dependent transcription, although this OCA-B isoform might regulate B cells through membrane-related signal transduction.

IQCJ-SCHIP1, a novel fusion transcript encoding a calmodulin-binding IQ motif protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kwasnicka-Crawford, Dorota A.; Carson, Andrew R.; Scherer, Stephen W.

The existence of transcripts that span two adjacent, independent genes is considered rare in the human genome. This study characterizes a novel human fusion gene named IQCJ-SCHIP1. IQCJ-SCHIP1 is the longest isoform of a complex transcriptional unit that bridges two separate genes that encode distinct proteins, IQCJ, a novel IQ motif containing protein and SCHIP1, a schwannomin interacting protein that has been previously shown to interact with the Neurofibromatosis type 2 (NF2) protein. IQCJ-SCHIP1 is located on the chromosome 3q25 and comprises a 1692-bp transcript encompassing 11 exons spanning 828 kb of the genomic DNA. We show that IQCJ-SCHIP1 mRNAmore » is highly expressed in the brain. Protein encoded by the IQCJ-SCHIP1 gene was localized to cytoplasm and actin-rich regions and in differentiated PC12 cells was also seen in neurite extensions.« less

Nesprin-2 epsilon: A novel nesprin isoform expressed in human ovary and Ntera-2 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lam, Le Thanh; Boehm, Sabrina V.; Roberts, Roland G.

2011-08-26

Highlights: {yields} A novel epsilon isoform of nesprin-2 has been discovered. {yields} This 120 kDa protein was predicted by bioinformatic analysis, but has not previously been observed. {yields} It is the main isoform expressed in a teratocarcinoma cell line and is also found in ovary. {yields} Like other nesprins, it is located at the nuclear envelope. {yields} We suggest it may have a role in very early development or in some ovary-specific function. -- Abstract: The nuclear envelope-associated cytoskeletal protein, nesprin-2, is encoded by a large gene containing several internal promoters that produce shorter isoforms. In a study of Ntera-2more » teratocarcinoma cells, a novel isoform, nesprin-2-epsilon, was found to be the major mRNA and protein product of the nesprin-2 gene. Its existence was predicted by bioinformatic analysis, but this is the first direct demonstration of both the mRNA and the 120 kDa protein which is located at the nuclear envelope. In a panel of 21 adult and foetal human tissues, the nesprin-2-epsilon mRNA was strongly expressed in ovary but was a minor isoform elsewhere. The expression pattern suggests a possible link with very early development and a likely physiological role in ovary.« less

Isoform Evolution in Primates through Independent Combination of Alternative RNA Processing Events

PubMed Central

Zhang, Shi-Jian; Wang, Chenqu; Yan, Shouyu; Fu, Aisi; Luan, Xuke; Li, Yumei; Sunny Shen, Qing; Zhong, Xiaoming; Chen, Jia-Yu; Wang, Xiangfeng; Chin-Ming Tan, Bertrand; He, Aibin; Li, Chuan-Yun

2017-01-01

Abstract Recent RNA-seq technology revealed thousands of splicing events that are under rapid evolution in primates, whereas the reliability of these events, as well as their combination on the isoform level, have not been adequately addressed due to its limited sequencing length. Here, we performed comparative transcriptome analyses in human and rhesus macaque cerebellum using single molecule long-read sequencing (Iso-seq) and matched RNA-seq. Besides 359 million RNA-seq reads, 4,165,527 Iso-seq reads were generated with a mean length of 14,875 bp, covering 11,466 human genes, and 10,159 macaque genes. With Iso-seq data, we substantially expanded the repertoire of alternative RNA processing events in primates, and found that intron retention and alternative polyadenylation are surprisingly more prevalent in primates than previously estimated. We then investigated the combinatorial mode of these alternative events at the whole-transcript level, and found that the combination of these events is largely independent along the transcript, leading to thousands of novel isoforms missed by current annotations. Notably, these novel isoforms are selectively constrained in general, and 1,119 isoforms have even higher expression than the previously annotated major isoforms in human, indicating that the complexity of the human transcriptome is still significantly underestimated. Comparative transcriptome analysis further revealed 502 genes encoding selectively constrained, lineage-specific isoforms in human but not in rhesus macaque, linking them to some lineage-specific functions. Overall, we propose that the independent combination of alternative RNA processing events has contributed to complex isoform evolution in primates, which provides a new foundation for the study of phenotypic difference among primates. PMID:28957512

Identification of BCAP, a new protein associated with basal bodies and centrioles.

PubMed

Ponsard, Cecile; Seltzer, Virginie; Perret, Eric; Tournier, Frederic; Middendorp, Sandrine

2007-05-01

Cilia exert critical functions in numerous organisms, including that of cell motility, fluid transport and protozoan locomotion. Defects in this organelle can lead to lethal pathologies in humans, including primary ciliary dyskinesia. An understanding of the cilia formation process would lead to better characterization of defects involved in such pathologies. In the present study, we identified a gene encoding a novel human protein, BCAP for Basal body Centriole-Associated Protein, which shares homologies with a previously described protein, Outer Dense Fiber 2 (ODF2). ODF2, a major component of the sperm tail cytoskeleton, is required for the formation of mother centriole distal/subdistal appendages and the generation of primary cilia. Here, we show that the bcap gene contains 18 alternatively spliced exons and encodes five different isoforms, three long and two short ones. BCAP is preferentially expressed in cilia/flagella containing tissues. Moreover, its expression is correlated with cilia formation during mucociliary differentiation of human nasal epithelial cells. Using immunofluorescence analyses, BCAP was localized within basal bodies of ciliated cells and within centrioles of proliferating cells. In light of the several spliced isoforms of BCAP and the particular localization of the protein, BCAP isoforms could play distinct roles in cilia and in centrosomes.

Expression Profiles of the Trehalose-6-Phosphate Synthase Gene Associated With Thermal Stress in Ostrinia furnacalis (Lepidoptera: Crambidae)

PubMed Central

Jin, Tingting; Gao, Yulin; He, Kanglai

2018-01-01

Abstract Trehalose is the major blood sugar in insects. Physiological significance of this compound has been extensively reported. Trehalose-6-phosphate synthase (TPS) is an important enzyme in the trehalose biosynthesis pathway. Full-length cDNAs of TPS (Of tps) and its alternative splicing isoform (Of tps_isoformI) were cloned from the Asian corn borer (ACB), Ostrinia furnacalis (Guenée; Lepidoptera: Crambidae) larvae. The Of tps and Of tps_isoformI transcripts were 2913 and 1689 bp long, contained 2529 and 1293 bp open reading frames encoding proteins of 842 and 430 amino acids with a molecular mass of 94.4 and 48.6 kDa, respectively. Transcriptional profiling and response to thermal stress of Of tps gene were determined by quantitative real-time PCR showing that the Of tps was predominantly expressed in the larval fat body, significantly enhanced during molting and transformation; and thermal stress also induced Of tps expression. Gene structure analysis is indicating that one TPS domain and one trehalose-6-phosphate phosphatase (TPP) domain were located at the N- and C-termini of Of        TPS, respectively, while only the TPS domain was detected in OfTPS_isoformI. Three-dimensional modeling and heterologous expression were developed to predict the putative functions of OfTPS and Of   TPS_isoformI. We infer that the expression of Of tps gene is thermally induced and might be crucial for larvae survival.

Alternative pre-mRNA splicing of Toll-like receptor signaling components in peripheral blood mononuclear cells from patients with ARDS.

PubMed

Blumhagen, Rachel Z; Hedin, Brenna R; Malcolm, Kenneth C; Burnham, Ellen L; Moss, Marc; Abraham, Edward; Huie, Tristan J; Nick, Jerry A; Fingerlin, Tasha E; Alper, Scott

2017-11-01

A key physiological feature of acute respiratory distress syndrome (ARDS) is inflammation. Toll-like receptor (TLR) signaling is required to combat the infection that underlies many ARDS cases but also contributes to pathological inflammation. Several TLR signaling pathway genes encoding positive effectors of inflammation also produce alternatively spliced mRNAs encoding negative regulators of inflammation. An imbalance between these isoforms could contribute to pathological inflammation and disease severity. To determine whether splicing in TLR pathways is altered in patients with ARDS, we monitored alternative splicing of MyD88 and IRAK1 , two genes that function in multiple TLR pathways. The MyD88 and IRAK1 genes produce long proinflammatory mRNAs (MyD88 L and IRAK1) and shorter anti-inflammatory mRNAs (MyD88 S and IRAK1c). We quantified mRNA encoding inflammatory cytokines and MyD88 and IRAK1 isoforms in peripheral blood mononuclear cells (PBMCs) from 104 patients with ARDS and 30 healthy control subjects. We found that MyD88 pre-mRNA splicing is altered in patients with ARDS in a proinflammatory direction. We also observed altered MyD88 isoform levels in a second critically ill patient cohort, suggesting that these changes may not be unique to ARDS. Early in ARDS, PBMC IRAK1c levels were associated with patient survival. Despite the similarities in MyD88 and IRAK1 alternative splicing observed in previous in vitro studies, there were differences in how MyD88 and IRAK1 alternative splicing was altered in patients with ARDS. We conclude that pre-mRNA splicing of TLR signaling genes is altered in patients with ARDS, and further investigation of altered splicing may lead to novel prognostic and therapeutic approaches. Copyright © 2017 the American Physiological Society.

Heterologous expression of laccase cDNA from Ceriporiopsis subvermispora yields copper-activated apoprotein and complex isoform patterns

Treesearch

Luis F. Larrondo; Marcela Avila; Loreto Salas; Dan Cullen; Rafael Vicuna

2003-01-01

Analysis of genomic clones encoding a putative laccase in homokaryon strains of Ceriporiopsis subvermispora led to the identification of an allelic variant of the previously described lcs-1 gene. A cDNA clone corresponding to this gene was expressed in Aspergillus nidulans and in Aspergillus niger. Enzyme assays and Western blots showed that both hosts secreted active...

Serotonin and Early Cognitive Development: Variation in the Tryptophan Hydroxylase 2 Gene Is Associated with Visual Attention in 7-Month-Old Infants

ERIC Educational Resources Information Center

Leppanen, Jukka M.; Peltola, Mikko J.; Puura, Kaija; Mantymaa, Mirjami; Mononen, Nina; Lehtimaki, Terho

2011-01-01

Background: Allelic variation in the promoter region of a gene that encodes tryptophan hydroxylase isoform 2 (TPH2), a rate-limiting enzyme of serotonin synthesis in the central nervous system, has been associated with variations in cognitive function and vulnerability to affective spectrum disorders. Little is known about the effects of this gene…

Identification of a Novel C-Terminal Truncated WT1 Isoform with Antagonistic Effects against Major WT1 Isoforms

PubMed Central

Tatsumi, Naoya; Hojo, Nozomi; Sakamoto, Hiroyuki; Inaba, Rena; Moriguchi, Nahoko; Matsuno, Keiko; Fukuda, Mari; Matsumura, Akihide; Hayashi, Seiji; Morimoto, Soyoko; Nakata, Jun; Fujiki, Fumihiro; Nishida, Sumiyuki; Nakajima, Hiroko; Tsuboi, Akihiro; Oka, Yoshihiro; Hosen, Naoki; Sugiyama, Haruo; Oji, Yusuke

2015-01-01

The Wilms’ tumor gene WT1 consists of 10 exons and encodes a zinc finger transcription factor. There are four major WT1 isoforms resulting from alternative splicing at two sites, exon 5 (17AA) and exon 9 (KTS). All major WT1 isoforms are overexpressed in leukemia and solid tumors and play oncogenic roles such as inhibition of apoptosis, and promotion of cell proliferation, migration and invasion. In the present study, a novel alternatively spliced WT1 isoform that had an extended exon 4 (designated as exon 4a) with an additional 153 bp (designated as 4a sequence) at the 3’ end was identified and designated as an Ex4a(+)WT1 isoform. The insertion of exon 4a resulted in the introduction of premature translational stop codons in the reading frame in exon 4a and production of C-terminal truncated WT1 proteins lacking zinc finger DNA-binding domain. Overexpression of the truncated Ex4a(+)WT1 isoform inhibited the major WT1-mediated transcriptional activation of anti-apoptotic Bcl-xL gene promoter and induced mitochondrial damage and apoptosis. Conversely, suppression of the Ex4a(+)WT1 isoform by Ex4a-specific siRNA attenuated apoptosis. These results indicated that the Ex4a(+)WT1 isoform exerted dominant negative effects on anti-apoptotic function of major WT1 isoforms. Ex4a(+)WT1 isoform was endogenously expressed as a minor isoform in myeloid leukemia and solid tumor cells and increased regardless of decrease in major WT1 isoforms during apoptosis, suggesting the dominant negative effects on anti-apoptotic function of major WT1 isoforms. These results indicated that Ex4a(+)WT1 isoform had an important physiological function that regulated oncogenic function of major WT1 isoforms. PMID:26090994

The gene transformer-2 of Sciara (Diptera, Nematocera) and its effect on Drosophila sexual development.

PubMed

Martín, Iker; Ruiz, María F; Sánchez, Lucas

2011-03-15

The gene transformer-2, which is involved in sex determination, has been studied in Drosophila, Musca, Ceratitis, Anastrepha and Lucilia. All these members of Diptera belong to the suborder Brachycera. In this work, it is reported the isolation and characterisation of genes transformer-2 of the dipterans Sciara ocellaris and Bradysia coprophila (formerly Sciara coprophila), which belong to the much less extensively analysed Sciaridae Family of the Suborder Nematocera, which is paraphyletic with respect to Suborder Brachycera. The transformer-2 genes of the studied Sciara species were found to be transcribed in both sexes during development and adult life, in both the soma and germ lines. They produced a single primary transcript, which follows the same alternative splicing in both sexes, giving rise to different mRNAs isoforms. In S. ocellaris the most abundant mRNA isoform encoded a full-length protein of 251 amino acids, while that of B. coprophila encoded a protein of 246 amino acids. Both showed the features of the SR protein family. The less significant mRNA isoforms of both species encoded truncated, presumably non-functional Transformer-2 proteins. The comparison of the functional Sciara Transformer-2 proteins among themselves and those of other insects revealed the greatest degree of conservation in the RRM domain and linker region. In contrast, the RS1 and RS2 domains showed extensive variation with respect to their number of amino acids and their arginine-serine (RS) dipeptide content. The expression of S. ocellaris Transformer-2 protein in Drosophila XX pseudomales lacking the endogenous transformer-2 function caused their partial feminisation. The transformer-2 genes of both Sciaridae species encode a single protein in both sexes that shares the characteristics of the Transformer-2 proteins of other insects. These proteins showed conserved sex-determination function in Drosophila; i.e., they were able to form a complex with the endogenous Drosophila Transformer protein that controls the female-specific splicing of the Drosophila doublesex pre-mRNA. However, it appears that the complex formed between the Drosophila Transformer protein and the Sciara Transformer-2 protein is less effective at inducing the female-specific splicing of the endogenous Drosophila doublesex pre-mRNA than the DrosophilaTransformer-Transformer2 complex. This suggests the existence of species-specific co-evolution of the Transformer and Transformer-2 proteins.

The gene transformer-2 of Sciara (Diptera, Nematocera) and its effect on Drosophila sexual development

PubMed Central

2011-01-01

Background The gene transformer-2, which is involved in sex determination, has been studied in Drosophila, Musca, Ceratitis, Anastrepha and Lucilia. All these members of Diptera belong to the suborder Brachycera. In this work, it is reported the isolation and characterisation of genes transformer-2 of the dipterans Sciara ocellaris and Bradysia coprophila (formerly Sciara coprophila), which belong to the much less extensively analysed Sciaridae Family of the Suborder Nematocera, which is paraphyletic with respect to Suborder Brachycera. Results The transformer-2 genes of the studied Sciara species were found to be transcribed in both sexes during development and adult life, in both the soma and germ lines. They produced a single primary transcript, which follows the same alternative splicing in both sexes, giving rise to different mRNAs isoforms. In S. ocellaris the most abundant mRNA isoform encoded a full-length protein of 251 amino acids, while that of B. coprophila encoded a protein of 246 amino acids. Both showed the features of the SR protein family. The less significant mRNA isoforms of both species encoded truncated, presumably non-functional Transformer-2 proteins. The comparison of the functional Sciara Transformer-2 proteins among themselves and those of other insects revealed the greatest degree of conservation in the RRM domain and linker region. In contrast, the RS1 and RS2 domains showed extensive variation with respect to their number of amino acids and their arginine-serine (RS) dipeptide content. The expression of S. ocellaris Transformer-2 protein in Drosophila XX pseudomales lacking the endogenous transformer-2 function caused their partial feminisation. Conclusions The transformer-2 genes of both Sciaridae species encode a single protein in both sexes that shares the characteristics of the Transformer-2 proteins of other insects. These proteins showed conserved sex-determination function in Drosophila; i.e., they were able to form a complex with the endogenous Drosophila Transformer protein that controls the female-specific splicing of the Drosophila doublesex pre-mRNA. However, it appears that the complex formed between the Drosophila Transformer protein and the Sciara Transformer-2 protein is less effective at inducing the female-specific splicing of the endogenous Drosophila doublesex pre-mRNA than the DrosophilaTransformer-Transformer2 complex. This suggests the existence of species-specific co-evolution of the Transformer and Transformer-2 proteins. PMID:21406087

Identification of a novel splice variant of human PD-L1 mRNA encoding an isoform-lacking Igv-like domain.

PubMed

He, Xian-hui; Xu, Li-hui; Liu, Yi

2005-04-01

To investigate the expression and regulation of PD-1 ligand 1 (PD-L1) in peripheral blood mononuclear cells (PBMC). The cDNA encoding human PD-L1 precursor was cloned from the total RNA extracted from the resting and phorbol dibutyrate plus ionomycin- or phytohemagglutinin-activated PBMC, by reverse transcription polymerase chain reaction (RT-PCR), and independent clones were sequenced and analyzed. The expression and subcellular localization were examined in transiently transfected cells. The PD-L1 gene expression in different PBMC was also analyzed by RT-PCR. A novel human PD-L1 splice variant was identified from the activated PBMC. It was generated by splicing out exon? encoding an immunoglobulin variable domain (Igv)-like domain but retaining all other exons without a frame-shift. Consequently, the putative translated protein contained all other domains including the transmembrane region except for the Igv-like domain. Furthermore, the conventional isoform was expressed on the plasma surface whereas the novel isoform showed a pattern of intracellular membrane distribution in transiently transfected K562 cells. In addition, the expression pattern of the PD-L1 splice variant was variable in different individuals and in different cellular status. PD-L1 expression may be regulated at the posttranscriptional level through alternative splicing, and modulation of the PD-L1 isoform expression may influence the outcome of specific immune responses in the peripheral tissues.

The GMD1 and GMD2 genes of Arabidopsis encode isoforms of GDP-D-mannose 4,6-dehydratase with cell type-specific expression patterns.

PubMed

Bonin, Christopher P; Freshour, Glenn; Hahn, Michael G; Vanzin, Gary F; Reiter, Wolf-Dieter

2003-06-01

l-Fucose (l-Fuc) is a monosaccharide constituent of plant cell wall polysaccharides and glycoproteins. The committing step in the de novo synthesis of l-Fuc is catalyzed by GDP-d-mannose 4,6-dehydratase, which, in Arabidopsis, is encoded by the GMD1 and GMD2 (MUR1) genes. To determine the functional significance of this genetic redundancy, the expression patterns of both genes were investigated via promoter-beta-glucuronidase fusions and immunolocalization of a Fuc-containing epitope. GMD2 is expressed in most cell types of the root, with the notable exception of the root tip where strong expression of GMD1 is observed. Within shoot organs, GMD1::GUS expression is confined to stipules and pollen grains leading to fucosylation of the walls of these cell types in the mur1 mutant. These results suggest that GMD2 represents the major housekeeping gene for the de novo synthesis of GDP-l-Fuc, whereas GMD1 expression is limited to a number of specialized cell types. We conclude that the synthesis of GDP-l-Fuc is controlled in a cell-autonomous manner by differential expression of two isoforms of the same enzyme.

Tissue-specific and pathogen-induced regulation of a Nicotiana plumbaginifolia beta-1,3-glucanase gene.

PubMed Central

Castresana, C; de Carvalho, F; Gheysen, G; Habets, M; Inzé, D; Van Montagu, M

1990-01-01

The Nicotiana plumbaginifolia gn1 gene encoding a beta-1,3-glucanase isoform has been characterized. The gn1 product represents an isoform distinct from the previously identified tobacco beta-1,3-glucanases. By expressing gn1 in Escherichia coli, we have determined directly that the encoded protein does, indeed, correspond to a beta-1,3-glucanase. In N. plumbaginifolia, gn1 was found to be expressed in roots and older leaves. Transgenic tobacco plants containing the 5'-noncoding region of gn1 fused to the beta-glucuronidase (GUS) reporter gene also showed maximum levels of GUS activity in roots and older leaves. No detectable activity was present in the upper part of the transgenic plants with the exception of stem cells at the bases of emerging shoots. The expression conferred by the gn1 promoter was differentially induced in response to specific plant stress treatments. Studies of three plant-bacteria interactions showed high levels of GUS activity when infection resulted in a hypersensitive reaction. Increased gene expression was confined to cells surrounding the necrotic lesions. The observed expression pattern suggests that the characterized beta-1,3-glucanase plays a role both in plant development and in the defense response against pathogen infection. PMID:2152158

Structural and functional characterisation of FOXO/Acan-DAF-16 from the parasitic nematode Angiostrongylus cantonensis.

PubMed

Yan, Baolong; Sun, Weiwei; Yan, Lanzhu; Zhang, Liangliang; Zheng, Yuan; Zeng, Yuzhen; Huang, Huicong; Liang, Shaohui

2016-12-01

Fork head box transcription factors subfamily O (FoxO) is regarded to be significant in cell-cycle control, cell differentiation, ageing, stress response, apoptosis, tumour formation and DNA damage repair. In the free-living nematode Caenorhabditis elegans, the FoxO transcription factor is encoded by Ce-daf-16, which is negatively regulated by insulin-like signaling (IIS) and involved in promoting dauer formation through bringing about its hundreds of downstream genes expression. In nematode parasites, orthologues of daf-16 from several species have been identified, with functions in rescue of dauer phenotypes determined in a surrogate system C. elegans. In this study, we identified the FoxO encoding gene, Acan-daf-16, from the parasitic nematode Angiostrongylus cantonensis, and determined the genomic structures, transcripts and functions far more thorough in longevity, stress resistance and dauer formation. Acan-daf-16 encodes two proteins, Acan-DAF-16A and Acan-DAF-16B, consisting of 555 and 491 amino acids, respectively. Both isoforms possess the highly conserved fork head domains. Acan-daf-16A and Acan-daf-16B are expressed from distinct promoters. The expression patterns of Acan-daf-16 isoforms in the C. elegans surrogate system showed that p Acan-daf-16a:gfp was expressed in all cells of C. elegans, including the pharynx, and the expression of p Acan-daf-16b:gfp was restricted to the pharynx. In addition to the same genomic organization to the orthologue in C. elegans, Ce-daf-16, both Acan-DAF-16 isoforms could restore the C. elegans daf-16(mg54) mutation in longevity, dauer formation and stress resistance, in spite of the partial complementation of Acan-DAF-16B isoform in longevity. These findings provide further evidence of the functional conservation of DAF-16s between parasitic nematodes and the free-living nematode C. elegans. Copyright © 2016. Published by Elsevier B.V.

Glutaminase and MMP-9 Downregulation in Cortex and Hippocampus of LPA1 Receptor Null Mice Correlate with Altered Dendritic Spine Plasticity

PubMed Central

Peñalver, Ana; Campos-Sandoval, José A.; Blanco, Eduardo; Cardona, Carolina; Castilla, Laura; Martín-Rufián, Mercedes; Estivill-Torrús, Guillermo; Sánchez-Varo, Raquel; Alonso, Francisco J.; Pérez-Hernández, Mercedes; Colado, María I.; Gutiérrez, Antonia; de Fonseca, Fernando Rodríguez; Márquez, Javier

2017-01-01

Lysophosphatidic acid (LPA) is an extracellular lipid mediator that regulates nervous system development and functions acting through G protein-coupled receptors (GPCRs). Here we explore the crosstalk between LPA1 receptor and glutamatergic transmission by examining expression of glutaminase (GA) isoforms in different brain areas isolated from wild-type (WT) and KOLPA1 mice. Silencing of LPA1 receptor induced a severe down-regulation of Gls-encoded long glutaminase protein variant (KGA) (glutaminase gene encoding the kidney-type isoforms, GLS) protein expression in several brain regions, particularly in brain cortex and hippocampus. Immunohistochemical assessment of protein levels for the second type of glutaminase (GA) isoform, glutaminase gene encoding the liver-type isoforms (GLS2), did not detect substantial differences with regard to WT animals. The regional mRNA levels of GLS were determined by real time RT-PCR and did not show significant variations, except for prefrontal and motor cortex values which clearly diminished in KO mice. Total GA activity was also significantly reduced in prefrontal and motor cortex, but remained essentially unchanged in the hippocampus and rest of brain regions examined, suggesting activation of genetic compensatory mechanisms and/or post-translational modifications to compensate for KGA protein deficit. Remarkably, Golgi staining of hippocampal regions showed an altered morphology of glutamatergic pyramidal cells dendritic spines towards a less mature filopodia-like phenotype, as compared with WT littermates. This structural change correlated with a strong decrease of active matrix-metalloproteinase (MMP) 9 in cerebral cortex and hippocampus of KOLPA1 mice. Taken together, these results demonstrate that LPA signaling through LPA1 influence expression of the main isoenzyme of glutamate biosynthesis with strong repercussions on dendritic spines maturation, which may partially explain the cognitive and learning defects previously reported for this colony of KOLPA1 mice. PMID:28928633

ELAV, a Drosophila neuron-specific protein, mediates the generation of an alternatively spliced neural protein isoform.

PubMed

Koushika, S P; Lisbin, M J; White, K

1996-12-01

Tissue-specific alternative pre-mRNA splicing is a widely used mechanism for gene regulation and the generation of different protein isoforms, but relatively little is known about the factors and mechanisms that mediate this process. Tissue-specific RNA-binding proteins could mediate alternative pre-mRNA splicing. In Drosophila melanogaster, the RNA-binding protein encoded by the elav (embryonic lethal abnormal visual system) gene is a candidate for such a role. The ELAV protein is expressed exclusively in neurons, and is important for the formation and maintenance of the nervous system. In this study, photoreceptor neurons genetically depleted of ELAV, and elav-null central nervous system neurons, were analyzed immunocytochemically for the expression of neural proteins. In both situations, the lack of ELAV corresponded with a decrease in the immunohistochemical signal of the neural-specific isoform of Neuroglian, which is generated by alternative splicing. Furthermore, when ELAV was expressed ectopically in cells that normally express only the non-neural isoform of Neuroglian, we observed the generation of the neural isoform of Neuroglian. Drosophila ELAV promotes the generation of the neuron-specific isoform of Neuroglian by the regulation of pre-mRNA splicing. The findings reported in this paper demonstrate that ELAV is necessary, and the ectopic expression of ELAV in imaginal disc cells is sufficient, to mediate neuron-specific alternative splicing.

Assignment and expression patterns of porcine muscle-specific isoform of phosphoglycerate mutase gene.

PubMed

Qiu, Haifang; Zhao, Shuhong; Xu, Xuewen; Yerle, Martine; Liu, Bang

2008-05-01

It has been reported that the muscle-specific isoform (type M, PGAM2) of phosphoglycerate mutase (PGAM) is a housekeeping enzyme; it catalyzes the conversion of 3-phosphoglycerate into 2-phosphoglycerate in the glycolysis process to release energy. It is encoded by the Pgam2 gene. In this study, the cDNA of the porcine Pgam2 was cloned. This gene contains an open reading frame of 765 bp encoding a protein of 253 residues, and the predicted protein sequences share high similarity with other mammalians, 96% identity with humans, and 94% identity with mouse and rats. Pgam2 was mapped to SSC18q13-q21 by the RH panel. In this region, there are several QTLs, such as fat ratio, lean percentage, and diameter of muscle fiber, which affect meat production and quality. The reverse transcriptase-polymerase chain reaction revealed that the porcine Pgam2 gene was mainly expressed in the muscle tissue (skeletal muscle and cardiac muscle), and was expressed highly at skeletal muscle development stages (embryonic periods: 33, 65, and 90 days post-conception (dpc); postnatal pigs: 4 days and adult). This indicates that the Pgam2 gene plays an important role in muscle growth and development. In addition, it was demonstrated that PGAM2 locates both in cytoplasm and nuclei, and takes part in the glycometabolism process of cytoplasm and nuclei.

Structure and polymorphism of the mouse prion protein gene.

PubMed Central

Westaway, D; Cooper, C; Turner, S; Da Costa, M; Carlson, G A; Prusiner, S B

1994-01-01

Missense mutations in the prion protein (PrP) gene, overexpression of the cellular isoform of PrP (PrPC), and infection with prions containing the scrapie isoform of PrP (PrPSc) all cause neurodegenerative disease. To understand better the physiology and expression of PrPC, we retrieved mouse PrP gene (Prn-p) yeast artificial chromosome (YAC), cosmid, phage, and cDNA clones. Physical mapping positions Prn-p approximately 300 kb from ecotropic virus integration site number 4 (Evi-4), compatible with failure to detect recombination between Prn-p and Evi-4 in genetic crosses. The Prn-pa allele encompasses three exons, with exons 1 and 2 encoding the mRNA 5' untranslated region. Exon 2 has no equivalent in the Syrian hamster and human PrP genes. The Prn-pb gene shares this intron/exon structure but harbors an approximately 6-kb deletion within intron 2. While the Prn-pb open reading frame encodes two amino acid substitutions linked to prolonged scrapie incubation periods, a deletion of intron 2 sequences also characterizes inbred strains such as RIII/S and MOLF/Ei with shorter incubation periods, making a relationship between intron 2 size and scrapie pathogenesis unlikely. The promoter regions of a and b Prn-p alleles include consensus Sp1 and AP-1 sites, as well as other conserved motifs which may represent binding sites for as yet unidentified transcription factors. Images PMID:7912827

The novel extremely psychrophilic luciferase from Metridia longa: Properties of a high-purity protein produced in insect cells.

PubMed

Larionova, Marina D; Markova, Svetlana V; Vysotski, Eugene S

2017-01-29

The bright bioluminescence of copepod Metridia longa is conditioned by a small secreted coelenterazine-dependent luciferase (MLuc). To date, three isoforms of MLuc differing in length, sequences, and some properties were cloned and successfully applied as high sensitive bioluminescent reporters. In this work, we report cloning of a novel group of genes from M. longa encoding extremely psychrophilic isoforms of MLuc (MLuc2-type). The novel isoforms share only ∼54-64% of protein sequence identity with the previously cloned isoforms and, consequently, are the product of a separate group of paralogous genes. The MLuc2 isoform with consensus sequence was produced as a natively folded protein using baculovirus/insect cell expression system, purified, and characterized. The MLuc2 displays a very high bioluminescent activity and high thermostability similar to those of the previously characterized M. longa luciferase isoform MLuc7. However, in contrast to MLuc7 revealing the highest activity at 12-17 °C and 0.5 M NaCl, the bioluminescence optima of MLuc2 isoforms are at ∼5 °C and 1 M NaCl. The MLuc2 adaptation to cold is also accompanied by decrease of melting temperature and affinity to substrate suggesting a more conformational flexibility of a protein structure. The luciferase isoforms with different temperature optima may provide adaptability of the M. longa bioluminescence to the changes of water temperature during diurnal vertical migrations. Copyright © 2016 Elsevier Inc. All rights reserved.

Molecular cloning of the Coch gene of guinea pig inner ear and its expression analysis in cultured fibrocytes of the spiral ligament.

PubMed

Li, Lishu; Ikezono, Tetsuo; Sekine, Kuwon; Shindo, Susumu; Matsumura, Tomohiro; Pawankar, Ruby; Ichimiya, Issei; Yagi, Toshiaki

2010-08-01

We have cloned guinea pig Coch cDNA and the sequence information will be useful for future molecular study combined with physiological experiments. Proper Coch gene expression appears to be dependent on the unique extracellular micro-environment of the inner ear in vivo. These results provide insight into the Coch gene expression and its regulation. To characterize the guinea pig Coch gene, we performed molecular cloning and expression analysis in the inner ear and cultured fibrocytes of the spiral ligament. The Coch cDNA was isolated using RACE. Cochlin isofoms were studied by Western blot using three different types of mammalian inner ear. The cochlear fibrocytes were cultured and characterized by immunostaining. Coch gene expression in the fibrocytes was investigated and the influence of cytokine stimulation was evaluated. The full-length 1991 bp Coch cDNA that encodes a 553 amino acid protein was isolated. The sequence had significant homology with other mammals, and the sizes of the Cochlin isoforms were identical. In the cultured fibrocytes, Coch mRNA was expressed in a very small amount and the isoform production was different, compared with the results in vivo. Cytokine stimulation did not alter the level of mRNA expression or isoform formation.

Myosin Heavy Chain Gene Expression in Developing Neonatal Skeletal Muscle: Involvement of the Nerve, Gravity, and Thyroid State

NASA Technical Reports Server (NTRS)

Baldwin, K. M.; Adams, G.; Haddad, F.; Zeng, M.; Qin, A.; Qin, L.; McCue, S.; Bodell, P.

1999-01-01

The myosin heavy chain (MHC) gene family encodes at least six MHC proteins (herein designated as neonatal, embryonic, slow type I (beta), and fast IIa, IIx, and IIb) that are expressed in skeletal muscle in a muscle-specific and developmentally-regulated fashion. At birth, both antigravity (e.g. soleus) and locomotor (e.g., plantaris) skeletal muscles are undifferentiated relative to the adult MHC phenotype such that the neonatal and embryonic MHC isoforms account for 80 - 90% of the MHC pool in a fast locomotor muscle; whereas, the embryonic and slow, type I isoforms account for approx. 90% of the pool in a typical antigravity muscle. The goal of this study was to investigate the role of an intact nerve, gravity and thyroid hormone (T3), as well as certain interactions of these interventions, on MHC gene expression in developing neonatal skeletal muscles of rodents.

Splice isoform-specific suppression of the CaV2.1 variant underlying Spinocerebellar ataxia type 6

PubMed Central

Tsou, Wei-Ling; Soong, Bing-Wen; Paulson, Henry L.; Rodríguez-Lebrón, Edgardo

2011-01-01

Spinocerebellar ataxia type 6 (SCA6) is an inherited neurodegenerative disease caused by a polyglutamine (polyQ) expansion in the CaV2.1 voltage-gated calcium channel subunit (CACNA1A). There is currently no treatment for this debilitating disorder and thus a pressing need to develop preventative therapies. RNA interference (RNAi) has proven effective at halting disease progression in several models of spinocerebellar ataxia (SCA), including SCA types 1 and 3. However, in SCA6 and other dominantly inherited neurodegenerative disorders, RNAi-based strategies that selectively suppress expression of mutant alleles may be required. Using a CaV2.1 mini-gene reporter system, we found that pathogenic CAG expansions in CaV2.1 enhance splicing activity at the 3′end of the transcript, leading to a CAG repeat length-dependent increase in the levels of a polyQ-encoding CaV2.1 mRNA splice isoform and the resultant disease protein. Taking advantage of this molecular phenomenon, we developed a novel splice isoform-specific (SIS)-RNAi strategy that selectively targets the polyQ-encoding CaV2.1 splice variant. Selective suppression of transiently expressed and endogenous polyQ-encoding CaV2.1 splice variants was achieved in a variety of cell-based models including a human neuronal cell line, using a new artificial miRNA-like delivery system. Moreover, the efficacy of gene silencing correlated with effective intracellular recognition and processing of SIS-RNAi miRNA mimics. These results lend support to the preclinical development of SIS-RNAi as a potential therapy for SCA6 and other dominantly inherited diseases. PMID:21550405

Splice isoform-specific suppression of the Cav2.1 variant underlying spinocerebellar ataxia type 6.

PubMed

Tsou, Wei-Ling; Soong, Bing-Wen; Paulson, Henry L; Rodríguez-Lebrón, Edgardo

2011-09-01

Spinocerebellar ataxia type 6 (SCA6) is an inherited neurodegenerative disease caused by a polyglutamine (polyQ) expansion in the Ca(V)2.1 voltage-gated calcium channel subunit (CACNA1A). There is currently no treatment for this debilitating disorder and thus a pressing need to develop preventative therapies. RNA interference (RNAi) has proven effective at halting disease progression in several models of spinocerebellar ataxia (SCA), including SCA types 1 and 3. However, in SCA6 and other dominantly inherited neurodegenerative disorders, RNAi-based strategies that selectively suppress expression of mutant alleles may be required. Using a Ca(V)2.1 mini-gene reporter system, we found that pathogenic CAG expansions in Ca(V)2.1 enhance splicing activity at the 3'end of the transcript, leading to a CAG repeat length-dependent increase in the levels of a polyQ-encoding Ca(V)2.1 mRNA splice isoform and the resultant disease protein. Taking advantage of this molecular phenomenon, we developed a novel splice isoform-specific (SIS)-RNAi strategy that selectively targets the polyQ-encoding Ca(V)2.1 splice variant. Selective suppression of transiently expressed and endogenous polyQ-encoding Ca(V)2.1 splice variants was achieved in a variety of cell-based models including a human neuronal cell line, using a new artificial miRNA-like delivery system. Moreover, the efficacy of gene silencing correlated with effective intracellular recognition and processing of SIS-RNAi miRNA mimics. These results lend support to the preclinical development of SIS-RNAi as a potential therapy for SCA6 and other dominantly inherited diseases. Copyright © 2011 Elsevier Inc. All rights reserved.

Protein Kinase A Regulatory Subunit Isoforms Regulate Growth and Differentiation in Mucor circinelloides: Essential Role of PKAR4

PubMed Central

Ocampo, J.; McCormack, B.; Navarro, E.; Moreno, S.; Garre, V.

2012-01-01

The protein kinase A (PKA) signaling pathway plays a role in regulating growth and differentiation in the dimorphic fungus Mucor circinelloides. PKA holoenzyme is comprised of two catalytic (C) and two regulatory (R) subunits. In M. circinelloides, four genes encode the PKAR1, PKAR2, PKAR3, and PKAR4 isoforms of R subunits. We have constructed null mutants and demonstrate that each isoform has a different role in growth and differentiation. The most striking finding is that pkaR4 is an essential gene, because only heterokaryons were obtained in knockout experiments. Heterokaryons with low levels of wild-type nuclei showed an impediment in the emission of the germ tube, suggesting a pivotal role of this gene in germ tube emergence. The remaining null strains showed different alterations in germ tube emergence, sporulation, and volume of the mother cell. The pkaR2 null mutant showed an accelerated germ tube emission and was the only mutant that germinated under anaerobic conditions when glycine was used as a nitrogen source, suggesting that pkaR2 participates in germ tube emergence by repressing it. From the measurement of the mRNA and protein levels of each isoform in the wild-type and knockout strains, it can be concluded that the expression of each subunit has its own mechanism of differential regulation. The PKAR1 and PKAR2 isoforms are posttranslationally modified by ubiquitylation, suggesting another regulation point in the specificity of the signal transduction. The results indicate that each R isoform has a different role in M. circinelloides physiology, controlling the dimorphism and contributing to the specificity of cyclic AMP (cAMP)-PKA pathway. PMID:22635921

Identification and Characterization of an Alternatively Spliced Isoform of the Human Protein Phosphatase 2Aα Catalytic Subunit*

PubMed Central

Migueleti, Deivid L. S.; Smetana, Juliana H. C.; Nunes, Hugo F.; Kobarg, Jörg; Zanchin, Nilson I. T.

2012-01-01

PP2A is the main serine/threonine-specific phosphatase in animal cells. The active phosphatase has been described as a holoenzyme consisting of a catalytic, a scaffolding, and a variable regulatory subunit, all encoded by multiple genes, allowing for the assembly of more than 70 different holoenzymes. The catalytic subunit can also interact with α4, TIPRL (TIP41, TOR signaling pathway regulator-like), the methyl-transferase LCMT-1, and the methyl-esterase PME-1. Here, we report that the gene encoding the catalytic subunit PP2Acα can generate two mRNA types, the standard mRNA and a shorter isoform, lacking exon 5, which we termed PP2Acα2. Higher levels of the PP2Acα2 mRNA, equivalent to the level of the longer PP2Acα mRNA, were detected in peripheral blood mononuclear cells that were left to rest for 24 h. After this time, the peripheral blood mononuclear cells are still viable and the PP2Acα2 mRNA decreases soon after they are transferred to culture medium, showing that generation of the shorter isoform depends on the incubation conditions. FLAG-tagged PP2Acα2 expressed in HEK293 is catalytically inactive. It displays a specific interaction profile with enhanced binding to the α4 regulatory subunit, but no binding to the scaffolding subunit and PME-1. Consistently, α4 out-competes PME-1 and LCMT-1 for binding to both PP2Acα isoforms in pulldown assays. Together with molecular modeling studies, this suggests that all three regulators share a common binding surface on the catalytic subunit. Our findings add important new insights into the complex mechanisms of PP2A regulation. PMID:22167190

Seed Dormancy in Arabidopsis Requires Self-Binding Ability of DOG1 Protein and the Presence of Multiple Isoforms Generated by Alternative Splicing.

PubMed

Nakabayashi, Kazumi; Bartsch, Melanie; Ding, Jia; Soppe, Wim J J

2015-12-01

The Arabidopsis protein DELAY OF GERMINATION 1 (DOG1) is a key regulator of seed dormancy, which is a life history trait that determines the timing of seedling emergence. The amount of DOG1 protein in freshly harvested seeds determines their dormancy level. DOG1 has been identified as a major dormancy QTL and variation in DOG1 transcript levels between accessions contributes to natural variation for seed dormancy. The DOG1 gene is alternatively spliced. Alternative splicing increases the transcriptome and proteome diversity in higher eukaryotes by producing transcripts that encode for proteins with altered or lost function. It can also generate tissue specific transcripts or affect mRNA stability. Here we suggest a different role for alternative splicing of the DOG1 gene. DOG1 produces five transcript variants encoding three protein isoforms. Transgenic dog1 mutant seeds expressing single DOG1 transcript variants from the endogenous DOG1 promoter did not complement because they were non-dormant and lacked DOG1 protein. However, transgenic plants overexpressing single DOG1 variants from the 35S promoter could accumulate protein and showed complementation. Simultaneous expression of two or more DOG1 transcript variants from the endogenous DOG1 promoter also led to increased dormancy levels and accumulation of DOG1 protein. This suggests that single isoforms are functional, but require the presence of additional isoforms to prevent protein degradation. Subsequently, we found that the DOG1 protein can bind to itself and that this binding is required for DOG1 function but not for protein accumulation. Natural variation for DOG1 binding efficiency was observed among Arabidopsis accessions and contributes to variation in seed dormancy.

A searchable, whole genome resource designed for protein variant analysis in diverse lineages of U.S. beef cattle

USDA-ARS?s Scientific Manuscript database

A key feature of a gene's function is the variety of protein isoforms it encodes in a population. However, the genetic diversity in bovine whole genome databases tends to be underrepresented because these databases contain an abundance of sequence from the most influential sires. Our first aim was ...

RNA splicing and splicing regulator changes in prostate cancer pathology.

PubMed

Munkley, Jennifer; Livermore, Karen; Rajan, Prabhakar; Elliott, David J

2017-09-01

Changes in mRNA splice patterns have been associated with key pathological mechanisms in prostate cancer progression. The androgen receptor (abbreviated AR) transcription factor is a major driver of prostate cancer pathology and activated by androgen steroid hormones. Selection of alternative promoters by the activated AR can critically alter gene function by switching mRNA isoform production, including creating a pro-oncogenic isoform of the normally tumour suppressor gene TSC2. A number of androgen-regulated genes generate alternatively spliced mRNA isoforms, including a prostate-specific splice isoform of ST6GALNAC1 mRNA. ST6GALNAC1 encodes a sialyltransferase that catalyses the synthesis of the cancer-associated sTn antigen important for cell mobility. Genetic rearrangements occurring early in prostate cancer development place ERG oncogene expression under the control of the androgen-regulated TMPRSS2 promoter to hijack cell behaviour. This TMPRSS2-ERG fusion gene shows different patterns of alternative splicing in invasive versus localised prostate cancer. Alternative AR mRNA isoforms play a key role in the generation of prostate cancer drug resistance, by providing a mechanism through which prostate cancer cells can grow in limited serum androgen concentrations. A number of splicing regulator proteins change expression patterns in prostate cancer and may help drive key stages of disease progression. Up-regulation of SRRM4 establishes neuronal splicing patterns in neuroendocrine prostate cancer. The splicing regulators Sam68 and Tra2β increase expression in prostate cancer. The SR protein kinase SRPK1 that modulates the activity of SR proteins is up-regulated in prostate cancer and has already given encouraging results as a potential therapeutic target in mouse models.

Cardiac-specific deletion of protein phosphatase 1β promotes increased myofilament protein phosphorylation and contractile alterations

PubMed Central

Liu, Ruijie; Correll, Robert N.; Davis, Jennifer; Vagnozzi, Ronald J.; York, Allen J.; Sargent, Michelle A.; Nairn, Angus C.; Molkentin, Jeffery D.

2015-01-01

There are 3 protein phosphatase 1 (PP1) catalytic isoforms (α, β and γ) encoded within the mammalian genome. These 3 gene products share ~90% amino acid homology within their catalytic domains but each has unique N- and C-termini that likely underlie distinctive subcellular localization or functionality. In this study, we assessed the effect associated with loss of each PP1 isoform in the heart using a conditional Cre-loxP targeting approach in mice. Ppp1ca-loxP, Ppp1cb-loxP and Ppp1cc-oxP alleles were crossed with either an Nkx2.5-Cre knock-in containing allele for early embryonic deletion or a tamoxifen inducible α-myosin heavy chain (αMHC)-MerCreMer transgene for adult and cardiac-specific deletion. We determined that while deletion of Ppp1ca (PP1α) or Ppp1cc (PP1γ) had little effect on the whole heart, deletion of Ppp1cb (PP1β) resulted in concentric remodeling of the heart, interstitial fibrosis and contractile dysregulation, using either the embryonic or adult-specific Cre-expressing alleles. However, myocytes isolated from Ppp1cb deleted hearts surprisingly showed enhanced contractility. Mechanistically we found that deletion of any of the 3 PP1 gene-encoding isoforms had no effect on phosphorylation of phospholamban, nor were Ca2+ handling dynamics altered in adult myocytes from Ppp1cb deleted hearts. However, loss of Ppp1cb from the heart, but not Ppp1ca or Ppp1cc, resulted in elevated phosphorylation of myofilament proteins such as myosin light chain 2 and cardiac myosin binding protein C, consistent with an enriched localization profile of this isoform to the sarcomeres. These results suggest a unique functional role for the PP1β isoform in affecting cardiac contractile function. PMID:26334248

The First Chameleon Transcriptome: Comparative Genomic Analysis of the OXPHOS System Reveals Loss of COX8 in Iguanian Lizards

PubMed Central

Bar-Yaacov, Dan; Bouskila, Amos; Mishmar, Dan

2013-01-01

Recently, we found dramatic mitochondrial DNA divergence of Israeli Chamaeleo chamaeleon populations into two geographically distinct groups. We aimed to examine whether the same pattern of divergence could be found in nuclear genes. However, no genomic resource is available for any chameleon species. Here we present the first chameleon transcriptome, obtained using deep sequencing (SOLiD). Our analysis identified 164,000 sequence contigs of which 19,000 yielded unique BlastX hits. To test the efficacy of our sequencing effort, we examined whether the chameleon and other available reptilian transcriptomes harbored complete sets of genes comprising known biochemical pathways, focusing on the nDNA-encoded oxidative phosphorylation (OXPHOS) genes as a model. As a reference for the screen, we used the human 86 (including isoforms) known structural nDNA-encoded OXPHOS subunits. Analysis of 34 publicly available vertebrate transcriptomes revealed orthologs for most human OXPHOS genes. However, OXPHOS subunit COX8 (Cytochrome C oxidase subunit 8), including all its known isoforms, was consistently absent in transcriptomes of iguanian lizards, implying loss of this subunit during the radiation of this suborder. The lack of COX8 in the suborder Iguania is intriguing, since it is important for cellular respiration and ATP production. Our sequencing effort added a new resource for comparative genomic studies, and shed new light on the evolutionary dynamics of the OXPHOS system. PMID:24009133

The first Chameleon transcriptome: comparative genomic analysis of the OXPHOS system reveals loss of COX8 in Iguanian lizards.

PubMed

Bar-Yaacov, Dan; Bouskila, Amos; Mishmar, Dan

2013-01-01

Recently, we found dramatic mitochondrial DNA divergence of Israeli Chamaeleo chamaeleon populations into two geographically distinct groups. We aimed to examine whether the same pattern of divergence could be found in nuclear genes. However, no genomic resource is available for any chameleon species. Here we present the first chameleon transcriptome, obtained using deep sequencing (SOLiD). Our analysis identified 164,000 sequence contigs of which 19,000 yielded unique BlastX hits. To test the efficacy of our sequencing effort, we examined whether the chameleon and other available reptilian transcriptomes harbored complete sets of genes comprising known biochemical pathways, focusing on the nDNA-encoded oxidative phosphorylation (OXPHOS) genes as a model. As a reference for the screen, we used the human 86 (including isoforms) known structural nDNA-encoded OXPHOS subunits. Analysis of 34 publicly available vertebrate transcriptomes revealed orthologs for most human OXPHOS genes. However, OXPHOS subunit COX8 (Cytochrome C oxidase subunit 8), including all its known isoforms, was consistently absent in transcriptomes of iguanian lizards, implying loss of this subunit during the radiation of this suborder. The lack of COX8 in the suborder Iguania is intriguing, since it is important for cellular respiration and ATP production. Our sequencing effort added a new resource for comparative genomic studies, and shed new light on the evolutionary dynamics of the OXPHOS system.

The molecular basis for relative physiological functionality of the ADP/ATP carrier isoforms in Saccharomyces cerevisiae.

PubMed

Smith, Christopher P; Thorsness, Peter E

2008-07-01

AAC2 is one of three paralogs encoding mitochondrial ADP/ATP carriers in the yeast Saccharomyces cerevisiae, and because it is required for respiratory growth it has been the most extensively studied. To comparatively examine the relative functionality of Aac1, Aac2, and Aac3 in vivo, the gene encoding each isoform was expressed from the native AAC2 locus in aac1Delta aac3Delta yeast. Compared to Aac2, Aac1 exhibited reduced capacity to support growth of yeast lacking mitochondrial DNA or of yeast lacking the ATP/Mg-P(i) carrier, both conditions requiring ATP import into the mitochondrial matrix through the ADP/ATP carrier. Sixteen AAC1/AAC2 chimeric genes were constructed and analyzed to determine the key differences between residues or sections of Aac1 and Aac2. On the basis of the growth rate differences of yeast expressing different chimeras, the C1 and M2 loops of the ADP/ATP carriers contain divergent residues that are responsible for the difference(s) between Aac1 and Aac2. One chimeric gene construct supported growth on nonfermentable carbon sources but failed to support growth of yeast lacking mitochondrial DNA. We identified nine independent intragenic mutations in this chimeric gene that suppressed the growth phenotype of yeast lacking mitochondrial DNA, identifying regions of the carrier important for nucleotide exchange activities.

Regulation and Functional Implications of Opioid Receptor Splicing in Opioid Pharmacology and HIV Pathogenesis

PubMed Central

Regan, Patrick M.; Langford, T. Dianne; Khalili, Kamel

2015-01-01

Despite the identification and characterization of four opioid receptor subtypes and the genes from which they are encoded, pharmacological data does not conform to the predications of a four opioid receptor model. Instead, current studies of opioid pharmacology suggest the existence of additional receptor subtypes; however, no additional opioid receptor subtype has been identified to date. It is now understood that this discrepancy is due to the generation of multiple isoforms of opioid receptor subtypes. While several mechanisms are utilized to generate these isoforms, the primary mechanism involves alternative splicing of the pre-mRNA transcript. Extensive alternative splicing patterns for opioid receptors have since been identified and discrepancies in opioid pharmacology are now partially attributed to variable expression of these isoforms. Recent studies have been successful in characterizing the localization of these isoforms as well as their specificity in ligand binding; however, the regulation of opioid receptor splicing specificity is poorly characterized. Furthermore, the functional significance of individual receptor isoforms and the extent to which opioid- and/or HIV-mediated changes in the opioid receptor isoform profile contributes to altered opioid pharmacology or the well-known physiological role of opioids in the exacerbation of HIV neurocognitive dysfunction is unknown. As such, the current review details constitutive splicing mechanisms as well as the specific architecture of opioid receptor genes, transcripts, and receptors in order to highlight the current understanding of opioid receptor isoforms, potential mechanisms of their regulation and signaling, and their functional significance in both opioid pharmacology and HIV-associated neuropathology. PMID:26529364

Role of Leptin Deficiency, Inefficiency, and Leptin Receptors in Obesity.

PubMed

Wasim, Muhammad; Awan, Fazli Rabbi; Najam, Syeda Sadia; Khan, Abdul Rehman; Khan, Haq Nawaz

2016-10-01

Leptin protein consists of 167 amino acids, which is mainly secreted from the white adipose tissue. This protein acts on the hypothalamic regions of the brain which control eating behavior, thus playing a significant role in maintaining body's metabolism. Leptin receptors belong to glycoprotein 130 (gp130) family of cytokine receptors and exist in six isoforms (LEPR a-f), and all the isoforms are encoded by LEPR gene; out of these isoforms, the LEPR-b receptor is the 'longest form,' and in most of the cases, mutations in this isoform cause severe obesity. Also, mutations in the leptin gene (LEP) or its receptors gene can lead to obesity. Some biochemical pathways affect the bioactivity of leptin and/or its receptors. To date, eleven pathogenic mutations have been reported in the LEP which are p.L72S, p.N103K, p.R105W, p.H118L, p.S141C, p.W121X c.104_106delTCA, c.135del3bp, c.398delG, c.481_482delCT, and c.163C>T. Different mutations in the LEPR have also been reported as c.2396-1 G>T, c.1675 G>A, p.P316T, etc. In some studies, where leptin was deficient, leptin replacement therapy has shown positive impact by preventing weight gain and obesity.

Genetic variation of six desaturase genes in flax and their impact on fatty acid composition.

PubMed

Thambugala, Dinushika; Duguid, Scott; Loewen, Evelyn; Rowland, Gordon; Booker, Helen; You, Frank M; Cloutier, Sylvie

2013-10-01

Flax (Linum usitatissimum L.) is one of the richest plant sources of omega-3 fatty acids praised for their health benefits. In this study, the extent of the genetic variability of genes encoding stearoyl-ACP desaturase (SAD), and fatty acid desaturase 2 (FAD2) and 3 (FAD3) was determined by sequencing the six paralogous genes from 120 flax accessions representing a broad range of germplasm including some EMS mutant lines. A total of 6 alleles for sad1 and sad2, 21 for fad2a, 5 for fad2b, 15 for fad3a and 18 for fad3b were identified. Deduced amino acid sequences of the alleles predicted 4, 2, 3, 4, 6 and 7 isoforms, respectively. Allele frequencies varied greatly across genes. Fad3a, with 110 SNPs and 19 indels, and fad3b, with 50 SNPs and 5 indels, showed the highest levels of genetic variations. While most of the SNPs and all the indels were silent mutations, both genes carried nonsense SNP mutations resulting in premature stop codons, a feature not observed in sad and fad2 genes. Some alleles and isoforms discovered in induced mutant lines were absent in the natural germplasm. Correlation of these genotypic data with fatty acid composition data of 120 flax accessions phenotyped in six field experiments revealed statistically significant effects of some of the SAD and FAD isoforms on fatty acid composition, oil content and iodine value. The novel allelic variants and isoforms identified for the six desaturases will be a resource for the development of oilseed flax with unique and useful fatty acid profiles.

Metabolic characterization of a mouse deficient in all known leptin receptor isoforms.

PubMed

Osborn, Olivia; Sanchez-Alavez, Manuel; Brownell, Sara E; Ross, Brendon; Klaus, Joe; Dubins, Jeffrey; Beutler, Bruce; Conti, Bruno; Bartfai, Tamas

2010-01-01

We have characterized a newly generated mouse model of obesity, a mouse strain deficient in all five previously described leptin receptor isoforms. These transgenic mice, named the db (333)/db (333) mice, were identified from an ENU mutagenesis screen and carry a point mutation in the seventh exon of the db gene encoding the leptin receptor, resulting in a premature stop codon (Y(333)Stop) and gene product that lacks STAT signaling domains. db (333)/db (333) mice have a morbidly obese phenotype, with body weights diverging from wild type as early as 4 weeks of age (P < 0.05). To determine the contribution of the short isoforms of the leptin receptor in this metabolic phenotype, we performed an extensive metabolic characterization of the db (333)/db (333) mouse in relation to the well-characterized db/db mouse lacking only the long form of the leptin receptor. db (333)/db (333) mice have similar endocrine and metabolic parameters as previously described in other leptin receptor transgenic mice including db/db mice that lack only the long isoform of the leptin receptor. However, db (333)/db (333) mice show a subtle trend toward higher body weight and insulin levels, lower oxygen, carbon dioxide production, respiratory exchange ratio (RER), and temperature than db/db mice suggesting the short isoforms may play an additional role in energy homeostasis.

FHL1B Interacts with Lamin A/C and Emerin at the Nuclear Lamina and is Misregulated in Emery-Dreifuss Muscular Dystrophy.

PubMed

Ziat, Esma; Mamchaoui, Kamel; Beuvin, Maud; Nelson, Isabelle; Azibani, Feriel; Spuler, Simone; Bonne, Gisèle; Bertrand, Anne T

2016-11-29

Emery-Dreifuss muscular dystrophy (EDMD) is associated with mutations in EMD and LMNA genes, encoding for the nuclear envelope proteins emerin and lamin A/C, indicating that EDMD is a nuclear envelope disease. We recently reported mutations in FHL1 gene in X-linked EDMD. FHL1 encodes FHL1A, and the two minor isoforms FHL1B and FHL1C. So far, none have been described at the nuclear envelope. To gain insight into the pathophysiology of EDMD, we focused our attention on the poorly characterized FHL1B isoform. The amount and the localisation of FHL1B were evaluated in control and diseased human primary myoblasts using immunofluorescence and western blotting. We found that in addition to a cytoplasmic localization, this isoform strongly accumulated at the nuclear envelope of primary human myoblasts, like but independently of lamin A/C and emerin. During myoblast differentiation, we observed a major reduction of FHL1B protein expression, especially in the nucleus. Interestingly, we found elevated FHL1B expression level in myoblasts from an FHL1-related EDMD patient where the FHL1 mutation only affects FHL1A, as well as in myoblasts from an LMNA-related EDMD patient. Altogether, the specific localization of FHL1B and its modulation in disease-patient's myoblasts confirmed FHL1-related EDMD as a nuclear envelope disease.

Comprehensive Analysis of Tropomyosin Isoforms in Skeletal Muscles by Top-down Proteomics

PubMed Central

Jin, Yutong; Peng, Ying; Lin, Ziqing; Chen, Yi-Chen; Wei, Liming; Hacker, Timothy A.; Larsson, Lars; Ge, Ying

2016-01-01

Mammalian skeletal muscles are heterogeneous in nature and are capable of performing various functions. Tropomyosin (Tpm) is a major component of the thin filament in skeletal muscles and plays an important role in controlling muscle contraction and relaxation. Tpm is known to consist of multiple isoforms resulting from different encoding genes and alternative splicing, along with post-translational modifications. However, a systematic characterization of Tpm isoforms in skeletal muscles is still lacking. Therefore, we employed top-down mass spectrometry (MS) to identify and characterize Tpm isoforms present in different skeletal muscles from multiple species, including swine, rat, and human. Our study revealed that Tpm1.1 and Tpm2.2 are the two major Tpm isoforms in swine and rat skeletal muscles, whereas Tpm1.1, Tpm2.2, and Tpm3.12 are present in human skeletal muscles. Tandem MS was utilized to identify the sequences of the major Tpm isoforms. Furthermore, quantitative analysis revealed muscle-type specific differences in the abundance of un-modified and modified Tpm isoforms in rat and human skeletal muscles. This study represents the first systematic investigation of Tpm isoforms in skeletal muscles, which not only demonstrates the capabilities of top-down MS for the comprehensive characterization of skeletal myofilament proteins but also provides the basis for further studies on these Tpm isoforms in muscle-related diseases. PMID:27090236

A proton pump ATPase with testis-specific E1-subunit isoform required for acrosome acidification.

PubMed

Sun-Wada, Ge-Hong; Imai-Senga, Yoko; Yamamoto, Akitsugu; Murata, Yoshiko; Hirata, Tomoyuki; Wada, Yoh; Futai, Masamitsu

2002-05-17

The vacuolar-type H(+)-ATPases (V-ATPases) are a family of multimeric proton pumps involved in a wide variety of physiological processes. We have identified two novel mouse genes, Atp6e1 and Atp6e2, encoding testis-specific (E1) and ubiquitous (E2) V-ATPase subunit E isoforms, respectively. The E1 transcript appears about 3 weeks after birth, corresponding to the start of meiosis, and is expressed specifically in round spermatids in seminiferous tubules. Immunohistochemistry with isoform-specific antibodies revealed that the V-ATPase with E1 and a2 isoforms is located specifically in developing acrosomes of spermatids and acrosomes in mature sperm. In contrast, the E2 isoform was expressed in all tissues examined and present in the perinuclear compartments of spermatocytes. The E1 isoform exhibits 70% identity with the E2, and both isoforms functionally complemented a null mutation of the yeast counterpart VMA4, indicating that they are bona fide V-ATPase subunits. The chimeric enzymes showed slightly lower K(m)(ATP) than yeast V-ATPase. Consistent with the temperature-sensitive growth of Deltavma4-expressing E1 isoform, vacuolar membrane vesicles exhibited temperature-sensitive coupling between ATP hydrolysis and proton transport. These results suggest that E1 isoform is essential for energy coupling involved in acidification of acrosome.

Nodule-enhanced expression of a sucrose phosphate synthase gene member (MsSPSA) has a role in carbon and nitrogen metabolism in the nodules of alfalfa (Medicago sativa L.)

PubMed Central

Aleman, Lorenzo; Ortega, Jose Luis; Martinez-Grimes, Martha; Seger, Mark; Holguin, Francisco Omar; Uribe, Diana J.; Garcia-Ibilcieta, David

2013-01-01

Sucrose phosphate synthase (SPS) catalyzes the first step in the synthesis of sucrose in photosynthetic tissues. We characterized the expression of three different isoforms of SPS belonging to two different SPS gene families in alfalfa (Medicago sativa L.), a previously identified SPS (MsSPSA) and two novel isoforms belonging to class B (MsSPSB and MsSPSB3). While MsSPSA showed nodule-enhanced expression, both MsSPSB genes exhibited leaf-enhanced expression. Alfalfa leaf and nodule SPS enzymes showed differences in chromatographic and electrophoretic migration and differences in Vmax and allosteric regulation. The root nodules in legume plants are a strong sink for photosynthates with its need for ATP, reducing power and carbon skeletons for dinitrogen fixation and ammonia assimilation. The expression of genes encoding SPS and other key enzymes in sucrose metabolism, sucrose phosphate phosphatase and sucrose synthase, was analyzed in the leaves and nodules of plants inoculated with Sinorhizobium meliloti. Based on the expression pattern of these genes, the properties of the SPS isoforms and the concentration of starch and soluble sugars in nodules induced by a wild type and a nitrogen fixation deficient strain, we propose that SPS has an important role in the control of carbon flux into different metabolic pathways in the symbiotic nodules. PMID:19898977

Sexual selection, genetic conflict, selfish genes, and the atypical patterns of gene expression in spermatogenic cells.

PubMed

Kleene, Kenneth C

2005-01-01

This review proposes that the peculiar patterns of gene expression in spermatogenic cells are the consequence of powerful evolutionary forces known as sexual selection. Sexual selection is generally characterized by intense competition of males for females, an enormous variety of the strategies to maximize male reproductive success, exaggerated male traits at all levels of biological organization, co-evolution of sexual traits in males and females, and conflict between the sexual advantage of the male trait and the reproductive fitness of females and the individual fitness of both sexes. In addition, spermatogenesis is afflicted by selfish genes that promote their transmission to progeny while causing deleterious effects. Sexual selection, selfish genes, and genetic conflict provide compelling explanations for many atypical features of gene expression in spermatogenic cells including the gross overexpression of certain mRNAs, transcripts encoding truncated proteins that cannot carry out basic functions of the proteins encoded by the same genes in somatic cells, the large number of gene families containing paralogous genes encoding spermatogenic cell-specific isoforms, the large number of testis-cancer-associated genes that are expressed only in spermatogenic cells and malignant cells, and the overbearing role of Sertoli cells in regulating the number and quality of spermatozoa.

Molecular cloning and biochemical characterization of three phosphoglycerate kinase isoforms from developing sunflower (Helianthus annuus L.) seeds.

PubMed

Troncoso-Ponce, M A; Rivoal, J; Venegas-Calerón, M; Dorion, S; Sánchez, R; Cejudo, F J; Garcés, R; Martínez-Force, E

2012-07-01

Three cDNAs encoding different phosphoglycerate kinase (PGK, EC 2.7.2.3) isoforms, two cytosolic (HacPGK1 and HacPGK2) and one plastidic (HapPGK), were cloned and characterized from developing sunflower (Helianthus annuus L.) seeds. The expression profiles of these genes showed differences in heterotrophic tissues, such as developing seeds and roots, where HacPGK1 was predominant, while HapPGK was highly expressed in photosynthetic tissues. The cDNAs were expressed in Escherichia coli, and the corresponding proteins purified to electrophoretic homogeneity, using immobilized metal ion affinity chromatography, and biochemically characterized. Despite the high level of identity between sequences, the HacPGK1 isoform showed strong differences in terms of specific activity, temperature stability and pH sensitivity in comparison to HacPGK2 and HapPGK. A polyclonal immune serum was raised against the purified HacPGK1 isoform, which showed cross-immunoreactivity with the other PGK isoforms. This serum allowed the localization of high expression levels of PGK isozymes in embryo tissues. Copyright © 2012 Elsevier Ltd. All rights reserved.

Potential involvement of N-terminal acetylation in the quantitative regulation of the ε subunit of chloroplast ATP synthase under drought stress.

PubMed

Hoshiyasu, Saki; Kohzuma, Kaori; Yoshida, Kazuo; Fujiwara, Masayuki; Fukao, Yoichiro; Yokota, Akiho; Akashi, Kinya

2013-01-01

In plants, modulation of photosynthetic energy conversion in varying environments is often accompanied by adjustment of the abundance of photosynthetic components. In wild watermelon (Citrullus lanatus L.), proteome analysis revealed that the ε subunit of chloroplast ATP synthase occurs as two distinct isoforms with largely-different isoelectric points, although encoded by a single gene. Mass spectrometry (MS) analysis of the ε isoforms indicated that the structural difference between the ε isoforms lies in the presence or absence of an acetyl group at the N-terminus. The protein level of the non-acetylated ε isoform preferentially decreased in drought, whereas the abundance of the acetylated ε isoform was unchanged. Moreover, metalloprotease activity that decomposed the ε subunit was detected in a leaf extract from drought-stressed plants. Furthermore, in vitro assay suggested that the non-acetylated ε subunit was more susceptible to degradation by metalloaminopeptidase. We propose a model in which quantitative regulation of the ε subunit involves N-terminal acetylation and stress-induced proteases.

Integrating Evolutionary and Functional Tests of Adaptive Hypotheses: A Case Study of Altitudinal Differentiation in Hemoglobin Function in an Andean Sparrow, Zonotrichia capensis

PubMed Central

Cheviron, Zachary A.; Natarajan, Chandrasekhar; Projecto-Garcia, Joana; Eddy, Douglas K.; Jones, Jennifer; Carling, Matthew D.; Witt, Christopher C.; Moriyama, Hideaki; Weber, Roy E.; Fago, Angela; Storz, Jay F.

2014-01-01

In air-breathing vertebrates, the physiologically optimal blood-O2 affinity is jointly determined by the prevailing partial pressure of atmospheric O2, the efficacy of pulmonary O2 transfer, and internal metabolic demands. Consequently, genetic variation in the oxygenation properties of hemoglobin (Hb) may be subject to spatially varying selection in species with broad elevational distributions. Here we report the results of a combined functional and evolutionary analysis of Hb polymorphism in the rufous-collared sparrow (Zonotrichia capensis), a species that is continuously distributed across a steep elevational gradient on the Pacific slope of the Peruvian Andes. We integrated a population genomic analysis that included all postnatally expressed Hb genes with functional studies of naturally occurring Hb variants, as well as recombinant Hb (rHb) mutants that were engineered through site-directed mutagenesis. We identified three clinally varying amino acid polymorphisms: Two in the αA-globin gene, which encodes the α-chain subunits of the major HbA isoform, and one in the αD-globin gene, which encodes the α-chain subunits of the minor HbD isoform. We then constructed and experimentally tested single- and double-mutant rHbs representing each of the alternative αA-globin genotypes that predominate at different elevations. Although the locus-specific patterns of altitudinal differentiation suggested a history of spatially varying selection acting on Hb polymorphism, the experimental tests demonstrated that the observed amino acid mutations have no discernible effect on respiratory properties of the HbA or HbD isoforms. These results highlight the importance of experimentally validating the hypothesized effects of genetic changes in protein function to avoid the pitfalls of adaptive storytelling. PMID:25135942

Tuning of RNA editing by ADAR is required in Drosophila

PubMed Central

Keegan, Liam P; Brindle, James; Gallo, Angela; Leroy, Anne; Reenan, Robert A; O'Connell, Mary A

2005-01-01

RNA editing increases during development in more than 20 transcripts encoding proteins involved in rapid synaptic neurotransmission in Drosophila central nervous system and muscle. Adar (adenosine deaminase acting on RNA) mutant flies expressing only genome-encoded, unedited isoforms of ion-channel subunits are viable but show severe locomotion defects. The Adar transcript itself is edited in adult wild-type flies to generate an isoform with a serine to glycine substitution close to the ADAR active site. We show that editing restricts ADAR function since the edited isoform of ADAR is less active in vitro and in vivo than the genome-encoded, unedited isoform. Ubiquitous expression in embryos and larvae of an Adar transcript that is resistant to editing is lethal. Expression of this transcript in embryonic muscle is also lethal, with above-normal, adult-like levels of editing at sites in a transcript encoding a muscle voltage-gated calcium channel. PMID:15920480

The human CD94 gene encodes multiple, expressible transcripts including a new partner of NKG2A/B.

PubMed

Lieto, L D; Maasho, K; West, D; Borrego, F; Coligan, J E

2006-01-01

CD94/NKG2A is an inhibitory receptor expressed by natural killer (NK) cells and a subset of CD8+ T cells. Ligation of CD94/NKG2A by its ligand HLA-E results in tyrosine phosphorylation of the NKG2A immunoreceptor tyrosine-based inhibitory motifs, and recruitment and activation of the SH2 domain-bearing tyrosine phosphatase-1, which in turn suppresses activation signals. The nkg2a gene encodes two isoforms, NKG2A and NKG2B, with the latter lacking the stem region. We identified three new alternative transcripts of the cd94 gene in addition to the originally described canonical CD94Full. One of the transcripts, termed CD94-T4, lacks the portion that encodes the stem region. CD94-T4 associates with both NKG2A and NKG2B, but preferentially associates with the latter. This is probably due to the absence of a stem region in both CD94-T4 and NKG2B. CD94-T4/NKG2B is capable of binding HLA-E and, when expressed in E6-1 Jurkat T cells, inhibits TCR mediated signals, demonstrating that this heterodimer is functional. Coevolution of stemless isoforms of CD94 and NKG2A that preferentially pair with each other to produce a functional heterodimer indicates that this may be more than a serendipitous event. CD94-T4/NKG2B may contribute to the plasticity of the NK immunological synapse by insuring an adequate inhibitory signal.

Germline Missense Mutations Affecting KRAS Isoform B Are Associated with a Severe Noonan Syndrome Phenotype

PubMed Central

Carta, Claudio; Pantaleoni, Francesca; Bocchinfuso, Gianfranco; Stella, Lorenzo; Vasta, Isabella; Sarkozy, Anna; Digilio, Cristina; Palleschi, Antonio; Pizzuti, Antonio; Grammatico, Paola; Zampino, Giuseppe; Dallapiccola, Bruno; Gelb, Bruce D.; Tartaglia, Marco

2006-01-01

Noonan syndrome (NS) is a developmental disorder characterized by short stature, facial dysmorphia, congenital heart disease, and multiple skeletal and hematologic defects. NS is an autosomal dominant trait and is genetically heterogeneous. Gain of function of SHP-2, a protein tyrosine phosphatase that positively modulates RAS signaling, is observed in nearly 50% of affected individuals. Here, we report the identification of heterozygous KRAS gene mutations in two subjects exhibiting a severe NS phenotype with features overlapping those of cardiofaciocutaneous and Costello syndromes. Both mutations were de novo and affected exon 6, which encodes the C-terminal portion of KRAS isoform B but does not contribute to KRAS isoform A. Structural analysis indicated that both substitutions (Val152Gly and Asp153Val) perturb the conformation of the guanine ring–binding pocket of the protein, predicting an increase in the guanine diphosphate/guanine triphosphate (GTP) dissociation rate that would favor GTP binding to the KRASB isoform and bypass the requirement for a guanine nucleotide exchange factor. PMID:16773572

A common polymorphism of the growth hormone receptor is associated with increased responsiveness to growth hormone.

PubMed

Dos Santos, Christine; Essioux, Laurent; Teinturier, Cécile; Tauber, Maïté; Goffin, Vincent; Bougnères, Pierre

2004-07-01

Growth hormone is used to increase height in short children who are not deficient in growth hormone, but its efficacy varies largely across individuals. The genetic factors responsible for this variation are entirely unknown. In two cohorts of short children treated with growth hormone, we found that an isoform of the growth hormone receptor gene that lacks exon 3 (d3-GHR) was associated with 1.7 to 2 times more growth acceleration induced by growth hormone than the full-length isoform (P < 0.0001). In transfection experiments, the transduction of growth hormone signaling through d3-GHR homo- or heterodimers was approximately 30% higher than through full-length GHR homodimers (P < 0.0001). One-half of Europeans are hetero- or homozygous with respect to the allele encoding the d3-GHR isoform, which is dominant over the full-length isoform. These observations suggest that the polymorphism in exon 3 of GHR is important in growth hormone pharmacogenetics.

Clarinet (CLA-1), a novel active zone protein required for synaptic vesicle clustering and release

PubMed Central

Nelson, Jessica; Richmond, Janet E; Colón-Ramos, Daniel A; Shen, Kang

2017-01-01

Active zone proteins cluster synaptic vesicles at presynaptic terminals and coordinate their release. In forward genetic screens, we isolated a novel Caenorhabditis elegans active zone gene, clarinet (cla-1). cla-1 mutants exhibit defects in synaptic vesicle clustering, active zone structure and synapse number. As a result, they have reduced spontaneous vesicle release and increased synaptic depression. cla-1 mutants show defects in vesicle distribution near the presynaptic dense projection, with fewer undocked vesicles contacting the dense projection and more docked vesicles at the plasma membrane. cla-1 encodes three isoforms containing common C-terminal PDZ and C2 domains with homology to vertebrate active zone proteins Piccolo and RIM. The C-termini of all isoforms localize to the active zone. Specific loss of the ~9000 amino acid long isoform results in vesicle clustering defects and increased synaptic depression. Our data indicate that specific isoforms of clarinet serve distinct functions, regulating synapse development, vesicle clustering and release. PMID:29160205

The pVHL172 isoform is not a tumor suppressor and up-regulates a subset of pro-tumorigenic genes including TGFB1 and MMP13

PubMed Central

Hascoet, Pauline; Chesnel, Franck; Jouan, Florence; Goff, Cathy Le; Couturier, Anne; Darrigrand, Eric; Mahe, Fabrice; Rioux-Leclercq, Nathalie; Goff, Xavier Le; Arlot-Bonnemains, Yannick

2017-01-01

The von Hippel-Lindau (VHL) tumor suppressor gene is often deleted or mutated in ccRCC (clear cell renal cell carcinoma) producing a non-functional protein. The gene encodes two mRNA, and three protein isoforms (pVHL213, pVHL160 and pVHL172). The pVHL protein is part of an E3 ligase complex involved in the ubiquitination and proteasomal degradation of different proteins, particularly hypoxia inducible factors (HIF) that drive the transcription of genes involved in the regulation of cell proliferation, angiogenesis or extracellular matrix remodelling. Other non-canonical (HIF-independent) pVHL functions have been described. A recent work reported the expression of the uncharacterized protein isoform pVHL172 which is translated from the variant 2 by alternative splicing of the exon 2. This splice variant is sometimes enriched in the ccRCCs and the protein has been identified in the respective samples of ccRCCs and different renal cell lines. Functional studies on pVHL have only concerned the pVHL213 and pVHL160 isoforms, but no function was assigned to pVHL172. Here we show that pVHL172 stable expression in renal cancer cells does not regulate the level of HIF, exacerbates tumorigenicity when 786-O-pVHL172 cells were xenografted in mice. The pVHL172-induced tumors developed a sarcomatoid phenotype. Moreover, pVHL172 expression was shown to up regulate a subset of pro-tumorigenic genes including TGFB1, MMP1 and MMP13. In summary we identified that pVHL172 is not a tumor suppressor. Furthermore our findings suggest an antagonistic function of this pVHL isoform in the HIF-independent aggressiveness of renal tumors compared to pVHL213. PMID:29100286

Molecular Cloning of Drebrin: Progress and Perspectives.

PubMed

Kojima, Nobuhiko

2017-01-01

Chicken drebrin isoforms were first identified in the optic tectum of developing brain. Although the time course of protein expression was different in each drebrin isoform, the similarity between their protein structures was suggested by biochemical analysis of purified protein. To determine their protein structures, the cloning of drebrin cDNAs was conducted. Comparison between the cDNA sequences shows that all drebrin cDNAs are identical except that the internal insertion sequences are present or absent in their sequences. Chicken drebrin are now classified into three isoforms, namely, drebrins E1, E2, and A. Genomic cloning demonstrated that the three isoforms are generated by an alternative splicing of individual exons encoding the insertion sequences from single drebrin gene. The mechanism should be precisely regulated in cell-type-specific and developmental stage-specific fashion. Drebrin protein, which is well conserved in various vertebrate species, although mammalian drebrin has only two isoforms, namely, drebrin E and drebrin A, is different from chicken drebrin that has three isoforms. Drebrin belongs to an actin-depolymerizing factor homology (ADF-H) domain protein family. Besides the ADF-H domain, drebrin has other domains, including the actin-binding domain and Homer-binding motifs. Diversity of protein isoform and multiple domains of drebrin could interact differentially with the actin cytoskeleton and other intracellular proteins and regulate diverse cellular processes.

CIITA promoter I CARD-deficient mice express functional MHC class II genes in myeloid and lymphoid compartments.

PubMed

Zinzow-Kramer, W M; Long, A B; Youngblood, B A; Rosenthal, K M; Butler, R; Mohammed, A-U-R; Skountzou, I; Ahmed, R; Evavold, B D; Boss, J M

2012-06-01

Three distinct promoters control the master regulator of major histocompatibility complex (MHC) class II expression, class II transactivator (CIITA), in a cell type-specific manner. Promoter I (pI) CIITA, expressed primarily by dendritic cells (DCs) and macrophages, expresses a unique isoform that contains a caspase-recruitment domain (CARD). The activity and function of this isoform are not understood, but are believed to enhance the function of CIITA in antigen-presenting cells. To determine whether isoform I of CIITA has specific functions, CIITA mutant mice were created in which isoform I was replaced with isoform III sequences. Mice in which pI and the CARD-encoding exon were deleted were also created. No defect in the formation of CD4 T cells, the ability to respond to a model antigen or bacterial or viral challenge was observed in mice lacking CIITA isoform I. Although CIITA and MHC-II expression was decreased in splenic DCs, pI knockout animals expressed CIITA from downstream promoters, suggesting that control of pI activity is mediated by unknown distal elements that could act at pIII, the B-cell promoter. Thus, no critical function is linked to the CARD domain of CIITA isoform I with respect to basic immune system development, function and challenge.

Detection of alternative splice variants at the proteome level in Aspergillus flavus.

PubMed

Chang, Kung-Yen; Georgianna, D Ryan; Heber, Steffen; Payne, Gary A; Muddiman, David C

2010-03-05

Identification of proteins from proteolytic peptides or intact proteins plays an essential role in proteomics. Researchers use search engines to match the acquired peptide sequences to the target proteins. However, search engines depend on protein databases to provide candidates for consideration. Alternative splicing (AS), the mechanism where the exon of pre-mRNAs can be spliced and rearranged to generate distinct mRNA and therefore protein variants, enable higher eukaryotic organisms, with only a limited number of genes, to have the requisite complexity and diversity at the proteome level. Multiple alternative isoforms from one gene often share common segments of sequences. However, many protein databases only include a limited number of isoforms to keep minimal redundancy. As a result, the database search might not identify a target protein even with high quality tandem MS data and accurate intact precursor ion mass. We computationally predicted an exhaustive list of putative isoforms of Aspergillus flavus proteins from 20 371 expressed sequence tags to investigate whether an alternative splicing protein database can assign a greater proportion of mass spectrometry data. The newly constructed AS database provided 9807 new alternatively spliced variants in addition to 12 832 previously annotated proteins. The searches of the existing tandem MS spectra data set using the AS database identified 29 new proteins encoded by 26 genes. Nine fungal genes appeared to have multiple protein isoforms. In addition to the discovery of splice variants, AS database also showed potential to improve genome annotation. In summary, the introduction of an alternative splicing database helps identify more proteins and unveils more information about a proteome.

The Characterization of GSDMB Splicing and Backsplicing Profiles Identifies Novel Isoforms and a Circular RNA That Are Dysregulated in Multiple Sclerosis.

PubMed

Cardamone, Giulia; Paraboschi, Elvezia Maria; Rimoldi, Valeria; Duga, Stefano; Soldà, Giulia; Asselta, Rosanna

2017-03-07

Abnormalities in alternative splicing (AS) are emerging as recurrent features in autoimmune diseases (AIDs). In particular, a growing body of evidence suggests the existence of a pathogenic association between a generalized defect in splicing regulatory genes and multiple sclerosis (MS). Moreover, several studies have documented an unbalance in alternatively-spliced isoforms in MS patients possibly contributing to the disease etiology. In this work, using a combination of PCR-based techniques (reverse-transcription (RT)-PCR, fluorescent-competitive, real-time, and digital RT-PCR assays), we investigated the alternatively-spliced gene encoding Gasdermin B, GSDMB , which was repeatedly associated with susceptibility to asthma and AIDs. The in-depth characterization of GSDMB AS and backsplicing profiles led us to the identification of an exonic circular RNA (ecircRNA) as well as of novel GSDMB in-frame and out-of-frame isoforms. The non-productive splicing variants were shown to be downregulated by the nonsense-mediated mRNA decay (NMD) in human cell lines, suggesting that GSDMB levels are significantly modulated by NMD. Importantly, both AS isoforms and the identified ecircRNA were significantly dysregulated in peripheral blood mononuclear cells of relapsing-remitting MS patients compared to controls, further supporting the notion that aberrant RNA metabolism is a characteristic feature of the disease.

The Splice Isoforms of the Drosophila Ecdysis Triggering Hormone Receptor Have Developmentally Distinct Roles

PubMed Central

Diao, Feici; Mena, Wilson; Shi, Jonathan; Park, Dongkook; Diao, Fengqiu; Taghert, Paul; Ewer, John; White, Benjamin H.

2016-01-01

To grow, insects must periodically shed their exoskeletons. This process, called ecdysis, is initiated by the endocrine release of Ecdysis Trigger Hormone (ETH) and has been extensively studied as a model for understanding the hormonal control of behavior. Understanding how ETH regulates ecdysis behavior, however, has been impeded by limited knowledge of the hormone’s neuronal targets. An alternatively spliced gene encoding a G-protein-coupled receptor (ETHR) that is activated by ETH has been identified, and several lines of evidence support a role in ecdysis for its A-isoform. The function of a second ETHR isoform (ETHRB) remains unknown. Here we use the recently introduced “Trojan exon” technique to simultaneously mutate the ETHR gene and gain genetic access to the neurons that express its two isoforms. We show that ETHRA and ETHRB are expressed in largely distinct subsets of neurons and that ETHRA- but not ETHRB-expressing neurons are required for ecdysis at all developmental stages. However, both genetic and neuronal manipulations indicate an essential role for ETHRB at pupal and adult, but not larval, ecdysis. We also identify several functionally important subsets of ETHR-expressing neurons including one that coexpresses the peptide Leucokinin and regulates fluid balance to facilitate ecdysis at the pupal stage. The general strategy presented here of using a receptor gene as an entry point for genetic and neuronal manipulations should be useful in establishing patterns of functional connectivity in other hormonally regulated networks. PMID:26534952

Uncovering the Rare Variants of DLC1 Isoform 1 and Their Functional Effects in a Chinese Sporadic Congenital Heart Disease Cohort

PubMed Central

Wang, Zhen; Tan, Huilian; Kong, Xianghua; Shu, Yang; Zhang, Yuchao; Huang, Yun; Zhu, Yufei; Xu, Heng; Wang, Zhiqiang; Wang, Ping; Ning, Guang; Kong, Xiangyin; Hu, Guohong; Hu, Landian

2014-01-01

Congenital heart disease (CHD) is the most common birth defect affecting the structure and function of fetal hearts. Despite decades of extensive studies, the genetic mechanism of sporadic CHD remains obscure. Deleted in liver cancer 1 (DLC1) gene, encoding a GTPase-activating protein, is highly expressed in heart and essential for heart development according to the knowledge of Dlc1-deficient mice. To determine whether DLC1 is a susceptibility gene for sporadic CHD, we sequenced the coding region of DLC1 isoform 1 in 151 sporadic CHD patients and identified 13 non-synonymous rare variants (including 6 private variants) in the case cohort. Importantly, these rare variants (8/13) were enriched in the N-terminal region of the DLC1 isoform 1 protein. Seven of eight amino acids at the N-terminal variant positions were conserved among the primates. Among the 9 rare variants that were predicted as “damaging”, five were located at the N-terminal region. Ensuing in vitro functional assays showed that three private variants (Met360Lys, Glu418Lys and Asp554Val) impaired the ability of DLC1 to inhibit cell migration or altered the subcellular location of the protein compared to wild-type DLC1 isoform 1. These data suggest that DLC1 might act as a CHD-associated gene in addition to its role as a tumor suppressor in cancer. PMID:24587289

Shark IgW C region diversification through RNA processing and isotype switching.

PubMed

Zhang, Cecilia; Du Pasquier, Louis; Hsu, Ellen

2013-09-15

Sharks and skates represent the earliest vertebrates with an adaptive immune system based on lymphocyte Ag receptors generated by V(D)J recombination. Shark B cells express two classical Igs, IgM and IgW, encoded by an early, alternative gene organization consisting of numerous autonomous miniloci, where the individual gene cluster carries a few rearranging gene segments and one C region, μ or ω. We have characterized eight distinct Ig miniloci encoding the nurse shark ω H chain. Each cluster consists of VH, D, and JH segments and six to eight C domain exons. Two interspersed secretory exons, in addition to the 3'-most C exon with tailpiece, provide the gene cluster with the ability to generate at least six secreted isoforms that differ as to polypeptide length and C domain combination. All clusters appear to be functional, as judged by the capability for rearrangement and absence of defects in the deduced amino acid sequence. We previously showed that IgW VDJ can perform isotype switching to μ C regions; in this study, we found that switching also occurs between ω clusters. Thus, C region diversification for any IgW VDJ can take place at the DNA level by switching to other ω or μ C regions, as well as by RNA processing to generate different C isoforms. The wide array of pathogens recognized by Abs requires different disposal pathways, and our findings demonstrate complex and unique pathways for C effector function diversity that evolved independently in cartilaginous fishes.

Genetic mapping of a caffeoyl-coenzyme A 3-O-methyltransferase gene in coffee trees. Impact on chlorogenic acid content.

PubMed

Campa, C; Noirot, M; Bourgeois, M; Pervent, M; Ky, C L; Chrestin, H; Hamon, S; de Kochko, A

2003-08-01

Chlorogenic acids (CGA) are involved in the bitterness of coffee due to their decomposition in phenolic compounds during roasting. CGA mainly include caffeoyl-quinic acids (CQA), dicaffeoyl-quinic acids (diCQA) and feruloyl-quinic acids (FQA), while CQA and diCQA constitute CGA sensu stricto (CGA s.s.). In the two cultivated species Coffea canephora and Coffea arabica, CGA s.s. represents 88% and 95% of total CGA, respectively. Among all enzymes involved in CGA biosynthesis, caffeoyl-coenzyme A 3-O-methyltransferase (CCoAOMT) is not directly involved in the CGA s.s. pathway, but rather in an upstream branch leading to FQA through feruloyl-CoA. We describe how a partial cDNA corresponding to a CCoAOMT encoding gene was obtained and sequenced. Specific primers were designed and used for studying polymorphism and locating the corresponding gene on a genetic map obtained from an interspecific backcross between Coffea liberica var. Dewevrei and Coffea pseudozanguebariae. Offspring of this backcross were also evaluated for the chlorogenic acid content in their green beans. A 10% decrease was observed in backcross progenies that possess one C. pseudozanguebariae allele of the CCoAOMT gene. This suggests that CGA s.s. accumulation is dependent on the CCoAMT allele present and consequently on the activity of the encoded isoform, whereby CGA accumulation increases as the isoform activity decreases. Possible implications in coffee breeding are discussed.

The anti-androgen drug dutasteride renders triple negative breast cancer cells more sensitive to chemotherapy via inhibition of HIF-1α-/VEGF-signaling.

PubMed

von Wahlde, Marie-Kristin; Hülsewig, Carolin; Ruckert, Christian; Götte, Martin; Kiesel, Ludwig; Bernemann, Christof

2015-02-01

Triple negative breast cancer (TNBC) is characterized by lack of expression of both estrogen and progesterone receptor as well as lack of amplification of HER2. Patients with TNBC carry an unfavorable prognosis compared to other breast cancer subtypes given that endocrine or HER2 targeted therapies are not effective, rendering chemotherapy the sole effective treatment option to date. Therefore, there is a high demand for additional novel treatment options. We previously published a list of genes showing both higher gene expression rates in TNBC and, in addition, are known to encode targets of non-oncologic drugs. SRD5A1, which encodes the type-1 isoform of the steroid-5alpha-reductase, which is involved in androgen metabolism, was found to be one of these genes. Dutasteride is a dual blocker of both the type-1 and type-2 isoform of SRD5A1 and is indicated in the treatment of benign prostate hyperplasia. Treatment of TNBC cell lines with dutasteride was associated with a dose-dependent decrease in cell viability, altered protein expression of VEGF and HIF-1α and increased chemosensitivity. Our results demonstrate that the SRD5A1-corresponding anti-androgenic drug dutasteride might act as a combinatorial therapeutic option besides standard chemotherapy in highly aggressive TNBC.

Aberrant alternative splicing is another hallmark of cancer.

PubMed

Ladomery, Michael

2013-01-01

The vast majority of human genes are alternatively spliced. Not surprisingly, aberrant alternative splicing is increasingly linked to cancer. Splice isoforms often encode proteins that have distinct and even antagonistic properties. The abnormal expression of splice factors and splice factor kinases in cancer changes the alternative splicing of critically important pre-mRNAs. Aberrant alternative splicing should be added to the growing list of cancer hallmarks.

The plastid casein kinase 2 phosphorylates Rubisco activase at the Thr-78 site but is not essential for regulation of Rubisco activation state

USDA-ARS?s Scientific Manuscript database

Rubisco activase (RCA) is essential for the activation of Rubisco, the carboxylating enzyme of photosynthesis. In Arabidopsis, RCA is encoded by a single gene (At2g39730) that is alternatively spliced to form a large alpha-RCA and small beta-RCA isoform. The activity of Rubisco is controlled in res...

The USH2A c.2299delG mutation: dating its common origin in a Southern European population

PubMed Central

Aller, Elena; Larrieu, Lise; Jaijo, Teresa; Baux, David; Espinós, Carmen; González-Candelas, Fernando; Nájera, Carmen; Palau, Francesc; Claustres, Mireille; Roux, Anne-Françoise; Millán, José M

2010-01-01

Usher syndrome type II is the most common form of Usher syndrome. USH2A is the main responsible gene of the three known to be disease causing. It encodes two isoforms of the protein usherin. This protein is part of an interactome that has an essential role in the development and function of inner ear hair cells and photoreceptors. The gene contains 72 exons spanning over a region of 800 kb. Although numerous mutations have been described, the c.2299delG mutation is the most prevalent in several populations. Its ancestral origin was previously suggested after the identification of a common core haplotype restricted to 250 kb in the 5′ region that encodes the short usherin isoform. By extending the haplotype analysis over the 800 kb region of the USH2A gene with a total of 14 intragenic single nucleotide polymorphisms, we have been able to define 10 different c.2299delG haplotypes, showing high variability but preserving the previously described core haplotype. An exhaustive c.2299delG/control haplotype study suggests that the major source of variability in the USH2A gene is recombination. Furthermore, we have evidenced twice the amount of recombination hotspots located in the 500 kb region that covers the 3′ end of the gene, explaining the higher variability observed in this region when compared with the 250 kb of the 5′ region. Our data confirm the common ancestral origin of the c.2299delG mutation. PMID:20145675

Drosophila female-specific Ilp7 motoneurons are generated by Fruitless-dependent cell death in males and by a double-assurance survival role for Transformer in females.

PubMed

Garner, Sarah Rose C; Castellanos, Monica C; Baillie, Katherine E; Lian, Tianshun; Allan, Douglas W

2018-01-08

Female-specific Ilp7 neuropeptide-expressing motoneurons (FS-Ilp7 motoneurons) are required in Drosophila for oviduct function in egg laying. Here, we uncover cellular and genetic mechanisms underlying their female-specific generation. We demonstrate that programmed cell death (PCD) eliminates FS-Ilp7 motoneurons in males, and that this requires male-specific splicing of the sex-determination gene fruitless ( fru ) into the Fru MC isoform. However, in females, fru alleles that only generate Fru M isoforms failed to kill FS-Ilp7 motoneurons. This blockade of Fru M -dependent PCD was not attributable to doublesex gene function but to a non-canonical role for transformer ( tra ), a gene encoding the RNA splicing activator that regulates female-specific splicing of fru and dsx transcripts. In both sexes, we show that Tra prevents PCD even when the Fru M isoform is expressed. In addition, we found that Fru MC eliminated FS-Ilp7 motoneurons in both sexes, but only when Tra was absent. Thus, Fru MC -dependent PCD eliminates female-specific neurons in males, and Tra plays a double-assurance function in females to establish and reinforce the decision to generate female-specific neurons. © 2018. Published by The Company of Biologists Ltd.

Identification and Characterization of Alternative Promoters, Transcripts and Protein Isoforms of Zebrafish R2 Gene

PubMed Central

Shang, Hanqiao; Li, Qing; Feng, Guohui; Cui, Zongbin

2011-01-01

Ribonucleotide reductase (RNR) is the rate-limiting enzyme in the de novo synthesis of deoxyribonucleoside triphosphates. Expression of RNR subunits is closely associated with DNA replication and repair. Mammalian RNR M2 subunit (R2) functions exclusively in DNA replication of normal cells due to its S phase-specific expression and late mitotic degradation. Herein, we demonstrate the control of R2 expression through alternative promoters, splicing and polyadenylation sites in zebrafish. Three functional R2 promoters were identified to generate six transcript variants with distinct 5′ termini. The proximal promoter contains a conserved E2F binding site and two CCAAT boxes, which are crucial for the transcription of R2 gene during cell cycle. Activity of the distal promoter can be induced by DNA damage to generate four transcript variants through alternative splicing. In addition, two novel splice variants were found to encode distinct N-truncated R2 isoforms containing residues for enzymatic activity but no KEN box essential for its proteolysis. These two N-truncated R2 isoforms remained in the cytoplasm and were able to interact with RNR M1 subunit (R1). Thus, our results suggest that multilayered mechanisms control the differential expression and function of zebrafish R2 gene during cell cycle and under genotoxic stress. PMID:21887375

Identification and characterization of alternative promoters, transcripts and protein isoforms of zebrafish R2 gene.

PubMed

Shang, Hanqiao; Li, Qing; Feng, Guohui; Cui, Zongbin

2011-01-01

Ribonucleotide reductase (RNR) is the rate-limiting enzyme in the de novo synthesis of deoxyribonucleoside triphosphates. Expression of RNR subunits is closely associated with DNA replication and repair. Mammalian RNR M2 subunit (R2) functions exclusively in DNA replication of normal cells due to its S phase-specific expression and late mitotic degradation. Herein, we demonstrate the control of R2 expression through alternative promoters, splicing and polyadenylation sites in zebrafish. Three functional R2 promoters were identified to generate six transcript variants with distinct 5' termini. The proximal promoter contains a conserved E2F binding site and two CCAAT boxes, which are crucial for the transcription of R2 gene during cell cycle. Activity of the distal promoter can be induced by DNA damage to generate four transcript variants through alternative splicing. In addition, two novel splice variants were found to encode distinct N-truncated R2 isoforms containing residues for enzymatic activity but no KEN box essential for its proteolysis. These two N-truncated R2 isoforms remained in the cytoplasm and were able to interact with RNR M1 subunit (R1). Thus, our results suggest that multilayered mechanisms control the differential expression and function of zebrafish R2 gene during cell cycle and under genotoxic stress.

Spliced synthetic genes as internal controls in RNA sequencing experiments.

PubMed

Hardwick, Simon A; Chen, Wendy Y; Wong, Ted; Deveson, Ira W; Blackburn, James; Andersen, Stacey B; Nielsen, Lars K; Mattick, John S; Mercer, Tim R

2016-09-01

RNA sequencing (RNA-seq) can be used to assemble spliced isoforms, quantify expressed genes and provide a global profile of the transcriptome. However, the size and diversity of the transcriptome, the wide dynamic range in gene expression and inherent technical biases confound RNA-seq analysis. We have developed a set of spike-in RNA standards, termed 'sequins' (sequencing spike-ins), that represent full-length spliced mRNA isoforms. Sequins have an entirely artificial sequence with no homology to natural reference genomes, but they align to gene loci encoded on an artificial in silico chromosome. The combination of multiple sequins across a range of concentrations emulates alternative splicing and differential gene expression, and it provides scaling factors for normalization between samples. We demonstrate the use of sequins in RNA-seq experiments to measure sample-specific biases and determine the limits of reliable transcript assembly and quantification in accompanying human RNA samples. In addition, we have designed a complementary set of sequins that represent fusion genes arising from rearrangements of the in silico chromosome to aid in cancer diagnosis. RNA sequins provide a qualitative and quantitative reference with which to navigate the complexity of the human transcriptome.

Multiple Scenarios of Transition to Chaos in the Alternative Splicing Model

NASA Astrophysics Data System (ADS)

Kogai, Vladislav V.; Likhoshvai, Vitaly A.; Fadeev, Stanislav I.; Khlebodarova, Tamara M.

We have investigated the scenarios of transition to chaos in the mathematical model of a genetic system constituted by a single transcription factor-encoding gene, the expression of which is self-regulated by a feedback loop that involves protein isoforms. Alternative splicing results in the synthesis of protein isoforms providing opposite regulatory outcomes — activation or repression. The model is represented by a differential equation with two delayed arguments. The possibility of transition to chaos dynamics via all classical scenarios: a cascade of period-doubling bifurcations, quasiperiodicity and type-I, type-II and type-III intermittencies, has been numerically demonstrated. The parametric features of each type of transition to chaos have been described.

A pseudogene long noncoding RNA network regulates PTEN transcription and translation in human cells

PubMed Central

Johnsson, Per; Ackley, Amanda; Vidarsdottir, Linda; Lui, Weng-Onn; Corcoran, Martin; Grandér, Dan; Morris, Kevin V.

2013-01-01

PTEN is a tumor suppressor gene that has been shown to be under the regulatory control of a PTEN pseudogene expressed noncoding RNA, PTENpg1. Here, we characterize a previously unidentified PTENpg1 encoded antisense RNA (asRNA), which regulates PTEN transcription and PTEN mRNA stability. We find two PTENpg1 asRNA isoforms, alpha and beta. The alpha isoform functions in trans, localizes to the PTEN promoter, and epigenetically modulates PTEN transcription by the recruitment of DNMT3a and EZH2. In contrast, the beta isoform interacts with PTENpg1 through an RNA:RNA pairing interaction, which affects PTEN protein output via changes of PTENpg1 stability and microRNA sponge activity. Disruption of this asRNA-regulated network induces cell cycle arrest and sensitizes cells to doxorubicin, suggesting a biological function for the respective PTENpg1 expressed asRNAs. PMID:23435381

A pseudogene long-noncoding-RNA network regulates PTEN transcription and translation in human cells.

PubMed

Johnsson, Per; Ackley, Amanda; Vidarsdottir, Linda; Lui, Weng-Onn; Corcoran, Martin; Grandér, Dan; Morris, Kevin V

2013-04-01

PTEN is a tumor-suppressor gene that has been shown to be under the regulatory control of a PTEN pseudogene expressed noncoding RNA, PTENpg1. Here, we characterize a previously unidentified PTENpg1-encoded antisense RNA (asRNA), which regulates PTEN transcription and PTEN mRNA stability. We find two PTENpg1 asRNA isoforms, α and β. The α isoform functions in trans, localizes to the PTEN promoter and epigenetically modulates PTEN transcription by the recruitment of DNA methyltransferase 3a and Enhancer of Zeste. In contrast, the β isoform interacts with PTENpg1 through an RNA-RNA pairing interaction, which affects PTEN protein output through changes of PTENpg1 stability and microRNA sponge activity. Disruption of this asRNA-regulated network induces cell-cycle arrest and sensitizes cells to doxorubicin, which suggests a biological function for the respective PTENpg1 expressed asRNAs.

Bridging the Synaptic Gap: Neuroligins and Neurexin I in Apis mellifera

PubMed Central

Biswas, Sunita; Russell, Robyn J.; Jackson, Colin J.; Vidovic, Maria; Ganeshina, Olga; Oakeshott, John G.; Claudianos, Charles

2008-01-01

Vertebrate studies show neuroligins and neurexins are binding partners in a trans-synaptic cell adhesion complex, implicated in human autism and mental retardation disorders. Here we report a genetic analysis of homologous proteins in the honey bee. As in humans, the honeybee has five large (31–246 kb, up to 12 exons each) neuroligin genes, three of which are tightly clustered. RNA analysis of the neuroligin-3 gene reveals five alternatively spliced transcripts, generated through alternative use of exons encoding the cholinesterase-like domain. Whereas vertebrates have three neurexins the bee has just one gene named neurexin I (400 kb, 28 exons). However alternative isoforms of bee neurexin I are generated by differential use of 12 splice sites, mostly located in regions encoding LNS subdomains. Some of the splice variants of bee neurexin I resemble the vertebrate α- and β-neurexins, albeit in vertebrates these forms are generated by alternative promoters. Novel splicing variations in the 3′ region generate transcripts encoding alternative trans-membrane and PDZ domains. Another 3′ splicing variation predicts soluble neurexin I isoforms. Neurexin I and neuroligin expression was found in brain tissue, with expression present throughout development, and in most cases significantly up-regulated in adults. Transcripts of neurexin I and one neuroligin tested were abundant in mushroom bodies, a higher order processing centre in the bee brain. We show neuroligins and neurexins comprise a highly conserved molecular system with likely similar functional roles in insects as vertebrates, and with scope in the honeybee to generate substantial functional diversity through alternative splicing. Our study provides important prerequisite data for using the bee as a model for vertebrate synaptic development. PMID:18974885

Systematically Differentiating Functions for Alternatively Spliced Isoforms through Integrating RNA-seq Data

PubMed Central

Menon, Rajasree; Wen, Yuchen; Omenn, Gilbert S.; Kretzler, Matthias; Guan, Yuanfang

2013-01-01

Integrating large-scale functional genomic data has significantly accelerated our understanding of gene functions. However, no algorithm has been developed to differentiate functions for isoforms of the same gene using high-throughput genomic data. This is because standard supervised learning requires ‘ground-truth’ functional annotations, which are lacking at the isoform level. To address this challenge, we developed a generic framework that interrogates public RNA-seq data at the transcript level to differentiate functions for alternatively spliced isoforms. For a specific function, our algorithm identifies the ‘responsible’ isoform(s) of a gene and generates classifying models at the isoform level instead of at the gene level. Through cross-validation, we demonstrated that our algorithm is effective in assigning functions to genes, especially the ones with multiple isoforms, and robust to gene expression levels and removal of homologous gene pairs. We identified genes in the mouse whose isoforms are predicted to have disparate functionalities and experimentally validated the ‘responsible’ isoforms using data from mammary tissue. With protein structure modeling and experimental evidence, we further validated the predicted isoform functional differences for the genes Cdkn2a and Anxa6. Our generic framework is the first to predict and differentiate functions for alternatively spliced isoforms, instead of genes, using genomic data. It is extendable to any base machine learner and other species with alternatively spliced isoforms, and shifts the current gene-centered function prediction to isoform-level predictions. PMID:24244129

Twelve actin-encoding cDNAs from the American lobster, Homarus americanus: cloning and tissue expression of eight skeletal muscle, one heart, and three cytoplasmic isoforms.

PubMed

Kim, Bo Kwang; Kim, Kyoung Sun; Oh, Chul-Woong; Mykles, Donald L; Lee, Sung Gu; Kim, Hak Jun; Kim, Hyun-Woo

2009-06-01

Lobster muscles express a diverse array of myofibrillar protein isoforms. Three fiber types (fast, slow-twitch or S1, and slow-tonic or S2) differ qualitatively and quantitatively in myosin heavy and light chains, troponin-T, -I, and -C, paramyosin, and tropomyosin variants. However, little is known about the diversity of actin isoforms present in crustacean tissues. In this report we characterized cDNAs that encode twelve actin isoforms in the American lobster, Homarus americanus: eight from skeletal muscle (Ha-ActinSK1-8), one from heart (Ha-ActinHT1), and three cytoplasmic type actins from hepatopancreas (Ha-ActinCT1-3). All twelve cDNAs were products of distinct genes, as indicated by differences in the 3'-untranslated regions (UTRs). The open reading frames specified polypeptides 376 or 377 amino acids in length. Although key amino residues are conserved in the lobster actins, variations in nearby sequences may affect actin polymerization and/or interactions with other myofibrillar proteins. Quantitative reverse transcription-polymerase chain reaction showed muscle fiber type- and tissue-specific expression patterns. Ha-Actin-HT1 was expressed exclusively in heart (87% of the total; 12% of the total was Ha-ActinCT1). Ha-ActinCT1 was expressed in all tissues, while CT2 and CT3 were expressed only in hepatopancreas, with Ha-ActinCT2 as the major isoform (93% of the total). Ha-ActinSK1 and SK2 were the major isoforms (88% and 12% of the total, respectively) in the S1 fibers of crusher claw closer muscle. Fast fibers in the cutter claw closer and deep abdominal muscles differed in SK isoforms. Ha-ActinSK3, SK4, and SK5 were the major isoforms in cutter claw closer muscle (12%, 48%, and 37% of the total, respectively). Ha-ActinSK5 and SK8 were the major isoforms in deep abdominal flexor (31% and 65% of the total, respectively) and extensor (46% and 53% of the total, respectively) muscles, with SK6 and SK7 expressed at low levels. These data indicate that fast fibers in cutter claw and abdominal muscles show a phenotypic plasticity with respect to the expression of actin isoforms and may constitute discrete subtypes that differ in contractile properties.

The 2p21 deletion syndrome: characterization of the transcription content.

PubMed

Parvari, Ruti; Gonen, Yael; Alshafee, Ismael; Buriakovsky, Sophia; Regev, Kfir; Hershkovitz, Eli

2005-08-01

The vast majority of small-deletion syndromes are caused by haploinsufficiency of one or several genes and are transmitted as dominant traits. We have previously identified a homozygous deletion of 179,311 bp on chromosome 2p21 as the cause of a unique syndrome, inherited in a recessive mode, consisting of cystinuria, neonatal seizures, hypotonia, severe somatic and developmental delay, facial dysmorphism, and reduced activity of all the respiratory chain enzymatic complexes that are encoded in the mitochondria. We now present the transcription content of this region: Multiple splicing variants of the genes protein phosphatase 1B (formerly 2C) magnesium-dependent, beta isoform (PPM1B), SLC3A1, and KIAA0436 (approved gene symbol PREPL) were identified and their patterns of expression analyzed. The spliced variants are predicted to have additional functions compared to the known variants and their patterns of expression fit the tissues affected by the syndrome. The first exon of an additional gene (C2orf34) is encoded in the deleted region and the gene is not expressed in the patients. In addition several transcripts with very short open reading frames are also encoded in the deletion. The identification of all transcripts encoded in the region deleted in the patients is the first step in the study of the genotype-phenotype correlation of the 2p21 patients.

Structural and transcriptional characterization of a novel member of the soybean urease gene family.

PubMed

Wiebke-Strohm, Beatriz; Ligabue-Braun, Rodrigo; Rechenmacher, Ciliana; De Oliveira-Busatto, Luisa Abruzzi; Carlini, Célia Regina; Bodanese-Zanettini, Maria Helena

2016-04-01

In plants, ureases have been related to urea degradation, to defense against pathogenic fungi and phytophagous insects, and to the soybean-Bradyrhizobium japonicum symbiosis. Two urease isoforms have been described for soybean: the embryo-specific, encoded by Eu1 gene, and the ubiquitous urease, encoded by Eu4. A third urease-encoding locus exists in the completed soybean genome. The gene was designated Eu5 and the putative product of its ORF as SBU-III. Phylogenetic analysis shows that 41 plant, moss and algal ureases have diverged from a common ancestor protein, but ureases from monocots, eudicots and ancient species have evolved independently. Genomes of ancient organisms present a single urease-encoding gene and urease-encoding gene duplication has occurred independently along the evolution of some eudicot species. SBU-III has a shorter amino acid sequence, since many gaps are found when compared to other sequences. A mutation in a highly conserved amino acid residue suggests absence of ureolytic activity, but the overall protein architecture remains very similar to the other ureases. The expression profile of urease-encoding genes in different organs and developmental stages was determined by RT-qPCR. Eu5 transcripts were detected in seeds one day after dormancy break, roots of young plants and embryos of developing seeds. Eu1 and Eu4 transcripts were found in all analyzed organs, but Eu4 expression was more prominent in seeds one day after dormancy break whereas Eu1 predominated in developing seeds. The evidence suggests that SBU-III may not be involved in nitrogen availability to plants, but it could be involved in other biological role(s). Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Developmental Regulation of Diacylglycerol Acyltransferase Family Gene Expression in Tung Tree Tissues

PubMed Central

Cao, Heping; Shockey, Jay M.; Klasson, K. Thomas; Chapital, Dorselyn C.; Mason, Catherine B.; Scheffler, Brian E.

2013-01-01

Diacylglycerol acyltransferases (DGAT) catalyze the final and rate-limiting step of triacylglycerol (TAG) biosynthesis in eukaryotic organisms. DGAT genes have been identified in numerous organisms. Multiple isoforms of DGAT are present in eukaryotes. We previously cloned DGAT1 and DGAT2 genes of tung tree (Vernicia fordii), whose novel seed TAGs are useful in a wide range of industrial applications. The objective of this study was to understand the developmental regulation of DGAT family gene expression in tung tree. To this end, we first cloned a tung tree gene encoding DGAT3, a putatively soluble form of DGAT that possesses 11 completely conserved amino acid residues shared among 27 DGAT3s from 19 plant species. Unlike DGAT1 and DGAT2 subfamilies, DGAT3 is absent from animals. We then used TaqMan and SYBR Green quantitative real-time PCR, along with northern and western blotting, to study the expression patterns of the three DGAT genes in tung tree tissues. Expression results demonstrate that 1) all three isoforms of DGAT genes are expressed in developing seeds, leaves and flowers; 2) DGAT2 is the major DGAT mRNA in tung seeds, whose expression profile is well-coordinated with the oil profile in developing tung seeds; and 3) DGAT3 is the major form of DGAT mRNA in tung leaves, flowers and immature seeds prior to active tung oil biosynthesis. These results suggest that DGAT2 is probably the major TAG biosynthetic isoform in tung seeds and that DGAT3 gene likely plays a significant role in TAG metabolism in other tissues. Therefore, DGAT2 should be a primary target for tung oil engineering in transgenic organisms. PMID:24146944

The ADAR RNA editing enzyme controls neuronal excitability in Drosophila melanogaster

PubMed Central

Li, Xianghua; Overton, Ian M.; Baines, Richard A.; Keegan, Liam P.; O’Connell, Mary A.

2014-01-01

RNA editing by deamination of specific adenosine bases to inosines during pre-mRNA processing generates edited isoforms of proteins. Recoding RNA editing is more widespread in Drosophila than in vertebrates. Editing levels rise strongly at metamorphosis, and Adar5G1 null mutant flies lack editing events in hundreds of CNS transcripts; mutant flies have reduced viability, severely defective locomotion and age-dependent neurodegeneration. On the other hand, overexpressing an adult dADAR isoform with high enzymatic activity ubiquitously during larval and pupal stages is lethal. Advantage was taken of this to screen for genetic modifiers; Adar overexpression lethality is rescued by reduced dosage of the Rdl (Resistant to dieldrin), gene encoding a subunit of inhibitory GABA receptors. Reduced dosage of the Gad1 gene encoding the GABA synthetase also rescues Adar overexpression lethality. Drosophila Adar5G1 mutant phenotypes are ameliorated by feeding GABA modulators. We demonstrate that neuronal excitability is linked to dADAR expression levels in individual neurons; Adar-overexpressing larval motor neurons show reduced excitability whereas Adar5G1 null mutant or targeted Adar knockdown motor neurons exhibit increased excitability. GABA inhibitory signalling is impaired in human epileptic and autistic conditions, and vertebrate ADARs may have a relevant evolutionarily conserved control over neuronal excitability. PMID:24137011

Organ-specific lymphangiectasia, arrested lymphatic sprouting, and maturation defects resulting from gene-targeting of the PI3K regulatory isoforms p85α, p55α, and p50α

PubMed Central

Mouta-Bellum, Carla; Kirov, Aleksander; Miceli-Libby, Laura; Mancini, Maria L.; Petrova, Tatiana V.; Liaw, Lucy; Prudovsky, Igor; Thorpe, Philip E.; Miura, Naoyuki; Cantley, Lewis C.; Alitalo, Kari; Fruman, David A.; Vary, Calvin P.H.

2010-01-01

The phosphoinositide 3-kinase (PI3K) family has multiple vascular functions, but the specific regulatory isoform supporting lymphangiogenesis remains unidentified. Here we report that deletion of the Pik3r1 gene, encoding the regulatory subunits p85α, p55α, and p50α impairs lymphatic sprouting and maturation, and causes abnormal lymphatic morphology, without major impact on blood vessels. Pik3r1 deletion had the most severe consequences among gut and diaphragm lymphatics, which share the retroperitoneal anlage, initially suggesting that the Pik3r1 role in this vasculature is anlage-dependent. However, whereas lymphatic sprouting toward the diaphragm was arrested, lymphatics invaded the gut, where remodeling and valve formation were impaired. Thus, cell-origin fails to explain the phenotype. Only the gut showed lymphangiectasia, lymphatic up-regulation of the TGFβ co-receptor endoglin, and reduced levels of mature VEGF-C protein. Our data suggest that Pik3r1 isoforms are required for distinct steps of embryonic lymphangiogenesis in different organ microenvironments, whereas they are largely dispensable for hemangiogenesis. PMID:19705443

The Allosterically Unregulated Isoform of ADP-Glucose Pyrophosphorylase from Barley Endosperm Is the Most Likely Source of ADP-Glucose Incorporated into Endosperm Starch.

PubMed

Doan; Rudi; Olsen

1999-11-01

We present the results of studies of an unmodified version of the recombinant major barley (Hordeum vulgare) endosperm ADP-glucose pyrophoshorylase (AGPase) expressed in insect cells, which corroborate previous data that this isoform of the enzyme acts independently of the allosteric regulators 3-phosphoglycerate and inorganic phosphate. We also present a characterization of the individual subunits expressed separately in insect cells, showing that the SS AGPase is active in the presence of 3-phosphoglycerate and is inhibited by inorganic phosphate. As a step toward the elucidation of the role of the two AGPase isoforms in barley, the temporal and spatial expression profile of the four barley AGPase transcripts encoding these isoforms were studied. The results show that the steady-state level of beps and bepl, the transcripts encoding the major endosperm isoform, correlated positively with the rate of endosperm starch accumulation. In contrast, blps and blpl, the transcripts encoding the major leaf isoform, were constitutively expressed at a very low steady-state level throughout the barley plant. The implications of these findings for the evolution of plant AGPases are discussed.

The Allosterically Unregulated Isoform of ADP-Glucose Pyrophosphorylase from Barley Endosperm Is the Most Likely Source of ADP-Glucose Incorporated into Endosperm Starch1

PubMed Central

Doan, Danny N.P.; Rudi, Heidi; Olsen, Odd-Arne

1999-01-01

We present the results of studies of an unmodified version of the recombinant major barley (Hordeum vulgare) endosperm ADP-glucose pyrophoshorylase (AGPase) expressed in insect cells, which corroborate previous data that this isoform of the enzyme acts independently of the allosteric regulators 3-phosphoglycerate and inorganic phosphate. We also present a characterization of the individual subunits expressed separately in insect cells, showing that the SS AGPase is active in the presence of 3-phosphoglycerate and is inhibited by inorganic phosphate. As a step toward the elucidation of the role of the two AGPase isoforms in barley, the temporal and spatial expression profile of the four barley AGPase transcripts encoding these isoforms were studied. The results show that the steady-state level of beps and bepl, the transcripts encoding the major endosperm isoform, correlated positively with the rate of endosperm starch accumulation. In contrast, blps and blpl, the transcripts encoding the major leaf isoform, were constitutively expressed at a very low steady-state level throughout the barley plant. The implications of these findings for the evolution of plant AGPases are discussed. PMID:10557246

Molecular analysis of two cDNA clones encoding acidic class I chitinase in maize.

PubMed Central

Wu, S; Kriz, A L; Widholm, J M

1994-01-01

The cloning and analysis of two different cDNA clones encoding putative maize (Zea mays L.) chitinases obtained by polymerase chain reaction (PCR) and cDNA library screening is described. The cDNA library was made from poly(A)+ RNA from leaves challenged with mercuric chloride for 2 d. The two clones, pCh2 and pCh11, appear to encode class I chitinase isoforms with cysteine-rich domains (not found in pCh11 due to the incomplete sequence) and proline-/glycine-rich or proline-rich hinge domains, respectively. The pCh11 clone resembles a previously reported maize seed chitinase; however, the deduced proteins were found to have acidic isoelectric points. Analysis of all monocot chitinase sequences available to date shows that not all class I chitinases possess the basic isoelectric points usually found in dicotyledonous plants and that monocot class II chitinases do not necessarily exhibit acidic isoelectric points. Based on sequence analysis, the pCh2 protein is apparently synthesized as a precursor polypeptide with a signal peptide. Although these two clones belong to class I chitinases, they share only about 70% amino acid homology in the catalytic domain region. Southern blot analysis showed that pCh2 may be encoded by a small gene family, whereas pCh11 was single copy. Northern blot analysis demonstrated that these genes are differentially regulated by mercuric chloride treatment. Mercuric chloride treatment caused rapid induction of pCh2 from 6 to 48 h, whereas pCh11 responded only slightly to the same treatment. During seed germination, embryos constitutively expressed both chitinase genes and the phytohormone abscisic acid had no effect on the expression. The fungus Aspergillus flavus was able to induce both genes to comparable levels in aleurone layers and embryos but not in endosperm tissue. Maize callus growth on the same plate with A. flavus for 1 week showed induction of the transcripts corresponding to pCh2 but not to pCh11. These studies indicate that the different chitinase isoforms in maize might have different functions in the plant, since they show differential expression patterns under different conditions. PMID:7972490

Adjustment of Trehalose Metabolism in Wine Saccharomyces cerevisiae Strains To Modify Ethanol Yields

PubMed Central

Rossouw, D.; Heyns, E. H.; Setati, M. E.; Bosch, S.

2013-01-01

The ability of Saccharomyces cerevisiae to efficiently produce high levels of ethanol through glycolysis has been the focus of much scientific and industrial activity. Despite the accumulated knowledge regarding glycolysis, the modification of flux through this pathway to modify ethanol yields has proved difficult. Here, we report on the systematic screening of 66 strains with deletion mutations of genes encoding enzymes involved in central carbohydrate metabolism for altered ethanol yields. Five of these strains showing the most prominent changes in carbon flux were selected for further investigation. The genes were representative of trehalose biosynthesis (TPS1, encoding trehalose-6-phosphate synthase), central glycolysis (TDH3, encoding glyceraldehyde-3-phosphate dehydrogenase), the oxidative pentose phosphate pathway (ZWF1, encoding glucose-6-phosphate dehydrogenase), and the tricarboxylic acid (TCA) cycle (ACO1 and ACO2, encoding aconitase isoforms 1 and 2). Two strains exhibited lower ethanol yields than the wild type (tps1Δ and tdh3Δ), while the remaining three showed higher ethanol yields. To validate these findings in an industrial yeast strain, the TPS1 gene was selected as a good candidate for genetic modification to alter flux to ethanol during alcoholic fermentation in wine. Using low-strength promoters active at different stages of fermentation, the expression of the TPS1 gene was slightly upregulated, resulting in a decrease in ethanol production and an increase in trehalose biosynthesis during fermentation. Thus, the mutant screening approach was successful in terms of identifying target genes for genetic modification in commercial yeast strains with the aim of producing lower-ethanol wines. PMID:23793638

Integrating evolutionary and functional tests of adaptive hypotheses: a case study of altitudinal differentiation in hemoglobin function in an Andean Sparrow, Zonotrichia capensis.

PubMed

Cheviron, Zachary A; Natarajan, Chandrasekhar; Projecto-Garcia, Joana; Eddy, Douglas K; Jones, Jennifer; Carling, Matthew D; Witt, Christopher C; Moriyama, Hideaki; Weber, Roy E; Fago, Angela; Storz, Jay F

2014-11-01

In air-breathing vertebrates, the physiologically optimal blood-O2 affinity is jointly determined by the prevailing partial pressure of atmospheric O2, the efficacy of pulmonary O2 transfer, and internal metabolic demands. Consequently, genetic variation in the oxygenation properties of hemoglobin (Hb) may be subject to spatially varying selection in species with broad elevational distributions. Here we report the results of a combined functional and evolutionary analysis of Hb polymorphism in the rufous-collared sparrow (Zonotrichia capensis), a species that is continuously distributed across a steep elevational gradient on the Pacific slope of the Peruvian Andes. We integrated a population genomic analysis that included all postnatally expressed Hb genes with functional studies of naturally occurring Hb variants, as well as recombinant Hb (rHb) mutants that were engineered through site-directed mutagenesis. We identified three clinally varying amino acid polymorphisms: Two in the α(A)-globin gene, which encodes the α-chain subunits of the major HbA isoform, and one in the α(D)-globin gene, which encodes the α-chain subunits of the minor HbD isoform. We then constructed and experimentally tested single- and double-mutant rHbs representing each of the alternative α(A)-globin genotypes that predominate at different elevations. Although the locus-specific patterns of altitudinal differentiation suggested a history of spatially varying selection acting on Hb polymorphism, the experimental tests demonstrated that the observed amino acid mutations have no discernible effect on respiratory properties of the HbA or HbD isoforms. These results highlight the importance of experimentally validating the hypothesized effects of genetic changes in protein function to avoid the pitfalls of adaptive storytelling. © The Author 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

The evolution of aryl hydrocarbon signaling proteins: diversity of ARNT isoforms among fish species.

PubMed

Powell, W H; Hahn, M E

2000-01-01

The aryl hydrocarbon receptor nuclear translocator (ARNT) mediates aryl hydrocarbon signaling and toxicity by dimerizing with the ligand-activated aryl hydrocarbon receptor (AHR), forming a complex that binds specific DNA elements and alters transcription of target genes. Two genes encode different forms of ARNT in rodents: ARNT1, which is widely expressed, and ARNT2, which exhibits a very restricted expression pattern. In an effort to characterize aryl hydrocarbon signaling mechanisms in fishes, we previously isolated an ARNT cDNA from Fundulus heteroclitus and discovered that this species expresses ARNT2 ubiquitously. This situation differs not only from mammals, but also from rainbow trout, which expresses a divergent ARNT gene that we hypothesized was peculiar to salmonids (rtARNTa/b). In this communication, we examine the ARNT sequences of multiple fish species, including a newly isolated cDNA from scup (Stenotomus chrysops). Our phylogenetic analysis demonstrates that zebrafish ARNT, like the Fundulus protein, is an ARNT2. Contrary to expectations, the scup ARNT is closely related to the rainbow trout protein, demonstrating that the existence of this ARNT isoform predates the divergence of salmonids from the other teleosts. Thus, different species of fish express distinct and highly conserved isoforms of ARNT. The number, type, and expression pattern of ARNT proteins may contribute to interspecies differences in aryl hydrocarbon toxicity, possibly through distinct interactions with additional PAS-family proteins.

The 21.5-kDa isoform of myelin basic protein has a non-traditional PY-nuclear-localization signal

DOE Office of Scientific and Technical Information (OSTI.GOV)

Smith, Graham S.T.; Seymour, Lauren V.; Boggs, Joan M.

2012-06-15

Highlights: Black-Right-Pointing-Pointer Full-length 21.5-kDa MBP isoform is translocated to the nucleus. Black-Right-Pointing-Pointer We hypothesized that the exon-II-encoded sequence contained the NLS. Black-Right-Pointing-Pointer We mutated this sequence in RFP-tagged constructs and transfected N19-cells. Black-Right-Pointing-Pointer Abolition of two key positively-charged residues resulted in loss of nuclear-trafficking. Black-Right-Pointing-Pointer The 21.5-kDa isoform of classic MBP contains a non-traditional PY-NLS. -- Abstract: The predominant 18.5-kDa classic myelin basic protein (MBP) is mainly responsible for compaction of the myelin sheath in the central nervous system, but is multifunctional, having numerous interactions with Ca{sup 2+}-calmodulin, actin, tubulin, and SH3-domains, and can tether these proteins to a lipidmore » membrane in vitro. The full-length 21.5-kDa MBP isoform has an additional 26 residues encoded by exon-II of the classic gene, which causes it to be trafficked to the nucleus of oligodendrocytes (OLGs). We have performed site-directed mutagenesis of selected residues within this segment in red fluorescent protein (RFP)-tagged constructs, which were then transfected into the immortalized N19-OLG cell line to view protein localization using epifluorescence microscopy. We found that 21.5-kDa MBP contains two non-traditional PY-nuclear-localization signals, and that arginine and lysine residues within these motifs were involved in subcellular trafficking of this protein to the nucleus, where it may have functional roles during myelinogenesis.« less

Exon Skipping in the RET Gene Encodes Novel Isoforms That Differentially Regulate RET Protein Signal Transduction*

PubMed Central

Gabreski, Nicole A.; Vaghasia, Janki K.; Novakova, Silvia S.; McDonald, Neil Q.; Pierchala, Brian A.

2016-01-01

Rearranged during transfection (RET), a receptor tyrosine kinase that is activated by the glial cell line-derived neurotrophic factor family ligands (GFLs), plays a crucial role in the development and function of the nervous system and additionally is required for kidney development and spermatogenesis. RET encodes a transmembrane receptor that is 20 exons long and produces two known protein isoforms differing in C-terminal amino acid composition, referred to as RET9 and RET51. Studies of human pheochromocytomas identified two additional novel transcripts involving the skipping of exon 3 or exons 3, 4, and 5 and are referred to as RETΔE3 and RETΔE345, respectively. Here we report the presence of RetΔE3 and RetΔE345 in zebrafish, mice, and rats and show that these transcripts are dynamically expressed throughout development of the CNS, peripheral nervous system, and kidneys. We further explore the biochemical properties of these isoforms, demonstrating that, like full-length RET, RETΔE3 and RETΔE345 are trafficked to the cell surface, interact with all four GFRα co-receptors, and have the ability to heterodimerize with full-length RET. Signaling experiments indicate that RETΔE3 is phosphorylated in a similar manner to full-length RET. RETΔE345, in contrast, displays higher baseline autophosphorylation, specifically on the catalytic tyrosine, Tyr905, and also on one of the most important signaling residues, Tyr1062. These data provide the first evidence for a physiologic role of these isoforms in RET pathway function. PMID:27226544

Photosynthetic Trichomes Contain a Specific Rubisco with a Modified pH-Dependent Activity.

PubMed

Laterre, Raphaëlle; Pottier, Mathieu; Remacle, Claire; Boutry, Marc

2017-04-01

Ribulose-1,5-biphosphate carboxylase/oxygenase (Rubisco) is the most abundant enzyme in plants and is responsible for CO 2 fixation during photosynthesis. This enzyme is assembled from eight large subunits (RbcL) encoded by a single chloroplast gene and eight small subunits (RbcS) encoded by a nuclear gene family. Rubisco is primarily found in the chloroplasts of mesophyll (C3 plants), bundle-sheath (C4 plants), and guard cells. In certain species, photosynthesis also takes place in the secretory cells of glandular trichomes, which are epidermal outgrowths (hairs) involved in the secretion of specialized metabolites. However, photosynthesis and, in particular, Rubisco have not been characterized in trichomes. Here, we show that tobacco ( Nicotiana tabacum ) trichomes contain a specific Rubisco small subunit, NtRbcS-T, which belongs to an uncharacterized phylogenetic cluster (T). This cluster contains RbcS from at least 33 species, including monocots, many of which are known to possess glandular trichomes. Cluster T is distinct from the cluster M, which includes the abundant, functionally characterized RbcS isoforms expressed in mesophyll or bundle-sheath cells. Expression of NtRbcS-T in Chlamydomonas reinhardtii and purification of the full Rubisco complex showed that this isoform conferred higher V max and K m values as well as higher acidic pH-dependent activity than NtRbcS-M, an isoform expressed in the mesophyll. This observation was confirmed with trichome extracts. These data show that an ancient divergence allowed for the emergence of a so-far-uncharacterized RbcS cluster. We propose that secretory trichomes have a particular Rubisco uniquely adapted to secretory cells where CO 2 is released by the active specialized metabolism. © 2017 American Society of Plant Biologists. All Rights Reserved.

A common variant in combination with a nonsense mutation in a member of the thioredoxin family causes primary ciliary dyskinesia

PubMed Central

Duriez, Bénédicte; Duquesnoy, Philippe; Escudier, Estelle; Bridoux, Anne-Marie; Escalier, Denise; Rayet, Isabelle; Marcos, Elisabeth; Vojtek, Anne-Marie; Bercher, Jean-François; Amselem, Serge

2007-01-01

Thioredoxins belong to a large family of enzymatic proteins that function as general protein disulfide reductases, therefore participating in several cellular processes via redox-mediated reactions. So far, none of the 18 members of this family has been involved in human pathology. Here we identified TXNDC3, which encodes a thioredoxin–nucleoside diphosphate kinase, as a gene implicated in primary ciliary dyskinesia (PCD), a genetic condition characterized by chronic respiratory tract infections, left–right asymmetry randomization, and male infertility. We show that the disease, which segregates as a recessive trait, results from the unusual combination of the following two transallelic defects: a nonsense mutation and a common intronic variant found in 1% of control chromosomes. This variant affects the ratio of two physiological TXNDC3 transcripts: the full-length isoform and a novel isoform, TXNDC3d7, carrying an in-frame deletion of exon 7. In vivo and in vitro expression data unveiled the physiological importance of TXNDC3d7 (whose expression was reduced in the patient) and the corresponding protein that was shown to bind microtubules. PCD is known to result from defects of the axoneme, an organelle common to respiratory cilia, embryonic nodal cilia, and sperm flagella, containing dynein arms, with, to date, the implication of genes encoding dynein proteins. Our findings, which identify a another class of molecules involved in PCD, disclose the key role of TXNDC3 in ciliary function; they also point to an unusual mechanism underlying a Mendelian disorder, which is an SNP-induced modification of the ratio of two physiological isoforms generated by alternative splicing. PMID:17360648

Alternative Splicing of Barley Clock Genes in Response to Low Temperature

PubMed Central

Calixto, Cristiane P. G.; Simpson, Craig G.; Waugh, Robbie; Brown, John W. S.

2016-01-01

Alternative splicing (AS) is a regulated mechanism that generates multiple transcripts from individual genes. It is widespread in eukaryotic genomes and provides an effective way to control gene expression. At low temperatures, AS regulates Arabidopsis clock genes through dynamic changes in the levels of productive mRNAs. We examined AS in barley clock genes to assess whether temperature-dependent AS responses also occur in a monocotyledonous crop species. We identify changes in AS of various barley core clock genes including the barley orthologues of Arabidopsis AtLHY and AtPRR7 which showed the most pronounced AS changes in response to low temperature. The AS events modulate the levels of functional and translatable mRNAs, and potentially protein levels, upon transition to cold. There is some conservation of AS events and/or splicing behaviour of clock genes between Arabidopsis and barley. In addition, novel temperature-dependent AS of the core clock gene HvPPD-H1 (a major determinant of photoperiod response and AtPRR7 orthologue) is conserved in monocots. HvPPD-H1 showed a rapid, temperature-sensitive isoform switch which resulted in changes in abundance of AS variants encoding different protein isoforms. This novel layer of low temperature control of clock gene expression, observed in two very different species, will help our understanding of plant adaptation to different environments and ultimately offer a new range of targets for plant improvement. PMID:27959947

PCR cloning and characterization of multiple ADP-glucose pyrophosphorylase cDNAs from tomato

NASA Technical Reports Server (NTRS)

Chen, B. Y.; Janes, H. W.; Gianfagna, T.

1998-01-01

Four ADP-glucose pyrophosphorylase (AGP) cDNAs were cloned from tomato fruit and leaves by the PCR techniques. Three of them (agp S1, agp S2, and agp S3) encode the large subunit of AGP, the fourth one (agp B) encodes the small subunit. The deduced amino acid sequences of the cDNAs show very high identities (96-98%) to the corresponding potato AGP isoforms, although there are major differences in tissue expression profiles. All four tomato AGP transcripts were detected in fruit and leaves; the predominant ones in fruit are agp B and agp S1, whereas in leaves they are agp B and agp S3. Genomic southern analysis suggests that the four AGP transcripts are encoded by distinct genes.

Differential expression of alternatively spliced transcripts related to energy metabolism in colorectal cancer.

PubMed

Snezhkina, Anastasiya Vladimirovna; Krasnov, George Sergeevich; Zaretsky, Andrew Rostislavovich; Zhavoronkov, Alex; Nyushko, Kirill Mikhailovich; Moskalev, Alexey Alexandrovich; Karpova, Irina Yurievna; Afremova, Anastasiya Isaevna; Lipatova, Anastasiya Valerievna; Kochetkov, Dmitriy Vladimitovich; Fedorova, Maria Sergeena; Volchenko, Nadezhda Nikolaevna; Sadritdinova, Asiya Fayazovna; Melnikova, Nataliya Vladimirovna; Sidorov, Dmitry Vladimirovich; Popov, Anatoly Yurievich; Kalinin, Dmitry Valerievich; Kaprin, Andrey Dmitrievich; Alekseev, Boris Yakovlevich; Dmitriev, Alexey Alexandrovich; Kudryavtseva, Anna Viktorovna

2016-12-28

Colorectal cancer (CRC) is one of the most common malignant tumors worldwide. CRC molecular pathogenesis is heterogeneous and may be followed by mutations in oncogenes and tumor suppressor genes, chromosomal and microsatellite instability, alternative splicing alterations, hypermethylation of CpG islands, oxidative stress, impairment of different signaling pathways and energy metabolism. In the present work, we have studied the alterations of alternative splicing patterns of genes related to energy metabolism in CRC. Using CrossHub software, we analyzed The Cancer Genome Atlas (TCGA) RNA-Seq datasets derived from colon tumor and matched normal tissues. The expression of 1014 alternative mRNA isoforms involved in cell energy metabolism was examined. We found 7 genes with differentially expressed alternative transcripts whereas overall expression of these genes was not significantly altered in CRC. A set of 8 differentially expressed transcripts of interest has been validated by qPCR. These eight isoforms encoded by OGDH, COL6A3, ICAM1, PHPT1, PPP2R5D, SLC29A1, and TRIB3 genes were up-regulated in colorectal tumors, and this is in concordance with the bioinformatics data. The alternative transcript NM_057167 of COL6A3 was also strongly up-regulated in breast, lung, prostate, and kidney tumors. Alternative transcript of SLC29A1 (NM_001078177) was up-regulated only in CRC samples, but not in the other tested tumor types. We identified tumor-specific expression of alternative spliced transcripts of seven genes involved in energy metabolism in CRC. Our results bring new knowledge on alternative splicing in colorectal cancer and suggest a set of mRNA isoforms that could be used for cancer diagnosis and development of treatment methods.

Identification of two frataxin isoforms in Zea mays: Structural and functional studies.

PubMed

Buchensky, Celeste; Sánchez, Manuel; Carrillo, Martin; Palacios, Oscar; Capdevila, Mercè; Domínguez-Vera, Jose M; Busi, Maria V; Atrian, Sílvia; Pagani, Maria A; Gomez-Casati, Diego F

2017-09-01

Frataxin is a ubiquitous protein that plays a role in Fe-S cluster biosynthesis and iron and heme metabolism, although its molecular functions are not entirely clear. In non-photosynthetic eukaryotes, frataxin is encoded by a single gene, and the protein localizes to mitochondria. Here we report the presence of two functional frataxin isoforms in Zea mays, ZmFH-1 and ZmFH-2. We confirmed our previous findings regarding plant frataxins: both proteins have dual localization in mitochondria and chloroplasts. Physiological, biochemical and biophysical studies show some differences in the expression pattern, protection against oxidants and in the aggregation state of both isoforms, suggesting that the two frataxin homologs would play similar but not identical roles in plant cell metabolism. In addition, two specific features of plant frataxins were evidenced: their ability to form dimers and their tendency to undergo conformational change under oxygen exposure. Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

New insights into plant glycoside hydrolase family 32 in Agave species

PubMed Central

Avila de Dios, Emmanuel; Gomez Vargas, Alan D.; Damián Santos, Maura L.; Simpson, June

2015-01-01

In order to optimize the use of agaves for commercial applications, an understanding of fructan metabolism in these species at the molecular and genetic level is essential. Based on transcriptome data, this report describes the identification and molecular characterization of cDNAs and deduced amino acid sequences for genes encoding fructosyltransferases, invertases and fructan exohydrolases (FEH) (enzymes belonging to plant glycoside hydrolase family 32) from four different agave species (A. tequilana, A. deserti, A. victoriae-reginae, and A. striata). Conserved amino acid sequences and a hypervariable domain allowed classification of distinct isoforms for each enzyme type. Notably however neither 1-FFT nor 6-SFT encoding cDNAs were identified. In silico analysis revealed that distinct isoforms for certain enzymes found in a single species, showed different levels and tissue specific patterns of expression whereas in other cases expression patterns were conserved both within the species and between different species. Relatively high levels of in silico expression for specific isoforms of both invertases and fructosyltransferases were observed in floral tissues in comparison to vegetative tissues such as leaves and stems and this pattern was confirmed by Quantitative Real Time PCR using RNA obtained from floral and leaf tissue of A. tequilana. Thin layer chromatography confirmed the presence of fructans with degree of polymerization (DP) greater than DP three in both immature buds and fully opened flowers also obtained from A. tequilana. PMID:26300895

New insights into plant glycoside hydrolase family 32 in Agave species.

PubMed

Avila de Dios, Emmanuel; Gomez Vargas, Alan D; Damián Santos, Maura L; Simpson, June

2015-01-01

In order to optimize the use of agaves for commercial applications, an understanding of fructan metabolism in these species at the molecular and genetic level is essential. Based on transcriptome data, this report describes the identification and molecular characterization of cDNAs and deduced amino acid sequences for genes encoding fructosyltransferases, invertases and fructan exohydrolases (FEH) (enzymes belonging to plant glycoside hydrolase family 32) from four different agave species (A. tequilana, A. deserti, A. victoriae-reginae, and A. striata). Conserved amino acid sequences and a hypervariable domain allowed classification of distinct isoforms for each enzyme type. Notably however neither 1-FFT nor 6-SFT encoding cDNAs were identified. In silico analysis revealed that distinct isoforms for certain enzymes found in a single species, showed different levels and tissue specific patterns of expression whereas in other cases expression patterns were conserved both within the species and between different species. Relatively high levels of in silico expression for specific isoforms of both invertases and fructosyltransferases were observed in floral tissues in comparison to vegetative tissues such as leaves and stems and this pattern was confirmed by Quantitative Real Time PCR using RNA obtained from floral and leaf tissue of A. tequilana. Thin layer chromatography confirmed the presence of fructans with degree of polymerization (DP) greater than DP three in both immature buds and fully opened flowers also obtained from A. tequilana.

A novel transcript of cyclin-dependent kinase-like 5 (CDKL5) has an alternative C-terminus and is the predominant transcript in brain.

PubMed

Williamson, Sarah L; Giudici, Laura; Kilstrup-Nielsen, Charlotte; Gold, Wendy; Pelka, Gregory J; Tam, Patrick P L; Grimm, Andrew; Prodi, Dionigio; Landsberger, Nicoletta; Christodoulou, John

2012-02-01

The X-linked cyclin-dependent kinase-like 5 (CDKL5) gene is an important molecular determinant of early-onset intractable seizures with infantile spasms and Rett syndrome-like phenotype. The gene encodes a kinase that may influence components of molecular pathways associated with MeCP2. In humans there are two previously reported splice variants that differ in the 5' untranslated exons and produce the same 115 kDa protein. Furthermore, very recently, a novel transcript including a novel exon (16b) has been described. By aligning both the human and mouse CDKL5 proteins to the orthologs of other species, we identified a theoretical 107 kDa isoform with an alternative C-terminus that terminates in intron 18. In human brain and all other tissues investigated except the testis, this novel isoform is the major CDKL5 transcript. The detailed characterisation of this novel isoform of CDKL5 reveals functional and subcellular localisation attributes that overlap greatly, but not completely, with that of the previously studied human CDKL5 protein. Considering its predominant expression in the human and mouse brain, we believe that this novel isoform is likely to be of primary pathogenic importance in human diseases associated with CDKL5 deficiency, and suggest that screening of the related intronic sequence should be included in the molecular genetic analyses of patients with a suggestive clinical phenotype.

Molecular characterization and expression profiles of four transformer-2 isoforms in the Chinese mitten crab Eriocheir sinensis

NASA Astrophysics Data System (ADS)

Luo, Danli; Liu, Yuan; Hui, Min; Song, Chengwen; Liu, Hourong; Cui, Zhaoxia

2017-07-01

The transformer-2 ( tra-2) gene plays a key role in the regulatory hierarchy of sexual differentiation in somatic tissues and in the germline of Drosophila melanogaster. In this study, sequences and expression profiles of tra-2 in the Chinese mitten crab Eriocheir sinensis were characterized. Four tra-2 isoforms, designated as Estra-2a, Estra-2b, Estra-2c, and Estra-2d, were isolated. They all contained an RNA-recognition motif (RRM) and a linker region, which shared high similarity with other reported tra-2s. Sequence analysis revealed that Estra-2a, Estra-2b and Estra-2c are encoded by the same genomic locus and are generated by alternative splicing of the pre-mRNA. Compared with the other three isoforms, Estra-2d lacks the RS2 domain. Quantitative real-time PCR showed that all four isoforms were highly expressed in the fertilized egg, and in the 2-4 cell and blastula stages compared with larval stages ( P≤0.01), suggesting their maternal origin in early embryonic developmental stages. Notably, Estra-2a was highly expressed in male somatic tissues, while Estra-2c was significantly highly expressed in the ovary. These results suggest that Estra-2c is involved in sexual differentiation of the Chinese mitten crab. Our findings provide basic information for further functional studies of the tra-2 gene/protein in this species.

Evidence of a bigenomic regulation of mitochondrial gene expression by thyroid hormone during rat brain development.

PubMed

Sinha, Rohit Anthony; Pathak, Amrita; Mohan, Vishwa; Babu, Satish; Pal, Amit; Khare, Drirh; Godbole, Madan M

2010-07-02

Hypothyroidism during early mammalian brain development is associated with decreased expression of various mitochondrial encoded genes along with evidence for mitochondrial dysfunction. However, in-spite of the similarities between neurological disorders caused by perinatal hypothyroidism and those caused by various genetic mitochondrial defects we still do not know as to how thyroid hormone (TH) regulates mitochondrial transcription during development and whether this regulation by TH is nuclear mediated or through mitochondrial TH receptors? We here in rat cerebellum show that hypothyroidism causes reduction in expression of nuclear encoded genes controlling mitochondrial biogenesis like PGC-1alpha, NRF-1alpha and Tfam. Also, we for the first time demonstrate a mitochondrial localization of thyroid hormone receptor (mTR) isoform in developing brain capable of binding a TH response element (DR2) present in D-loop region of mitochondrial DNA. These results thus indicate an integrated nuclear-mitochondrial cross talk in regulation of mitochondrial transcription by TH during brain development. Copyright 2010 Elsevier Inc. All rights reserved.

Evidence of a bigenomic regulation of mitochondrial gene expression by thyroid hormone during rat brain development

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sinha, Rohit Anthony; Pathak, Amrita; Mohan, Vishwa

Hypothyroidism during early mammalian brain development is associated with decreased expression of various mitochondrial encoded genes along with evidence for mitochondrial dysfunction. However, in-spite of the similarities between neurological disorders caused by perinatal hypothyroidism and those caused by various genetic mitochondrial defects we still do not know as to how thyroid hormone (TH) regulates mitochondrial transcription during development and whether this regulation by TH is nuclear mediated or through mitochondrial TH receptors? We here in rat cerebellum show that hypothyroidism causes reduction in expression of nuclear encoded genes controlling mitochondrial biogenesis like PGC-1{alpha}, NRF-1{alpha} and Tfam. Also, we for themore » first time demonstrate a mitochondrial localization of thyroid hormone receptor (mTR) isoform in developing brain capable of binding a TH response element (DR2) present in D-loop region of mitochondrial DNA. These results thus indicate an integrated nuclear-mitochondrial cross talk in regulation of mitochondrial transcription by TH during brain development.« less

The gene for the alpha 1 subunit of the skeletal muscle dihydropyridine-sensitive calcium channel (Cchl1a3) maps to mouse chromosome 1.

PubMed

Chin, H; Krall, M; Kim, H L; Kozak, C A; Mock, B

1992-12-01

Cchl1a3 encodes the dihydropyridine-sensitive calcium channel alpha 1 subunit isoform predominantly expressed in skeletal muscle. mdg (muscular dysgenesis) has previously been implicated as a mutant allele of this gene. Hybridization of a rat brain cDNA probe for Cchl1a3 to Southern blots of DNAs from a panel of Chinese hamster x mouse somatic cell hybrids suggested that this gene maps to mouse Chromosome 1. Analysis of the progeny of an inbred strain cross-positioned Cchl1a3 1.3 cM proximal to the Pep-3 locus on Chr 1.

Post-Training Intrahippocampal Injection of Synthetic Poly-Alpha-2,8-Sialic Acid-Neural Cell Adhesion Molecule Mimetic Peptide Improves Spatial Long-Term Performance in Mice

ERIC Educational Resources Information Center

Florian, Cedrick; Foltz, Jane; Norreel, Jean-Chretien; Rougon, Genevieve; Roullet, Pascal

2006-01-01

Several data have shown that the neural cell adhesion molecule (NCAM) is necessary for long-term memory formation and might play a role in the structural reorganization of synapses. The NCAM, encoded by a single gene, is represented by several isoforms that differ with regard to their content of alpha-2,8-linked sialic acid residues (PSA) on their…

Genome-based insights into the resistome and mobilome of multidrug-resistant Aeromonas sp. ARM81 isolated from wastewater.

PubMed

Adamczuk, Marcin; Dziewit, Lukasz

2017-01-01

The draft genome of multidrug-resistant Aeromonas sp. ARM81 isolated from a wastewater treatment plant in Warsaw (Poland) was obtained. Sequence analysis revealed multiple genes conferring resistance to aminoglycosides, β-lactams or tetracycline. Three different β-lactamase genes were identified, including an extended-spectrum β-lactamase gene bla PER-1 . The antibiotic susceptibility was experimentally tested. Genome sequencing also allowed us to investigate the plasmidome and transposable mobilome of ARM81. Four plasmids, of which two carry phenotypic modules (i.e., genes encoding a zinc transporter ZitB and a putative glucosyltransferase), and 28 putative transposase genes were identified. The mobility of three insertion sequences (isoforms of previously identified elements ISAs12, ISKpn9 and ISAs26) was confirmed using trap plasmids.

Cloning and expression of hepatic synaptotagmin 1 in mouse.

PubMed

Sancho-Knapik, Sara; Guillén, Natalia; Osada, Jesús

2015-05-15

Mouse hepatic synaptotagmin 1 (SYT1) cDNA was cloned, characterized and compared to the brain one. The hepatic transcript was 1807 bp in length, smaller than the brain, and only encoded by 9 of 11 gene exons. In this regard, 5'-and 3'-untranslated regions were 66 and 476 bp, respectively; the open reading frame of 1266 bp codified for a protein of 421 amino acids, identical to the brain, with a predicted molecular mass of 47.4 kDa and highly conserved across different species. Immunoblotting of protein showed two isoforms of higher molecular masses than the theoretical prediction based on amino acid sequence suggesting posttranslational modifications. Subcellular distribution of protein isoforms corresponded to plasma membrane, lysosomes and microsomes and was identical between the brain and liver. Nonetheless, the highest molecular weight isoform was smaller in the liver, irrespective of subcellular location. Quantitative mRNA tissue distribution showed that it was widely expressed and that the highest values corresponded to the brain, followed by the liver, spleen, abdominal fat, intestine and skeletal muscle. These findings indicate tissue-specific splicing of the gene and posttranslational modification and the variation in expression in the different tissues might suggest a different requirement of SYT1 for the specific function in each organ. Copyright © 2015 Elsevier B.V. All rights reserved.

Identification and characterization of doublesex in Bemisia tabaci.

PubMed

Guo, L; Xie, W; Liu, Y; Yang, Z; Yang, X; Xia, J; Wang, S; Wu, Q; Zhang, Y

2018-04-16

Bemisia tabaci (Gennadius) is an important agricultural pest with a worldwide distribution. Although B. tabaci is known to have a unique haplodiploid reproductive strategy, its sex determination mechanism is largely unknown. In this study, we cloned the full-length sequence of B. tabaci doublesex (Btdsx) and found that Btdsx has 28 splicing isoforms. We found two new splicing isoforms of transformer 2 (Bttra2), which encode two proteins. We also confirmed that both genes lack sex-specific splicing isoforms. Real-time quantitative PCR analysis showed that the expression of Btdsx and Bttra2 is higher in males than in females. RNA interference of Bttra2 affected the expression of Btdsx and vice versa. Furthermore, silencing of Bttra2 or Btdsx caused malformation of the male genitalia (anal style). It did not affect the female phenotype, but reduced the expression of vitellogenin gene in females. These results indicate that Btdsx is associated with sex determination in B. tabaci and that Btdsx and Bttra2 affect each other and are important for male genitalia formation. In addition to increasing our understanding of the roles of dsx and tra2 in the sex determination of B. tabaci, the results will be useful for studies of sex determination in other haplodiploid species. © 2018 The Royal Entomological Society.

[Characterization of a malic enzyme isoform V from Mucor circinelloides].

PubMed

Zhang, Yingtong; Chen, Haiqin; Song, Yuanda; Zhang, Hao; Chen, Yongquan; Chen, Wei

2016-02-04

We aimed at characterizing a malic enzyme isoform V from Mucor circinelloides. me1 gene encoding malic enzyme isoform V was amplified and cloned into expression vector pET28a. High-purity recombinant protein BLME1 was obtained by affinity chromatography using. Ni-NTA column and characterized subsequently. The optimum conditions were pH at 8.0 and temperature at 33 degrees C. Under optimum conditions, BLME1 activity achieved 92.8 U/mg. The K(m) for L-malate and NADP+ were 0.74960 ± 0.06120 mmol/L and 0.22070 ± 0.01810 mmol/L, the V(max) for L-malate and NADP+ were 72.820 ± 1.077 U/mg and 86.110 ± 1.665 U/mg, respectively. In addition, ions played important roles in BLME1 activity; several ions such as Mn2+, Mg2+, Co2+, Ni2+ could activate BLME1, whereas Ca2+, Cu2+ could be used as inhibitors. Additionally, the metabolic intermediates such as oxaloacetic acid and α-ketoglutaric acid inhibited the activity of BLME1, whereas succinic acid activated it. A malic enzyme isoform V from Mucor circinelloides was characterized, providing the references for further studies on this enzyme.

Ankyrin-G isoform imbalance and interneuronopathy link epilepsy and bipolar disorder.

PubMed

Lopez, A Y; Wang, X; Xu, M; Maheshwari, A; Curry, D; Lam, S; Adesina, A M; Noebels, J L; Sun, Q-Q; Cooper, E C

2017-10-01

ANK3, encoding the adaptor protein Ankyrin-G (AnkG), has been implicated in bipolar disorder by genome-wide association studies. ANK3 has multiple alternative first exons, and a bipolar disorder-associated ANK3 variant has been shown to reduce the expression of exon 1b. Here we identify mechanisms through which reduced ANK3 exon 1b isoform expression disrupts neuronal excitation-inhibition balance. We find that parvalbumin (PV) interneurons and principal cells differentially express ANK3 first exon subtypes. PV interneurons express only isoforms containing exon 1b, whereas excitatory principal cells express exon 1e alone or both 1e and 1b. In transgenic mice deficient for exon 1b, PV interneurons lack voltage-gated sodium channels at their axonal initial segments and have increased firing thresholds and diminished action potential dynamic range. These mice exhibit an Ank3 gene dosage-dependent phenotype including behavior changes modeling bipolar disorder, epilepsy and sudden death. Thus ANK3's important association with human bipolar susceptibility may arise from imbalance between AnkG function in interneurons and principal cells and resultant excessive circuit sensitivity and output. AnkG isoform imbalance is a novel molecular endophenotype and potential therapeutic target.

RhoGAP18B Isoforms Act on Distinct Rho-Family GTPases and Regulate Behavioral Responses to Alcohol via Cofilin

PubMed Central

Kalahasti, Geetha; Rodan, Aylin R.; Rothenfluh, Adrian

2015-01-01

Responses to the effects of ethanol are highly conserved across organisms, with reduced responses to the sedating effects of ethanol being predictive of increased risk for human alcohol dependence. Previously, we described that regulators of actin dynamics, such as the Rho-family GTPases Rac1, Rho1, and Cdc42, alter Drosophila’s sensitivity to ethanol-induced sedation. The GTPase activating protein RhoGAP18B also affects sensitivity to ethanol. To better understand how different RhoGAP18B isoforms affect ethanol sedation, we examined them for their effects on cell shape, GTP-loading of Rho-family GTPase, activation of the actin-severing cofilin, and actin filamentation. Our results suggest that the RhoGAP18B-PA isoform acts on Cdc42, while PC and PD act via Rac1 and Rho1 to activate cofilin. In vivo, a loss-of-function mutation in the cofilin-encoding gene twinstar leads to reduced ethanol-sensitivity and acts in concert with RhoGAP18B. Different RhoGAP18B isoforms, therefore, act on distinct subsets of Rho-family GTPases to modulate cofilin activity, actin dynamics, and ethanol-induced behaviors. PMID:26366560

Ankyrin-G isoform imbalance and interneuronopathy link epilepsy and bipolar disorder

PubMed Central

Lopez, Angel Y.; Wang, Xinjun; Xu, Mingxuan; Maheshwari, Atul; Curry, Daniel; Lam, Sandi; Adesina, Adekunle M.; Noebels, Jeffrey L.; Sun, Qian-Quan; Cooper, Edward C.

2016-01-01

ANK3, encoding the adaptor protein Ankyrin-G, has been implicated in bipolar disorder by genome wide association studies. ANK3 has multiple alternative first exons, and a bipolar disorder-associated ANK3 variant has been shown to reduce expression of exon 1b. Here we identify mechanisms through which reduced ANK3 exon 1b isoform expression disrupts neuronal excitation-inhibition balance. We find that parvalbumin interneurons and principal cells differentially express ANK3 first exon subtypes. Parvalbumin interneurons express only isoforms containing exon 1b, whereas excitatory principal cells express exon 1e alone, or both 1e and 1b. In transgenic mice deficient for exon 1b, parvalbumin interneurons lack voltage-gated sodium channels at their axonal initial segments and have increased firing thresholds and diminished action potential dynamic range. These mice exhibit an Ank3 gene dosage-dependent phenotype including behavior changes modeling bipolar disorder, epilepsy, and sudden death. Thus, ANK3’s important association with human bipolar susceptibility may arise from imbalance between ankyrin-G function in interneurons and principal cells and resultant excessive circuit sensitivity and output. Ankyrin-G isoform imbalance is a novel molecular endophenotype and potential therapeutic target. PMID:27956739

The long and the short of SAD-1 kinase.

PubMed

Kim, Joanne S M; Hung, Wesley; Zhen, Mei

2010-05-01

The Ser/Thr SAD kinases are evolutionarily conserved, critical regulators of neural development. Exciting findings in recent years have significantly advanced our understanding of the mechanism through which SAD kinases regulate neural development. Mammalian SAD-A and SAD-B, activated by a master kinase LKB1, regulate microtubule dynamics and polarize neurons. In C. elegans, the sad-1 gene encodes two isoforms, namely the long and the short, which exhibit overlapping and yet distinct functions in neuronal polarity and synaptic organization. Surprisingly, our most recent findings in C. elegans revealed a SAD-1-independent LKB1 activity in neuronal polarity. We also found that the long SAD-1 isoform directly interacts with a STRADalpha pseudokinase, STRD-1, to regulate neuronal polarity and synaptic organization. We elaborate here a working model of SAD-1 in which the two isoforms dimer/oligomerize to form a functional complex, and STRD-1 clusters and localizes the SAD-1 complex to synapses. While the mechanistic difference between the vertebrate and invertebrate SAD kinases may be puzzling, a recent discovery of the functionally distinct SAD-B isoforms predicts that the difference likely arises from our incomplete understanding of the SAD kinase mechanism and may eventually be reconciled as the revelation continues.

α2-COP is involved in early secretory traffic in Arabidopsis and is required for plant growth

PubMed Central

Gimeno-Ferrer, Fátima; Pastor-Cantizano, Noelia; Bernat-Silvestre, César; Selvi-Martínez, Pilar; Vera-Sirera, Francisco; Gao, Caiji; Perez-Amador, Miguel Angel; Jiang, Liwen; Aniento, Fernando

2017-01-01

Abstract COP (coat protein) I-coated vesicles mediate intra-Golgi transport and retrograde transport from the Golgi to the endoplasmic reticulum. These vesicles form through the action of the small GTPase ADP-ribosylation factor 1 (ARF1) and the COPI heptameric protein complex (coatomer), which consists of seven subunits (α-, β-, β′-, γ-, δ-, ε- and ζ-COP). In contrast to mammals and yeast, several isoforms for coatomer subunits, with the exception of γ and δ, have been identified in Arabidopsis. To understand the role of COPI proteins in plant biology, we have identified and characterized a loss-of-function mutant of α2-COP, an Arabidopsis α-COP isoform. The α2-cop mutant displayed defects in plant growth, including small rosettes, stems and roots and mislocalization of p24δ5, a protein of the p24 family containing a C-terminal dilysine motif involved in COPI binding. The α2-cop mutant also exhibited abnormal morphology of the Golgi apparatus. Global expression analysis of the α2-cop mutant revealed altered expression of plant cell wall-associated genes. In addition, a strong upregulation of SEC31A, which encodes a subunit of the COPII coat, was observed in the α2-cop mutant; this also occurs in a mutant of a gene upstream of COPI assembly, GNL1, which encodes an ARF-guanine nucleotide exchange factor (GEF). These findings suggest that loss of α2-COP affects the expression of secretory pathway genes. PMID:28025315

Organization and alternative splicing of the Caenorhabditis elegans cAMP-dependent protein kinase catalytic-subunit gene (kin-1).

PubMed

Tabish, M; Clegg, R A; Rees, H H; Fisher, M J

1999-04-01

The cAMP-dependent protein kinase (protein kinase A, PK-A) is multifunctional in nature, with key roles in the control of diverse aspects of eukaryotic cellular activity. In the case of the free-living nematode, Caenorhabditis elegans, a gene encoding the PK-A catalytic subunit has been identified and two isoforms of this subunit, arising from a C-terminal alternative-splicing event, have been characterized [Gross, Bagchi, Lu and Rubin (1990) J. Biol. Chem. 265, 6896-6907]. Here we report the occurrence of N-terminal alternative-splicing events that, in addition to generating a multiplicity of non-myristoylatable isoforms, also generate the myristoylated variant(s) of the catalytic subunit that we have recently characterized [Aspbury, Fisher, Rees and Clegg (1997) Biochem. Biophys. Res. Commun. 238, 523-527]. The gene spans more than 36 kb and is divided into a total of 13 exons. Each of the mature transcripts contains only 7 exons. In addition to the already characterized exon 1, the 5'-untranslated region and first intron actually contain 5 other exons, any one of which may be alternatively spliced on to exon 2 at the 5' end of the pre-mRNA. This N-terminal alternative splicing occurs in combination with either of the already characterized C-terminal alternative exons. Thus, C. elegans expresses at least 12 different isoforms of the catalytic subunit of PK-A. The significance of this unprecedented structural diversity in the family of PK-A catalytic subunits is discussed.

Flies lacking all synapsins are unexpectedly healthy but are impaired in complex behaviour.

PubMed

Godenschwege, Tanja A; Reisch, Dietmar; Diegelmann, Sören; Eberle, Kai; Funk, Natalja; Heisenberg, Martin; Hoppe, Viviane; Hoppe, Jürgen; Klagges, Bert R E; Martin, Jean-René; Nikitina, Ekaterina A; Putz, Gabi; Reifegerste, Rita; Reisch, Natascha; Rister, Jens; Schaupp, Michael; Scholz, Henrike; Schwärzel, Martin; Werner, Ursula; Zars, Troy D; Buchner, Sigrid; Buchner, Erich

2004-08-01

Vertebrate synapsins are abundant synaptic vesicle phosphoproteins that have been proposed to fine-regulate neurotransmitter release by phosphorylation-dependent control of synaptic vesicle motility. However, the consequences of a total lack of all synapsin isoforms due to a knock-out of all three mouse synapsin genes have not yet been investigated. In Drosophila a single synapsin gene encodes several isoforms and is expressed in most synaptic terminals. Thus the targeted deletion of the synapsin gene of Drosophila eliminates the possibility of functional knock-out complementation by other isoforms. Unexpectedly, synapsin null mutant flies show no obvious defects in brain morphology, and no striking qualitative changes in behaviour are observed. Ultrastructural analysis of an identified 'model' synapse of the larval nerve muscle preparation revealed no difference between wild-type and mutant, and spontaneous or evoked excitatory junction potentials at this synapse were normal up to a stimulus frequency of 5 Hz. However, when several behavioural responses were analysed quantitatively, specific differences between mutant and wild-type flies are noted. Adult locomotor activity, optomotor responses at high pattern velocities, wing beat frequency, and visual pattern preference are modified. Synapsin mutant flies show faster habituation of an olfactory jump response, enhanced ethanol tolerance, and significant defects in learning and memory as measured using three different paradigms. Larval behavioural defects are described in a separate paper. We conclude that Drosophila synapsins play a significant role in nervous system function, which is subtle at the cellular level but manifests itself in complex behaviour.

Genome-wide analysis of starch metabolism genes in potato (Solanum tuberosum L.).

PubMed

Van Harsselaar, Jessica K; Lorenz, Julia; Senning, Melanie; Sonnewald, Uwe; Sonnewald, Sophia

2017-01-05

Starch is the principle constituent of potato tubers and is of considerable importance for food and non-food applications. Its metabolism has been subject of extensive research over the past decades. Despite its importance, a description of the complete inventory of genes involved in starch metabolism and their genome organization in potato plants is still missing. Moreover, mechanisms regulating the expression of starch genes in leaves and tubers remain elusive with regard to differences between transitory and storage starch metabolism, respectively. This study aimed at identifying and mapping the complete set of potato starch genes, and to study their expression pattern in leaves and tubers using different sets of transcriptome data. Moreover, we wanted to uncover transcription factors co-regulated with starch accumulation in tubers in order to get insight into the regulation of starch metabolism. We identified 77 genomic loci encoding enzymes involved in starch metabolism. Novel isoforms of many enzymes were found. Their analysis will help to elucidate mechanisms of starch biosynthesis and degradation. Expression analysis of starch genes led to the identification of tissue-specific isoenzymes suggesting differences in the transcriptional regulation of starch metabolism between potato leaf and tuber tissues. Selection of genes predominantly expressed in developing potato tubers and exhibiting an expression pattern indicative for a role in starch biosynthesis enabled the identification of possible transcriptional regulators of tuber starch biosynthesis by co-expression analysis. This study provides the annotation of the complete set of starch metabolic genes in potato plants and their genomic localizations. Novel, so far undescribed, enzyme isoforms were revealed. Comparative transcriptome analysis enabled the identification of tuber- and leaf-specific isoforms of starch genes. This finding suggests distinct regulatory mechanisms in transitory and storage starch metabolism. Putative regulatory proteins of starch biosynthesis in potato tubers have been identified by co-expression and their expression was verified by quantitative RT-PCR.

Characterization of the apolipoprotein AI and CIII genes in the domestic pig

DOE Office of Scientific and Technical Information (OSTI.GOV)

Birchbauer, A.; Knipping, G.; Juritsch, B.

1993-03-01

The apolipoproteins (apo) AI and CIII are important constituents of triglyceride-rich lipoproteins and high-density lipoproteins. In humans, apo AI is believed to play an important protective role in the pathogenesis of arteriosclerosis, whereas apo CIII might be involved in the development of hypertriglyceridemia. Both human genes are located within a gene cluster on chromosome 11. Although the domestic pig has been widely used as an animal model in arteriosclerosis and lipid research, the porcine apolipoproteins genes are poorly characterized. In this report, the complete nucleotide sequences of the porcine apo AI and CIII genes are presented and the authors demonstrate,more » for the first time, apo CIII expression in the pig. Both genes are composed of four exons and three introns and resemble closely their human counterparts with regard to the transcriptional start sites, exon sizes, intron sizes, exon-intron borders, and the size of the intergenic region. The predicted pig apo AI is a protein of 241 amino acids, which is 2 amino acids shorter than human apo AI. The protein sequence was found to be very homologous to apo AI sequences in other mammalian species. Apo AI expression was detected on the mRNA level in porcine liver and intestine. The apo CIII gene encodes a protein with 73 amino acids, which is 6 amino acids shorter than human apo CIII. In contrast to the three isoforms of apo CIII found in humans, only one major isoform was detected in the pig. Presumably this isoform is unglycosylated. In addition to apo CIII expression in the liver and the intestine, a truncated form of apo CIII mRNA was also found in porcine kidney. The studies demonstrate the presence of an apo CIII gene, an apo CIII mRNA, and an apo CIII protein in the pig and, therefore, exclude a hypothesized apo CIII deficiency in these animals. 53 refs., 5 figs.« less

Functional expression and characterization of the Trypanosoma brucei procyclic glucose transporter, THT2.

PubMed

Barrett, M P; Tetaud, E; Seyfang, A; Bringaud, F; Baltz, T

1995-12-15

The gene encoding THT2, one of two hexose-transporter isoforms present in Trypanosoma brucei, has been expressed in both Xenopus laevis oocytes and a stably transfected line of Chinese hamster ovary (CHO) cells. The heterologously expressed gene encodes a protein with pharmacological and kinetic parameters similar to those of the hexose transporter measured in procyclic-culture-form trypanosomes. The substrate recognition of the THT2 transporter differed from that of the THT1 isoform, which is expressed only in bloodstream forms, in that: (i) it has a relatively high affinity for substrate with a Km of 59 microM for 2-deoxy-D-glucose (2-DOG) and a similar high affinity for D-glucose (compared with Km of 0.5 mM for 2-DOG in bloodstream forms); (ii) the affinity for 6-deoxy-D-glucose (6-DOG) is two orders of magnitude lower than that for D-glucose, whereas the bloodstream-form transporter recognizes D-glucose and its 6-DOG analogue with similar affinity; (iii) the bloodstream-form transporter, but not THT2, recognizes 3-fluoro-3-deoxy-D-glucose. D-Fructose-transport capacity and insensitivity to D-galactose was also found in THT2-expressing CHO cells and procyclic trypanosomes. We conclude from these cumulative results that the THT2 gene encodes the transporter responsible for hexose transport in procyclic trypanosomes. The transport of 2-DOG in procyclic organisms was inhibited by both the protonophore, carbonyl cyanide 4-trifluoromethoxy phenylhydrazone (FCCP), and KCN, suggesting a requirement for a protonmotive force. However, sensitivity to these reagents depended on the external substrate concentration, with uptake being unaffected at substrate concentrations higher than 2 mM. THT2 expressed in CHO cells behaved as a facilitated transporter, and was unaffected by FCCP or KCN over the whole substrate concentration range tested.

Basic anatomy and tumor biology of the RPS6KA6 gene that encodes the p90 ribosomal S6 kinase-4

PubMed Central

Sun, Yuan; Cao, Shousong; Yang, Min; Wu, Sihong; Wang, Zhe; Lin, Xiukun; Song, Xiangrang; Liao, D.J.

2012-01-01

The RPS6KA6 gene encodes the p90 ribosomal S6 kinase-4 (RSK4) that is still largely uncharacterized. In this study we identified a new RSK4 transcription initiation site and several alternative splice sites with a 5’RACE approach. The resulting mRNA variants encompass four possible first start codons. The first 15 nucleotides (nt) of exon 22 in mouse and the penultimate exon in both human (exon 21) and mouse (exon 24) RSK4 underwent alternative splicing, although the penultimate exon deleted variant appeared mainly in cell clines, but not in most normal tissues. Demethylation agent 5-azacytidine inhibited the deletion of the penultimate exon whereas two indolocarbazole-derived inhibitors of cyclin dependent kinase 4 or 6 induced deletion of the first 39 nt from exon 21 of human RSK4. In all human cancer cell lines studied, the 90-kD wild type RSK4 was sparse but, surprisingly, several isoforms at or smaller than 72-kD were expressed as detected by seven different antibodies. On immunoblots, each of these smaller isoforms often appeared as a duplet or triplet and the levels of these isoforms varied greatly among different cell lines and culture conditions. Cyclin D1 inhibited RSK4 expression and serum starvation enhanced the inhibition, whereas c-Myc and RSK4 inhibited cyclin D1. The effects of RSK4 on cell growth, cell death and chemoresponse depended on the mRNA variant or the protein isoform expressed, on the specificity of the cell lines, as well as on the anchorage-dependent or -independent growth conditions and the in vivo situation. Moreover, we also observed that even a given cDNA might be expressed to multiple proteins; therefore, when using a cDNA, one needs to exclude this possibility before attribution of the biological results from the cDNA to the anticipated protein. Collectively, our results suggest that whether RSK4 is oncogenic or tumor suppressive depends on many factors. PMID:22614021

Gene structure and mutant alleles of PCDH15: nonsyndromic deafness DFNB23 and type 1 Usher syndrome.

PubMed

Ahmed, Zubair M; Riazuddin, Saima; Aye, Sandar; Ali, Rana A; Venselaar, Hanka; Anwar, Saima; Belyantseva, Polina P; Qasim, Muhammad; Riazuddin, Sheikh; Friedman, Thomas B

2008-10-01

Mutations of PCDH15, encoding protocadherin 15, can cause either combined hearing and vision impairment (type 1 Usher syndrome; USH1F) or nonsyndromic deafness (DFNB23). Human PCDH15 is reported to be composed of 35 exons and encodes a variety of isoforms with 3-11 ectodomains (ECs), a transmembrane domain and a carboxy-terminal cytoplasmic domain (CD). Building on these observations, we describe an updated gene structure that has four additional exons of PCDH15 and isoforms that can be subdivided into four classes. Human PCDH15 encodes three alternative, evolutionarily conserved unique cytoplasmic domains (CD1, CD2 or CD3). Families ascertained on the basis of prelingual hearing loss were screened for linkage of this phenotype to markers for PCDH15 on chromosome 10q21.1. In seven of twelve families segregating USH1, we identified homozygous mutant alleles (one missense, one splice site, three nonsense and two deletion mutations) of which six are novel. One family was segregating nonsyndromic deafness DFNB23 due to a homozygous missense mutation. To date, in our cohort of 557 Pakistani families, we have found 11 different PCDH15 mutations that account for deafness in 13 families. Molecular modeling provided mechanistic insight into the phenotypic variation in severity of the PCDH15 missense mutations. We did not find pathogenic mutations in five of the twelve USH1 families linked to markers for USH1F, which suggest either the presence of mutations of yet additional undiscovered exons of PCDH15, mutations in the introns or regulatory elements of PCDH15, or an additional locus for type I USH at chromosome 10q21.1.

Gene structure and mutant alleles of PCDH15: nonsyndromic deafness DFNB23 and type 1 Usher syndrome

PubMed Central

Ahmed, Zubair M.; Riazuddin, Saima; Aye, Sandar; Ali, Rana A.; Venselaar, Hanka; Anwar, Saima; Belyantseva, Polina P.; Qasim, Muhammad; Riazuddin, Sheikh; Friedman, Thomas B.

2009-01-01

Mutations of PCDH15, encoding protocadherin 15, can cause either combined hearing and vision impairment (type 1 Usher syndrome; USH1F) or nonsyndromic deafness (DFNB23). Human PCDH15 is reported to be comprised of 35 exons and encodes a variety of isoforms with 3 to 11 ectodomains (EC), a transmembrane domain and a carboxy-terminal cytoplasmic domain (CD). Building on these observations we describe an updated gene structure that has four additional exons of PCDH15 and isoforms that can be subdivided into four classes. Human PCDH15 encodes three alternative, evolutionarily conserved unique cytoplasmic domains (CD1, CD2 or CD3). Families ascertained on the basis of prelingual hearing loss were screened for linkage of this phenotype to markers for PCDH15 on chromosome 10q21.1. In seven of twelve families segregating USH1 we identified homozygous mutant alleles (1 missense, 1 splice site, 3 nonsense and 2 deletion mutations) of which six are novel. One family was segregating nonsyndromic deafness DFNB23 due to a homozygous missense mutation. To date in our cohort of 557 Pakistani families, we have found 11 different PCDH15 mutations that account for deafness in 13 families. Molecular modeling provided mechanistic insight into the phenotypic variation in severity of the PCDH15 missense mutations. We did not find pathogenic mutations in five of the twelve USH1 families linked to markers for USH1F, which suggest either the presence of mutations of yet additional undiscovered exons of PCDH15, mutations in the introns or regulatory elements of PCDH15, or an additional locus for type I USH at chromosome 10q21.1. PMID:18719945

Mutations in TMEM260 Cause a Pediatric Neurodevelopmental, Cardiac, and Renal Syndrome.

PubMed

Ta-Shma, Asaf; Khan, Tahir N; Vivante, Asaf; Willer, Jason R; Matak, Pavle; Jalas, Chaim; Pode-Shakked, Ben; Salem, Yishay; Anikster, Yair; Hildebrandt, Friedhelm; Katsanis, Nicholas; Elpeleg, Orly; Davis, Erica E

2017-04-06

Despite the accelerated discovery of genes associated with syndromic traits, the majority of families affected by such conditions remain undiagnosed. Here, we employed whole-exome sequencing in two unrelated consanguineous kindreds with central nervous system (CNS), cardiac, renal, and digit abnormalities. We identified homozygous truncating mutations in TMEM260, a locus predicted to encode numerous splice isoforms. Systematic expression analyses across tissues and developmental stages validated two such isoforms, which differ in the utilization of an internal exon. The mutations in both families map uniquely to the long isoform, raising the possibility of an isoform-specific disorder. Consistent with this notion, RT-PCR of lymphocyte cell lines from one of the kindreds showed reduced levels of only the long isoform, which could be ameliorated by emetine, suggesting that the mutation induces nonsense-mediated decay. Subsequent in vivo testing supported this hypothesis. First, either transient suppression or CRISPR/Cas9 genome editing of zebrafish tmem260 recapitulated key neurological phenotypes. Second, co-injection of morphants with the long human TMEM260 mRNA rescued CNS pathology, whereas the short isoform was significantly less efficient. Finally, immunocytochemical and biochemical studies showed preferential enrichment of the long TMEM260 isoform to the plasma membrane. Together, our data suggest that there is overall reduced, but not ablated, functionality of TMEM260 and that attenuation of the membrane-associated functions of this protein is a principal driver of pathology. These observations contribute to an appreciation of the roles of splice isoforms in genetic disorders and suggest that dissection of the functions of these transcripts will most likely inform pathomechanism. Copyright © 2017 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

Malate valves: Old shuttles with new perspectives.

PubMed

Selinski, Jennifer; Scheibe, Renate

2018-06-22

Malate valves act as powerful systems for balancing the ATP/NAD(P)H ratio required in various subcellular compartments in plant cells. As components of malate valves, isoforms of malate dehydrogenases (MDHs) and dicarboxylate translocators catalyze the reversible interconversion of malate and oxaloacetate and their transport. Depending on the coenzyme specificity of the MDH isoforms, either NADH or NADPH can be transported indirectly. Arabidopsis thaliana possesses nine genes encoding MDH isoenzymes: Activities of NAD-dependent MDHs have been detected in mitochondria, peroxisomes, cytosol and plastids, respectively. In addition, chloroplasts possess a NADP-dependent MDH isoform. The NADP-MDH as part of the "light malate valve" plays an important role as a poising mechanism to adjust the ATP/NADPH ratio in the stroma. Its activity is strictly regulated by post-translational redox-modification mediated via the ferredoxin-thioredoxin system and fine control via the NADP + /NADP(H) ratio, thereby maintaining redox homeostasis under changing conditions. In contrast, the plastid NAD-MDH ("dark malate valve") is constitutively active and its lack leads to failure in early embryo development. While redox regulation of the main cytosolic MDH isoform has been shown, the knowledge about regulation of the other two cytosolic MDHs as well as NAD-MDH isoforms from peroxisomes and mitochondria is still lacking. Knockout mutants lacking the isoforms from chloroplasts, mitochondria, and peroxisomes have been characterized, but not much is known about cytosolic NAD-MDH isoforms and their role in planta. This review updates the current knowledge on MDH isoforms and the shuttle systems for intercompartmental dicarboxylate exchange focusing on the various metabolic functions of these valves. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Striated muscle preferentially expressed genes alpha and beta are two serine/threonine protein kinases derived from the same gene as the aortic preferentially expressed gene-1.

PubMed

Hsieh, C M; Fukumoto, S; Layne, M D; Maemura, K; Charles, H; Patel, A; Perrella, M A; Lee, M E

2000-11-24

Aortic preferentially expressed gene (APEG)-1 is a 1.4-kilobase pair (kb) mRNA expressed in vascular smooth muscle cells and is down-regulated by vascular injury. An APEG-1 5'-end cDNA probe identified three additional isoforms. The 9-kb striated preferentially expressed gene (SPEG)alpha and the 11-kb SPEGbeta were found in skeletal muscle and heart. The 4-kb brain preferentially expressed gene was detected in the brain and aorta. We report here cloning of the 11-kb SPEGbeta cDNA. SPEGbeta encodes a 355-kDa protein that contains two serine/threonine kinase domains and is homologous to proteins of the myosin light chain kinase family. At least one kinase domain is active and capable of autophosphorylation. In the genome, all four isoforms share the middle three of the five exons of APEG-1, and they differ from each other by using different 5'- and 3'-ends and alternative splicing. We show that the expression of SPEGalpha and SPEGbeta is developmentally regulated in the striated muscle during C2C12 myoblast to myotube differentiation in vitro and cardiomyocyte maturation in vivo. This developmental regulation suggests that both SPEGalpha and SPEGbeta can serve as sensitive markers for striated muscle differentiation and that they may be important for adult striated muscle function.

Three new members of the RNP protein family in Xenopus.

PubMed Central

Good, P J; Rebbert, M L; Dawid, I B

1993-01-01

Many RNP proteins contain one or more copies of the RNA recognition motif (RRM) and are thought to be involved in cellular RNA metabolism. We have previously characterized in Xenopus a nervous system specific gene, nrp1, that is more similar to the hnRNP A/B proteins than to other known proteins (K. Richter, P. J. Good, and I. B. Dawid (1990), New Biol. 2, 556-565). PCR amplification with degenerate primers was used to identify additional cDNAs encoding two RRMs in Xenopus. Three previously uncharacterized genes were identified. Two genes encode hnRNP A/B proteins with two RRMs and a glycine-rich domain. One of these is the Xenopus homolog of the human A2/B1 gene; the other, named hnRNP A3, is similar to both the A1 and A2 hnRNP genes. The Xenopus hnRNP A1, A2 and A3 genes are expressed throughout development and in all adult tissues. Multiple protein isoforms for the hnRNP A2 gene are predicted that differ by the insertion of short peptide sequences in the glycine-rich domain. The third newly isolated gene, named xrp1, encodes a protein that is related by sequence to the nrp1 protein but is expressed ubiquitously. Despite the similarity to nuclear RNP proteins, both the nrp1 and xrp1 proteins are localized to the cytoplasm in the Xenopus oocyte. The xrp1 gene may have a function in all cells that is similar to that executed by nrp1 specifically within the nervous system. Images PMID:8451200

The translation initiation factor 3 subunit eIF3K interacts with PML and associates with PML nuclear bodies

DOE Office of Scientific and Technical Information (OSTI.GOV)

Salsman, Jayme; Pinder, Jordan; Tse, Brenda

2013-10-15

The promyelocytic leukemia protein (PML) is a tumor suppressor protein that regulates a variety of important cellular processes, including gene expression, DNA repair and cell fate decisions. Integral to its function is the ability of PML to form nuclear bodies (NBs) that serve as hubs for the interaction and modification of over 90 cellular proteins. There are seven canonical isoforms of PML, which encode diverse C-termini generated by alternative pre-mRNA splicing. Recruitment of specific cellular proteins to PML NBs is mediated by protein–protein interactions with individual PML isoforms. Using a yeast two-hybrid screen employing peptide sequences unique to PML isoformmore » I (PML-I), we identified an interaction with the eukaryotic initiation factor 3 subunit K (eIF3K), and in the process identified a novel eIF3K isoform, which we term eIF3K-2. We further demonstrate that eIF3K and PML interact both in vitro via pull-down assays, as well as in vivo within human cells by co-immunoprecipitation and co-immunofluorescence. In addition, eIF3K isoform 2 (eIF3K-2) colocalizes to PML bodies, particularly those enriched in PML-I, while eIF3K isoform 1 associates poorly with PML NBs. Thus, we report eIF3K as the first known subunit of the eIF3 translation pre-initiation complex to interact directly with the PML protein, and provide data implicating alternative splicing of both PML and eIF3K as a possible regulatory mechanism for eIF3K localization at PML NBs. - Highlights: • The PML-I C-terminus, encoded by exon 9, interacts with translation factor eIF3K. • We identify a novel eIF3K isoform that excludes exon 2 (eIF3K-2). • eIF3K-2 preferentially associates with PML bodies enriched in PML-I vs. PML-IV. • Alternative splicing of eIF3K regulates association with PML bodies.« less

Single cell transcriptome analysis of MCF-7 reveals consistently and inconsistently expressed gene groups each associated with distinct cellular localization and functions

PubMed Central

Chen, Tzu-Han; Shiau, Hsin-Chieh

2018-01-01

Single cell transcriptome (SCT) analysis provides superior resolution to illustrate tumor cell heterogeneity for clinical implications. We characterized four SCTs of MCF-7 using 143 housekeeping genes (HKGs) as control, of which lactate dehydrogenase B (LDHB) expression is silenced. These SCT libraries mapped to 11,423, 11,486, 10,380, and 11,306 RefSeq genes (UCSC), respectively. High consistency in HKG expression levels across all four SCTs, along with transcriptional silencing of LDHB, was observed, suggesting a high sensitivity and reproducibility of the SCT analysis. Cross-library comparison on expression levels by scatter plotting revealed a linear correlation and an 83–94% overlap in transcript isoforms and expressed genes were also observed. To gain insight of transcriptional diversity among the SCTs, expressed genes were split into consistently expressed (CE) (expressed in all SCTs) and inconsistently expressed (IE) (expressed in some but not all SCTs) genes for further characterization, along with the 142 expressed HKGs as a reference. Distinct transcriptional strengths were found among these groups, with averages of 1,612.0, 88.0 and 1.2 FPKM for HKGs, CE and IE, respectively. Comparison between CE and IE groups further indicated that expressions of CE genes vary more significantly than that of IE genes. Gene Ontology analysis indicated that proteins encoded by CE genes are mainly involved in fundamental intracellular activities, while proteins encoded by IE genes are mainly for extracellular activities, especially acting as receptors or ion channels. The diversified gene expressions, especially for those encoded by IE genes, may contribute to cancer drug resistance. PMID:29920548

Characterisation of Reproduction-Associated Genes and Peptides in the Pest Land Snail, Theba pisana.

PubMed

Stewart, Michael J; Wang, Tianfang; Harding, Bradley I; Bose, U; Wyeth, Russell C; Storey, Kenneth B; Cummins, Scott F

2016-01-01

Increased understanding of the molecular components involved in reproduction may assist in understanding the evolutionary adaptations used by animals, including hermaphrodites, to produce offspring and retain a continuation of their lineage. In this study, we focus on the Mediterranean snail, Theba pisana, a hermaphroditic land snail that has become a highly invasive pest species within agricultural areas throughout the world. Our analysis of T. pisana CNS tissue has revealed gene transcripts encoding molluscan reproduction-associated proteins including APGWamide, gonadotropin-releasing hormone (GnRH) and an egg-laying hormone (ELH). ELH isoform 1 (ELH1) is known to be a potent reproductive peptide hormone involved in ovulation and egg-laying in some aquatic molluscs. Two other non-CNS ELH isoforms were also present in T. pisana (Tpi-ELH2 and Tpi-ELH3) within the snail dart sac and mucous glands. Bioactivity of a synthetic ELH1 on sexually mature T. pisana was confirmed through bioassay, with snails showing ELH1-induced egg-laying behaviours, including soil burrowing and oviposition. In summary, this study presents a detailed molecular analysis of reproductive neuropeptide genes in a land snail and provides a foundation for understanding ELH function.

Identification of the Phosphorylated Residues in TveIF5A by Mass Spectrometry

PubMed Central

Quintas-Granados, Laura Itzel; López-Camarillo, César; Armas, Jesús Fandiño; Mendoza Hernandez, Guillermo; Alvarez-Sánchez, María Elizbeth

2013-01-01

The initiation factor eIF5A in Trichomonas vaginalis (TveIF5A) is previously shown to undergo hypusination, phosphorylation and glycosylation. Three different pI isoforms of TveIF5A have been reported. The most acidic isoform (pI 5.2) corresponds to the precursor TveIF5A, whereas the mature TveIF5A appears to be the most basic isoform (pI 5.5). In addition, the intermediary isoform (pI 5.3) is found only under polyamine-depleted conditions and restored with exogenous putrescine. We propose that differences in PI are due to phosphorylation of the TveIF5A isoforms. Here, we have identified phosphorylation sites using mass spectrometry. The mature TveIF5A contains four phosphorylated residues (S3, T55, T78 and T82). Phosphorylation at S3 and T82 is also identified in the intermediary TveIF5A, while no phosphorylated residues are found in the precursor TveIF5A. It has been demonstrated that eIF5A proteins from plants and yeast are phosphorylated by a casein kinase 2 (CK2). Interestingly, a gene encoding a protein highly similar to CK2 (TvCK2) is found in T. vaginalis, which might be involved in the phosphorylation of TveIF5A in T. vaginalis. PMID:24308916

Identification of a core set of rhizobial infection genes using data from single cell-types.

PubMed

Chen, Da-Song; Liu, Cheng-Wu; Roy, Sonali; Cousins, Donna; Stacey, Nicola; Murray, Jeremy D

2015-01-01

Genome-wide expression studies on nodulation have varied in their scale from entire root systems to dissected nodules or root sections containing nodule primordia (NP). More recently efforts have focused on developing methods for isolation of root hairs from infected plants and the application of laser-capture microdissection technology to nodules. Here we analyze two published data sets to identify a core set of infection genes that are expressed in the nodule and in root hairs during infection. Among the genes identified were those encoding phenylpropanoid biosynthesis enzymes including Chalcone-O-Methyltransferase which is required for the production of the potent Nod gene inducer 4',4-dihydroxy-2-methoxychalcone. A promoter-GUS analysis in transgenic hairy roots for two genes encoding Chalcone-O-Methyltransferase isoforms revealed their expression in rhizobially infected root hairs and the nodule infection zone but not in the nitrogen fixation zone. We also describe a group of Rhizobially Induced Peroxidases whose expression overlaps with the production of superoxide in rhizobially infected root hairs and in nodules and roots. Finally, we identify a cohort of co-regulated transcription factors as candidate regulators of these processes.

Introducing a Null Mutation in the Mouse K6α and K6β Genes Reveals Their Essential Structural Role in the Oral Mucosa

PubMed Central

Wong, Pauline; Colucci-Guyon, Emma; Takahashi, Kenzo; Gu, Changhong; Babinet, Charles; Coulombe, Pierre A.

2000-01-01

Mammalian genomes feature multiple genes encoding highly related keratin 6 (K6) isoforms. These type II keratins show a complex regulation with constitutive and inducible components in several stratified epithelia, including the oral mucosa and skin. Two functional genes, K6α and K6β, exist in a head-to-tail tandem array in mouse genomes. We inactivated these two genes simultaneously via targeting and homologous recombination. K6 null mice are viable and initially indistinguishable from their littermates. Starting at two to three days after birth, they show a growth delay associated with reduced milk intake and the presence of white plaques in the posterior region of dorsal tongue and upper palate. These regions are subjected to greater mechanical stress during suckling. Morphological analyses implicate the filiform papillae as being particularly sensitive to trauma in K6α/K6β null mice, and establish the complete absence of keratin filaments in their anterior compartment. All null mice die about a week after birth. These studies demonstrate an essential structural role for K6 isoforms in the oral mucosa, and implicate filiform papillae as being the major stress bearing structures in dorsal tongue epithelium. PMID:10953016

APPRIS 2017: principal isoforms for multiple gene sets

PubMed Central

Rodriguez-Rivas, Juan; Di Domenico, Tomás; Vázquez, Jesús; Valencia, Alfonso

2018-01-01

Abstract The APPRIS database (http://appris-tools.org) uses protein structural and functional features and information from cross-species conservation to annotate splice isoforms in protein-coding genes. APPRIS selects a single protein isoform, the ‘principal’ isoform, as the reference for each gene based on these annotations. A single main splice isoform reflects the biological reality for most protein coding genes and APPRIS principal isoforms are the best predictors of these main proteins isoforms. Here, we present the updates to the database, new developments that include the addition of three new species (chimpanzee, Drosophila melangaster and Caenorhabditis elegans), the expansion of APPRIS to cover the RefSeq gene set and the UniProtKB proteome for six species and refinements in the core methods that make up the annotation pipeline. In addition APPRIS now provides a measure of reliability for individual principal isoforms and updates with each release of the GENCODE/Ensembl and RefSeq reference sets. The individual GENCODE/Ensembl, RefSeq and UniProtKB reference gene sets for six organisms have been merged to produce common sets of splice variants. PMID:29069475

Axial level-specific regulation of neuronal development: lessons from PITX2.

PubMed

Waite, Mindy R; Martin, Donna M

2015-02-01

Transcriptional regulation of gene expression is vital for proper control of proliferation, migration, differentiation, and survival of developing neurons. Pitx2 encodes a homeodomain transcription factor that is highly expressed in the developing and adult mammalian brain. In humans, mutations in PITX2 result in Rieger syndrome, characterized by defects in the development of the eyes, umbilicus, and teeth and variable abnormalities in the brain, including hydrocephalus and cerebellar hypoplasia. Alternative splicing of Pitx2 in the mouse results in three isoforms, Pitx2a, Pitx2b, and Pitx2c, each of which is expressed symmetrically along the left-right axis of the brain throughout development. Here, we review recent evidence for axial and brain region-specific requirements for Pitx2 during neuronal migration and differentiation, highlighting known isoform contributions. © 2014 Wiley Periodicals, Inc.

LEFPS1, a Tomato Farnesyl Pyrophosphate Gene Highly Expressed during Early Fruit Development1

PubMed Central

Gaffe, Joel; Bru, Jean-Philippe; Causse, Mathilde; Vidal, Alain; Stamitti-Bert, Linda; Carde, Jean-Pierre; Gallusci, Philippe

2000-01-01

Farnesyl pyrophosphate synthase (FPS) catalyzes the synthesis of farnesyl pyrophosphate, a key intermediate in sterol and sesquiterpene biosynthesis. Using a polymerase chain reaction-based approach, we have characterized LeFPS1, a tomato (Lycoperscion esculentum cv Wva 106) fruit cDNA, which encodes a functional FPS. We demonstrate that tomato FPSs are encoded by a small multigenic family with genes located on chromosomes 10 and 12. Consistent with farnesyl pyrophosphate requirement in sterol biosynthesis, FPS genes are ubiquitously expressed in tomato plants. Using an LeFPS1 specific probe, we show that the corresponding gene can account for most of FPS mRNA in most plant organs, but not during young seedling development, indicating a differential regulation of FPS genes in tomato. FPS gene expression is also under strict developmental control: FPS mRNA was mainly abundant in young organs and decreased as organs matured with the exception of fruits that presented a biphasic accumulation pattern. In this latter case in situ hybridization studies have shown that FPS mRNA is similarly abundant in all tissues of young fruit. Taken together our results suggest that several FPS isoforms are involved in tomato farnesyl pyrophosphate metabolism and that FPS genes are mostly expressed in relation to cell division and enlargement. PMID:10938353

Xcat, a novel mouse model for Nance-Horan syndrome inhibits expression of the cytoplasmic-targeted Nhs1 isoform.

PubMed

Huang, Kristen M; Wu, Junhua; Duncan, Melinda K; Moy, Chris; Dutra, Amalia; Favor, Jack; Da, Tong; Stambolian, Dwight

2006-01-15

Nance-Horan syndrome (NHS) is an X-linked disorder characterized by congenital cataracts, dental anomalies, dysmorphic features and mental retardation. A recent report suggests that the novel gene NHS1 is involved in this disorder due to the presence of point mutations in NHS patients. A possible mouse model for NHS, Xcat, was mapped to a 2.11 Mb interval on the X-chromosome. Sequence and FISH analysis of the X-chromosome region containing the Xcat mutation reveal a large insertion between exons 1 and 2 of the mouse Nhs1 gene. The insertion inhibits the expression of the Nhs1 isoform containing exon 1 and results in exclusive expression of the alternative isoform containing exon 1A. Quantitative RT-PCR of Xcat cDNA shows reduced levels of Nhs1 transcripts. The Nhs1 protein is strongly expressed within the cytoplasm of elongating lens fiber cells from wild-type neonate lens, but is significantly reduced within the Xcat lens. Transient transfection studies of CHO cells with Nhs1-GFP fusion proteins were done to determine whether the amino acids encoded by exon 1 were critical for protein localization. We found the presence of Nhs1 exon 1 critical for localization of the fusion protein to the cytoplasm, whereas fusion proteins lacking Nhs1 exon 1 are predominantly nuclear. These results indicate that the first exon of Nhs1 contains crucial information required for the proper expression and localization of Nhs1 protein. Inhibition of expression of the exon 1 containing isoform results in the abnormal phenotype of Xcat.

Loss of an Androgen-Inactivating and Isoform-Specific HSD17B4 Splice Form Enables Emergence of Castration-Resistant Prostate Cancer.

PubMed

Ko, Hyun-Kyung; Berk, Michael; Chung, Yoon-Mi; Willard, Belinda; Bareja, Rohan; Rubin, Mark; Sboner, Andrea; Sharifi, Nima

2018-01-16

Castration-resistant prostate cancer (CRPC) requires tumors to engage metabolic mechanisms that allow sustained testosterone and/or dihydrotestosterone to stimulate progression. 17β-Hydroxysteroid dehydrogenase type 4 (17βHSD4), encoded by HSD17B4, is thought to inactivate testosterone and dihydrotestosterone by converting them to their respective inert 17-keto steroids. Counterintuitively, HSD17B4 expression increases in CRPC and predicts poor prognosis. Here, we show that, of five alternative splice forms, only isoform 2 encodes an enzyme capable of testosterone and dihydrotestosterone inactivation. In contrast with other transcripts, functional expression of isoform 2 is specifically suppressed in development of CRPC in patients. Genetically silencing isoform 2 shifts the metabolic balance toward 17β-OH androgens (testosterone and dihydrotestosterone), stimulating androgen receptor (AR) and CRPC development. Our studies specifically implicate HSD17B4 isoform 2 loss in lethal prostate cancer. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Plasma-membrane H(+)-ATPase gene expression is regulated by NaCl in cells of the halophyte Atriplex nummularia L.

PubMed

Niu, X; Zhu, J K; Narasimhan, M L; Bressan, R A; Hasegawa, P M

1993-01-01

An Atriplex nummularia L. cDNA probe encoding the partial sequence of an isoform of the plasma-membrane H(+)-ATPase was isolated, and used to characterize the NaCl regulation of mRNA accumulation in cultured cells of this halophyte. The peptide (477 amino acids) translated from the open reading frame has the highest sequence homology to the Nicotiana plumbaginifolia plasma-membrane H(+)-ATPase isoform pma4 (greater than 80% identity) and detected a transcript of approximately 3.7 kb on Northern blots of both total and poly(A)+ RNA. The mRNA levels were comparable in unadapted cells, adapted cells (cells adapted to and growing in 342 mM NaCl) and deadapted cells (cells previously adapted to 342 mM NaCl that are now growing without salt). Increased mRNA abundance was detected in deadapted cells within 24 h after exposure to NaCl but not in unadapted cells with similar salt treatments. The NaCl up-regulation of message abundance in deadapted cells was subject to developmental control. Analogous to those reported for glycophytes, the plasma-membrane H(+)-ATPase are encoded by a multigene family in the halophyte.

Amino-termini isoforms of the Slack K+ channel, regulated by alternative promoters, differentially modulate rhythmic firing and adaptation.

PubMed

Brown, Maile R; Kronengold, Jack; Gazula, Valeswara-Rao; Spilianakis, Charalampos G; Flavell, Richard A; von Hehn, Christian A A; Bhattacharjee, Arin; Kaczmarek, Leonard K

2008-11-01

The rates of activation and unitary properties of Na+-activated K+ (K(Na)) currents have been found to vary substantially in different types of neurones. One class of K(Na) channels is encoded by the Slack gene. We have now determined that alternative RNA splicing gives rise to at least five different transcripts for Slack, which produce Slack channels that differ in their predicted cytoplasmic amino-termini and in their kinetic properties. Two of these, termed Slack-A channels, contain an amino-terminus domain closely resembling that of another class of K(Na) channels encoded by the Slick gene. Neuronal expression of Slack-A channels and of the previously described Slack isoform, now called Slack-B, are driven by independent promoters. Slack-A mRNAs were enriched in the brainstem and olfactory bulb and detected at significant levels in four different brain regions. When expressed in CHO cells, Slack-A channels activate rapidly upon depolarization and, in single channel recordings in Xenopus oocytes, are characterized by multiple subconductance states with only brief transient openings to the fully open state. In contrast, Slack-B channels activate slowly over hundreds of milliseconds, with openings to the fully open state that are approximately 6-fold longer than those for Slack-A channels. In numerical simulations, neurones in which outward currents are dominated by a Slack-A-like conductance adapt very rapidly to repeated or maintained stimulation over a wide range of stimulus strengths. In contrast, Slack-B currents promote rhythmic firing during maintained stimulation, and allow adaptation rate to vary with stimulus strength. Using an antibody that recognizes all amino-termini isoforms of Slack, Slack immunoreactivity is present at locations that have no Slack-B-specific staining, including olfactory bulb glomeruli and the dendrites of hippocampal neurones, suggesting that Slack channels with alternate amino-termini such as Slack-A channels are present at these locations. Our data suggest that alternative promoters of the Slack gene differentially modulate the properties of neurones.

Probing the Boundaries of Orthology: The Unanticipated Rapid Evolution of Drosophila centrosomin

PubMed Central

Eisman, Robert C.; Kaufman, Thomas C.

2013-01-01

The rapid evolution of essential developmental genes and their protein products is both intriguing and problematic. The rapid evolution of gene products with simple protein folds and a lack of well-characterized functional domains typically result in a low discovery rate of orthologous genes. Additionally, in the absence of orthologs it is difficult to study the processes and mechanisms underlying rapid evolution. In this study, we have investigated the rapid evolution of centrosomin (cnn), an essential gene encoding centrosomal protein isoforms required during syncytial development in Drosophila melanogaster. Until recently the rapid divergence of cnn made identification of orthologs difficult and questionable because Cnn violates many of the assumptions underlying models for protein evolution. To overcome these limitations, we have identified a group of insect orthologs and present conserved features likely to be required for the functions attributed to cnn in D. melanogaster. We also show that the rapid divergence of Cnn isoforms is apparently due to frequent coding sequence indels and an accelerated rate of intronic additions and eliminations. These changes appear to be buffered by multi-exon and multi-reading frame maximum potential ORFs, simple protein folds, and the splicing machinery. These buffering features also occur in other genes in Drosophila and may help prevent potentially deleterious mutations due to indels in genes with large coding exons and exon-dense regions separated by small introns. This work promises to be useful for future investigations of cnn and potentially other rapidly evolving genes and proteins. PMID:23749319

IDP-ASE: haplotyping and quantifying allele-specific expression at the gene and gene isoform level by hybrid sequencing

PubMed Central

Deonovic, Benjamin; Wang, Yunhao; Weirather, Jason; Wang, Xiu-Jie; Au, Kin Fai

2017-01-01

Abstract Allele-specific expression (ASE) is a fundamental problem in studying gene regulation and diploid transcriptome profiles, with two key challenges: (i) haplotyping and (ii) estimation of ASE at the gene isoform level. Existing ASE analysis methods are limited by a dependence on haplotyping from laborious experiments or extra genome/family trio data. In addition, there is a lack of methods for gene isoform level ASE analysis. We developed a tool, IDP-ASE, for full ASE analysis. By innovative integration of Third Generation Sequencing (TGS) long reads with Second Generation Sequencing (SGS) short reads, the accuracy of haplotyping and ASE quantification at the gene and gene isoform level was greatly improved as demonstrated by the gold standard data GM12878 data and semi-simulation data. In addition to methodology development, applications of IDP-ASE to human embryonic stem cells and breast cancer cells indicate that the imbalance of ASE and non-uniformity of gene isoform ASE is widespread, including tumorigenesis relevant genes and pluripotency markers. These results show that gene isoform expression and allele-specific expression cooperate to provide high diversity and complexity of gene regulation and expression, highlighting the importance of studying ASE at the gene isoform level. Our study provides a robust bioinformatics solution to understand ASE using RNA sequencing data only. PMID:27899656

CK2(beta)tes gene encodes a testis-specific isoform of the regulatory subunit of casein kinase 2 in Drosophila melanogaster.

PubMed

Kalmykova, Alla I; Shevelyov, Yuri Y; Polesskaya, Oksana O; Dobritsa, Anna A; Evstafieva, Alexandra G; Boldyreff, Brigitte; Issinger, Olaf-Georg; Gvozdev, Vladimir A

2002-03-01

An earlier described CK2(beta)tes gene of Drosophila melanogaster is shown to encode a male germline specific isoform of regulatory beta subunit of casein kinase 2. Western-analysis using anti-CK2(beta)tes Ig revealed CK2(beta)tes protein in Drosophila testes extract. Expression of a CK2(beta)tes-beta-galactosidase fusion protein driven by the CK2(beta)tes promoter was found in transgenic flies at postmitotic stages of spermatogenesis. Examination of biochemical characteristics of a recombinant CK2(beta)tes protein expressed in Escherichia coli revealed properties similar to those of CK2beta: (a) CK2(beta)tes protein stimulates CK2alpha catalytic activity toward synthetic peptide; (b) it inhibits phosphorylation of calmodulin and mediates stimulation of CK2alpha by polylysine; (c) it is able to form (CK2(beta)tes)2 dimers, as well as (CK2alpha)2(CK2(beta)tes)2 tetramers. Using the yeast two-hybrid system and coimmunoprecipitation analysis of protein extract from Drosophila testes, we demonstrated an association between CK2(beta)tes and CK2alpha. Northern-analysis has shown that another regulatory (beta') subunit found recently in D. melanogaster genome is also testis-specific. Thus, we describe the first example of two tissue-specific regulatory subunits of CK2 which might serve to provide CK2 substrate recognition during spermatogenesis.

Fluorescence Reporter-Based Genome-Wide RNA Interference Screening to Identify Alternative Splicing Regulators.

PubMed

Misra, Ashish; Green, Michael R

2017-01-01

Alternative splicing is a regulated process that leads to inclusion or exclusion of particular exons in a pre-mRNA transcript, resulting in multiple protein isoforms being encoded by a single gene. With more than 90 % of human genes known to undergo alternative splicing, it represents a major source for biological diversity inside cells. Although in vitro splicing assays have revealed insights into the mechanisms regulating individual alternative splicing events, our global understanding of alternative splicing regulation is still evolving. In recent years, genome-wide RNA interference (RNAi) screening has transformed biological research by enabling genome-scale loss-of-function screens in cultured cells and model organisms. In addition to resulting in the identification of new cellular pathways and potential drug targets, these screens have also uncovered many previously unknown mechanisms regulating alternative splicing. Here, we describe a method for the identification of alternative splicing regulators using genome-wide RNAi screening, as well as assays for further validation of the identified candidates. With modifications, this method can also be adapted to study the splicing regulation of pre-mRNAs that contain two or more splice isoforms.

Loss of predominant Shank3 isoforms results in hippocampus-dependent impairments in behavior and synaptic transmission.

PubMed

Kouser, Mehreen; Speed, Haley E; Dewey, Colleen M; Reimers, Jeremy M; Widman, Allie J; Gupta, Natasha; Liu, Shunan; Jaramillo, Thomas C; Bangash, Muhammad; Xiao, Bo; Worley, Paul F; Powell, Craig M

2013-11-20

The Shank3 gene encodes a scaffolding protein that anchors multiple elements of the postsynaptic density at the synapse. Previous attempts to delete the Shank3 gene have not resulted in a complete loss of the predominant naturally occurring Shank3 isoforms. We have now characterized a homozygous Shank3 mutation in mice that deletes exon 21, including the Homer binding domain. In the homozygous state, deletion of exon 21 results in loss of the major naturally occurring Shank3 protein bands detected by C-terminal and N-terminal antibodies, allowing us to more definitively examine the role of Shank3 in synaptic function and behavior. This loss of Shank3 leads to an increased localization of mGluR5 to both synaptosome and postsynaptic density-enriched fractions in the hippocampus. These mice exhibit a decrease in NMDA/AMPA excitatory postsynaptic current ratio in area CA1 of the hippocampus, reduced long-term potentiation in area CA1, and deficits in hippocampus-dependent spatial learning and memory. In addition, these mice also exhibit motor-coordination deficits, hypersensitivity to heat, novelty avoidance, altered locomotor response to novelty, and minimal social abnormalities. These data suggest that Shank3 isoforms are required for normal synaptic transmission/plasticity in the hippocampus, as well as hippocampus-dependent spatial learning and memory.

Structure–Function Analysis of the Non-Muscle Myosin Light Chain Kinase (nmMLCK) Isoform by NMR Spectroscopy and Molecular Modeling: Influence of MYLK Variants

PubMed Central

Shen, Kui; Ramirez, Benjamin; Mapes, Brandon; Shen, Grace R.; Gokhale, Vijay; Brown, Mary E.; Santarsiero, Bernard; Ishii, Yoshitaka; Dudek, Steven M.; Wang, Ting; Garcia, Joe G. N.

2015-01-01

The MYLK gene encodes the multifunctional enzyme, myosin light chain kinase (MLCK), involved in isoform-specific non-muscle and smooth muscle contraction and regulation of vascular permeability during inflammation. Three MYLK SNPs (P21H, S147P, V261A) alter the N-terminal amino acid sequence of the non-muscle isoform of MLCK (nmMLCK) and are highly associated with susceptibility to acute lung injury (ALI) and asthma, especially in individuals of African descent. To understand the functional effects of SNP associations, we examined the N-terminal segments of nmMLCK by 1H-15N heteronuclear single quantum correlation (HSQC) spectroscopy, a 2-D NMR technique, and by in silico molecular modeling. Both NMR analysis and molecular modeling indicated SNP localization to loops that connect the immunoglobulin-like domains of nmMLCK, consistent with minimal structural changes evoked by these SNPs. Molecular modeling analysis identified protein-protein interaction motifs adversely affected by these MYLK SNPs including binding by the scaffold protein 14-3-3, results confirmed by immunoprecipitation and western blot studies. These structure-function studies suggest novel mechanisms for nmMLCK regulation, which may confirm MYLK as a candidate gene in inflammatory lung disease and advance knowledge of the genetic underpinning of lung-related health disparities. PMID:26111161

Neutropenia-associated ELANE mutations disrupting translation initiation produce novel neutrophil elastase isoforms

PubMed Central

Tidwell, Timothy; Wechsler, Jeremy; Nayak, Ramesh C.; Trump, Lisa; Salipante, Stephen J.; Cheng, Jerry C.; Donadieu, Jean; Glaubach, Taly; Corey, Seth J.; Grimes, H. Leighton; Lutzko, Carolyn; Cancelas, Jose A.

2014-01-01

Hereditary neutropenia is usually caused by heterozygous germline mutations in the ELANE gene encoding neutrophil elastase (NE). How mutations cause disease remains uncertain, but two hypotheses have been proposed. In one, ELANE mutations lead to mislocalization of NE. In the other, ELANE mutations disturb protein folding, inducing an unfolded protein response in the endoplasmic reticulum (ER). In this study, we describe new types of mutations that disrupt the translational start site. At first glance, they should block translation and are incompatible with either the mislocalization or misfolding hypotheses, which require mutant protein for pathogenicity. We find that start-site mutations, instead, force translation from downstream in-frame initiation codons, yielding amino-terminally truncated isoforms lacking ER-localizing (pre) and zymogen-maintaining (pro) sequences, yet retain essential catalytic residues. Patient-derived induced pluripotent stem cells recapitulate hematopoietic and molecular phenotypes. Expression of the amino-terminally deleted isoforms in vitro reduces myeloid cell clonogenic capacity. We define an internal ribosome entry site (IRES) within ELANE and demonstrate that adjacent mutations modulate IRES activity, independently of protein-coding sequence alterations. Some ELANE mutations, therefore, appear to cause neutropenia via the production of amino-terminally deleted NE isoforms rather than by altering the coding sequence of the full-length protein. PMID:24184683

Anti-angiogenic VEGFAxxxb transcripts are not expressed in the medio-basal hypothalamus of the seasonal sheep

PubMed Central

Lomet, Didier; Piégu, Benoît; Wood, Shona H.

2018-01-01

This study investigated Vegfa expression in the pars tuberalis (PT) of the pituitary and medio-basal hypothalamus (MBH) of sheep, across seasons and reproductive states. It has recently been proposed that season impacts alternative splicing of Vegfa mRNA in the PT, which shifts the balance between angiogenic VEGFAxxx and anti-angiogenic VEGFAxxxb isoforms (with xxx the number of amino acids of the mature VEGFA proteins) to modulate seasonal breeding. Here, we used various RT-PCR methodologies and analysis of RNAseq datasets to investigate seasonal variation in expression and splicing of the ovine Vegfa gene. Collectively, we identify 5 different transcripts for Vegfa within the ewe PT/MBH, which correspond to splicing events previously described in mouse and human. All identified transcripts encode angiogenic VEGFAxxx isoforms, with no evidence for alternative splicing within exon 8. These findings led us to investigate in detail how “Vegfaxxxb-like” PCR products could be generated by RT-PCR and misidentified as endogenous transcripts, in sheep and human HEK293 cells. In conclusion, our findings do not support the existence of anti-angiogenic VEGFAxxxb isoforms in the ovine PT/MBH and shed new light on the interpretation of prior studies, which claimed to identify Vegfaxxxb isoforms by RT-PCR. PMID:29746548

Nitric oxide as an initiator of brain lesions during the development of Alzheimer disease.

PubMed

Aliev, Gjumrakch; Palacios, Hector H; Lipsitt, Amanda E; Fischbach, Kathryn; Lamb, Bruce T; Obrenovich, Mark E; Morales, Ludis; Gasimov, Eldar; Bragin, Valentin

2009-10-01

Nitric oxide (NO) is an important regulatory molecule for the host defense that plays a fundamental role in the cardiovascular, immune, and nervous systems. NO is synthesized through the conversion of L-arginine to L-citrulline by the enzyme NO synthase (NOS), which is found in three isoforms classified as neuronal (nNOS), inducible (iNOS), and endothelial (eNOS). Recent evidence supports the theory that this bioactive molecule has an influential role in the disruption of normal brain and vascular homeostasis, a condition known to elucidate chronic hypoperfusion which ultimately causes the development of brain lesions and the pathology that typify Alzheimer disease (AD). In addition, vascular NO activity appears to be a major contributor to this pathology before any overexpression of NOS isoforms is observed in the neuron, glia, and microglia of the brain tree, where the overexpression the NOS isoforms causes the formation of a large amount of NO. We hypothesize that since an imbalance between the NOS isoforms and endothelin-1 (ET-1), a human gene that encodes for blood vessel constriction, can cause antioxidant system insufficiency; by using pharmacological intervention with NO donors and/or NO suppressors, the brain lesions and the downstream progression of brain pathology and dementia in AD should be delayed or minimized.

Inhibition of class IA PI3K enzymes in non-small cell lung cancer cells uncovers functional compensation among isoforms.

PubMed

Stamatkin, Christopher; Ratermann, Kelley L; Overley, Colleen W; Black, Esther P

2015-01-01

Deregulation of the phosphatidylinositol 3-kinase (PI3K) pathway is central to many human malignancies while normal cell proliferation requires pathway functionality. Although inhibitors of the PI3K pathway are in clinical trials or approved for therapy, an understanding of the functional activities of pathway members in specific malignancies is needed. In lung cancers, the PI3K pathway is often aberrantly activated by mutation of genes encoding EGFR, KRAS, and PIK3CA proteins. We sought to understand whether class IA PI3K enzymes represent rational therapeutic targets in cells of non-squamous lung cancers by exploring pharmacological and genetic inhibitors of PI3K enzymes in a non-small cell lung cancer (NSCLC) cell line system. We found that class IA PI3K enzymes were expressed in all cell lines tested, but treatment of NSCLC lines with isoform-selective inhibitors (A66, TGX-221, CAL-101 and IC488743) had little effect on cell proliferation or prolonged inhibition of AKT activity. Inhibitory pharmacokinetic and pharmacodynamic responses were observed using these agents at non-isoform selective concentrations and with the pan-class I (ZSTK474) agent. Response to pharmacological inhibition suggested that PI3K isoforms may functionally compensate for one another thus limiting efficacy of single agent treatment. However, combination of ZSTK474 and an EGFR inhibitor (erlotinib) in NSCLC resistant to each single agent reduced cellular proliferation. These studies uncovered unanticipated cellular responses to PI3K isoform inhibition in NSCLC that does not correlate with PI3K mutations, suggesting that patients bearing tumors with wildtype EGFR and KRAS are unlikely to benefit from inhibitors of single isoforms but may respond to pan-isoform inhibition.

A Short Isoform of Human Cytomegalovirus US3 Functions as a Dominant Negative Inhibitor of the Full-Length Form

PubMed Central

Shin, Jinwook; Park, Boyoun; Lee, Sungwook; Kim, Youngkyun; Biegalke, Bonita J.; Kang, Seongman; Ahn, Kwangseog

2006-01-01

Human cytomegalovirus encodes four unique short (US) region proteins, each of which is independently sufficient for causing the down-regulation of major histocompatibility complex (MHC) class I molecules on the cell surface. This down-regulation enables infected cells to evade recognition by cytotoxic T lymphocytes (CTLs) but makes them vulnerable to lysis by natural killer (NK) cells, which lyse those cells that lack MHC class I molecules. The 22-kDa US3 glycoprotein is able to down-regulate the surface expression of MHC class I molecules by dual mechanisms: direct endoplasmic reticulum retention by physical association and/or tapasin inhibition. The alternative splicing of the US3 gene generates two additional products, including 17-kDa and 3.5-kDa truncated isoforms; however, the functional significance of these isoforms during viral infection is unknown. Here, we describe a novel mode of self-regulation of US3 function that uses the endogenously produced truncated isoform. The truncated isoform itself neither binds to MHC class I molecules nor prevents the full-length US3 from interacting with MHC class I molecules. Instead, the truncated isoform associates with tapasin and competes with full-length US3 for binding to tapasin; thus, it suppresses the action of US3 that causes the disruption of the function of tapasin. Our results indicate that the truncated isoform of the US3 locus acts as a dominant negative regulator of full-length US3 activity. These data reflect the manner in which the virus has developed temporal survival strategies during viral infection against immune surveillance involving both CTLs and NK cells. PMID:16699020

A short isoform of human cytomegalovirus US3 functions as a dominant negative inhibitor of the full-length form.

PubMed

Shin, Jinwook; Park, Boyoun; Lee, Sungwook; Kim, Youngkyun; Biegalke, Bonita J; Kang, Seongman; Ahn, Kwangseog

2006-06-01

Human cytomegalovirus encodes four unique short (US) region proteins, each of which is independently sufficient for causing the down-regulation of major histocompatibility complex (MHC) class I molecules on the cell surface. This down-regulation enables infected cells to evade recognition by cytotoxic T lymphocytes (CTLs) but makes them vulnerable to lysis by natural killer (NK) cells, which lyse those cells that lack MHC class I molecules. The 22-kDa US3 glycoprotein is able to down-regulate the surface expression of MHC class I molecules by dual mechanisms: direct endoplasmic reticulum retention by physical association and/or tapasin inhibition. The alternative splicing of the US3 gene generates two additional products, including 17-kDa and 3.5-kDa truncated isoforms; however, the functional significance of these isoforms during viral infection is unknown. Here, we describe a novel mode of self-regulation of US3 function that uses the endogenously produced truncated isoform. The truncated isoform itself neither binds to MHC class I molecules nor prevents the full-length US3 from interacting with MHC class I molecules. Instead, the truncated isoform associates with tapasin and competes with full-length US3 for binding to tapasin; thus, it suppresses the action of US3 that causes the disruption of the function of tapasin. Our results indicate that the truncated isoform of the US3 locus acts as a dominant negative regulator of full-length US3 activity. These data reflect the manner in which the virus has developed temporal survival strategies during viral infection against immune surveillance involving both CTLs and NK cells.

Genetic structure and regulation of isoprene synthase in Poplar (Populus spp.).

PubMed

Vickers, Claudia E; Possell, Malcolm; Nicholas Hewitt, C; Mullineaux, Philip M

2010-07-01

Isoprene is a volatile 5-carbon hydrocarbon derived from the chloroplastic methylerythritol 2-C-methyl-D: -erythritol 4-phosphate isoprenoid pathway. In plants, isoprene emission is controlled by the enzyme isoprene synthase; however, there is still relatively little known about the genetics and regulation of this enzyme. Isoprene synthase gene structure was analysed in three poplar species. It was found that genes encoding stromal isoprene synthase exist as a small gene family, the members of which encode virtually identical proteins and are differentially regulated. Accumulation of isoprene synthase protein is developmentally regulated, but does not differ between sun and shade leaves and does not increase when heat stress is applied. Our data suggest that, in mature leaves, isoprene emission rates are primarily determined by substrate (dimethylallyl diphosphate, DMADP) availability. In immature leaves, where isoprene synthase levels are variable, emission levels are also influenced by the amount of isoprene synthase protein. No thylakoid isoforms could be identified in Populus alba or in Salix babylonica. Together, these data show that control of isoprene emission at the genetic level is far more complicated than previously assumed.

Loss of the Chloroplast Transit Peptide from an Ancestral C3 Carbonic Anhydrase Is Associated with C4 Evolution in the Grass Genus Neurachne.

PubMed

Clayton, Harmony; Saladié, Montserrat; Rolland, Vivien; Sharwood, Robert; Macfarlane, Terry; Ludwig, Martha

2017-03-01

Neurachne is the only known grass lineage containing closely related C 3 , C 3 -C 4 intermediate, and C 4 species, making it an ideal taxon with which to study the evolution of C 4 photosynthesis in the grasses. To begin dissecting the molecular changes that led to the evolution of C 4 photosynthesis in this group, the complementary DNAs encoding four distinct β-carbonic anhydrase (CA) isoforms were characterized from leaf tissue of Neurachne munroi (C 4 ), Neurachne minor (C 3 -C 4 ), and Neurachne alopecuroidea (C 3 ). Two genes ( CA1 and CA2 ) each encode two different isoforms: CA1a/CA1b and CA2a/CA2b. Transcript analyses found that CA1 messenger RNAs were significantly more abundant than transcripts from the CA2 gene in the leaves of each species examined, constituting ∼99% of all β-CA transcripts measured. Localization experiments using green fluorescent protein fusion constructs showed that, while CA1b is a cytosolic CA in all three species, the CA1a proteins are differentially localized. The N. alopecuroidea and N. minor CA1a isoforms were imported into chloroplasts of Nicotiana benthamiana leaf cells, whereas N. munroi CA1a localized to the cytosol. Sequence analysis indicated an 11-amino acid deletion in the amino terminus of N. munroi CA1a relative to the C 3 and C 3 -C 4 proteins, suggesting that chloroplast targeting of CA1a is the ancestral state and that loss of a functional chloroplast transit peptide in N. munroi CA1a is associated with the evolution of C 4 photosynthesis in Neurachne spp. Remarkably, this mechanism is homoplastic with the evolution of the C 4 -associated CA in the dicotyledonous genus Flaveria , although the actual mutations in the two lineages differ. © 2017 American Society of Plant Biologists. All Rights Reserved.

Loss of the Chloroplast Transit Peptide from an Ancestral C3 Carbonic Anhydrase Is Associated with C4 Evolution in the Grass Genus Neurachne1[OPEN

PubMed Central

Clayton, Harmony; Saladié, Montserrat; Sharwood, Robert; Macfarlane, Terry

2017-01-01

Neurachne is the only known grass lineage containing closely related C3, C3-C4 intermediate, and C4 species, making it an ideal taxon with which to study the evolution of C4 photosynthesis in the grasses. To begin dissecting the molecular changes that led to the evolution of C4 photosynthesis in this group, the complementary DNAs encoding four distinct β-carbonic anhydrase (CA) isoforms were characterized from leaf tissue of Neurachne munroi (C4), Neurachne minor (C3-C4), and Neurachne alopecuroidea (C3). Two genes (CA1 and CA2) each encode two different isoforms: CA1a/CA1b and CA2a/CA2b. Transcript analyses found that CA1 messenger RNAs were significantly more abundant than transcripts from the CA2 gene in the leaves of each species examined, constituting ∼99% of all β-CA transcripts measured. Localization experiments using green fluorescent protein fusion constructs showed that, while CA1b is a cytosolic CA in all three species, the CA1a proteins are differentially localized. The N. alopecuroidea and N. minor CA1a isoforms were imported into chloroplasts of Nicotiana benthamiana leaf cells, whereas N. munroi CA1a localized to the cytosol. Sequence analysis indicated an 11-amino acid deletion in the amino terminus of N. munroi CA1a relative to the C3 and C3-C4 proteins, suggesting that chloroplast targeting of CA1a is the ancestral state and that loss of a functional chloroplast transit peptide in N. munroi CA1a is associated with the evolution of C4 photosynthesis in Neurachne spp. Remarkably, this mechanism is homoplastic with the evolution of the C4-associated CA in the dicotyledonous genus Flaveria, although the actual mutations in the two lineages differ. PMID:28153918

Specific role of LeMAN2 in the control of seed germination exposed by overexpression of the LeMAN3 gene in tomato plants.

PubMed

Belotserkovsky, Harel; Berger, Yael; Shahar, Ron; Wolf, Shmuel

2007-12-01

Endo-beta-mannanase is one of the key enzymes involved in the hydrolysis of the mannan-rich cell walls of tomato (Solanum lycopersicon) seeds. Two isoforms of endo-beta-mannanase have been characterized in tomato seeds: LeMAN2 is active in the micropylar area prior to germination and LeMAN1 is active after germination in all endosperm cells surrounding the cotyledons. To explore whether general mannanase activity in the endosperm cap is sufficient to promote germination, the gene encoding LeMAN3 was inserted into transgenic tomato plants under the control of a CaMV-35S promoter. Expression of LeMAN3 was evident in the endosperm cap and in the lateral endosperm of the transgenic seeds 10 min after imbibition. An activity test indicated increased activity of endo-beta-mannanase in the transgenic lines relative to the control line in all seed parts, during the first 20 h of imbibition. However, overexpression of LeMAN3 in transgenic seeds inhibited seed germination at both optimal and suboptimal temperatures. Detailed RT-PCR analyses revealed the transcription patterns of the genes encoding the various mannanase isoforms, and indicated a delay in LeMAN2 transcription in the endosperm cap of the transgenic seeds. Interestingly, tissue-print assays indicated similar mannanase activity in the micropylar areas for both transgenic and control seeds. These results indicate that overexpression of active endo-beta-mannanase in the endosperm cap is not sufficient to enable hydrolysis of the cell walls or to promote germination of tomato seeds. Cell-wall hydrolysis in these endosperm cells is under tight control and requires the specific activity of LeMAN2.

Transcriptomic investigation of meat tenderness in two Italian cattle breeds.

PubMed

Bongiorni, S; Gruber, C E M; Bueno, S; Chillemi, G; Ferrè, F; Failla, S; Moioli, B; Valentini, A

2016-06-01

Our objectives for this study were to understand the biological basis of meat tenderness and to provide an overview of the gene expression profiles related to meat quality as a tool for selection. Through deep mRNA sequencing, we analyzed gene expression in muscle tissues of two Italian cattle breeds: Maremmana and Chianina. We uncovered several differentially expressed genes that encode for proteins belonging to a family of tripartite motif proteins, which are involved in growth, cell differentiation and apoptosis, such as TRIM45, or play an essential role in regulating skeletal muscle differentiation and the regeneration of adult skeletal muscle, such as TRIM32. Other differentially expressed genes (SCN2B, SLC9A7 and KCNK3) emphasize the involvement of potassium-sodium pumps in tender meat. By mapping splice junctions in RNA-Seq reads, we found significant differences in gene isoform expression levels. The PRKAG3 gene, which is involved in the regulation of energy metabolism, showed four isoforms that were differentially expressed. This distinct pattern of PRKAG3 gene expression could indicate impaired glycogen storage in skeletal muscle, and consequently, this gene very likely has a role in the tenderization process. Furthermore, with this deep RNA-sequencing, we captured a high number of expressed SNPs, for example, we found 1462 homozygous SNPs showing the alternative allele with a 100% frequency when comparing tender and tough meat. SNPs were then classified into categories by their position and also by their effect on gene coding (174 non-synonymous polymorphisms) based on the available UMD_3.1 annotations. © 2016 Stichting International Foundation for Animal Genetics.

Myosin isoform switching during assembly of the Drosophila flight muscle thick filament lattice.

PubMed

Orfanos, Zacharias; Sparrow, John C

2013-01-01

During muscle development myosin molecules form symmetrical thick filaments, which integrate with the thin filaments to produce the regular sarcomeric lattice. In Drosophila indirect flight muscles (IFMs) the details of this process can be studied using genetic approaches. The weeP26 transgenic line has a GFP-encoding exon inserted into the single Drosophila muscle myosin heavy chain gene, Mhc. The weeP26 IFM sarcomeres have a unique MHC-GFP-labelling pattern restricted to the sarcomere core, explained by non-translation of the GFP exon following alternative splicing. Characterisation of wild-type IFM MHC mRNA confirmed the presence of an alternately spliced isoform, expressed earlier than the major IFM-specific isoform. The two wild-type IFM-specific MHC isoforms differ by the presence of a C-terminal 'tailpiece' in the minor isoform. The sequential expression and assembly of these two MHCs into developing thick filaments suggest a role for the tailpiece in initiating A-band formation. The restriction of the MHC-GFP sarcomeric pattern in weeP26 is lifted when the IFM lack the IFM-specific myosin binding protein flightin, suggesting that it limits myosin dissociation from thick filaments. Studies of flightin binding to developing thick filaments reveal a progressive binding at the growing thick filament tips and in a retrograde direction to earlier assembled, proximal filament regions. We propose that this flightin binding restricts myosin molecule incorporation/dissociation during thick filament assembly and explains the location of the early MHC isoform pattern in the IFM A-band.

An intact SAM-dependent methyltransferase fold is encoded by the human endothelin-converting enzyme-2 gene

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tempel, W.; Wu, H.; Dombrovsky, L.

2010-08-17

A recent survey of protein expression patterns in patients with Alzheimer's disease (AD) has identified ece2 (chromosome: 3; Locations: 3q27.1) as the most significantly downregulated gene within the tested group. ece2 encodes endothelin-converting enzyme ECE2, a metalloprotease with a role in neuropeptide processing. Deficiency in the highly homologous ECE1 has earlier been linked to increased levels of AD-related {beta}-amyloid peptide in mice, consistent with a role for ECE in the degradation of that peptide. Initially, ECE2 was presumed to resemble ECE1, in that it comprises a single transmembrane region of {approx}20 residues flanked by a small amino-terminal cytosolic segment andmore » a carboxy-terminal lumenar peptidase domain. The carboxy-terminal domain has significant sequence similarity to both neutral endopeptidase, for which an X-ray structure has been determined, and Kell blood group protein. After their initial discovery, multiple isoforms of ECE1 and ECE2 were discovered, generated by alternative splicing of multiple exons. The originally described ece2 transcript, RefSeq NM{_}174046, contains the amino-terminal cytosolic portion followed by the transmembrane region and peptidase domain (Fig. 1, isoform B). Another ece2 transcript, available from the Mammalian Gene Collection under MGC2408 (Fig. 1, isoform C), RefSeq accession NM{_}032331, is predicted to be translated into a 255 residue peptide with low but detectable sequence similarity to known S-adenosyl-L-methionine (SAM)-dependent methyltransferases (SAM-MTs), such as the hypothetical protein TT1324 from Thermus thermophilis, PDB code 2GS9, which shares 30% amino acid sequence identity with ECE2 over 138 residues of the sequence. Intriguingly, another 'elongated' ece2 transcript (Fig. 1, isoform A) (RefSeq NM{_}014693) contains an amino-terminal portion of the putative SAM-MT domain, the transmembrane domain, and the protease domain. This suggests the possibility for coexistence of the putative SAM-MT and protease domains in a single polypeptide and their transmembrane interplay. Although sequence conservation across the SAM-MT family is weak, the structural fold is highly conserved. The most conserved part of this fold is the SAM-binding subdomain, which is shared between MGC2408 and hypothetical protein TT1324. Typically, the SAM-binding subdomain is flanked by a variable Nterminal extension and, at the C-terminus, by a substrate- binding subdomain, which varies enormously in size but preserves a conserved topology with three antiparallel b-strands. The 'elongated' transcript of ece2 lacks this substrate-binding subdomain. To test the hypothesis that the 255 residue ece2 gene product MGC2408 represents a complete SAM-MT fold, we have determined a crystal structure of this protein in the presence of SAH.« less

Overlapping Role of Dynamin Isoforms in Synaptic Vesicle Endocytosis

PubMed Central

Raimondi, Andrea; Ferguson, Shawn M.; Lou, Xuelin; Armbruster, Moritz; Paradise, Summer; Giovedi, Silvia; Messa, Mirko; Kono, Nao; Takasaki, Junko; Cappello, Valentina; O’Toole, Eileen; Ryan, Timothy A.; De Camilli, Pietro

2011-01-01

The existence of neuron specific endocytic protein isoforms raises questions about their importance for specialized neuronal functions. Dynamin, a GTPase implicated in the fission reaction of endocytosis, is encoded by three genes, two of which, dynamin 1 and 3, are highly expressed in neurons. We show that dynamin 3, thought to play a predominantly postsynaptic role, has a major presynaptic function. While lack of dynamin 3 does not produce an overt phenotype in mice, it worsens the dynamin 1 KO phenotype, leading to perinatal lethality and a more severe defect in activity-dependent synaptic vesicle endocytosis. Thus, dynamin 1 and 3, which together account for the overwhelming majority of brain dynamin, cooperate in supporting optimal rates of synaptic vesicle endocytosis. Persistence of synaptic transmission in their absence indicates that if dynamin plays essential functions in neurons, such functions can be achieved by the very low levels of dynamin 2. PMID:21689597

Function of alternative splicing

PubMed Central

Kelemen, Olga; Convertini, Paolo; Zhang, Zhaiyi; Wen, Yuan; Shen, Manli; Falaleeva, Marina; Stamm, Stefan

2017-01-01

Almost all polymerase II transcripts undergo alternative pre-mRNA splicing. Here, we review the functions of alternative splicing events that have been experimentally determined. The overall function of alternative splicing is to increase the diversity of mRNAs expressed from the genome. Alternative splicing changes proteins encoded by mRNAs, which has profound functional effects. Experimental analysis of these protein isoforms showed that alternative splicing regulates binding between proteins, between proteins and nucleic acids as well as between proteins and membranes. Alternative splicing regulates the localization of proteins, their enzymatic properties and their interaction with ligands. In most cases, changes caused by individual splicing isoforms are small. However, cells typically coordinate numerous changes in ‘splicing programs’, which can have strong effects on cell proliferation, cell survival and properties of the nervous system. Due to its widespread usage and molecular versatility, alternative splicing emerges as a central element in gene regulation that interferes with almost every biological function analyzed. PMID:22909801

GABAA receptor subtype involvement in addictive behaviour.

PubMed

Stephens, D N; King, S L; Lambert, J J; Belelli, D; Duka, T

2017-01-01

GABA A receptors form the major class of inhibitory neurotransmitter receptors in the mammalian brain. This review sets out to summarize the evidence that variations in genes encoding GABA A receptor isoforms are associated with aspects of addictive behaviour in humans, while animal models of addictive behaviour also implicate certain subtypes of GABA A receptor. In addition to outlining the evidence for the involvement of specific subtypes in addiction, we summarize the particular contributions of these isoforms in control over the functioning of brain circuits, especially the mesolimbic system, and make a first attempt to bring together evidence from several fields to understanding potential involvement of GABA A receptor subtypes in addictive behaviour. While the weight of the published literature is on alcohol dependency, the underlying principles outlined are relevant across a number of different aspects of addictive behaviour. © 2016 John Wiley & Sons Ltd and International Behavioural and Neural Genetics Society.

Exclusion of pituitary homeobox 2 gene polymorphism in vertical mandibular asymmetry patients: a preliminary study

NASA Astrophysics Data System (ADS)

Sofyanti, Ervina; Boel, Trelia; Soegiharto, Benny; Ilyas, Syafruddin; Irani Nainggolan, Lidya; Auerkari, Elza Ibrahim

2018-03-01

Pituitary Homeobox 2 (PITX2), is an active gene as a paired-related homeobox gene that encodes multiple isoforms. Its Nodal pathway in determination of left-right patterning during embryogenesis has been reported in satellite cells and expressed in adult human skeletal muscle. PITX2A and PITX2B are produced by alternative splicing and used of different promoters. PITX2C uses an alternative promoter located upstream of exon 4. PITX2D is produced by PITX2C alternative promoter and differential splicing. The 5’-primers and 3’- antisense primer were unique for each isoforms. Variability measurement in vertical dimension showed stronger genetic component than sagittal. This study aims to obtain the genotype marker of vertical mandibular asymmetry related to PITX2A and PITX2D isoform by visualization of the amplified product on stained gel to allele specific oligonucleotide between the case and control with Restriction Fragment Length Polymorphism (RFLP). Determination of vertical mandibular asymmetry based on condylar height asymmetry index of pre-treatment panoramic radiograph using Kjellberg’s technique whilst vertical mandibular growth pattern using lateral cephalogram. The differences of condylar height asymmetry in case-control based on vertical growth pattern was compared using Pearson’s chi-squared test. DNA extraction of 129 out-coming orthodontic patients in Universitas Sumatera Utara Dental Hospital were obtained from Buccal swab. Then DNA samples were amplified by Polymerase chain reaction (PCR) and digested with NciI restriction enzyme prior to electrophoresis visualization. There was no significant statistical difference in vertical mandibular asymmetry compared to vertical mandibular growth pattern. The RFLP analysis did not show any polymorphism for PITX2A and PITX2D isoform. All of the samples showed wild type homozygote. Further analysis method, except RFLP, were required to understand the genetic factor in the variance of vertical mandibular asymmetry.

Evidence that "brain-specific" FOX-1, FOX-2, and nPTB alternatively spliced isoforms are produced in the lens.

PubMed

Bitel, Claudine L; Nathan, Rachel; Wong, Patrick; Kuppasani, Sunil; Matsushita, Masafumi; Kanazawa, Hrioshi; Frederikse, Peter H

2011-04-01

Alternative RNA splicing is essential in development and more rapid physiological processes that include disease mechanisms. Studies over the last 20 years demonstrated that RNA binding protein families, which mediate the alternative splicing of a large percentage of genes in mammals, contain isoforms with mutually exclusive expression in non-neural and neural progenitor cells vs. post-mitotic neurons, and regulate the comprehensive reprogramming of alternative splicing during neurogenesis. Polypyrimidine tract binding (PTB) proteins and Fox-1 proteins also undergo mutually exclusive alternative splicing in neural and non-neural cells that regulates their tissue-specific expression and splicing activities. Over the past 50 years, striking morphological similarities noted between lens fiber cells and neurons suggested that cell biology processes and gene expression profiles may be shared as well. Here, we examined mouse and rat lenses to determine if alternative splicing of neuronal nPTB and Fox-1/Fox-2 isoforms also occurs in lenses. Immunoblot, immunofluorescence, and RT-PCR were used to examine expression and alternative splicing of transcripts in lens and brain. We demonstrated that exon 10 is predominantly included in nPTB transcripts consistent with nPTB protein in lenses, and that alternatively spliced Fox-1/-2 lens transcripts contain exons that have been considered neuron-specific. We identified a 3' alternative Fox-1 exon in lenses that encodes a nuclear localization signal consistent with its protein distribution detected in fiber cells. Neuronal alternative splicing of kinesin KIF1Bβ2 has been associated with PTB/nPTB and Fox-2, and we found that two 'neuron-specific' exons are also included in lenses. The present study provides evidence that alternative neuronal nPTB and Fox-1/Fox-2 isoforms are also produced in lenses. These findings raise questions regarding the extent these factors contribute to a similar reprogramming of alternative splicing during lens differentiation, and the degree that alternative gene transcripts produced during neurogenesis are also expressed in the lens.

Molecular cloning and developmental expression of the catalytic and 65-kDa regulatory subunits of protein phosphatase 2A in Drosophila.

PubMed Central

Mayer-Jaekel, R E; Baumgartner, S; Bilbe, G; Ohkura, H; Glover, D M; Hemmings, B A

1992-01-01

cDNA clones encoding the catalytic subunit and the 65-kDa regulatory subunit of protein phosphatase 2A (PR65) from Drosophila melanogaster have been isolated by homology screening with the corresponding human cDNAs. The Drosophila clones were used to analyze the spatial and temporal expression of the transcripts encoding these two proteins. The Drosophila PR65 cDNA clones contained an open reading frame of 1773 nucleotides encoding a protein of 65.5 kDa. The predicted amino acid sequence showed 75 and 71% identity to the human PR65 alpha and beta isoforms, respectively. As previously reported for the mammalian PR65 isoforms, Drosophila PR65 is composed of 15 imperfect repeating units of approximately 39 amino acids. The residues contributing to this repeat structure show also the highest sequence conservation between species, indicating a functional importance for these repeats. The gene encoding Drosophila PR65 was located at 29B1,2 on the second chromosome. A major transcript of 2.8 kilobase (kb) encoding the PR65 subunit and two transcripts of 1.6 and 2.5 kb encoding the catalytic subunit could be detected throughout Drosophila development. All of these mRNAs were most abundant during early embryogenesis and were expressed at lower levels in larvae and adult flies. In situ hybridization of different developmental stages showed a colocalization of the PR65 and catalytic subunit transcripts. The mRNA expression is high in the nurse cells and oocytes, consistent with a high equally distributed expression in early embryos. In later embryonal development, the expression remains high in the nervous system and the gonads but the overall transcript levels decrease. In third instar larvae, high levels of mRNA could be observed in brain, imaginal discs, and in salivary glands. These results indicate that protein phosphatase 2A transcript levels change during development in a tissue and in a time-specific manner. Images PMID:1320961

[Regulation of the β-globin gene family expression, useful in the search for new therapeutic targets for hemoglobinopathies].

PubMed

Scheps, Karen G; Varela, Viviana

Different hemoglobin isoforms are expressed during the embryonic, fetal and postnatal stages. They are formed by combination of polypeptide chains synthesized from the α- and β-globin gene clusters. Based on the fact that the presence of high hemoglobin F levels is beneficial in both sickle cell disease and severe thalassemic syndromes, a revision of the regulation of the β-globin cluster expression is proposed, especially regarding the genes encoding the y-globin chains (HBG1 and HBG2). In this review we describe the current knowledge about transcription factors and epigenetic regulators involved in the switches of the β-globin cluster. It is expected that the consolidation of knowledge in this field will allow finding new therapeutic targets for the treatment of hemoglobinopathies.

A unique gene expression signature associated with serotonin 2C receptor RNA editing in the prefrontal cortex and altered in suicide

PubMed Central

Di Narzo, Antonio Fabio; Kozlenkov, Alexey; Roussos, Panos; Hao, Ke; Hurd, Yasmin; Lewis, David A.; Sibille, Etienne; Siever, Larry J.; Koonin, Eugene; Dracheva, Stella

2014-01-01

Editing of the pre-mRNA for the serotonin receptor 2C (5-HT2CR) by site-specific adenosine deamination (A-to-I pre-mRNA editing) substantially increases the functional plasticity of this key neurotransmitter receptor and is thought to contribute to homeostatic mechanisms in neurons. 5-HT2CR mRNA editing generates up to 24 different receptor isoforms. The extent of editing correlates with 5-HT2CR functional activity: more highly edited isoforms exhibit the least function. Altered 5-HT2CR editing has been reported in postmortem brains of suicide victims. We report a comparative analysis of the connections among 5-HT2CR editing, genome-wide gene expression and DNA methylation in suicide victims, individuals with major depressive disorder and non-psychiatric controls. The results confirm previous findings of an overrepresentation of highly edited mRNA variants (which encode hypoactive 5-HT2CR receptors) in the brains of suicide victims. A large set of genes for which the expression level is associated with editing was detected. This signature set of editing-associated genes is significantly enriched for genes that are involved in synaptic transmission, genes that are preferentially expressed in neurons, and genes whose expression is correlated with the level of DNA methylation. Notably, we report that the link between 5-HT2CR editing and gene expression is disrupted in suicide victims. The results suggest that the postulated homeostatic function of 5-HT2CR editing is dysregulated in individuals who committed suicide. PMID:24781207

Progesterone receptor isoforms in the mammary gland of cats and dogs.

PubMed

Gracanin, A; de Gier, J; Zegers, K; Bominaar, M; Rutteman, G R; Schaefers-Okkens, A C; Kooistra, H S; Mol, J A

2012-12-01

Progesterone exerts its effect by binding to specific progesterone receptors (PR) within the cell. In dogs and cats, no data are available on PR isoforms as found in other species. We therefore investigated the sequence of the PR gene and encoded protein in dogs and cats, the expression of PR isoforms in mammary tissue using Western blots and the presence of PR in mammary tissue using immunohistochemistry. Comparison of the amino acid sequence of the canine and feline PR with human PR revealed major differences in the PR-B-specific upstream segment (BUS). However, the essential activation function 3 (AF3) domain was intact in the cat but mutated in the dog. The DNA and ligand-binding domains were highly similar among the species. In cats with fibroadenomatous hyperplasia (FAH), high expression of PR mRNA together with growth hormone (GH), GH receptor (GHR) and IGF-I mRNA was found in comparison with feline mammary carcinomas. Immunohistochemical analysis showed strong nuclear as well as cytoplasmic staining for PR in FAH. Western blot analysis revealed expression of the PR-A and PR-B isoforms in the feline mammary gland. In canine mammary tissue, the most abundant PR staining was found in proliferative zones of the mammary gland. Western blot analyses showed mainly staining for PR-A with lower PR-B staining. It is concluded that in dogs and cats both PR isoforms are expressed. The role of mutations found in the canine PR-B is discussed. © 2012 Blackwell Verlag GmbH.

ACA12 Is a Deregulated Isoform of Plasma Membrane Ca2+-ATPase of Arabidopsis thaliana

PubMed Central

Limonta, Margherita; Romanowsky, Shawn; Olivari, Claudio; Bonza, Maria Cristina; Luoni, Laura; Rosenberg, Alexa; Harper, Jeffrey F.; De Michelis, Maria Ida

2014-01-01

Plant auto-inhibited Ca2+-ATPases (ACA) are crucial in defining the shape of calcium transients and therefore in eliciting plant responses to various stimuli. Arabidopsis thaliana genome encodes ten ACA isoforms that can be divided into four clusters based on gene structure and sequence homology. While isoforms from clusters 1, 2 and 4 have been characterized, virtually nothing is known about members of cluster 3 (ACA12 and ACA13). Here we show that a GFP-tagged ACA12 localizes at the plasma membrane and that expression of ACA12 rescues the phenotype of partial male sterility of a null mutant of the plasma membrane isoform ACA9, thus providing genetic evidence that ACA12 is a functional plasma membrane-resident Ca2+-ATPase. By ACA12 expression in yeast and purification by CaM-affinity chromatography, we show that, unlike other ACAs, the activity of ACA12 is not stimulated by CaM. Moreover, full length ACA12 is able to rescue a yeast mutant deficient in calcium pumps. Analysis of single point ACA12 mutants suggests that ACA12 loss of auto-inhibition can be ascribed to the lack of two acidic residues - highly conserved in other ACA isoforms - localized at the cytoplasmic edge of the second and third transmembrane segments. Together, these results support a model in which the calcium pump activity of ACA12 is primarily regulated by increasing or decreasing mRNA expression and/or protein translation and degradation. PMID:24101142

IL-10 production by B cells expressing CD5 with the alternative exon 1B.

PubMed

Garaud, Soizic; Le Dantec, Christelle; de Mendoza, Agnès Revol; Mageed, Rizgar A; Youinou, Pierre; Renaudineau, Yves

2009-09-01

B lymphocytes are divided into two subpopulations, B1 and B2 cells based on expression of the T cell-associated protein CD5. Natural B1 cells are further divided into B1a cells that express CD5 on their membrane and B1b cells that do not but share most other biological characteristics of B1a cells. Recent studies from our laboratory have revealed, in humans, the existence of two alternative isoforms of the CD5 protein. A cell surface CD5 isoform which uses exon 1A (E1A) of the gene in B1a cells, and an intracellular isoform which uses exon 1B (E1B) mainly in human B1b cells. Indeed, the protein isoform encoded by transcripts containing E1B lack the leader peptide and is, thus, retained in the cytoplasm of B cells. The restriction of interleukin (IL)-10 to B1 lymphocytes in the mouse raises the possibility that the human CD5-E1B-expressing B cells produce IL-10. This prediction was confirmed in the CD5 negative Jok-1 B cells transfected with cDNA for either isoforms resulted in high level IL-10 production. Our data indicate that E1B-CD5-expressing B cells have the capacity to interfere with the immune response through their ability to produce high levels of IL-10.

Genomic organization of the rat alpha 2u-globulin gene cluster.

PubMed

McFadyen, D A; Addison, W; Locke, J

1999-05-01

The alpha 2u-globulin are a group of similar proteins, belonging to the lipocalin superfamily of proteins, that are synthesized in a subset of secretory tissues in rats. The many alpha 2u-globulin isoforms are encoded by a multigene family that exhibits extensive homology. Despite a high degree of sequence identity, individual family members show diverse expression patterns involving complex hormonal, tissue-specific, and developmental regulation. Analysis suggests that there are approximately 20 alpha 2u-globulin genes in the rat genome. We have used fluorescence in situ hybridization (FISH) to show that the alpha 2u-globulin genes are clustered at a single site on rat Chromosome (Chr) 5 (5q22-24). Southern blots of rat genomic DNA separated by pulsed field gel electrophoresis indicated that the alpha 2u-globulin genes are contained on two NruI fragments with a total size of 880 kbp. Analysis of three P1 clones containing alpha 2u-globulin genes indicated that the alpha 2u-globulin genes are tandemly arranged in a head-to-tail fashion. The organization of the alpha 2u-globulin genes in the rat as a tandem array of single genes differs from the homologous major urinary protein genes in the mouse, which are organized as tandem arrays of divergently oriented gene pairs. The structure of these gene clusters may have consequences for the proposed function, as a pheromone transporter, for the protein products encoded by these genes.

Characterization of two chitin synthase genes of the red flour beetle, Tribolium castaneum, and alternate exon usage in one of the genes during development.

PubMed

Arakane, Yasuyuki; Hogenkamp, David G; Zhu, Yu Cheng; Kramer, Karl J; Specht, Charles A; Beeman, Richard W; Kanost, Michael R; Muthukrishnan, Subbaratnam

2004-03-01

Two chitin synthase (CHS) genes of the red flour beetle, Tribolium castaneum, were sequenced and their transcription patterns during development examined. By screening a BAC library of genomic DNA from T. castaneum (Tc) with a DNA probe encoding the catalytic domain of a putative Tribolium CHS, several clones that contained CHS genes were identified. Two distinct PCR products were amplified from these BAC clones and confirmed to be highly similar to CHS genes from other insects, nematodes and fungi. The DNA sequences of these genes, TcCHS1 and TcCHS2, were determined by amplification of overlapping PCR fragments from two of the BAC DNAs and mapped to different linkage groups. Each ORF was identified and full-length cDNAs were also amplified, cloned and sequenced. TcCHS1 and TcCHS2 encode transmembrane proteins of 1558 and 1464 amino acids, respectively. The TcCHS1 gene was found to use alternate exons, each encoding 59 amino acids, a feature not found in the TcCHS2 gene. During development, Tribolium expressed TcCHS1 predominantly in the embryonic and pupal stages, whereas TcCHS2 was prevalent in the late larval and adult stages. The alternate exon 8a of TcCHS1 was utilized over a much broader range of development than exon 8b. We propose that the two isoforms of the TcCHS1 enzyme are used predominantly for the formation of chitin in embryonic and pupal cuticles, whereas TcCHS2 is utilized primarily for the synthesis of peritrophic membrane-associated chitin in the midgut.

Histone Code Modulation by Oncogenic PWWP-Domain Protein in Breast Cancers

DTIC Science & Technology

2010-06-01

athanogene 4 * DDHD2 DDHD domain containing 2 * PPAPDC1B phosphatidic acid phosphatase type 2 domain containing 1B * WHSC1L1 Wolf-Hirschhorn syndrome...from alternative splicing of exon 10. The WHSC1L1 long isoform encodes a 1437 amino acid protein containing 2 PWWP domains, 2 PHD-type zinc finger...motifs, a TANG2 domain, an AWS domain and a SET domain. The short isoform encodes a 645 amino acid protein containing a PWWP domain only. Our western

Expression of the TPα and TPβ isoforms of the thromboxane prostanoid receptor (TP) in prostate cancer: clinical significance and diagnostic potential.

PubMed

Mulvaney, Eamon P; Shilling, Christine; Eivers, Sarah B; Perry, Antoinette S; Bjartell, Anders; Kay, Elaine W; Watson, R William; Kinsella, B Therese

2016-11-08

The prostanoid thromboxane (TX)A2 plays a central role in haemostasis and is increasingly implicated in cancer progression. TXA2 signals through two T Prostanoid receptor (TP) isoforms termed TPα and TPβ, with both encoded by the TBXA2R gene. Despite exhibiting several functional and regulatory differences, the role of the individual TP isoforms in neoplastic diseases is largely unknown.This study evaluated expression of the TPα and TPβ isoforms in tumour microarrays of the benign prostate and different pathological (Gleason) grades of prostate cancer (PCa). Expression of TPβ was significantly increased in PCa relative to benign tissue and strongly correlated with increasing Gleason grade. Furthermore, higher TPβ expression was associated with increased risk of biochemical recurrence (BCR) and significantly shorter disease-free survival time in patients post-surgery. While TPα was more variably expressed than TPβ in PCa, increased/high TPα expression within the tumour also trended toward increased BCR and shorter disease-free survival time. Comparative genomic CpG DNA methylation analysis revealed substantial differences in the extent of methylation of the promoter regions of the TBXA2R that specifically regulate expression of TPα and TPβ, respectively, both in benign prostate and in clinically-derived tissue representative of precursor lesions and progressive stages of PCa. Collectively, TPα and TPβ expression is differentially regulated both in the benign and tumourigenic prostate, and coincides with clinical pathology and altered CpG methylation of the TBXA2R gene. Analysis of TPβ, or a combination of TPα/TPβ, expression levels may have significant clinical potential as a diagnostic biomarker and predictor of PCa disease recurrence.

The Arabidopsis 14-3-3 Protein RARE COLD INDUCIBLE 1A Links Low-Temperature Response and Ethylene Biosynthesis to Regulate Freezing Tolerance and Cold Acclimation[C][W

PubMed Central

Catalá, Rafael; López-Cobollo, Rosa; Mar Castellano, M.; Angosto, Trinidad; Alonso, José M.; Ecker, Joseph R.; Salinas, Julio

2014-01-01

In plants, the expression of 14-3-3 genes reacts to various adverse environmental conditions, including cold, high salt, and drought. Although these results suggest that 14-3-3 proteins have the potential to regulate plant responses to abiotic stresses, their role in such responses remains poorly understood. Previously, we showed that the RARE COLD INDUCIBLE 1A (RCI1A) gene encodes the 14-3-3 psi isoform. Here, we present genetic and molecular evidence implicating RCI1A in the response to low temperature. Our results demonstrate that RCI1A functions as a negative regulator of constitutive freezing tolerance and cold acclimation in Arabidopsis thaliana by controlling cold-induced gene expression. Interestingly, this control is partially performed through an ethylene (ET)-dependent pathway involving physical interaction with different ACC SYNTHASE (ACS) isoforms and a decreased ACS stability. We show that, consequently, RCI1A restrains ET biosynthesis, contributing to establish adequate levels of this hormone in Arabidopsis under both standard and low-temperature conditions. We further show that these levels are required to promote proper cold-induced gene expression and freezing tolerance before and after cold acclimation. All these data indicate that RCI1A connects the low-temperature response with ET biosynthesis to modulate constitutive freezing tolerance and cold acclimation in Arabidopsis. PMID:25122152

The Wilms tumor protein WT1 stimulates transcription of the gene encoding insulin-like growth factor binding protein 5 (IGFBP5).

PubMed

Müller, Miriam; Persson, Anja Bondke; Krueger, Katharina; Kirschner, Karin M; Scholz, Holger

2017-07-01

Insulin-like growth factor (IGF) binding proteins (IGFBPs) constitute a family of six secreted proteins that regulate the signaling of insulin-like growth factors (IGFs). IGFBP5 is the most conserved family member in vertebrates and the major IGF binding protein in bone. IGFBP5 is required for normal development of the musculoskeletal system, and various types of cancer frequently express high levels of IGFP5. Here we identify the gene encoding IGFBP5 as a novel downstream target of the Wilms tumor protein WT1. IGFBP5 and WT1 are expressed in an overlapping pattern in the condensing metanephric mesenchyme of embryonic murine kidneys. Down-regulation of WT1 by transfection with antisense vivo-morpholino significantly decreased Igfbp5 transcripts in murine embryonic kidney explants. Likewise, silencing of Wt1 in a mouse mesonephros-derived cell line reduced Igfbp5 mRNA levels by approximately 80%. Conversely, induction of the WT1(-KTS) isoform, whose role as transcriptional regulator has been firmly established, significantly increased IGFBP5 mRNA and protein levels in osteosarcoma cells. IGFBP5 expression was not significantly changed by WT1(+KTS) protein, which exhibits lower DNA binding affinity than the WT1(-KTS) isoform and has a presumed role in post-transcriptional gene regulation. Luciferase reporter constructs harboring 0.8 and 1.6 kilobases of the murine Igfbp5 promoter, respectively, were stimulated approximately 5-fold by co-transfection of WT1(-KTS). The WT1(+KTS) variant had no significant effect on IGFBP5 promoter activity. Binding of WT1(-KTS), but not of WT1(+KTS) protein, to the IGFBP5 promoter in human osteosarcoma cells was proven by chromatin immunoprecipitation (ChIP) and confirmed by electrophoretic mobility shift assay. These findings demonstrate that WT1 activates transcription of the IGFBP5 gene with possible implications for kidney development and bone (patho)physiology. Copyright © 2017 Elsevier B.V. All rights reserved.

The human serotonin 5-HT{sub 2C} receptor: Complete cDNA, genomic structure, and alternatively spliced variant

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xie, Enzhong; Zhu, Lingyu; Zhao, Lingyun

1996-08-01

The complete 4775-nt cDNA encoding the human serotonin 5-HT{sub 2C} receptor (5-HT{sub 2C}R), a G-protein-coupled receptor, has been isolated. It contains a 1377-nt coding region flanked by a 728-nt 5{prime}-untranslated region and a 2670-nt 3{prime}-untranslated region. By using the cloned 5-HT{sub 2C}R cDNA probe, the complete human gene for this receptor has been isolated and shown to contain six exons and five introns spanning at least 230 kb of DNA. The coding region of the human 5-HT{sub 2C}R gene is interrupted by three introns, and the positions of the intron/exon junctions are conserved between the human and the rodent genes.more » In addition, an alternatively spliced 5-HT{sub 2C}R RNA that contains a 95-nt deletion in the region coding for the second intracellular loop and the fourth transmembrane domain of the receptor has been identified. This deletion leads to a frameshift and premature termination so that the short isoform RNA encodes a putative protein of 248 amino acids. The ratio for the short isoform over the 5-HT{sub 2C}R RNA was found to be higher in choroid plexus tumor than in normal brain tissue, suggesting the possibility of differential regulation of the 5-HT{sub 2C}R gene in different neural tissues or during tumorigenesis. Transcription of the human 5-HT{sub 2C}R gene was found to be initiated at multiple sites. No classical TATA-box sequence was found at the appropriate location, and the 5{prime}-flanking sequence contains many potential transcription factor-binding sites. A 7.3-kb 5{prime}-flanking 5-HT{sub 2C}R DNA directed the efficient expression of a luciferase reported gene in SK-N-SH and IMR32 neuroblastoma cells, indicating that is contains a functional promoter. 69 refs., 8 figs., 1 tab.« less

The alpha1-fetoprotein locus is activated by a nuclear receptor of the Drosophila FTZ-F1 family.

PubMed

Galarneau, L; Paré, J F; Allard, D; Hamel, D; Levesque, L; Tugwood, J D; Green, S; Bélanger, L

1996-07-01

The alpha1-fetoprotein (AFP) gene is located between the albumin and alpha-albumin genes and is activated by transcription factor FTF (fetoprotein transcription factor), presumed to transduce early developmental signals to the albumin gene cluster. We have identified FTF as an orphan nuclear receptor of the Drosophila FTZ-F1 family. FTF recognizes the DNA sequence 5'-TCAAGGTCA-3', the canonical recognition motif for FTZ-F1 receptors. cDNA sequence homologies indicate that rat FTF is the ortholog of mouse LRH-1 and Xenopus xFF1rA. Rodent FTF is encoded by a single-copy gene, related to the gene encoding steroidogenic factor 1 (SF-1). The 5.2-kb FTF transcript is translated from several in-frame initiator codons into FTF isoforms (54 to 64 kDa) which appear to bind DNA as monomers, with no need for a specific ligand, similar KdS (approximately equal 3 x 10(-10) M), and similar transcriptional effects. FTF activates the AFP promoter without the use of an amino-terminal activation domain; carboxy-terminus-truncated FTF exerts strong dominant negative effects. In the AFP promoter, FTF recruits an accessory trans-activator which imparts glucocorticoid reactivity upon the AFP gene. FTF binding sites are found in the promoters of other liver-expressed genes, some encoding liver transcription factors; FTF, liver alpha1-antitrypsin promoter factor LFB2, and HNF-3beta promoter factor UF2-H3beta are probably the same factor. FTF is also abundantly expressed in the pancreas and may exert differentiation functions in endodermal sublineages, similar to SF-1 in steroidogenic tissues. HepG2 hepatoma cells seem to express a mutated form of FTF.

Two rat brain staufen isoforms differentially bind RNA.

PubMed

Monshausen, M; Putz, U; Rehbein, M; Schweizer, M; DesGroseillers, L; Kuhl, D; Richter, D; Kindler, S

2001-01-01

In neurones, a limited number of mRNAs is found in dendrites, including transcripts encoding the microtubule-associated protein 2 (MAP2). Recently, we identified a cis-acting dendritic targeting element (DTE) in MAP2 mRNAs. Here we used the yeast tri-hybrid system to identify potential trans-acting RNA-binding factors of the DTE. A cDNA clone was isolated that encodes a member of a mammalian protein family that is highly homologous to the Drosophila RNA-binding protein Staufen. Mammalian Staufen appears to be expressed in most tissues and brain areas. Two distinct rat brain Staufen isoforms, rStau+I6 and rStau-I6, are encoded by alternatively spliced mRNAs. Both isoforms contain four double-stranded RNA-binding domains (dsRBD). In the larger rStau+I6 isoform, six additional amino acids are inserted in the second dsRBD. Although both isoforms interacted with the MAP2-DTE and various additional RNA fragments in an in vitro north-western assay, rStau-I6 exhibited a stronger signal of bound radioactively labelled RNAs as compared with rStau+I6. Using an antibody directed against mammalian Staufen, the protein was detected in somata and dendrites of neurones of the adult rat hippocampus and cerebral cortex. Ultrastructural studies revealed that in dendrites, rat Staufen accumulates along microtubules. Thus in neurones, rat Staufen may serve to link RNAs to the dendritic microtubular cytoskeleton and may thereby regulate their subcellular localization.

A survey of the sorghum transcriptome using single-molecule long reads

DOE PAGES

Abdel-Ghany, Salah E.; Hamilton, Michael; Jacobi, Jennifer L.; ...

2016-06-24

Alternative splicing and alternative polyadenylation (APA) of pre-mRNAs greatly contribute to transcriptome diversity, coding capacity of a genome and gene regulatory mechanisms in eukaryotes. Second-generation sequencing technologies have been extensively used to analyse transcriptomes. However, a major limitation of short-read data is that it is difficult to accurately predict full-length splice isoforms. Here we sequenced the sorghum transcriptome using Pacific Biosciences single-molecule real-time long-read isoform sequencing and developed a pipeline called TAPIS (Transcriptome Analysis Pipeline for Isoform Sequencing) to identify full-length splice isoforms and APA sites. Our analysis reveals transcriptome-wide full-length isoforms at an unprecedented scale with over 11,000 novelmore » splice isoforms. Additionally, we uncover APA ofB11,000 expressed genes and more than 2,100 novel genes. Lastly, these results greatly enhance sorghum gene annotations and aid in studying gene regulation in this important bioenergy crop. The TAPIS pipeline will serve as a useful tool to analyse Iso-Seq data from any organism.« less

A survey of the sorghum transcriptome using single-molecule long reads

PubMed Central

Abdel-Ghany, Salah E.; Hamilton, Michael; Jacobi, Jennifer L.; Ngam, Peter; Devitt, Nicholas; Schilkey, Faye; Ben-Hur, Asa; Reddy, Anireddy S. N.

2016-01-01

Alternative splicing and alternative polyadenylation (APA) of pre-mRNAs greatly contribute to transcriptome diversity, coding capacity of a genome and gene regulatory mechanisms in eukaryotes. Second-generation sequencing technologies have been extensively used to analyse transcriptomes. However, a major limitation of short-read data is that it is difficult to accurately predict full-length splice isoforms. Here we sequenced the sorghum transcriptome using Pacific Biosciences single-molecule real-time long-read isoform sequencing and developed a pipeline called TAPIS (Transcriptome Analysis Pipeline for Isoform Sequencing) to identify full-length splice isoforms and APA sites. Our analysis reveals transcriptome-wide full-length isoforms at an unprecedented scale with over 11,000 novel splice isoforms. Additionally, we uncover APA of ∼11,000 expressed genes and more than 2,100 novel genes. These results greatly enhance sorghum gene annotations and aid in studying gene regulation in this important bioenergy crop. The TAPIS pipeline will serve as a useful tool to analyse Iso-Seq data from any organism. PMID:27339290

Developmental expression of high molecular weight tropomyosin isoforms in Mesocestoides corti.

PubMed

Koziol, Uriel; Costábile, Alicia; Domínguez, María Fernanda; Iriarte, Andrés; Alvite, Gabriela; Kun, Alejandra; Castillo, Estela

2011-02-01

Tropomyosins are a family of actin-binding proteins with diverse roles in actin filament function. One of the best characterized roles is the regulation of muscle contraction. Tropomyosin isoforms can be generated from different genes, and from alternative promoters and alternative splicing from the same gene. In this work, we have isolated sequences for tropomyosin isoforms from the cestode Mesocestoides corti, and searched for tropomyosin genes and isoforms in other flatworms. Two genes are conserved in the cestodes M. corti and Echinococcus multilocularis, and in the trematode Schistosoma mansoni. Both genes have the same structure, and each gene gives rise to at least two different isoforms, a high molecular weight (HMW) and a low molecular weight (LMW) one. Because most exons are duplicated and spliced in a mutually exclusive fashion, isoforms from one gene only share one exon and are highly divergent. The gene duplication preceded the divergence of neodermatans and the planarian Schmidtea mediterranea. Further duplications occurred in Schmidtea, coupled to the selective loss of duplicated exons, resulting in genes that only code for HMW or LMW isoforms. A polyclonal antibody raised against a HMW tropomyosin from Echinococcus granulosus was demonstrated to specifically recognize HMW tropomyosin isoforms of M. corti, and used to study their expression during segmentation. HMW tropomyosins are expressed in muscle layers, with very low or absent levels in other tissues. No expression of HMW tropomyosins is present in early or late genital primordia, and expression only begins once muscle fibers develop in the genital ducts. Therefore, HMW tropomyosins are markers for the development of muscles during the final differentiation of genital primordia. Copyright © 2010 Elsevier B.V. All rights reserved.

A single nucleotide polymorphism in MGEA5 encoding O-GlcNAc-selective N-acetyl-beta-D glucosaminidase is associated with type 2 diabetes in Mexican Americans.

PubMed

Lehman, Donna M; Fu, Dong-Jing; Freeman, Angela B; Hunt, Kelly J; Leach, Robin J; Johnson-Pais, Teresa; Hamlington, Jeanette; Dyer, Thomas D; Arya, Rector; Abboud, Hanna; Göring, Harald H H; Duggirala, Ravindranath; Blangero, John; Konrad, Robert J; Stern, Michael P

2005-04-01

Excess O-glycosylation of proteins by O-linked beta-N-acetylglucosamine (O-GlcNAc) may be involved in the pathogenesis of type 2 diabetes. The enzyme O-GlcNAc-selective N-acetyl-beta-d glucosaminidase (O-GlcNAcase) encoded by MGEA5 on 10q24.1-q24.3 reverses this modification by catalyzing the removal of O-GlcNAc. We have previously reported the linkage of type 2 diabetes and age at diabetes onset to an overlapping region on chromosome 10q in the San Antonio Family Diabetes Study (SAFADS). In this study, we investigated menangioma-expressed antigen-5 (MGEA5) as a positional candidate gene. Twenty-four single nucleotide polymorphisms (SNPs), identified by sequencing 44 SAFADS subjects, were genotyped in 436 individuals from 27 families whose data were used in the original linkage report. Association tests indicated significant association of a novel SNP with the traits diabetes (P = 0.0128, relative risk = 2.77) and age at diabetes onset (P = 0.0017). The associated SNP is located in intron 10, which contains an alternate stop codon and may lead to decreased expression of the 130-kDa isoform, the isoform predicted to contain the O-GlcNAcase activity. We investigated whether this variant was responsible for the original linkage signal. The variance attributed to this SNP accounted for approximately 25% of the logarithm of odds. These results suggest that this variant within the MGEA5 gene may increase diabetes risk in Mexican Americans.

Cloning and sequencing of a laccase gene from the lignin-degrading basidiomycete Pleurotus ostreatus.

PubMed Central

Giardina, P; Cannio, R; Martirani, L; Marzullo, L; Palmieri, G; Sannia, G

1995-01-01

The gene (pox1) encoding a phenol oxidase from Pleurotus ostreatus, a lignin-degrading basidiomycete, was cloned and sequenced, and the corresponding pox1 cDNA was also synthesized and sequenced. The isolated gene consists of 2,592 bp, with the coding sequence being interrupted by 19 introns and flanked by an upstream region in which putative CAAT and TATA consensus sequences could be identified at positions -174 and -84, respectively. The isolation of a second cDNA (pox2 cDNA), showing 84% similarity, and of the corresponding truncated genomic clones demonstrated the existence of a multigene family coding for isoforms of laccase in P. ostreatus. PCR amplifications of specific regions on the DNA of isolated monokaryons proved that the two genes are not allelic forms. The POX1 amino acid sequence deduced was compared with those of other known laccases from different fungi. PMID:7793961

Novel isoforms of Dlg are fundamental for neuronal development in Drosophila.

PubMed

Mendoza, Carolina; Olguín, Patricio; Lafferte, Gabriela; Thomas, Ulrich; Ebitsch, Susanne; Gundelfinger, Eckart D; Kukuljan, Manuel; Sierralta, Jimena

2003-03-15

Drosophila discs-large (dlg) mutants exhibit multiple developmental abnormalities, including severe defects in neuronal differentiation and synaptic structure and function. These defects have been ascribed to the loss of a single gene product, Dlg-A, a scaffold protein thought to be expressed in many cell types. Here, we describe that additional isoforms arise as a consequence of different transcription start points and alternative splicing of dlg. At least five different dlg gene products are predicted. We identified a subset of dlg-derived cDNAs that include novel exons encoding a peptide homologous to the N terminus of the mammalian protein SAP97/hDLG (S97N). Dlg isoforms containing the S97N domain are expressed at larval neuromuscular junctions and within the CNS of both embryos and larvae but are not detectable in epithelial tissues. Strong hypomorphic dlg alleles exhibit decreased expression of S97N, which may account for neural-specific aspects of the pleiomorphic dlg mutant phenotype. Selective inhibition of the expression of S97N-containing proteins in embryos by double-strand RNA leads to severe defects in neuronal differentiation and axon guidance, without overt perturbations in epithelia. These results indicate that the differential expression of dlg products correlates with distinct functions in non-neural and neural cells. During embryonic development, proteins that include the S97N domain are essential for proper neuronal differentiation and organization, acting through mechanisms that may include the adequate localization of cell fate determinants.

Competitive regulation of alternative splicing and alternative polyadenylation by hnRNP H and CstF64 determines acetylcholinesterase isoforms

PubMed Central

Nazim, Mohammad; Masuda, Akio; Rahman, Mohammad Alinoor; Nasrin, Farhana; Takeda, Jun-ichi; Ohe, Kenji; Ohkawara, Bisei; Ito, Mikako

2017-01-01

Abstract Acetylcholinesterase (AChE), encoded by the ACHE gene, hydrolyzes the neurotransmitter acetylcholine to terminate synaptic transmission. Alternative splicing close to the 3΄ end generates three distinct isoforms of AChET, AChEH and AChER. We found that hnRNP H binds to two specific G-runs in exon 5a of human ACHE and activates the distal alternative 3΄ splice site (ss) between exons 5a and 5b to generate AChET. Specific effect of hnRNP H was corroborated by siRNA-mediated knockdown and artificial tethering of hnRNP H. Furthermore, hnRNP H competes for binding of CstF64 to the overlapping binding sites in exon 5a, and suppresses the selection of a cryptic polyadenylation site (PAS), which additionally ensures transcription of the distal 3΄ ss required for the generation of AChET. Expression levels of hnRNP H were positively correlated with the proportions of the AChET isoform in three different cell lines. HnRNP H thus critically generates AChET by enhancing the distal 3΄ ss and by suppressing the cryptic PAS. Global analysis of CLIP-seq and RNA-seq also revealed that hnRNP H competitively regulates alternative 3΄ ss and alternative PAS in other genes. We propose that hnRNP H is an essential factor that competitively regulates alternative splicing and alternative polyadenylation. PMID:28180311

Competitive regulation of alternative splicing and alternative polyadenylation by hnRNP H and CstF64 determines acetylcholinesterase isoforms.

PubMed

Nazim, Mohammad; Masuda, Akio; Rahman, Mohammad Alinoor; Nasrin, Farhana; Takeda, Jun-Ichi; Ohe, Kenji; Ohkawara, Bisei; Ito, Mikako; Ohno, Kinji

2017-02-17

Acetylcholinesterase (AChE), encoded by the ACHE gene, hydrolyzes the neurotransmitter acetylcholine to terminate synaptic transmission. Alternative splicing close to the 3΄ end generates three distinct isoforms of AChET, AChEH and AChER. We found that hnRNP H binds to two specific G-runs in exon 5a of human ACHE and activates the distal alternative 3΄ splice site (ss) between exons 5a and 5b to generate AChET. Specific effect of hnRNP H was corroborated by siRNA-mediated knockdown and artificial tethering of hnRNP H. Furthermore, hnRNP H competes for binding of CstF64 to the overlapping binding sites in exon 5a, and suppresses the selection of a cryptic polyadenylation site (PAS), which additionally ensures transcription of the distal 3΄ ss required for the generation of AChET. Expression levels of hnRNP H were positively correlated with the proportions of the AChET isoform in three different cell lines. HnRNP H thus critically generates AChET by enhancing the distal 3΄ ss and by suppressing the cryptic PAS. Global analysis of CLIP-seq and RNA-seq also revealed that hnRNP H competitively regulates alternative 3΄ ss and alternative PAS in other genes. We propose that hnRNP H is an essential factor that competitively regulates alternative splicing and alternative polyadenylation.

Induction of 1-acylglycerophosphocholine acyltransferase genes by fibrates in the liver of rats.

PubMed

Yamazaki, Tohru; Wakabayashi, Michiko; Ikeda, Erika; Tanaka, Shizuyo; Sakamoto, Takeshi; Mitsumoto, Atsushi; Kudo, Naomi; Kawashima, Yoichi

2012-01-01

The effect of fibrates (clofibric acid, bezafibrate and fenofibrate) on the gene expression and activity of 1-acylglycerophosphocholine acyltransferase (LPCAT) was investigated. The administration of 0.1% (w/w) clofibric acid, bezafibrate or fenofibrate in diet for 14 d to rats induced LPCAT activity in hepatic microsomes in the following order: fenofibrate>bezafibrate>clofibric acid. The LPCAT induced by fenofibrate preferred to arachidonoyl-CoA and linoleoyl-CoA to a greater extent than did LPCAT in control microsomes. The treatment with the fibrates resulted in upregulation of the relative expression of mRNAs encoding LPCAT3 and LPCAT4 in the following order: fenofibrate>bezafibrate>clofibric acid. The administration of fibrates did not change the expression of genes encoding either LPCAT1 or LPCAT2. The treatment with fibrates elevated relative levels of both mRNAs encoding Δ6 desaturase (Fads2) and Δ5 desaturase (Fads1) in the order of fenofibrate>bezafibrate>clofibric acid, and the extent of the increase in the level of Δ6 desaturase mRNA was greater than that of Δ5 desaturase. Fatty acid profile in hepatic phosphatidylcholine (PC) was significantly changed by the treatments with fibrates. These results suggest (i) that fibrates induce LPCAT activity in hepatic microsomes by elevating the expression of genes encoding LPCAT3 and LPCAT4, (ii) that the changes in fatty acid profile of hepatic PC are, in part, due to the elevated expression of two isoforms, LPCAT3 and LPCAT4, and (iii) that the ability of fibrates to induce these changes are in the order of fenofibrate>bezafibrate>clofibric acid.

Microaerobic glycerol formation in Saccharomyces cerevisiae.

PubMed

Costenoble, R; Valadi, H; Gustafsson, L; Niklasson, C; Franzén, C J

2000-12-01

The yeast Saccharomyces cerevisiae produces large amounts of glycerol as an osmoregulator during hyperosmotic stress and as a redox sink at low oxygen availability. NAD(+)-dependent glycerol-3-phosphate dehydrogenase in S. cerevisiae is present in two isoforms, coded for by two different genes, GPD1 and GPD2. Mutants for either one or both of these genes were investigated under carefully controlled static and dynamic conditions in continuous cultures at low oxygen transfer rates. Our results show that S. cerevisiae controls the production of glycerol in response to hypoxic conditions by regulating the expression of several genes. At high demand for NADH reoxidation, a strong induction was seen not only of the GPD2 gene, but also of GPP1, encoding one of the molecular forms of glycerol-3-phosphatase. Induction of the GPP1 gene appears to play a decisive role at elevated growth rates. At low demand for NADH reoxidation via glycerol formation, the GPD1, GPD2, GPP1, and GPP2 genes were all expressed at basal levels. The dynamics of the gene induction and the glycerol formation at low demand for NADH reoxidation point to an important role of the Gpd1p; deletion of the GPD1 gene strongly altered the expression patterns of the GPD2 and GPP1 genes under such conditions. Furthermore, our results indicate that GCY1 and DAK1, tentatively encoding glycerol dehydrogenase and dihydroxyacetone kinase, respectively, may be involved in the redox regulation of S. cerevisiae. Copyright 2000 John Wiley & Sons, Ltd.

Diversity in genomic organisation, developmental regulation and distribution of the murine PR72/B" subunits of protein phosphatase 2A

PubMed Central

Zwaenepoel, Karen; Louis, Justin V; Goris, Jozef; Janssens, Veerle

2008-01-01

Background Protein phosphatase 2A (PP2A) is a serine/threonine-specific phosphatase displaying vital functions in growth and development through its role in various signalling pathways. PP2A holoenzymes comprise a core dimer composed of a catalytic C and a structural A subunit, which can associate with a variable B-type subunit. The importance of the B-type subunits for PP2A regulation cannot be overestimated as they determine holoenzyme localisation, activity and substrate specificity. Three B-type subunit families have been identified: PR55/B, PR61/B' and PR72/B", of which the latter is currently the least characterised. Results We deduced the sequences and genomic organisation of the different murine PR72/B" isoforms: three genes encode nine isoforms, five of which are abundantly expressed and give rise to genuine PP2A subunits. Thereby, one novel subunit was identified. Using Northern blotting, we examined the tissue-specific and developmental expression of these subunits. All subunits are highly expressed in heart, suggesting an important cardiac function. Immunohistochemical analysis revealed a striated expression pattern of PR72 and PR130 in heart and skeletal muscle, but not in bladder smooth muscle. The subcellular localisation and cell cycle regulatory ability of several PR72/B" isoforms were determined, demonstrating differences as well as similarities. Conclusion In contrast to PR55/B and PR61/B', the PR72/B" family seems evolutionary more divergent, as only two of the murine genes have a human orthologue. We have integrated these results in a more consistent nomenclature of both human and murine PR72/B" genes and their transcripts/proteins. Our results provide a platform for the future generation of PR72/B" knockout mice. PMID:18715506

ASGR1 and ASGR2, the Genes that Encode the Asialoglycoprotein Receptor (Ashwell Receptor), Are Expressed in Peripheral Blood Monocytes and Show Interindividual Differences in Transcript Profile

PubMed Central

Harris, Rebecca Louise; van den Berg, Carmen Wilma; Bowen, Derrick John

2012-01-01

Background. The asialoglycoprotein receptor (ASGPR) is a hepatic receptor that mediates removal of potentially hazardous glycoconjugates from blood in health and disease. The receptor comprises two proteins, asialoglycoprotein receptor 1 and 2 (ASGR1 and ASGR2), encoded by the genes ASGR1 and ASGR2. Design and Methods. Using reverse transcription amplification (RT-PCR), expression of ASGR1 and ASGR2 was investigated in human peripheral blood monocytes. Results. Monocytes were found to express ASGR1 and ASGR2 transcripts. Correctly spliced transcript variants encoding different isoforms of ASGR1 and ASGR2 were present in monocytes. The profile of transcript variants from both ASGR1 and ASGR2 differed among individuals. Transcript expression levels were compared with the hepatocyte cell line HepG2 which produces high levels of ASGPR. Monocyte transcripts were 4 to 6 orders of magnitude less than in HepG2 but nonetheless readily detectable using standard RT-PCR. The monocyte cell line THP1 gave similar results to monocytes harvested from peripheral blood, indicating it may provide a suitable model system for studying ASGPR function in this cell type. Conclusions. Monocytes transcribe and correctly process transcripts encoding the constituent proteins of the ASGPR. Monocytes may therefore represent a mobile pool of the receptor, capable of reaching sites remote from the liver. PMID:22919488

Angiomodulin is required for cardiogenesis of embryonic stem cells and is maintained by a feedback loop network of p63 and Activin-A.

PubMed

Wolchinsky, Zohar; Shivtiel, Shoham; Kouwenhoven, Evelyn Nathalie; Putin, Daria; Sprecher, Eli; Zhou, Huiqing; Rouleau, Matthieu; Aberdam, Daniel

2014-01-01

The transcription factor p63, member of the p53 gene family, encodes for two main isoforms, TAp63 and ΔNp63 with distinct functions on epithelial homeostasis and cancer. Recently, we discovered that TAp63 is essential for in vitro cardiogenesis and heart development in vivo. TAp63 is expressed by embryonic endoderm and acts on cardiac progenitors by a cell-non-autonomous manner. In the present study, we search for cardiogenic secreted factors that could be regulated by TAp63 and, by ChIP-seq analysis, identified Angiomodulin (AGM), also named IGFBP7 or IGFBP-rP1. We demonstrate that AGM is necessary for cardiac commitment of embryonic stem cells (ESCs) and its regulation depends on TAp63 isoform. TAp63 directly activates both AGM and Activin-A during ESC cardiogenesis while these secreted factors modulate TAp63 gene expression by a feedback loop mechanism. The molecular circuitry controlled by TAp63 on AGM/Activin-A signaling pathway and thus on cardiogenesis emphasizes the importance of p63 during early cardiac development. © 2013.

Embryonic expression of zebrafish MiT family genes tfe3b, tfeb, and tfec.

PubMed

Lister, James A; Lane, Brandon M; Nguyen, Anhthu; Lunney, Katherine

2011-11-01

The MiT family comprises four genes in mammals: Mitf, Tfe3, Tfeb, and Tfec, which encode transcription factors of the basic-helix-loop-helix/leucine zipper class. Mitf is well-known for its essential role in the development of melanocytes, however the functions of the other members of this family, and of interactions between them, are less well understood. We have now characterized the complete set of MiT genes from zebrafish, which totals six instead of four. The zebrafish genome contain two mitf (mitfa and mitfb), two tfe3 (tfe3a and tfe3b), and single tfeb and tfec genes; this distribution is shared with other teleosts. We present here the sequence and embryonic expression patterns for the zebrafish tfe3b, tfeb, and tfec genes, and identify a new isoform of tfe3a. These findings will assist in elucidating the roles of the MiT gene family over the course of vertebrate evolution. Copyright © 2011 Wiley-Liss, Inc.

Osteoblast gene expression is differentially regulated by TGF-beta isoforms.

PubMed

Fagenholz, P J; Warren, S M; Greenwald, J A; Bouletreau, P J; Spector, J A; Crisera, F E; Longaker, M T

2001-03-01

The transforming growth factor beta (TGF-beta) superfamily encompasses a number of important growth factors including several TGF-beta isoforms, the bone morphogenetic proteins, activins, inhibins, and growth and differentiation factors. TGF-beta 1, -beta 2, and -beta 3 are three closely related isoforms that are widely expressed during skeletal morphogenesis and bone repair. Numerous studies suggest that each isoform has unique in vivo functions; however, the effects of these TGF-beta isoforms on osteoblast gene expression and maturation have never been directly compared. In the current study, we treated undifferentiated neonatal rat calvaria osteoblast-enriched cell cultures with 2.5 ng/ml of each TGF-beta isoform and analyzed gene expression at 0, 3, 6, and 24 hours. We demonstrated unique isoform-specific regulation of endogenous TGF-beta 1 and type I collagen mRNA transcription. To assess the effects of extended TGF-beta treatment on osteoblast maturation, we differentiated osteoblast cultures in the presence of 2.5 ng/ml of each TGF-beta isoform. Analysis of collagen I, alkaline phosphatase, and osteocalcin demonstrated that each TGF-beta isoform uniquely suppressed the transcription of these osteoblast differentiation markers. Interestingly, TGF-beta isoform treatment increased osteopontin expression in primary osteoblasts after 4 and 10 days of differentiation. To our knowledge, these data provide the first direct comparison of the effects of the TGF-beta isoforms on osteoblast gene expression in vitro. Furthermore, these data suggest that TGF-beta isoforms may exert their unique in vivo effects by differentially regulating osteoblast cytokine secretion, extracellular matrix production, and the rate of cellular maturation.

Biochemical and Molecular Characterization of a Laccase from Marasmius quercophilus

PubMed Central

Dedeyan, Boghos; Klonowska, Agnieszka; Tagger, Simone; Tron, Thierry; Iacazio, Gilles; Gil, Gérard; Le Petit, Jean

2000-01-01

The basidiomycete Marasmius quercophilus is commonly found during autumn on the decaying litter of the evergreen oak (Quercus ilex L.), a plant characteristic of Mediterranean forest. This white-rot fungus colonizes the leaf surface with rhizomorphs, causing a total bleaching of the leaf. In synthetic liquid media, this white-rot fungus has strong laccase activity. From a three-step chromatographic procedure, we purified a major isoform to homogeneity. The gene encodes a monomeric glycoprotein of approximately 63 kDa, with a 3.6 isoelectric point, that contains 12% carbohydrate. Spectroscopic analysis of the purified enzyme (UV/visible and electron paramagnetic resonance, atomic absorption) confirmed that it belongs to the “blue copper oxidase” family. With syringaldazine as the substrate, the enzyme's pH optimum was 4.5, the optimal temperature was 75°C, and the Km was 7.1 μM. The structural gene, lac1, was cloned and sequenced. This gene encodes a 517-amino-acid protein 99% identical to a laccase produced by PM1, an unidentified basidiomycete previously isolated from wastewater from a paper factory in Spain. This similarity may be explained by the ecological distribution of the evergreen oak in Mediterranean forest. PMID:10698753

Evolution and Distribution of Teleost myomiRNAs: Functionally Diversified myomiRs in Teleosts.

PubMed

Siddique, Bhuiyan Sharmin; Kinoshita, Shigeharu; Wongkarangkana, Chaninya; Asakawa, Shuichi; Watabe, Shugo

2016-06-01

Myosin heavy chain (MYH) genes belong to a multigene family, and the regulated expression of each member determines the physiological and contractile muscle properties. Among these, MYH6, MYH7, and MYH14 occupy unique positions in the mammalian MYH gene family because of their specific expression in slow/cardiac muscles and the existence of intronic micro(mi) RNAs. MYH6, MYH7, and MYH14 encode miR-208a, miR-208b, and miR-499, respectively. These MYH encoded miRNAs are designated as myomiRs because of their muscle-specific expression and functions. In mammals, myomiRs and host MYHs form a transcription network involved in muscle fiber-type specification; thus, genomic positions and expression patterns of them are well conserved. However, our previous studies revealed divergent distribution and expression of MYH14/miR-499 among teleosts, suggesting the unique evolution of myomiRs and host MYHs in teleosts. Here, we examined distribution and expression of myomiRs and host MYHs in various teleost species. The major cardiac MYH isoforms in teleosts are an intronless gene, atrial myosin heavy chain (amhc), and ventricular myosin heavy chain (vmhc) gene that encodes an intronic miRNA, miR-736. Phylogenetic analysis revealed that vmhc/miR-736 is a teleost-specific myomiR that differed from tetrapoda MYH6/MYH7/miR-208s. Teleost genomes also contain species-specific orthologs in addition to vmhc and amhc, indicating complex gene duplication and gene loss events during teleost evolution. In medaka and torafugu, miR-499 was highly expressed in slow/cardiac muscles whereas the expression of miR-736 was quite low and not muscle specific. These results suggest functional diversification of myomiRs in teleost with the diversification of host MYHs.

Mitochondrial glutamate carriers from Drosophila melanogaster: biochemical, evolutionary and modeling studies.

PubMed

Lunetti, Paola; Cappello, Anna Rita; Marsano, René Massimiliano; Pierri, Ciro Leonardo; Carrisi, Chiara; Martello, Emanuela; Caggese, Corrado; Dolce, Vincenza; Capobianco, Loredana

2013-10-01

The mitochondrial carriers are members of a family of transport proteins that mediate solute transport across the inner mitochondrial membrane. Two isoforms of the glutamate carriers, GC1 and GC2 (encoded by the SLC25A22 and SLC25A18 genes, respectively), have been identified in humans. Two independent mutations in SLC25A22 are associated with severe epileptic encephalopathy. In the present study we show that two genes (CG18347 and CG12201) phylogenetically related to the human GC encoding genes are present in the D. melanogaster genome. We have functionally characterized the proteins encoded by CG18347 and CG12201, designated as DmGC1p and DmGC2p respectively, by overexpression in Escherichia coli and reconstitution into liposomes. Their transport properties demonstrate that DmGC1p and DmGC2p both catalyze the transport of glutamate across the inner mitochondrial membrane. Computational approaches have been used in order to highlight residues of DmGC1p and DmGC2p involved in substrate binding. Furthermore, gene expression analysis during development and in various adult tissues reveals that CG18347 is ubiquitously expressed in all examined D. melanogaster tissues, while the expression of CG12201 is strongly testis-biased. Finally, we identified mitochondrial glutamate carrier orthologs in 49 eukaryotic species in order to attempt the reconstruction of the evolutionary history of the glutamate carrier function. Comparison of the exon/intron structure and other key features of the analyzed orthologs suggests that eukaryotic glutamate carrier genes descend from an intron-rich ancestral gene already present in the common ancestor of lineages that diverged as early as bilateria and radiata. © 2013.

IUTA: a tool for effectively detecting differential isoform usage from RNA-Seq data.

PubMed

Niu, Liang; Huang, Weichun; Umbach, David M; Li, Leping

2014-10-06

Most genes in mammals generate several transcript isoforms that differ in stability and translational efficiency through alternative splicing. Such alternative splicing can be tissue- and developmental stage-specific, and such specificity is sometimes associated with disease. Thus, detecting differential isoform usage for a gene between tissues or cell lines/types (differences in the fraction of total expression of a gene represented by the expression of each of its isoforms) is potentially important for cell and developmental biology. We present a new method IUTA that is designed to test each gene in the genome for differential isoform usage between two groups of samples. IUTA also estimates isoform usage for each gene in each sample as well as averaged across samples within each group. IUTA is the first method to formulate the testing problem as testing for equal means of two probability distributions under the Aitchison geometry, which is widely recognized as the most appropriate geometry for compositional data (vectors that contain the relative amount of each component comprising the whole). Evaluation using simulated data showed that IUTA was able to provide test results for many more genes than was Cuffdiff2 (version 2.2.0, released in Mar. 2014), and IUTA performed better than Cuffdiff2 for the limited number of genes that Cuffdiff2 did analyze. When applied to actual mouse RNA-Seq datasets from six tissues, IUTA identified 2,073 significant genes with clear patterns of differential isoform usage between a pair of tissues. IUTA is implemented as an R package and is available at http://www.niehs.nih.gov/research/resources/software/biostatistics/iuta/index.cfm. Both simulation and real-data results suggest that IUTA accurately detects differential isoform usage. We believe that our analysis of RNA-seq data from six mouse tissues represents the first comprehensive characterization of isoform usage in these tissues. IUTA will be a valuable resource for those who study the roles of alternative transcripts in cell development and disease.

A unique gene expression signature associated with serotonin 2C receptor RNA editing in the prefrontal cortex and altered in suicide.

PubMed

Di Narzo, Antonio Fabio; Kozlenkov, Alexey; Roussos, Panos; Hao, Ke; Hurd, Yasmin; Lewis, David A; Sibille, Etienne; Siever, Larry J; Koonin, Eugene; Dracheva, Stella

2014-09-15

Editing of the pre-mRNA for the serotonin receptor 2C (5-HT2CR) by site-specific adenosine deamination (A-to-I pre-mRNA editing) substantially increases the functional plasticity of this key neurotransmitter receptor and is thought to contribute to homeostatic mechanisms in neurons. 5-HT2CR mRNA editing generates up to 24 different receptor isoforms. The extent of editing correlates with 5-HT2CR functional activity: more highly edited isoforms exhibit the least function. Altered 5-HT2CR editing has been reported in postmortem brains of suicide victims. We report a comparative analysis of the connections among 5-HT2CR editing, genome-wide gene expression and DNA methylation in suicide victims, individuals with major depressive disorder and non-psychiatric controls. The results confirm previous findings of an overrepresentation of highly edited mRNA variants (which encode hypoactive 5-HT2CR receptors) in the brains of suicide victims. A large set of genes for which the expression level is associated with editing was detected. This signature set of editing-associated genes is significantly enriched for genes that are involved in synaptic transmission, genes that are preferentially expressed in neurons, and genes whose expression is correlated with the level of DNA methylation. Notably, we report that the link between 5-HT2CR editing and gene expression is disrupted in suicide victims. The results suggest that the postulated homeostatic function of 5-HT2CR editing is dysregulated in individuals who committed suicide. Published by Oxford University Press 2014. This work is written by (a) US Government employee(s) and is in the public domain in the US.

The BG21 isoform of Golli myelin basic protein is intrinsically disordered with a highly flexible amino-terminal domain.

PubMed

Ahmed, Mumdooh A M; Bamm, Vladimir V; Harauz, George; Ladizhansky, Vladimir

2007-08-28

The genes of the oligodendrocyte lineage (Golli) encode a family of developmentally regulated isoforms of myelin basic protein. The "classic" MBP isoforms arise from transcription start site 3, whereas Golli-specific isoforms arise from transcription start site 1, and comprise both Golli-specific and classic MBP sequences. The Golli isoform BG21 has been suggested to play roles in myelination and T cell activation pathways. It is an intrinsically disordered protein, thereby presenting a large effective surface area for interaction with other proteins such as Golli-interacting protein. We have used multidimensional heteronuclear NMR spectroscopy to achieve sequence-specific resonance assignments of the recombinant murine BG21 in physiologically relevant buffer, to analyze its secondary structure using chemical shift indexing (CSI), and to investigate its backbone dynamics using 15N spin relaxation measurements. We have assigned 184 out of 199 residues unambiguously. The CSI analysis revealed little ordered secondary structure under these conditions, with only some small fragments having a slight tendency toward alpha-helicity, which may represent putative recognition motifs. The 15N relaxation and NOE measurements confirmed the general behavior of the protein as an extended polypeptide chain, with the N-terminal Golli-specific portion (residues S5-T69) being exceptionally flexible, even in comparison to other intrinsically disordered proteins that have been studied this way. The high degree of flexibility of this N-terminal region may be to provide additional plasticity, or conformational adaptability, in protein-protein interactions. Another highly mobile segment, A126-S127-G128-G129, may function as a hinge.

ACA12 is a deregulated isoform of plasma membrane Ca²⁺-ATPase of Arabidopsis thaliana.

PubMed

Limonta, Margherita; Romanowsky, Shawn; Olivari, Claudio; Bonza, Maria Cristina; Luoni, Laura; Rosenberg, Alexa; Harper, Jeffrey F; De Michelis, Maria Ida

2014-03-01

Plant auto-inhibited Ca²⁺-ATPases (ACA) are crucial in defining the shape of calcium transients and therefore in eliciting plant responses to various stimuli. Arabidopsis thaliana genome encodes ten ACA isoforms that can be divided into four clusters based on gene structure and sequence homology. While isoforms from clusters 1, 2 and 4 have been characterized, virtually nothing is known about members of cluster 3 (ACA12 and ACA13). Here we show that a GFP-tagged ACA12 localizes at the plasma membrane and that expression of ACA12 rescues the phenotype of partial male sterility of a null mutant of the plasma membrane isoform ACA9, thus providing genetic evidence that ACA12 is a functional plasma membrane-resident Ca²⁺-ATPase. By ACA12 expression in yeast and purification by CaM-affinity chromatography, we show that, unlike other ACAs, the activity of ACA12 is not stimulated by CaM. Moreover, full length ACA12 is able to rescue a yeast mutant deficient in calcium pumps. Analysis of single point ACA12 mutants suggests that ACA12 loss of auto-inhibition can be ascribed to the lack of two acidic residues--highly conserved in other ACA isoforms--localized at the cytoplasmic edge of the second and third transmembrane segments. Together, these results support a model in which the calcium pump activity of ACA12 is primarily regulated by increasing or decreasing mRNA expression and/or protein translation and degradation.

Molecular and functional characterization of an invertase secreted by Ashbya gossypii.

PubMed

Aguiar, Tatiana Q; Dinis, Cláudia; Magalhães, Frederico; Oliveira, Carla; Wiebe, Marilyn G; Penttilä, Merja; Domingues, Lucília

2014-06-01

The repertoire of hydrolytic enzymes natively secreted by the filamentous fungus Ashbya (Eremothecium) gossypii has been poorly explored. Here, an invertase secreted by this flavinogenic fungus was for the first time molecularly and functionally characterized. Invertase activity was detected in A. gossypii culture supernatants and cell-associated fractions. Extracellular invertase migrated in a native polyacrylamide gel as diffuse protein bands, indicating the occurrence of at least two invertase isoforms. Hydrolytic activity toward sucrose was approximately 10 times higher than toward raffinose. Inulin and levan were not hydrolyzed. Production of invertase by A. gossypii was repressed by the presence of glucose in the culture medium. The A. gossypii invertase was demonstrated to be encoded by the AFR529W (AgSUC2) gene, which is highly homologous to the Saccharomyces cerevisiae SUC2 (ScSUC2) gene. Agsuc2 null mutants were unable to hydrolyze sucrose, proving that invertase is encoded by a single gene in A. gossypii. This mutation was functionally complemented by the ScSUC2 and AgSUC2 genes, when expressed from a 2-μm-plasmid. The signal sequences of both AgSuc2p and ScSuc2p were able to direct the secretion of invertase into the culture medium in A. gossypii.

Mitochondrial O-GlcNAc Transferase (mOGT) Regulates Mitochondrial Structure, Function, and Survival in HeLa Cells*

PubMed Central

Sacoman, Juliana L.; Dagda, Raul Y.; Burnham-Marusich, Amanda R.; Dagda, Ruben K.; Berninsone, Patricia M.

2017-01-01

O-Linked N-acetylglucosamine transferase (OGT) catalyzes O-GlcNAcylation of target proteins and regulates numerous biological processes. OGT is encoded by a single gene that yields nucleocytosolic and mitochondrial isoforms. To date, the role of the mitochondrial isoform of OGT (mOGT) remains largely unknown. Using high throughput proteomics, we identified 84 candidate mitochondrial glycoproteins, of which 44 are novel. Notably, two of the candidate glycoproteins identified (cytochrome oxidase 2 (COX2) and NADH:ubiquinone oxidoreductase core subunit 4 (MT-ND4)) are encoded by mitochondrial DNA. Using siRNA in HeLa cells, we found that reducing endogenous mOGT expression leads to alterations in mitochondrial structure and function, including Drp1-dependent mitochondrial fragmentation, reduction in mitochondrial membrane potential, and a significant loss of mitochondrial content in the absence of mitochondrial ROS. These defects are associated with a compensatory increase in oxidative phosphorylation per mitochondrion. mOGT is also critical for cell survival; siRNA-mediated knockdown of endogenous mOGT protected cells against toxicity mediated by rotenone, a complex I inhibitor. Conversely, reduced expression of both nucleocytoplasmic (ncOGT) and mitochondrial (mOGT) OGT isoforms is associated with increased mitochondrial respiration and elevated glycolysis, suggesting that ncOGT is a negative regulator of cellular bioenergetics. Last, we determined that mOGT is probably involved in the glycosylation of a restricted set of mitochondrial targets. We identified four proteins implicated in mitochondrial biogenesis and metabolism regulation as candidate substrates of mOGT, including leucine-rich PPR-containing protein and mitochondrial aconitate hydratase. Our findings suggest that mOGT is catalytically active in vivo and supports mitochondrial structure, health, and survival, whereas ncOGT predominantly regulates cellular bioenergetics. PMID:28100784

Molecular evolution and diversification of snake toxin genes, revealed by analysis of intron sequences.

PubMed

Fujimi, T J; Nakajyo, T; Nishimura, E; Ogura, E; Tsuchiya, T; Tamiya, T

2003-08-14

The genes encoding erabutoxin (short chain neurotoxin) isoforms (Ea, Eb, and Ec), LsIII (long chain neurotoxin) and a novel long chain neurotoxin pseudogene were cloned from a Laticauda semifasciata genomic library. Short and long chain neurotoxin genes were also cloned from the genome of Laticauda laticaudata, a closely related species of L. semifasciata, by PCR. A putative matrix attached region (MAR) sequence was found in the intron I of the LsIII gene. Comparative analysis of 11 structurally relevant snake toxin genes (three-finger-structure toxins) revealed the molecular evolution of these toxins. Three-finger-structure toxin genes diverged from a common ancestor through two types of evolutionary pathways (long and short types), early in the course of evolution. At a later stage of evolution in each gene, the accumulation of mutations in the exons, especially exon II, by accelerated evolution may have caused the increased diversification in their functions. It was also revealed that the putative MAR sequence found in the LsIII gene was integrated into the gene after the species-level divergence.

A novel gene for Usher syndrome type 2: mutations in the long isoform of whirlin are associated with retinitis pigmentosa and sensorineural hearing loss.

PubMed

Ebermann, Inga; Scholl, Hendrik P N; Charbel Issa, Peter; Becirovic, Elvir; Lamprecht, Jürgen; Jurklies, Bernhard; Millán, José M; Aller, Elena; Mitter, Diana; Bolz, Hanno

2007-04-01

Usher syndrome is an autosomal recessive condition characterized by sensorineural hearing loss, variable vestibular dysfunction, and visual impairment due to retinitis pigmentosa (RP). The seven proteins that have been identified for Usher syndrome type 1 (USH1) and type 2 (USH2) may interact in a large protein complex. In order to identify novel USH genes, we followed a candidate strategy, assuming that mutations in proteins interacting with this "USH network" may cause Usher syndrome as well. The DFNB31 gene encodes whirlin, a PDZ scaffold protein with expression in both hair cell stereocilia and retinal photoreceptor cells. Whirlin represents an excellent candidate for USH2 because it binds to Usherin (USH2A) and VLGR1b (USH2C). Genotyping of microsatellite markers specific for the DFNB31 gene locus on chromosome 9q32 was performed in a German USH2 family that had been excluded for all known USH loci. Patients showed common haplotypes. Sequence analysis of DFNB31 revealed compound heterozygosity for a nonsense mutation, p.Q103X, in exon 1, and a mutation in the splice donor site of exon 2, c.837+1G>A. DFNB31 mutations appear to be a rare cause of Usher syndrome, since no mutations were identified in an additional 96 USH2 patients. While mutations in the C-terminal half of whirlin have previously been reported in non-syndromic deafness (DFNB31), both alterations identified in our USH2 family affect the long protein isoform. We propose that mutations causing Usher syndrome are probably restricted to exons 1-6 that are specific for the long isoform and probably crucial for retinal function. We describe a novel genetic subtype for Usher syndrome, which we named USH2D and which is caused by mutations in whirlin. Moreover, this is the first case of USH2 that is allelic to non-syndromic deafness.

Expression analysis of two gene subfamilies encoding the plasma membrane H+-ATPase in Nicotiana plumbaginifolia reveals the major transport functions of this enzyme.

PubMed

Moriau, L; Michelet, B; Bogaerts, P; Lambert, L; Michel, A; Oufattole, M; Boutry, M

1999-07-01

The plasma membrane H+-ATPase couples ATP hydrolysis to proton transport, thereby establishing the driving force for solute transport across the plasma membrane. In Nicotiana plumbaginifolia, this enzyme is encoded by at least nine pma (plasma membrane H+-ATPase) genes. Four of these are classified into two gene subfamilies, pma1-2-3 and pma4, which are the most highly expressed in plant species. We have isolated genomic clones for pma2 and pma4. Mapping of their transcript 5' end revealed the presence of a long leader that contained small open reading frames, regulatory features typical of other pma genes. The gusA reporter gene was then used to determine the expression of pma2, pma3 and pma4 in N. tabacum. These data, together with those obtained previously for pma1, led to the following conclusions. (i) The four pma-gusA genes were all expressed in root, stem, leaf and flower organs, but each in a cell-type specific manner. Expression in these organs was confirmed at the protein level, using subfamily-specific antibodies. (ii) pma4-gusA was expressed in many cell types and notably in root hair and epidermis, in companion cells, and in guard cells, indicating that in N. plumbaginifolia the same H+-ATPase isoform might be involved in mineral nutrition, phloem loading and control of stomata aperture. (iii) The second gene subfamily is composed, in N. plumbaginifolia, of a single gene (pma4) with a wide expression pattern and, in Arabidopsis thaliana, of three genes (aha1, aha2, aha3), at least two of them having a more restrictive expression pattern. (iv) Some cell types expressed pma2 and pma4 at the same time, which encode H+-ATPases with different enzymatic properties.

Tobacco LSU-like protein couples sulphur-deficiency response with ethylene signalling pathway.

PubMed

Moniuszko, Grzegorz; Skoneczny, Marek; Zientara-Rytter, Katarzyna; Wawrzyńska, Anna; Głów, Dawid; Cristescu, Simona M; Harren, Frans J M; Sirko, Agnieszka

2013-11-01

Most genes from the plant-specific family encoding Response to Low Sulphur (LSU)-like proteins are strongly induced in sulphur (S)-deficient conditions. The exact role of these proteins remains unclear; however, some data suggest their importance for plants' adjustment to nutrient deficiency and other environmental stresses. This work established that the regulation of ethylene signalling is a part of plants' response to S deficiency and showed the interaction between UP9C, a tobacco LSU family member, and one of the tobacco isoforms of 1-aminocyclopropane-1-carboxylic acid oxidase (ACO2A). Increase in ethylene level induced by S deficiency does not take place in tobacco plants with UP9C expressed in an antisense orientation. Based on transcriptomics data, this work also demonstrated that the majority of tobacco's response to S deficiency is misregulated in plants expressing UP9C-antisense. A link between response to S deficiency, ethylene sensing, and LSU-like proteins was emphasized by changes in expression of the genes encoding ethylene receptors and F-box proteins specific for the ethylene pathway.

Major Protein of Resting Rhizomes of Calystegia sepium (Hedge Bindweed) Closely Resembles Plant RNases But Has No Enzymatic Activity1

PubMed Central

Van Damme, Els J.M.; Hao, Qiang; Barre, Annick; Rougé, Pierre; Van Leuven, Fred; Peumans, Willy J.

2000-01-01

The most abundant protein of resting rhizomes of Calystegia sepium (L.) R.Br. (hedge bindweed) has been isolated and its corresponding cDNA cloned. The native protein consists of a single polypeptide of 212 amino acid residues and occurs as a mixture of glycosylated and unglycosylated isoforms. Both forms are derived from the same preproprotein containing a signal peptide and a C-terminal propeptide. Analysis of the deduced amino acid sequence indicated that the C. sepium protein shows high sequence identity and structural similarity with plant RNases. However, no RNase activity could be detected in highly purified preparations of the protein. This apparent lack of activity results most probably from the replacement of a conserved His residue, which is essential for the catalytic activity of plant RNases. Our findings not only demonstrate the occurrence of a catalytically inactive variant of an S-like RNase, but also provide further evidence that genes encoding storage proteins may have evolved from genes encoding enzymes or other biologically active proteins. PMID:10677436

O-acetylserine(thiol)lyase from spinach (Spinacia oleracea L.) leaf: cDNA cloning, characterization, and overexpression in Escherichia coli of the chloroplast isoform.

PubMed

Rolland, N; Droux, M; Lebrun, M; Douce, R

1993-01-01

The last enzymatic step for L-cysteine biosynthesis is catalyzed by O-acetylserine(thiol)lyase (OASTL, EC 4.2.99.8) which synthesizes L-cysteine from O-acetylserine and "sulfide." We have isolated and characterized a full-length cDNA (1432 bp) from a lambda gt11 library of spinach leaf encoding the complete precursor of the chloroplast isoform. The 1149-nucleotide open reading frame coding for O-acetylserine(thiol)lyase was in the direction opposite that of the lambda gt11 beta-galactosidase gene. The derived amino acid sequence indicates that the protein precursor consists of 383 amino acid residues including a N-terminal presequence peptide of 52 residues. The amino acid sequence of mature spinach chloroplast O-acetylserine(thiol)lyase shows 40 and 57% homology with its bacterial counterparts. Sequence comparison with several pyridoxal 5'-phosphate-containing proteins reveals the presence of a lysine residue assumed to be involved in cofactor binding. A synthetic cDNA was constructed, coding for the entire 331-amino-acid mature O-acetylserine(thiol)lyase and for an initiating methionine. A high level of expression of the active mature chloroplast isoform was achieved in an Escherichia coli strain carrying the T7 RNA polymerase system (F. W. Studier, A. H. Rosenberg, J. J. Dunn, and J. W. Dubendorff, 1990, in Methods in Enzymology, D. V. Goeddel, Ed., Vol. 185, pp. 60-89, Academic Press, San Diego, CA). Addition of pyridoxine to the bacterial growth medium enhanced the enzyme activity due to the recombinant protein. The extent of production is 25-fold higher than in chloroplast from spinach leaves and the recombinant protein presents the relative molecular mass and immunological properties of the natural enzyme from spinach leaf chloroplast. This work, together with our previous biochemical studies, are in accordance with a prokaryotic type enzyme for L-cysteine biosynthesis in higher plant chloroplasts. Southern blot analysis indicated that O-acetylserine(thiol)lyase is encoded by multiple genes in the spinach leaf genomic DNA.

Cloning and characterization of mouse ACF7, a novel member of the dystonin subfamily of actin binding proteins.

PubMed

Bernier, G; Mathieu, M; De Repentigny, Y; Vidal, S M; Kothary, R

1996-11-15

We have recently cloned the gene responsible for the mouse neurological disorder dystonia musculorum. The predicted product of this gene, dystonin (Dst), is a neural isoform of bullous pemphigoid antigen 1 (Bpag1) with an N-terminal actin binding domain. Here we report on the cloning and characterization of mouse ACF7. Sequence analysis revealed extended homology of mACF7 with both the actin binding domain (ABD) and the Bpag1 portions of dystonin. Moreover, mACF7 and Dst display similar isoform diversity and encode similar sized transcripts in the nervous system. Phylogenetic analysis of mACF7 and dystonin ABD sequences suggests a recent evolutionary origin and that these proteins form a separate novel subfamily within the beta-spectrin superfamily of actin binding proteins. Given the implication of several actin binding proteins in genetic disorders, it is important to know the pattern of mACF7 expression. mACF7 transcripts are detected principally in lung, brain, spinal cord, skeletal and cardiac muscle, and skin. Intriguingly, mACF7 expression in lung is strongly induced just before birth and is restricted to type II alveolar cells. To determine whether spontaneous mutants that may be defective in mACF7 exist, we have mapped the mACF7 gene to mouse chromosome 4.

Characterization and pharmacological analysis of two adipokinetic hormone receptor variants of the tsetse fly, Glossina morsitans morsitans.

PubMed

Caers, Jelle; Janssen, Tom; Van Rompay, Liesbeth; Broeckx, Valérie; Van Den Abbeele, Jan; Gäde, Gerd; Schoofs, Liliane; Beets, Isabel

2016-03-01

Adipokinetic hormones (AKH) are well known regulators of energy metabolism in insects. These neuropeptides are produced in the corpora cardiaca and perform their hormonal function by interacting with specific G protein-coupled receptors (GPCRs) at the cell membranes of target tissues, mainly the fat body. Here, we investigated the sequences, spatial and temporal distributions, and pharmacology of AKH neuropeptides and receptors in the tsetse fly, Glossina morsitans morsitans. The open reading frames of two splice variants of the Glomo-akh receptor (Glomo-akhr) gene and of the AKH neuropeptide encoding genes, gmmhrth and gmmakh, were cloned. Both tsetse AKHR isoforms show strong sequence conservation when compared to other insect AKHRs. Glomo-AKH prepropeptides also have the typical architecture of AKH precursors. In an in vitro Ca(2+) mobilization assay, Glomo-AKH neuropeptides activated each receptor isoform up to nanomolar concentrations. We identified structural features of tsetse AKH neuropeptides essential for receptor activation in vitro. Gene expression profiles suggest a function for AKH signaling in regulating Glossina energy metabolism, where AKH peptides are released from the corpora cardiaca and activate receptors mainly expressed in the fat body. This analysis of the ligand-receptor coupling, expression, and pharmacology of the two Glomo-AKHR variants facilitates further elucidation of the function of AKH in G. m. morsitans. Copyright © 2015 Elsevier Ltd. All rights reserved.

RBFOX2 protein domains and cellular activities.

PubMed

Arya, Anurada D; Wilson, David I; Baralle, Diana; Raponi, Michaela

2014-08-01

RBFOX2 (RNA-binding protein, Fox-1 homologue 2)/RBM9 (RNA-binding-motif protein 9)/RTA (repressor of tamoxifen action)/HNRBP2 (hexaribonucleotide-binding protein 2) encodes an RNA-binding protein involved in tissue specific alternative splicing regulation and steroid receptors transcriptional activity. Its ability to regulate specific splicing profiles depending on context has been related to different expression levels of the RBFOX2 protein itself and that of other splicing regulatory proteins involved in the shared modulation of specific genes splicing. However, this cannot be the sole explanation as to why RBFOX2 plays a widespread role in numerous cellular mechanisms from development to cell survival dependent on cell/tissue type. RBFOX2 isoforms with altered protein domains exist. In the present article, we describe the main RBFOX2 protein domains, their importance in the context of splicing and transcriptional regulation and we propose that RBFOX2 isoform distribution may play a fundamental role in RBFOX2-specific cellular effects.

Decoding the disease-associated proteins encoded in the human chromosome 4.

PubMed

Chen, Lien-Chin; Liu, Mei-Ying; Hsiao, Yung-Chin; Choong, Wai-Kok; Wu, Hsin-Yi; Hsu, Wen-Lian; Liao, Pao-Chi; Sung, Ting-Yi; Tsai, Shih-Feng; Yu, Jau-Song; Chen, Yu-Ju

2013-01-04

Chromosome 4 is the fourth largest chromosome, containing approximately 191 megabases (~6.4% of the human genome) with 757 protein-coding genes. A number of marker genes for many diseases have been found in this chromosome, including genetic diseases (e.g., hepatocellular carcinoma) and biomedical research (cardiac system, aging, metabolic disorders, immune system, cancer and stem cell) related genes (e.g., oncogenes, growth factors). As a pilot study for the chromosome 4-centric human proteome project (Chr 4-HPP), we present here a systematic analysis of the disease association, protein isoforms, coding single nucleotide polymorphisms of these 757 protein-coding genes and their experimental evidence at the protein level. We also describe how the findings from the chromosome 4 project might be used to drive the biomarker discovery and validation study in disease-oriented projects, using the examples of secretomic and membrane proteomic approaches in cancer research. By integrating with cancer cell secretomes and several other existing databases in the public domain, we identified 141 chromosome 4-encoded proteins as cancer cell-secretable/shedable proteins. Additionally, we also identified 54 chromosome 4-encoded proteins that have been classified as cancer-associated proteins with successful selected or multiple reaction monitoring (SRM/MRM) assays developed. From literature annotation and topology analysis, 271 proteins were recognized as membrane proteins while 27.9% of the 757 proteins do not have any experimental evidence at the protein-level. In summary, the analysis revealed that the chromosome 4 is a rich resource for cancer-associated proteins for biomarker verification projects and for drug target discovery projects.

PKG in honey bees: spatial expression, Amfor gene expression, sucrose responsiveness, and division of labor.

PubMed

Thamm, Markus; Scheiner, Ricarda

2014-06-01

Division of labor is a hallmark of social insects. In honey bees, division of labor involves transition of female workers from one task to the next. The most distinct tasks are nursing (providing food for the brood) and foraging (collecting pollen and nectar). The brain mechanisms regulating this form of behavioral plasticity have largely remained elusive. Recently, it was suggested that division of labor is based on nutrition-associated signaling pathways. One highly conserved gene associated with food-related behavior across species is the foraging gene, which encodes a cyclic guanosine monophosphate (cGMP)-dependent protein kinase (PKG). Our analysis of this gene reveals the presence of alternative splicing in the honey bee. One isoform is expressed in the brain. Expression of this isoform is most pronounced in the mushroom bodies, the subesophageal ganglion, and the corpora allata. Division of labor and sucrose responsiveness in honey bees correlate significantly with foraging gene expression in distinct brain regions. Activating PKG selectively increases sucrose responsiveness in nurse bees to the level of foragers, whereas the same treatment does not affect responsiveness to light. These findings demonstrate a direct link between PKG signaling in distinct brain areas and division of labor. Furthermore, they demonstrate that the difference in sensory responsiveness between nurse bees and foragers can be compensated for by activating PKG. Our findings on the function of PKG in regulating specific sensory responsiveness and social organization offer valuable indications for the function of the cGMP/PKG pathway in many other insects and vertebrates. Copyright © 2013 Wiley Periodicals, Inc.

Sequence of the cDNA of a human dihydrodiol dehydrogenase isoform (AKR1C2) and tissue distribution of its mRNA.

PubMed Central

Shiraishi, H; Ishikura, S; Matsuura, K; Deyashiki, Y; Ninomiya, M; Sakai, S; Hara, A

1998-01-01

Human liver contains three isoforms (DD1, DD2 and DD4) of dihydrodiol dehydrogenase with 20alpha- or 3alpha-hydroxysteroid dehydrogenase activity; the dehydrogenases belong to the aldo-oxo reductase (AKR) superfamily. cDNA species encoding DD1 and DD4 have been identified. However, four cDNA species with more than 99% sequence identity have been cloned and are compatible with a partial amino acid sequence of DD2. In this study we have isolated a cDNA clone encoding DD2, which was confirmed by comparison of the properties of the recombinant and hepatic enzymes. This cDNA showed differences of one, two, four and five nucleotides from the previously reported four cDNA species for a dehydrogenase of human colon carcinoma HT29 cells, human prostatic 3alpha-hydroxysteroid dehydrogenase, a human liver 3alpha-hydroxysteroid dehydrogenase-like protein and chlordecone reductase-like protein respectively. Expression of mRNA species for the five similar cDNA species in 20 liver samples and 10 other different tissue samples was examined by reverse transcriptase-mediated PCR with specific primers followed by diagnostic restriction with endonucleases. All the tissues expressed only one mRNA species corresponding to the newly identified cDNA for DD2: mRNA transcripts corresponding to the other cDNA species were not detected. We suggest that the new cDNA is derived from the principal gene for DD2, which has been named AKR1C2 by a new nomenclature for the AKR superfamily. It is possible that some of the other cDNA species previously reported are rare allelic variants of this gene. PMID:9716498

Characterization of a highly conserved human homolog to the chicken neural cell surface protein Bravo/Nr-CAM that maps to chromosome band 7q31

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lane, R.P.; Vielmetter, J.; Dreyer, W.J.

1996-08-01

The neuronal cell adhesion molecule Bravo/Nr-CAM is a cell surface protein of the immunoglobulin (Ig) superfamily and is closely related to the L1/NgCAM and neurofascin molecules, all of which contain six immunoglobulin domains, five fibronectin repeats, a transmembrane region, and an intracellular domain. Chicken Bravo/Nr-CAM has been shown to interact with other cell surface molecules of the Ig superfamily and has been implicated in specific pathfinding roles of axonal growth cones in the developing nervous system. We now report the characterization of cDNA clones encoding the human Bravo/Nr-CAM protein, which, like its chicken homolog, is composed of six V-like Igmore » domains and five fibronectin type III repeats. The human Bravo/Nr-CAM homolog also contains a transmembrane and intracellular domain, both of which are 100% conserved at the amino acid level compared to its chicken homolog. Overall, the human Bravo/Nr-CAM homolog is 82% identical to the chicken Bravo/Nr-CAM amino acid sequence. Independent cDNAs encoding four different isoforms were also identified, all of which contain alternatively spliced variants around the fifth fibronectin type III repeat, including one isoform that had been previously identified for chicken Bravo/Nr-CAM. Northern blot analysis reveals one mRNA species of approximately 7.0 kb in adult human brain tissue. Fluorescence in situ hybridization maps the gene for human Bravo/Nr-CAM to human chromosome 7q31.1-q31.2. This chromosomal locus has been previously identified as containing a tumore suppressor candidate gene commonly deleted in certain human cancer tissues. 38 refs., 5 figs.« less

Cleavage and polyadenylation: Ending the message expands gene regulation

PubMed Central

Neve, Jonathan

2017-01-01

ABSTRACT Cleavage and polyadenylation (pA) is a fundamental step that is required for the maturation of primary protein encoding transcripts into functional mRNAs that can be exported from the nucleus and translated in the cytoplasm. 3′end processing is dependent on the assembly of a multiprotein processing complex on the pA signals that reside in the pre-mRNAs. Most eukaryotic genes have multiple pA signals, resulting in alternative cleavage and polyadenylation (APA), a widespread phenomenon that is important to establish cell state and cell type specific transcriptomes. Here, we review how pA sites are recognized and comprehensively summarize how APA is regulated and creates mRNA isoform profiles that are characteristic for cell types, tissues, cellular states and disease. PMID:28453393

Improving RNA-Seq expression estimation by modeling isoform- and exon-specific read sequencing rate.

PubMed

Liu, Xuejun; Shi, Xinxin; Chen, Chunlin; Zhang, Li

2015-10-16

The high-throughput sequencing technology, RNA-Seq, has been widely used to quantify gene and isoform expression in the study of transcriptome in recent years. Accurate expression measurement from the millions or billions of short generated reads is obstructed by difficulties. One is ambiguous mapping of reads to reference transcriptome caused by alternative splicing. This increases the uncertainty in estimating isoform expression. The other is non-uniformity of read distribution along the reference transcriptome due to positional, sequencing, mappability and other undiscovered sources of biases. This violates the uniform assumption of read distribution for many expression calculation approaches, such as the direct RPKM calculation and Poisson-based models. Many methods have been proposed to address these difficulties. Some approaches employ latent variable models to discover the underlying pattern of read sequencing. However, most of these methods make bias correction based on surrounding sequence contents and share the bias models by all genes. They therefore cannot estimate gene- and isoform-specific biases as revealed by recent studies. We propose a latent variable model, NLDMseq, to estimate gene and isoform expression. Our method adopts latent variables to model the unknown isoforms, from which reads originate, and the underlying percentage of multiple spliced variants. The isoform- and exon-specific read sequencing biases are modeled to account for the non-uniformity of read distribution, and are identified by utilizing the replicate information of multiple lanes of a single library run. We employ simulation and real data to verify the performance of our method in terms of accuracy in the calculation of gene and isoform expression. Results show that NLDMseq obtains competitive gene and isoform expression compared to popular alternatives. Finally, the proposed method is applied to the detection of differential expression (DE) to show its usefulness in the downstream analysis. The proposed NLDMseq method provides an approach to accurately estimate gene and isoform expression from RNA-Seq data by modeling the isoform- and exon-specific read sequencing biases. It makes use of a latent variable model to discover the hidden pattern of read sequencing. We have shown that it works well in both simulations and real datasets, and has competitive performance compared to popular methods. The method has been implemented as a freely available software which can be found at https://github.com/PUGEA/NLDMseq.

A fast and cost-effective method for apolipoprotein E isotyping as an alternative to APOE genotyping for patient screening and stratification.

PubMed

Calero, Olga; García-Albert, Luis; Rodríguez-Martín, Andrés; Veiga, Sergio; Calero, Miguel

2018-04-13

Apolipoprotein E (apoE) is a 34 kDa glycoprotein involved in lipid metabolism. The human APOE gene encodes for three different apoE protein isoforms: E2, E3 and E4. The interest in apoE isoforms is high for epidemiological research, patient stratification and identification of those at increased risk for clinical trials and prevention. The isoform apoE4 is associated with increased risk for coronary heart and Alzheimer's diseases. This paper describes a method for specifically detecting the apoE4 isoform from biological fluids by taking advantage of the capacity of apoE to bind "specifically" to polystyrene surfaces as capture and a specific anti-apoE4 monoclonal antibody as reporter. Our results indicate that the apoE-polystyrene binding interaction is highly stable, resistant to detergents and acid and basic washes. The methodology here described is accurate, easily implementable, fast and cost-effective. Although at present, our technique is unable to discriminate homozygous APOE ε4/ε4 from APOE ε3/ε4 and ε2/ε4 heterozygous, it opens new avenues for the development of inexpensive, yet effective, tests for the detection of apoE4 for patients' stratification. Preliminary results indicated that this methodology is also adaptable into turbidimetric platforms, which make it a good candidate for clinical implementation through its translation to the clinical analysis routine.

Differential expression of choline kinase isoforms in skeletal muscle explains the phenotypic variability in the rostrocaudal muscular dystrophy mouse.

PubMed

Wu, Gengshu; Sher, Roger B; Cox, Gregory A; Vance, Dennis E

2010-04-01

Choline kinase in mammals is encoded by two genes, Chka and Chkb. Disruption of murine Chka leads to embryonic lethality, whereas a spontaneous genomic deletion in murine Chkb results in neonatal forelimb bone deformity and hindlimb muscular dystrophy. Surprisingly, muscular dystrophy isn't significantly developed in the forelimb. We have investigated the mechanism by which a lack of choline kinase beta, encoded by Chkb, results in minimal muscular dystrophy in forelimbs. We have found that choline kinase beta is the major isoform in hindlimb muscle and contributes more to choline kinase activity, while choline kinase alpha is predominant in forelimb muscle and contributes more to choline kinase activity. Although choline kinase activity is decreased in forelimb muscles of Chkb(-/-) mice, the activity of CTP:phosphocholine cytidylyltransferase is increased, resulting in enhanced phosphatidylcholine biosynthesis. The activity of phosphatidylcholine phospholipase C is up-regulated while the activity of phospholipase A(2) in forelimb muscle is not altered. Regeneration of forelimb muscles of Chkb(-/-) mice is normal when challenged with cardiotoxin. In contrast to hindlimb muscle, mega-mitochondria are not significantly formed in forelimb muscle of Chkb(-/-) mice. We conclude that the relative lack of muscle degeneration in forelimbs of Chkb(-/-) mice is due to abundant choline kinase alpha and the stable homeostasis of phosphatidylcholine. 2009 Elsevier B.V. All rights reserved.

Mechanisms and Regulation of Alternative Pre-mRNA Splicing

PubMed Central

Lee, Yeon

2015-01-01

Precursor messenger RNA (pre-mRNA) splicing is a critical step in the posttranscriptional regulation of gene expression, providing significant expansion of the functional proteome of eukaryotic organisms with limited gene numbers. Split eukaryotic genes contain intervening sequences or introns disrupting protein-coding exons, and intron removal occurs by repeated assembly of a large and highly dynamic ribonucleoprotein complex termed the spliceosome, which is composed of five small nuclear ribonucleoprotein particles, U1, U2, U4/U6, and U5. Biochemical studies over the past 10 years have allowed the isolation as well as compositional, functional, and structural analysis of splicing complexes at distinct stages along the spliceosome cycle. The average human gene contains eight exons and seven introns, producing an average of three or more alternatively spliced mRNA isoforms. Recent high-throughput sequencing studies indicate that 100% of human genes produce at least two alternative mRNA isoforms. Mechanisms of alternative splicing include RNA–protein interactions of splicing factors with regulatory sites termed silencers or enhancers, RNA–RNA base-pairing interactions, or chromatin-based effects that can change or determine splicing patterns. Disease-causing mutations can often occur in splice sites near intron borders or in exonic or intronic RNA regulatory silencer or enhancer elements, as well as in genes that encode splicing factors. Together, these studies provide mechanistic insights into how spliceosome assembly, dynamics, and catalysis occur; how alternative splicing is regulated and evolves; and how splicing can be disrupted by cis- and trans-acting mutations leading to disease states. These findings make the spliceosome an attractive new target for small-molecule, antisense, and genome-editing therapeutic interventions. PMID:25784052

In vivo and in vitro studies support that a new splicing isoform of OLR1 gene is protective against acute myocardial infarction.

PubMed

Mango, Ruggiero; Biocca, Silvia; del Vecchio, Francesca; Clementi, Fabrizio; Sangiuolo, Federica; Amati, Francesca; Filareto, Antonio; Grelli, Sandro; Spitalieri, Paola; Filesi, Ilaria; Favalli, Cartesio; Lauro, Renato; Mehta, Jawahar L; Romeo, Francesco; Novelli, Giuseppe

2005-07-22

Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), encoded by the OLR1 gene, is a scavenger receptor that plays a fundamental role in the pathogenesis of atherosclerosis. LOX-1 activation is associated with apoptosis of endothelial cells, smooth muscle cells (SMCs), and macrophages. This process is an important underlying mechanism that contributes to plaque instability and subsequent development of acute coronary syndromes. Independent association genetic studies have implicated OLR1 gene variants in myocardial infarction (MI) susceptibility. Because single nucleotide polymorphisms (SNPs) linked to MI are located in intronic sequences of the gene, it remains unclear as to how they determine their biological effects. Using quantitative real-time PCR and minigene approach, we show that intronic SNPs, linked to MI, regulate the expression of a new functional splicing isoform of the OLR1 gene, LOXIN, which lacks exon 5. Macrophages from subjects carrying the "non-risk" disease haplotype at OLR1 gene have an increased expression of LOXIN at mRNA and protein level, which results in a significant reduction of apoptosis in response to oxLDL. Expression of LOXIN in different cell types results in loss of surface staining, indicating that truncation of the C-terminal portion of the protein has a profound effect on its cellular trafficking. Furthermore, the proapoptotic effect of LOX-1 receptor in cell culture is specifically rescued by the coexpression of LOXIN in a dose-dependent manner. The demonstration that increasing levels of LOXIN protect cells from LOX-1 induced apoptosis sets a groundwork for developing therapeutic approaches for prevention of plaque instability.

Sustained expression of a neuron-specific isoform of the Taf1 gene in development stages and aging in mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jambaldorj, Jamiyansuren; Advanced Molecular Epidemiology Research Institute, Yamagata University Faculty of Medicine, Yamagata 990-9585; Central Scientific Research Laboratory, Institute of Medical Sciences, Ulaanbaatar

2012-08-24

Highlights: Black-Right-Pointing-Pointer We identified the mouse homologue of neuron-specific TAF1 (N-Taf1). Black-Right-Pointing-Pointer Taf1 mRNA was expressed in most tissues and cell lines. Black-Right-Pointing-Pointer N-Taf1 mRNA was expressed in the brain and Neuroblastoma N2a cell lines. Black-Right-Pointing-Pointer Taf1 and N-Taf1 showed different expression profile in development stage and aging. -- Abstract: TATA-box binding protein associated factor 1 (TAF1) protein is the largest and the essential component of the TFIID complex in the pathway of RNA polymerase II-mediated gene transcription, and it regulates transcription of a large number of genes related to cell division. The neuron-specific isoform of the TAF1 gene (N-TAF1),more » which we reported previously, may have an essential role in neurons through transcriptional regulation of many neuron-specific genes. In the present study, we cloned the full-length cDNA that encodes the mouse homologue of N-TAF1 (N-Taf1) protein. By carrying out of real time RT-PCR, we investigated the expression analysis of the N-Taf1 mRNA in mouse tissues and cell lines. As well as the human N-TAF1, the N-Taf1 showed limited expression in the brain and neuroblastoma, whereas Taf1 expressed elsewhere. Furthermore, in mouse embryo head or mouse brain, mRNA expression of TAF1 changes dramatically during development but N-Taf1 showed sustained expression. Our result suggests that the N-Taf1 gene has an important role in non-dividing neuronal cell rather than in cell division and proliferation during neurogenesis.« less

The smallest natural high-active luciferase: cloning and characterization of novel 16.5-kDa luciferase from copepod Metridia longa.

PubMed

Markova, Svetlana V; Larionova, Marina D; Burakova, Ludmila P; Vysotski, Eugene S

2015-01-30

Coelenterazine-dependent copepod luciferases containing natural signal peptide for secretion are a very convenient analytical tool as they enable monitoring of intracellular events with high sensitivity, without destroying cells or tissues. This property is well suited for application in biomedical research and development of cell-based assays for high throughput screening. We report the cloning of cDNA gene encoding a novel secreted non-allelic 16.5-kDa isoform (MLuc7) of Metridia longa luciferase, which, in fact, is the smallest natural luciferase of known for today. Despite the small size, isoform contains 10 conservative Cys residues suggesting the presence of up to 5 SS bonds. This hampers the efficient production of functionally active recombinant luciferase in bacterial expression systems. With the use of the baculovirus expression system, we produced substantial amounts of the proper folded MLuc7 luciferase with a yield of ∼3 mg/L of a high purity protein. We demonstrate that MLuc7 produced in insect cells is highly active and extremely thermostable, and is well suited as a secreted reporter when expressed in mammalian cells ensuring higher sensitivity of detection as compared to another Metridia luciferase isoform (MLuc164) which is widely employed in real-time imaging. Copyright © 2014 Elsevier Inc. All rights reserved.

Differential Response to Risperidone in Schizophrenia Patients by KCNH2 Genotype and Drug Metabolizer Status.

PubMed

Heide, Juliane; Zhang, Fengyu; Bigos, Kristin L; Mann, Stefan A; Carr, Vaughan J; Shannon Weickert, Cynthia; Green, Melissa J; Weinberger, Daniel R; Vandenberg, Jamie I

2016-01-01

Antipsychotic drugs target dopamine and serotonin receptors as well as Kv11.1 potassium channels encoded by KCNH2. Variable patient responses and a wide range of side effects, however, limit their efficacy. Slow metabolizer status and gene variants in KCNH2 associated with increased expression of Kv11.1-3.1, an alternatively spliced isoform of Kv11.1, are correlated with improved responses to antipsychotic medications. Here, the authors test the hypothesis that these effects may be influenced by differential drug binding to Kv11.1 channel isoforms. Drug block of Kv11.1 isoforms was tested in cellular electrophysiology assays. The effects of drug metabolism and KCNH2 genotypes on clinical responses were assessed in patients enrolled in the multicenter Clinical Antipsychotic Trials of Intervention Effectiveness (CATIE). Risperidone caused greater in vitro block of the alternatively spliced Kv11.1-3.1 isoform than full-length Kv11.1-1A channels, whereas its metabolite paliperidone and other atypical antipsychotics have similar potencies for the two isoforms. In the CATIE study (N=362), patients with genotypes associated with increased Kv11.1-3.1 expression (N=52) showed a better treatment response to risperidone compared with other drugs, but this association was dependent on metabolism status. Patients with KCNH2 risk genotypes and slow metabolizer status (approximately 7% of patients) showed marked improvement in symptoms when treated with risperidone compared with patients with fast metabolizer status or without the KCNH2 risk genotypes. These data support the hypothesis that Kv11.1 channels play a role in the therapeutic action of antipsychotic drugs, particularly risperidone, and further highlight the promise of optimizing response with genotype-guided therapy for schizophrenia patients.

Active Sites of Reduced Epidermal Fluorescence1 (REF1) Isoforms Contain Amino Acid Substitutions That Are Different between Monocots and Dicots

PubMed Central

Missihoun, Tagnon D.; Kotchoni, Simeon O.; Bartels, Dorothea

2016-01-01

Plant aldehyde dehydrogenases (ALDHs) play important roles in cell wall biosynthesis, growth, development, and tolerance to biotic and abiotic stresses. The Reduced Epidermal Fluorescence1 is encoded by the subfamily 2C of ALDHs and was shown to oxidise coniferaldehyde and sinapaldehyde to ferulic acid and sinapic acid in the phenylpropanoid pathway, respectively. This knowledge has been gained from works in the dicotyledon model species Arabidopsis thaliana then used to functionally annotate ALDH2C isoforms in other species, based on the orthology principle. However, the extent to which the ALDH isoforms differ between monocotyledons and dicotyledons has rarely been accessed side-by-side. In this study, we used a phylogenetic approach to address this question. We have analysed the ALDH genes in Brachypodium distachyon, alongside those of other sequenced monocotyledon and dicotyledon species to examine traits supporting either a convergent or divergent evolution of the ALDH2C/REF1-type proteins. We found that B. distachyon, like other grasses, contains more ALDH2C/REF1 isoforms than A. thaliana and other dicotyledon species. Some amino acid residues in ALDH2C/REF1 isoforms were found as being conserved in dicotyledons but substituted by non-equivalent residues in monocotyledons. One example of those substitutions concerns a conserved phenylalanine and a conserved tyrosine in monocotyledons and dicotyledons, respectively. Protein structure modelling suggests that the presence of tyrosine would widen the substrate-binding pocket in the dicotyledons, and thereby influence substrate specificity. We discussed the importance of these findings as new hints to investigate why ferulic acid contents and cell wall digestibility differ between the dicotyledon and monocotyledon species. PMID:27798665

Active Sites of Reduced Epidermal Fluorescence1 (REF1) Isoforms Contain Amino Acid Substitutions That Are Different between Monocots and Dicots.

PubMed

Missihoun, Tagnon D; Kotchoni, Simeon O; Bartels, Dorothea

2016-01-01

Plant aldehyde dehydrogenases (ALDHs) play important roles in cell wall biosynthesis, growth, development, and tolerance to biotic and abiotic stresses. The Reduced Epidermal Fluorescence1 is encoded by the subfamily 2C of ALDHs and was shown to oxidise coniferaldehyde and sinapaldehyde to ferulic acid and sinapic acid in the phenylpropanoid pathway, respectively. This knowledge has been gained from works in the dicotyledon model species Arabidopsis thaliana then used to functionally annotate ALDH2C isoforms in other species, based on the orthology principle. However, the extent to which the ALDH isoforms differ between monocotyledons and dicotyledons has rarely been accessed side-by-side. In this study, we used a phylogenetic approach to address this question. We have analysed the ALDH genes in Brachypodium distachyon, alongside those of other sequenced monocotyledon and dicotyledon species to examine traits supporting either a convergent or divergent evolution of the ALDH2C/REF1-type proteins. We found that B. distachyon, like other grasses, contains more ALDH2C/REF1 isoforms than A. thaliana and other dicotyledon species. Some amino acid residues in ALDH2C/REF1 isoforms were found as being conserved in dicotyledons but substituted by non-equivalent residues in monocotyledons. One example of those substitutions concerns a conserved phenylalanine and a conserved tyrosine in monocotyledons and dicotyledons, respectively. Protein structure modelling suggests that the presence of tyrosine would widen the substrate-binding pocket in the dicotyledons, and thereby influence substrate specificity. We discussed the importance of these findings as new hints to investigate why ferulic acid contents and cell wall digestibility differ between the dicotyledon and monocotyledon species.

Differential Expression of Metallothionein Isoforms in Terrestrial Snail Embryos Reflects Early Life Stage Adaptation to Metal Stress

PubMed Central

Baurand, Pierre-Emmanuel; Pedrini-Martha, Veronika; de Vaufleury, Annette; Niederwanger, Michael; Capelli, Nicolas; Scheifler, Renaud; Dallinger, Reinhard

2015-01-01

The aim of this study was to analyze the expression of three metallothionein (MT) isoform genes (CdMT, CuMT and Cd/CuMT), already known from adults, in the Early Life Stage (ELS) of Cantareus aspersus. This was accomplished by detection of the MT isoform-specific transcription adopting Polymerase Chain Reaction (PCR) amplification and quantitative Real Time (qRT)-PCR of the three MT genes. Freshly laid eggs were kept for 24 hours under control conditions or exposed to three cadmium (Cd) solutions of increasing concentration (5, 10, and 15 mg Cd/L). The transcription of the three MT isoform genes was detected via PCR in 1, 6 and 12-day-old control or Cd-exposed embryos. Moreover, the transcription of this isoform genes during development was followed by qRT-PCR in 6 and 12-day-old embryos. Our results showed that the CdMT and Cd/CuMT genes, but not the CuMT gene, are expressed in embryos at the first day of development. The transcription of the 3 MT genes in control embryos increased with development time, suggesting that the capacities of metal regulation and detoxification may have gradually increased throughout embryogenesis. However in control embryos, the most highly expressed MT gene was that of the Cd/CuMT isoform, whose transcription levels greatly exceeded those of the other two MT genes. This contrasts with the minor significance of this gene in adult snails and suggests that in embryos, this isoform may play a comparatively more important role in metal physiology compared to adult individuals. This function in adult snails appears not to be related to Cd detoxification. Instead, snail embryos responded to Cd exposure by over-expression of the CdMT gene in a concentration-dependent manner, whereas the expression of the Cd/CuMT gene remained unaffected. Moreover, our study demonstrates the ability of snail embryos to respond very early to Cd exposure by up-regulation of the CdMT gene. PMID:25706953

Mechanisms of Surface Antigenic Variation in the Human Pathogenic Fungus Pneumocystis jirovecii.

PubMed

Schmid-Siegert, Emanuel; Richard, Sophie; Luraschi, Amanda; Mühlethaler, Konrad; Pagni, Marco; Hauser, Philippe M

2017-11-07

Microbial pathogens commonly escape the human immune system by varying surface proteins. We investigated the mechanisms used for that purpose by Pneumocystis jirovecii This uncultivable fungus is an obligate pulmonary pathogen that in immunocompromised individuals causes pneumonia, a major life-threatening infection. Long-read PacBio sequencing was used to assemble a core of subtelomeres of a single P. jirovecii strain from a bronchoalveolar lavage fluid specimen from a single patient. A total of 113 genes encoding surface proteins were identified, including 28 pseudogenes. These genes formed a subtelomeric gene superfamily, which included five families encoding adhesive glycosylphosphatidylinositol (GPI)-anchored glycoproteins and one family encoding excreted glycoproteins. Numerical analyses suggested that diversification of the glycoproteins relies on mosaic genes created by ectopic recombination and occurs only within each family. DNA motifs suggested that all genes are expressed independently, except those of the family encoding the most abundant surface glycoproteins, which are subject to mutually exclusive expression. PCR analyses showed that exchange of the expressed gene of the latter family occurs frequently, possibly favored by the location of the genes proximal to the telomere because this allows concomitant telomere exchange. Our observations suggest that (i) the P. jirovecii cell surface is made of a complex mixture of different surface proteins, with a majority of a single isoform of the most abundant glycoprotein, (ii) genetic mosaicism within each family ensures variation of the glycoproteins, and (iii) the strategy of the fungus consists of the continuous production of new subpopulations composed of cells that are antigenically different. IMPORTANCE Pneumocystis jirovecii is a fungus causing severe pneumonia in immunocompromised individuals. It is the second most frequent life-threatening invasive fungal infection. We have studied the mechanisms of antigenic variation used by this pathogen to escape the human immune system, a strategy commonly used by pathogenic microorganisms. Using a new DNA sequencing technology generating long reads, we could characterize the highly repetitive gene families encoding the proteins that are present on the cellular surface of this pest. These gene families are localized in the regions close to the ends of all chromosomes, the subtelomeres. Such chromosomal localization was found to favor genetic recombinations between members of each gene family and to allow diversification of these proteins continuously over time. This pathogen seems to use a strategy of antigenic variation consisting of the continuous production of new subpopulations composed of cells that are antigenically different. Such a strategy is unique among human pathogens. Copyright © 2017 Schmid-Siegert et al.

Intron retention and transcript chimerism conserved across mammals: Ly6g5b and Csnk2b-Ly6g5b as examples

PubMed Central

2013-01-01

Background Alternative splicing (AS) is a major mechanism for modulating gene expression of an organism, allowing the synthesis of several structurally and functionally distinct mRNAs and protein isoforms from a unique gene. Related to AS is the Transcription Induced Chimerism (TIC) or Tandem Chimerism, by which chimeric RNAs between adjacent genes can be found, increasing combinatorial complexity of the proteome. The Ly6g5b gene presents particular behaviours in its expression, involving an intron retention event and being capable to form RNA chimera transcripts with the upstream gene Csnk2b. We wanted to characterise these events more deeply in four tissues in six different mammals and analyse their protein products. Results While canonical Csnk2b isoform was widely expressed, Ly6g5b canonical isoform was less ubiquitous, although the Ly6g5b first intron retained transcript was present in all the tissues and species analysed. Csnk2b-Ly6g5b chimeras were present in all the samples analysed, but with restricted expression patterns. Some of these chimeric transcripts maintained correct structural domains from Csnk2b and Ly6g5b. Moreover, we found Csnk2b, Ly6g5b, and Csnk2b-Ly6g5b transcripts that present exon skipping, alternative 5' and 3' splice site and intron retention events. These would generate truncated or aberrant proteins whose role remains unknown. Some chimeric transcripts would encode CSNK2B proteins with an altered C-terminus, which could affect its biological function broadening its substrate specificity. Over-expression of human CSNK2B, LY6G5B, and CSNK2B-LY6G5B proteins, show different patterns of post-translational modifications and cell distribution. Conclusions Ly6g5b intron retention and Csnk2b-Ly6g5b transcript chimerism are broadly distributed in tissues of different mammals. PMID:23521802

Casein Kinase I Isoform Hrr25 Is a Negative Regulator of Haa1 in the Weak Acid Stress Response Pathway in Saccharomyces cerevisiae

PubMed Central

Collins, Morgan E.; Black, Joshua J.

2017-01-01

ABSTRACT Haa1 is a transcription factor that adapts Saccharomyces cerevisiae cells to weak organic acid stresses by activating the expression of various genes. Many of these genes encode membrane proteins, such as TPO2 and YRO2. How Haa1 is activated by weak acids is not clear. Here, we show that casein kinase I isoform Hrr25 is an important negative regulator of Haa1. Haa1 is known to be multiply phosphorylated. We found that mutations in HRR25 lead to reduced Haa1 phosphorylation and increased expression of Haa1 target genes and that Hrr25 interacts with Haa1. The other three casein kinase I isoforms, Yck1, Yck2, and Yck3, do not seem to play critical roles in Haa1 regulation. Hrr25 has a 200-residue C-terminal region, including a proline- and glutamine-rich domain. Our data suggest that the C-terminal region of Hrr25 is required for normal inhibition of expression of Haa1 target genes TPO2 and YRO2 and is important for cell growth but is not required for cell morphogenesis. We propose that Hrr25 is an important regulator of cellular adaptation to weak acid stress by inhibiting Haa1 through phosphorylation. IMPORTANCE Our study has revealed the casein kinase I protein Hrr25 to be a negative regulator of Haa1, a transcription factor mediating the cellular response to stresses caused by weak acids. Many studies have focused on the target genes of Haa1 and their roles in weak acid stress responses, but little has been reported on the regulatory mechanism of Haa1. Weak acids, such as acetic acid, have long been used for food preservation by slowing down the growth of fungal species, including S. cerevisiae. In the biofuel industry, acetic acid in the lignocellulosic hydrolysates limits the production of ethanol, which is undesirable. By understanding how Haa1 is regulated, we can make advances in the field of food sciences to better preserve food and engineer acetic acid-resistant strains that will increase productivity in the biofuel industry. PMID:28432100

Differential gene expression of CYP3A isoforms in equine liver and intestines.

PubMed

Tydén, E; Löfgren, M; Pegolo, S; Capolongo, F; Tjälve, H; Larsson, P

2012-12-01

Recently, seven CYP3A isoforms - CYP3A89, CYP3A93, CYP3A94, CYP3A95, CYP3A96, CYP3A97 and CYP129 - have been isolated from the horse genome. In this study, we have examined the hepatic and intestinal gene expression of these CYP3A isoforms using TaqMan probes. We have also studied the enzyme activity using luciferin-isopropyl acetal (LIPA) as a substrate. The results show a differential gene expression of the CYP3A isoforms in the liver and intestines in horses. In the liver, CYP3A89, CYP3A94, CYP3A96 and CYP3A97 were highly expressed, while in the intestine there were only two dominating isoforms, CYP3A93 and CYP3A96. The isoform CYP3A129 was not detected in the liver or the intestine, although this gene consists of a complete set of exons and should therefore code for a functional protein. It is possible that this gene is expressed in tissues other than the liver and intestines. In the intestine, both CYP3A96 and CYP3A93 showed the highest gene expression in the duodenum and the proximal parts of the jejunum. This correlated with a high protein expression in these tissues. Studies of the enzyme activity showed the same K(m) for the LIPA substrate in the liver and the intestine, while the maximum velocity (V(max)) in the liver was higher than in the intestine. Our finding of a differential gene expression of the CYP3A isoforms in the liver and the intestines contributes to a better understanding of drug metabolism in horses. © 2012 Blackwell Publishing Ltd.

Molecular biology of C4 phosphoenolpyruvate carboxylase: Structure, regulation and genetic engineering.

PubMed

Rajagopalan, A V; Devi, M T; Raghavendra, A S

1994-02-01

Three to four families of nuclear genes encode different isoforms of phosphoenolpyruvate (PEP) carboxylase (PEPC): C4-specific, C3 or etiolated, CAM and root forms. C4 leaf PEPC is encoded by a single gene (ppc) in sorghum and maize, but multiple genes in the C4-dicot Flaveria trinervia. Selective expression of ppc in only C4-mesophyll cells is proposed to be due to nuclear factors, DNA methylation and a distinct gene promoter. Deduced amino acid sequences of C4-PEPC pinpoint the phosphorylatable serine near the N-terminus, C4-specific valine and serine residues near the C-terminus, conserved cysteine, lysine and histidine residues and PEP binding/catalytic sites. During the PEPC reaction, PEP and bicarbonate are first converted into carboxyphosphate and the enolate of pyruvate. Carboxyphosphate decomposes within the active site into Pi and CO2, the latter combining with the enolate to form oxalacetate. Besides carboxylation, PEPC catalyzes a HCO3 (-)-dependent hydrolysis of PEP to yield pyruvate and Pi. Post-translational regulation of PEPC occurs by a phosphorylation/dephosphorylation cascade in vivo and by reversible enzyme oligomerization in vitro. The interrelation between phosphorylation and oligomerization of the enzyme is not clear. PEPC-protein kinase (PEPC-PK), the enzyme responsible for phosphorylation of PEPC, has been studied extensively while only limited information is available on the protein phosphatase 2A capable of dephosphorylating PEPC. The C4 ppc was cloned and expressed in Escherichia coli as well as tobacco. The transformed E. coli produced a functional/phosphorylatable C4 PEPC and the transgenic tobacco plants expressed both C3 and C4 isoforms. Site-directed mutagenesis of ppc indicates the importance of His(138), His(579) and Arg(587) in catalysis and/or substrate-binding by the E. coli enzyme, Ser(8) in the regulation of sorghum PEPC. Important areas for further research on C4 PEPC are: mechanism of transduction of light signal during photoactivation of PEPC-PK and PEPC in leaves, extensive use of site-directed mutagenesis to precisely identify other key amino acid residues, changes in quarternary structure of PEPC in vivo, a high-resolution crystal structure, and hormonal regulation of PEPC expression.

Cloning and genomic characterization of sytdep, a new synaptotagmin XIV-related gene.

PubMed

Herrero-Turrión, M Javier; Fukuda, Mitsunori; Mollinedo, Faustino

2006-02-10

We have identified a new human gene coined sytdep (synaptotagmin XIV-derived protein) in human neutrophils. Sytdep encodes a 188-amino acid sequence with a 21.435kDa deduced molecular mass, showing 75% identity to human synaptotagmin (syt) XIV. Human neutrophils express sytdep, but not syt XIV. Sytdep was upregulated during HL-60 neutrophil differentiation. Sytdep gene is located in human chromosome 4 and contains a unique exon, whereas syt XIV gene, located in chromosome 1, comprises 10 exons with 9 introns. Mouse genome did not contain sytdep. The N-terminal region of sytdep shows no homology with any known protein and, unlike synaptotagmin XIV isoforms, sytdep shows a unique C-terminal C2B domain. Polyclonal antibodies against the C2B domain of syt XIV recognized sytdep as a 27-kDa protein in human neutrophils. Genomic analyses suggest that human sytdep could derive from a retrotranslocation of a syt XIV transcript into chromosome 4.

Shared and novel molecular responses of mandarin to drought.

PubMed

Gimeno, Jacinta; Gadea, José; Forment, Javier; Pérez-Valle, Jorge; Santiago, Julia; Martínez-Godoy, María A; Yenush, Lynne; Bellés, José M; Brumós, Javier; Colmenero-Flores, José M; Talón, Manuel; Serrano, Ramón

2009-07-01

Drought is the most important stress experienced by citrus crops. A citrus cDNA microarray of about 6.000 genes has been utilized to identify transcriptomic responses of mandarin to water stress. As observed in other plant species challenged with drought stress, key genes for lysine catabolism, proline and raffinose synthesis, hydrogen peroxide reduction, vacuolar malate transport, RCI2 proteolipids and defence proteins such as osmotin, dehydrins and heat-shock proteins are induced in mandarin. Also, some aquaporin genes are repressed. The osmolyte raffinose could be detected in stressed roots while the dehydrin COR15 protein only accumulated in stressed leaves but not in roots. Novel drought responses in mandarin include the induction of genes encoding a new miraculin isoform, chloroplast beta-carotene hydroxylase, oleoyl desaturase, ribosomal protein RPS13A and protein kinase CTR1. These results suggest that drought tolerance in citrus may benefit from inhibition of proteolysis, activation of zeaxanthin and linolenoyl synthesis, reinforcement of ribosomal structure and down-regulation of the ethylene response.

Cloning, characterization and sequence comparison of the gene coding for IMP dehydrogenase from Pyrococcus furiosus.

PubMed

Collart, F R; Osipiuk, J; Trent, J; Olsen, G J; Huberman, E

1996-10-03

We have cloned and characterized the gene encoding inosine monophosphate dehydrogenase (IMPDH) from Pyrococcus furiosus (Pf), a hyperthermophillic archeon. Sequence analysis of the Pf gene indicated an open reading frame specifying a protein of 485 amino acids (aa) with a calculated M(r) of 52900. Canonical Archaea promoter elements, Box A and Box B, are located -49 and -17 nucleotides (nt), respectively, upstream of the putative start codon. The sequence of the putative active-site region conforms to the IMPDH signature motif and contains a putative active-site cysteine. Phylogenetic relationships derived by using all available IMPDH sequences are consistent with trees developed for other molecules; they do not precisely resolve the history of Pf IMPDH but indicate a close similarity to bacterial IMPDH proteins. The phylogenetic analysis indicates that a gene duplication occurred prior to the division between rodents and humans, accounting for the Type I and II isoforms identified in mice and humans.

Assessment and Reconstruction of Novel HSP90 Genes: Duplications, Gains and Losses in Fungal and Animal Lineages

PubMed Central

Pantzartzi, Chrysoula N.; Drosopoulou, Elena; Scouras, Zacharias G.

2013-01-01

Hsp90s, members of the Heat Shock Protein class, protect the structure and function of proteins and play a significant task in cellular homeostasis and signal transduction. In order to determine the number of hsp90 gene copies and encoded proteins in fungal and animal lineages and through that key duplication events that this family has undergone, we collected and evaluated Hsp90 protein sequences and corresponding Expressed Sequence Tags and analyzed available genomes from various taxa. We provide evidence for duplication events affecting either single species or wider taxonomic groups. With regard to Fungi, duplicated genes have been detected in several lineages. In invertebrates, we demonstrate key duplication events in certain clades of Arthropoda and Mollusca, and a possible gene loss event in a hymenopteran family. Finally, we infer that the duplication event responsible for the two (a and b) isoforms in vertebrates occurred probably shortly after the split of Hyperoartia and Gnathostomata. PMID:24066039

Isolation of complementary DNA clones encoding pathogenesis-related proteins P and Q, two acidic chitinases from tobacco.

PubMed Central

Payne, G; Ahl, P; Moyer, M; Harper, A; Beck, J; Meins, F; Ryals, J

1990-01-01

Complementary DNA clones encoding two isoforms of the acidic endochitinase (chitinase, EC 3.2.1.14) from tobacco were isolated. Comparison of amino acid sequences deduced from the cDNA clones and the sequence of peptides derived from purified proteins show that these clones encode the pathogenesis-related proteins PR-P and PR-Q. The cDNA inserts were not homologous to either the bacterial form of chitinase or the form from cucumber but shared significant homology to the basic form of chitinase from tobacco and bean. The acidic isoforms of tobacco chitinase did not contain the amino-terminal, cysteine-rich "hevein" domain found in the basic isoforms, indicating that this domain, which binds chitin, is not essential for chitinolytic activity. The accumulation of mRNA for the pathogenesis-related proteins PR-1, PR-R, PR-P, and PR-Q in Xanthi.nc tobacco leaves following infection with tobacco mosaic virus was measured by primer extension. The results indicate that the induction of these proteins during the local necrotic lesion response to the virus is coordinated at the mRNA level. Images PMID:2296608

Expression and Stress-Dependent Induction of Potassium Channel Transcripts in the Common Ice Plant1

PubMed Central

Su, Hua; Golldack, Dortje; Katsuhara, Maki; Zhao, Chengsong; Bohnert, Hans J.

2001-01-01

We have characterized transcripts for three potassium channel homologs in the AKT/KAT subfamily (Shaker type) from the common ice plant (Mesembryanthemum crystallinum), with a focus on their expression during salt stress (up to 500 mm NaCl). Mkt1 and 2, Arabidopsis AKT homologs, and Kmt1, a KAT homolog, are members of small gene families with two to three isoforms each. Mkt1 is root specific; Mkt2 is found in leaves, flowers, and seed capsules; and Kmt1 is expressed in leaves and seed capsules. Mkt1 is present in all cells of the root, and in leaves a highly conserved isoform is detected present in all cells with highest abundance in the vasculature. MKT1 for which antibodies were made is localized to the plasma membrane. Following salt stress, MKT1 (transcripts and protein) is drastically down-regulated, Mkt2 transcripts do not change significantly, and Kmt1 is strongly and transiently (maximum at 6 h) up-regulated in leaves and stems. The detection and stress-dependent behavior of abundant transcripts representing subfamilies of potassium channels provides information about tissue specificity and the complex regulation of genes encoding potassium uptake systems in a halophytic plant. PMID:11161018

Mitochondrial genome-maintaining activity of mouse mitochondrial transcription factor A and its transcript isoform in Saccharomyces cerevisiae.

PubMed

Yoon, Young Geol; Koob, Michael D; Yoo, Young Hyun

2011-09-15

Mitochondrial transcription factor A (Tfam) binds to and organizes mitochondrial DNA (mtDNA) genome into a mitochondrial nucleoid (mt-nucleoid) structure, which is necessary for mtDNA transcription and maintenance. Here, we demonstrate the mtDNA-organizing activity of mouse Tfam and its transcript isoform (Tfam(iso)), which has a smaller high-mobility group (HMG)-box1 domain, using a yeast model system that contains a deletion of the yeast homolog of mouse Tfam protein, Abf2p. When the mouse Tfam genes were introduced into the ABF2 locus of yeast genome, the corresponding mouse proteins, Tfam and Tfam(iso), can functionally replace the yeast Abf2p and support mtDNA maintenance and mitochondrial biogenesis in yeast. Growth properties, mtDNA content and mitochondrial protein levels of genes encoded in the mtDNA were comparable in the strains expressing mouse proteins and the wild-type yeast strain, indicating that the proteins have robust mtDNA-maintaining and -expressing function in yeast mitochondria. These results imply that the mtDNA-organizing activities of the mouse mt-nucleoid proteins are structurally and evolutionary conserved, thus they can maintain the mtDNA of distantly related and distinctively different species, such as yeast. Copyright © 2011 Elsevier B.V. All rights reserved.

Importin-α7 Is Involved in the Formation of Ebola Virus Inclusion Bodies but Is Not Essential for Pathogenicity in Mice

PubMed Central

Gabriel, Gülsah; Feldmann, Friederike; Reimer, Rudolph; Thiele, Swantje; Fischer, Meike; Hartmann, Enno; Bader, Michael; Ebihara, Hideki; Hoenen, Thomas; Feldmann, Heinz

2015-01-01

Ebola virus (EBOV) protein 24 antagonizes the host interferon (IFN) response by hijacking select nuclear importin-α isoforms. Thereby, it blocks STAT1-mediated IFN-α/β and IFN-γ synthesis. However, owing to the lack of importin-α knockout animal models in the past, their role in EBOV pathogenesis remained largely unknown. Here, we demonstrate that importin-α7 is involved in the formation of EBOV inclusion bodies and replication. However, deletion of the gene encoding importin-α7 was not sufficient to increase survival rates among mice infected with EBOV. PMID:26185094

Evidence for light perception in a bioluminescent organ

PubMed Central

Tong, Deyan; Rozas, Natalia S.; Oakley, Todd H.; Mitchell, Jane; Colley, Nansi J.; McFall-Ngai, Margaret J.

2009-01-01

Here we show that bioluminescent organs of the squid Euprymna scolopes possess the molecular, biochemical, and physiological capability for light detection. Transcriptome analyses revealed expression of genes encoding key visual transduction proteins in light-organ tissues, including the same isoform of opsin that occurs in the retina. Electroretinograms demonstrated that the organ responds physiologically to light, and immunocytochemistry experiments localized multiple proteins of visual transduction cascades to tissues housing light-producing bacterial symbionts. These data provide evidence that the light-organ tissues harboring the symbionts serve as extraocular photoreceptors, with the potential to perceive directly the bioluminescence produced by their bacterial partners. PMID:19509343

TFIIH Subunit Alterations Causing Xeroderma Pigmentosum and Trichothiodystrophy Specifically Disturb Several Steps during Transcription

PubMed Central

Singh, Amita; Compe, Emanuel; Le May, Nicolas; Egly, Jean-Marc

2015-01-01

Mutations in genes encoding the ERCC3 (XPB), ERCC2 (XPD), and GTF2H5 (p8 or TTD-A) subunits of the transcription and DNA-repair factor TFIIH lead to three autosomal-recessive disorders: xeroderma pigmentosum (XP), XP associated with Cockayne syndrome (XP/CS), and trichothiodystrophy (TTD). Although these diseases were originally associated with defects in DNA repair, transcription deficiencies might be also implicated. By using retinoic acid receptor beta isoform 2 (RARB2) as a model in several cells bearing mutations in genes encoding TFIIH subunits, we observed that (1) the recruitment of the TFIIH complex was altered at the activated RARB2 promoter, (2) TFIIH participated in the recruitment of nucleotide excision repair (NER) factors during transcription in a manner different from that observed during NER, and (3) the different TFIIH variants disturbed transcription by having distinct consequences on post-translational modifications of histones, DNA-break induction, DNA demethylation, and gene-loop formation. The transition from heterochromatin to euchromatin was disrupted depending on the variant, illustrating the fact that TFIIH, by contributing to NER factor recruitment, orchestrates chromatin remodeling. The subtle transcriptional differences found between various TFIIH variants thus participate in the phenotypic variability observed among XP, XP/CS, and TTD individuals. PMID:25620205

The root application of a purified leonardite humic acid modifies the transcriptional regulation of the main physiological root responses to Fe deficiency in Fe-sufficient cucumber plants.

PubMed

Aguirre, Elena; Elena, Aguirre; Leménager, Diane; Diane, Leménager; Bacaicoa, Eva; Eva, Bacaicoa; Fuentes, Marta; Marta, Fuentes; Baigorri, Roberto; Roberto, Baigorri; Zamarreño, Angel Ma; García-Mina, José Ma

2009-03-01

The aim of this study is to investigate the effect of a well-characterized purified humic acid (non-measurable concentrations of the main plant hormones were detected) on the transcriptional regulation of the principal molecular agents involved in iron assimilation. To this end, non-deficient cucumber plants were treated with different concentrations of a purified humic acid (PHA) (2, 5, 100 and 250 mg of organic carbonL(-1)) and harvested 4, 24, 48, 76 and 92 h from the onset of the treatment. At harvest times, the mRNA transcript accumulation of CsFRO1 encoding for Fe(III) chelate-reductase (EC 1.16.1.7); CsHa1 and CsHa2 encoding for plasma membrane H+-ATPase (EC 3.6.3.6); and CsIRT1 encoding for Fe(II) high-affinity transporter, was quantified by real-time RT-PCR. Meanwhile, the respective enzyme activity of the Fe(III) chelate-reductase and plasma membrane H+-ATPase was also investigated. The results obtained indicated that PHA root treatments affected the regulation of the expression of the studied genes, but this effect was transient and differed (up-regulation or down-regulation) depending on the genes studied. Thus, principally the higher doses of PHA caused a transient increase in the expression of the CsHa2 isoform for 24 and 48 h whereas the CsHa1 isoform was unaffected or down-regulated. These effects were accompanied by an increase in the plasma membrane H+-ATPase activity for 4, 48 and 96 h. Likewise, PHA root treatments (principally the higher doses) up-regulated CsFRO1 and CsIRT1 expression for 48 and 72 h; whereas these genes were down-regulated by PHA for 96 h. These effects were associated with an increase in the Fe(III) chelate-reductase activity for 72 h. These effects were not associated with a significant decrease in the Fe root or leaf concentrations, although an eventual effect on the Fe root assimilation pattern cannot be ruled out. These results stress the close relationships between the effects of humic substances on plant development and iron nutrition. However, further studies are needed in order to elucidate if these effects at molecular level are caused by mechanisms involving hormone-like actions and/or nutritional factors.

A hitchhiker's guide to the human Hsp70 family

PubMed Central

Tavaria, Michael; Gabriele, Tim; Kola, Ismail; Anderson, Robin L.

1996-01-01

The human Hsp70 family encompasses at least 11 genes which encode a group of highly related proteins. These proteins include both cognate and highly inducible members, at least some of which act as molecular chaperones. The location of cognate Hsp70s within all the major subcellular compartments is an indication of the importance of these proteins. The expression of several inducible Hsp70 genes is also an indication of the importance of these proteins in the stres response. The existence of multiple genes and protein isoforms has created confusion in the identification and naming of particular family members. We have compiled, from the literature, a list of genes and genetic loci and produced a two-dimensional protein map of the known human Hsp70 family members. This will enable researchers in the field to quickly and reliably identify human Hsp70s. We have also devised a more rational nomenclature for these genes and gene products which, subject to general acceptance, could be extended to Hsp70 families from other species. PMID:9222585

Deep Sequencing Reveals Uncharted Isoform Heterogeneity of the Protein-Coding Transcriptome in Cerebral Ischemia.

PubMed

Bhattarai, Sunil; Aly, Ahmed; Garcia, Kristy; Ruiz, Diandra; Pontarelli, Fabrizio; Dharap, Ashutosh

2018-06-03

Gene expression in cerebral ischemia has been a subject of intense investigations for several years. Studies utilizing probe-based high-throughput methodologies such as microarrays have contributed significantly to our existing knowledge but lacked the capacity to dissect the transcriptome in detail. Genome-wide RNA-sequencing (RNA-seq) enables comprehensive examinations of transcriptomes for attributes such as strandedness, alternative splicing, alternative transcription start/stop sites, and sequence composition, thus providing a very detailed account of gene expression. Leveraging this capability, we conducted an in-depth, genome-wide evaluation of the protein-coding transcriptome of the adult mouse cortex after transient focal ischemia at 6, 12, or 24 h of reperfusion using RNA-seq. We identified a total of 1007 transcripts at 6 h, 1878 transcripts at 12 h, and 1618 transcripts at 24 h of reperfusion that were significantly altered as compared to sham controls. With isoform-level resolution, we identified 23 splice variants arising from 23 genes that were novel mRNA isoforms. For a subset of genes, we detected reperfusion time-point-dependent splice isoform switching, indicating an expression and/or functional switch for these genes. Finally, for 286 genes across all three reperfusion time-points, we discovered multiple, distinct, simultaneously expressed and differentially altered isoforms per gene that were generated via alternative transcription start/stop sites. Of these, 165 isoforms derived from 109 genes were novel mRNAs. Together, our data unravel the protein-coding transcriptome of the cerebral cortex at an unprecedented depth to provide several new insights into the flexibility and complexity of stroke-related gene transcription and transcript organization.

Divergent functional isoforms drive niche specialisation for nutrient acquisition and use in rumen microbiome.

PubMed

Rubino, Francesco; Carberry, Ciara; M Waters, Sinéad; Kenny, David; McCabe, Matthew S; Creevey, Christopher J

2017-04-01

Many microbes in complex competitive environments share genes for acquiring and utilising nutrients, questioning whether niche specialisation exists and if so, how it is maintained. We investigated the genomic signatures of niche specialisation in the rumen microbiome, a highly competitive, anaerobic environment, with limited nutrient availability determined by the biomass consumed by the host. We generated individual metagenomic libraries from 14 cows fed an ad libitum diet of grass silage and calculated functional isoform diversity for each microbial gene identified. The animal replicates were used to calculate confidence intervals to test for differences in diversity of functional isoforms between microbes that may drive niche specialisation. We identified 153 genes with significant differences in functional isoform diversity between the two most abundant bacterial genera in the rumen (Prevotella and Clostridium). We found Prevotella possesses a more diverse range of isoforms capable of degrading hemicellulose, whereas Clostridium for cellulose. Furthermore, significant differences were observed in key metabolic processes indicating that isoform diversity plays an important role in maintaining their niche specialisation. The methods presented represent a novel approach for untangling complex interactions between microorganisms in natural environments and have resulted in an expanded catalogue of gene targets central to rumen cellulosic biomass degradation.

Gene expression and activity of antioxidant enzymes in rice plants, cv. BRS AG, under saline stress.

PubMed

Rossatto, Tatiana; do Amaral, Marcelo Nogueira; Benitez, Letícia Carvalho; Vighi, Isabel Lopes; Braga, Eugenia Jacira Bolacel; de Magalhães Júnior, Ariano Martins; Maia, Mara Andrade Colares; da Silva Pinto, Luciano

2017-10-01

The rice cultivar ( Oryza sativa L.) BRS AG, developed by Embrapa Clima Temperado, is the first cultivar designed for purposes other than human consumption. It may be used in ethanol production and animal feed. Different abiotic stresses negatively affect plant growth. Soil salinity is responsible for a serious reduction in productivity. Therefore, the objective of this study was to evaluate the gene expression and the activity of antioxidant enzymes (SOD, CAT, APX and GR) and identify their functions in controlling ROS levels in rice plants, cultivar BRS AG, after a saline stress period. The plants were grown in vitro with two NaCl concentrations (0 and 136 mM), collected at 10, 15 and 20 days of cultivation. The results indicated that the activity of the enzymes evaluated promotes protection against oxidative stress. Although, there was an increase of reactive oxygen species, there was no increase in MDA levels. Regarding genes encoding isoforms of antioxidant enzymes, it was observed that OsSOD3 - CU/Zn , OsSOD2 - Cu/Zn , OsSOD - Cu/Zn , OsSOD4 - Cu/Zn , OsSODCc1 - Cu/Zn , OsSOD - Fe , OsAPX1 , OsCATB and OsGR2 were the most responsive. The increase in the transcription of all genes among evaluated isoforms, except for OsAPX6 , which remained stable, contributed to the increase or the maintenance of enzyme activity. Thus, it is possible to infer that the cv. BRS AG has defense mechanisms against salt stress.

The activities of progesterone receptor isoform A and B are differentially modulated by their ligands in a gene-selective manner.

PubMed

Leo, Joyce C L; Lin, Valerie C L

2008-01-01

It is known that progesterone receptor (PR) isoform A (PR-A) and isoform B (PR-B) may mediate different effects of progesterone. The objective of this study was to determine if the functions of PR isoforms also vary in response to different PR modulators (PRM). The effects of 7 synthetic PRM were tested in MDA-MB-231 cells engineered to express PR-A, PR-B, or both PR isoforms. The effects of progesterone were similar in cells expressing PR-A or PR-B in which it inhibited growth and induced focal adhesion. On the other hand, synthetic PRM modulated the activity of the PR isoforms differently. RU486, CDB4124, 17alpha-hydroxy CDB4124 and VA2914 exerted agonist activities on cell growth and adhesion via PR-B. Via PR-A, however, these compounds displayed agonist effect on cell growth but induced stellate morphology which was distinct from the agonist's effect. Their dual properties via PR-A were also displayed at the gene expression level: the compounds acted as agonists on cell cycle genes but exhibited antagonistic effect on cell adhesion genes. Introduction of ERalpha by adenoviral vector to these cells did not change PR-A or PR-B mediated effect of PRM radically, but it causes significant cell rounding and modified the magnitudes of the responses to PRM. The findings suggest that the activities of PR isoforms may be modulated by different PRM through gene-specific regulatory mechanisms. This raises an interesting possibility that PRM may be designed to be PR isoform and cellular pathway selective to achieve targeted therapy in breast cancer. Copyright 2007 Wiley-Liss, Inc.

The Oxytricha trifallax Macronuclear Genome: A Complex Eukaryotic Genome with 16,000 Tiny Chromosomes

PubMed Central

Swart, Estienne C.; Bracht, John R.; Magrini, Vincent; Minx, Patrick; Chen, Xiao; Zhou, Yi; Khurana, Jaspreet S.; Goldman, Aaron D.; Nowacki, Mariusz; Schotanus, Klaas; Jung, Seolkyoung; Fulton, Robert S.; Ly, Amy; McGrath, Sean; Haub, Kevin; Wiggins, Jessica L.; Storton, Donna; Matese, John C.; Parsons, Lance; Chang, Wei-Jen; Bowen, Michael S.; Stover, Nicholas A.; Jones, Thomas A.; Eddy, Sean R.; Herrick, Glenn A.; Doak, Thomas G.; Wilson, Richard K.; Mardis, Elaine R.; Landweber, Laura F.

2013-01-01

The macronuclear genome of the ciliate Oxytricha trifallax displays an extreme and unique eukaryotic genome architecture with extensive genomic variation. During sexual genome development, the expressed, somatic macronuclear genome is whittled down to the genic portion of a small fraction (∼5%) of its precursor “silent” germline micronuclear genome by a process of “unscrambling” and fragmentation. The tiny macronuclear “nanochromosomes” typically encode single, protein-coding genes (a small portion, 10%, encode 2–8 genes), have minimal noncoding regions, and are differentially amplified to an average of ∼2,000 copies. We report the high-quality genome assembly of ∼16,000 complete nanochromosomes (∼50 Mb haploid genome size) that vary from 469 bp to 66 kb long (mean ∼3.2 kb) and encode ∼18,500 genes. Alternative DNA fragmentation processes ∼10% of the nanochromosomes into multiple isoforms that usually encode complete genes. Nucleotide diversity in the macronucleus is very high (SNP heterozygosity is ∼4.0%), suggesting that Oxytricha trifallax may have one of the largest known effective population sizes of eukaryotes. Comparison to other ciliates with nonscrambled genomes and long macronuclear chromosomes (on the order of 100 kb) suggests several candidate proteins that could be involved in genome rearrangement, including domesticated MULE and IS1595-like DDE transposases. The assembly of the highly fragmented Oxytricha macronuclear genome is the first completed genome with such an unusual architecture. This genome sequence provides tantalizing glimpses into novel molecular biology and evolution. For example, Oxytricha maintains tens of millions of telomeres per cell and has also evolved an intriguing expansion of telomere end-binding proteins. In conjunction with the micronuclear genome in progress, the O. trifallax macronuclear genome will provide an invaluable resource for investigating programmed genome rearrangements, complementing studies of rearrangements arising during evolution and disease. PMID:23382650

Characterization of big bang, a novel gene encoding for PDZ domain-containing proteins that are dynamically expressed throughout Drosophila development.

PubMed

Kim, Sabrina Y; Renihan, Maia K; Boulianne, Gabrielle L

2006-06-01

PDZ (PSD-95, Discs-large, ZO-1) domain proteins often function as scaffolding proteins and have been shown to play important roles in diverse cellular processes such as the establishment and maintenance of cell polarity, and signal transduction. Here, we report the identification and cloning of a novel Drosophila melanogaster gene that is predicted to produce several different PDZ domain-containing proteins through alternative promoter usage and alternative splicing. This gene, that we have named big bang (bbg), was first identified as C96-GAL4, a GAL4 enhancer trap line that was generated in our lab. To further characterize bbg, its expression pattern was examined in ovaries, embryos, and late third instar larvae using UAS reporter gene constructs, in situ hybridization, or immunocytochemistry. In addition, the expression of alternatively spliced transcripts was examined in more detail using in situ hybridization. We find that during embryogenesis bbg is predominantly expressed in the developing gut, but it is also expressed in external sensory organs found in the epidermis. In the late third instar larva, bbg is expressed along the presumptive wing margin in the wing disc, broadly in the eye disc, and in other imaginal discs as well as in the brain. The expression patterns observed are dynamic and specific during development, suggesting that like other genes that encode for several different PDZ domain protein isoforms, bbg likely plays important roles in multiple developmental processes.

Evidence of IgY subclass diversification in snakes: evolutionary implications.

PubMed

Wang, Tao; Sun, Yi; Shao, Wenwei; Cheng, Gang; Li, Lingxiao; Cao, Zubing; Yang, Zhi; Zou, Huiying; Zhang, Wei; Han, Binyue; Hu, Yang; Ren, Liming; Hu, Xiaoxiang; Guo, Ying; Fei, Jing; Hammarström, Lennart; Li, Ning; Zhao, Yaofeng

2012-10-01

Mammalian IgG and IgE are thought to have evolved from IgY of nonmammalian tetrapods; however, no diversification of IgY subclasses has been reported in reptiles or birds, which are phylogenetically close to mammals. To our knowledge, we report the first evidence of the presence of multiple IgY-encoding (υ) genes in snakes. Two υ genes were identified in the snake Elaphe taeniura, and three υ genes were identified in the Burmese python (Python molurus bivittatus). Although four of the υ genes displayed a conventional four-H chain C region exon structure, one of the υ genes in the Burmese python lacked the H chain C region 2 exon, thus exhibiting a structure similar to that of the mammalian γ genes. We developed mouse mAbs specific for the IgY1 and IgY2 of E. taeniura and showed that both were expressed in serum; each had two isoforms: one full-length and one truncated at the C terminus. The truncation was not caused by alternative splicing or transcriptional termination. We also identified the μ and δ genes, but no α gene, in both snakes. This study provides valuable clues for our understanding of Ig gene evolution in tetrapods.

The single fgf receptor gene in the beetle Tribolium castaneum codes for two isoforms that integrate FGF8- and Branchless-dependent signals.

PubMed

Sharma, Rahul; Beer, Katharina; Iwanov, Katharina; Schmöhl, Felix; Beckmann, Paula Indigo; Schröder, Reinhard

2015-06-15

The precise regulation of cell-cell communication by numerous signal-transduction pathways is fundamental for many different processes during embryonic development. One important signalling pathway is the evolutionary conserved fibroblast-growth-factor (FGF)-pathway that controls processes like cell migration, axis specification and mesoderm formation in vertebrate and invertebrate animals. In the model insect Drosophila, the FGF ligand / receptor combinations of FGF8 (Pyramus and Thisbe) / Heartless (Htl) and Branchless (Bnl) / Breathless (Btl) are required for the migration of mesodermal cells and for the formation of the tracheal network respectively with both the receptors functioning independently of each other. However, only a single fgf-receptor gene (Tc-fgfr) has been identified in the genome of the beetle Tribolium. We therefore asked whether both the ligands Fgf8 and Bnl could transduce their signal through a common FGF-receptor in Tribolium. Indeed, we found that the function of the single Tc-fgfr gene is essential for mesoderm differentiation as well as for the formation of the tracheal network during early development. Ligand specific RNAi for Tc-fgf8 and Tc-bnl resulted in two distinct non-overlapping phenotypes of impaired mesoderm differentiation and abnormal formation of the tracheal network in Tc-fgf8- and Tc-bnl(RNAi) embryos respectively. We further show that the single Tc-fgfr gene encodes at least two different receptor isoforms that are generated through alternative splicing. We in addition demonstrate through exon-specific RNAi their distinct tissue-specific functions. Finally, we discuss the structure of the fgf-receptor gene from an evolutionary perspective. Copyright © 2015 Elsevier Inc. All rights reserved.

Fungal histidine phosphotransferase plays a crucial role in photomorphogenesis and pathogenesis in Magnaporthe oryzae

NASA Astrophysics Data System (ADS)

Mohanan, Varsha C.; Chandarana, Pinal M.; Chattoo, Bharat. B.; Patkar, Rajesh N.; Manjrekar, Johannes

2017-05-01

Two-component signal transduction (TCST) pathways play crucial roles in many cellular functions such as stress responses, biofilm formation and sporulation. The histidine phosphotransferase (HPt), which is an intermediate phosphotransfer protein in a two-component system, transfers a phosphate group to a phosphorylatable aspartate residue in the target protein(s), and up-regulates stress-activated MAP kinase cascades. Most fungal genomes carry a single copy of the gene coding for HPt, which are potential antifungal targets. However, unlike the histidine kinases (HK) or the downstream response regulators (RR) in two-component system, the HPts have not been well studied in phytopathogenic fungi. In this study, we investigated the role of HPt in the model rice-blast fungal pathogen Magnaporthe oryzae. We found that in M. oryzae an additional isoform of the HPT gene YPD1 was expressed specifically in response to light. Further, the expression of light-regulated genes such as those encoding envoy and blue-light-harvesting protein, and PAS domain containing HKs was significantly reduced upon down-regulation of YPD1 in M. oryzae. Importantly, down-regulation of YPD1 led to a significant decrease in the ability to penetrate the host cuticle and in light-dependent conidiation in M. oryzae. Thus, our results indicate that Ypd1 plays an important role in asexual development and host invasion, and suggest that YPD1 isoforms likely have distinct roles to play in the rice-blast pathogen M. oryzae.

Distinct Transcript Isoforms of the Atypical Chemokine Receptor 1 (ACKR1) / Duffy Antigen Receptor for Chemokines (DARC) Gene Are Expressed in Lymphoblasts and Altered Isoform Levels Are Associated with Genetic Ancestry and the Duffy-Null Allele

PubMed Central

Davis, Melissa B.; Walens, Andrea; Hire, Rupali; Mumin, Kauthar; Brown, Andrea M.; Ford, DeJuana; Howerth, Elizabeth W.; Monteil, Michele

2015-01-01

The Atypical ChemoKine Receptor 1 (ACKR1) gene, better known as Duffy Antigen Receptor for Chemokines (DARC or Duffy), is responsible for the Duffy Blood Group and plays a major role in regulating the circulating homeostatic levels of pro-inflammatory chemokines. Previous studies have shown that one common variant, the Duffy Null (Fy-) allele that is specific to African Ancestry groups, completely removes expression of the gene on erythrocytes; however, these individuals retain endothelial expression. Additional alleles are associated with a myriad of clinical outcomes related to immune responses and inflammation. In addition to allele variants, there are two distinct transcript isoforms of DARC which are expressed from separate promoters, and very little is known about the distinct transcriptional regulation or the distinct functionality of these protein isoforms. Our objective was to determine if the African specific Fy- allele alters the expression pattern of DARC isoforms and therefore could potentially result in a unique signature of the gene products, commonly referred to as antigens. Our work is the first to establish that there is expression of DARC on lymphoblasts. Our data indicates that people of African ancestry have distinct relative levels of DARC isoforms expressed in these cells. We conclude that the expression of both isoforms in combination with alternate alleles yields multiple Duffy antigens in ancestry groups, depending upon the haplotypes across the gene. Importantly, we hypothesize that DARC isoform expression patterns will translate into ancestry-specific inflammatory responses that are correlated with the axis of pro-inflammatory chemokine levels and distinct isoform-specific interactions with these chemokines. Ultimately, this work will increase knowledge of biological mechanisms underlying disparate clinical outcomes of inflammatory-related diseases among ethnic and geographic ancestry groups. PMID:26473357

Predicting the Impact of Alternative Splicing on Plant MADS Domain Protein Function

PubMed Central

Severing, Edouard I.; van Dijk, Aalt D. J.; Morabito, Giuseppa; Busscher-Lange, Jacqueline; Immink, Richard G. H.; van Ham, Roeland C. H. J.

2012-01-01

Several genome-wide studies demonstrated that alternative splicing (AS) significantly increases the transcriptome complexity in plants. However, the impact of AS on the functional diversity of proteins is difficult to assess using genome-wide approaches. The availability of detailed sequence annotations for specific genes and gene families allows for a more detailed assessment of the potential effect of AS on their function. One example is the plant MADS-domain transcription factor family, members of which interact to form protein complexes that function in transcription regulation. Here, we perform an in silico analysis of the potential impact of AS on the protein-protein interaction capabilities of MIKC-type MADS-domain proteins. We first confirmed the expression of transcript isoforms resulting from predicted AS events. Expressed transcript isoforms were considered functional if they were likely to be translated and if their corresponding AS events either had an effect on predicted dimerisation motifs or occurred in regions known to be involved in multimeric complex formation, or otherwise, if their effect was conserved in different species. Nine out of twelve MIKC MADS-box genes predicted to produce multiple protein isoforms harbored putative functional AS events according to those criteria. AS events with conserved effects were only found at the borders of or within the K-box domain. We illustrate how AS can contribute to the evolution of interaction networks through an example of selective inclusion of a recently evolved interaction motif in the MADS AFFECTING FLOWERING1-3 (MAF1–3) subclade. Furthermore, we demonstrate the potential effect of an AS event in SHORT VEGETATIVE PHASE (SVP), resulting in the deletion of a short sequence stretch including a predicted interaction motif, by overexpression of the fully spliced and the alternatively spliced SVP transcripts. For most of the AS events we were able to formulate hypotheses about the potential impact on the interaction capabilities of the encoded MIKC proteins. PMID:22295091

Functional understanding of the diverse exon-intron structures of human GPCR genes.

PubMed

Hammond, Dorothy A; Olman, Victor; Xu, Ying

2014-02-01

The GPCR genes have a variety of exon-intron structures even though their proteins are all structurally homologous. We have examined all human GPCR genes with at least two functional protein isoforms, totaling 199, aiming to gain an understanding of what may have contributed to the large diversity of the exon-intron structures of the GPCR genes. The 199 genes have a total of 808 known protein splicing isoforms with experimentally verified functions. Our analysis reveals that 1301 (80.6%) adjacent exon-exon pairs out of the total of 1,613 in the 199 genes have either exactly one exon skipped or the intron in-between retained in at least one of the 808 protein splicing isoforms. This observation has a statistical significance p-value of 2.051762 * e(-09), assuming that the observed splicing isoforms are independent of the exon-intron structures. Our interpretation of this observation is that the exon boundaries of the GPCR genes are not randomly determined; instead they may be selected to facilitate specific alternative splicing for functional purposes.

Characterisation of Cdkl5 transcript isoforms in rat.

PubMed

Hector, Ralph D; Dando, Owen; Ritakari, Tuula E; Kind, Peter C; Bailey, Mark E S; Cobb, Stuart R

2017-03-01

CDKL5 deficiency is a severe neurological disorder caused by mutations in the X-linked Cyclin-Dependent Kinase-Like 5 gene (CDKL5). The predominant human CDKL5 brain isoform is a 9.7kb transcript comprised of 18 exons with a large 6.6kb 3'-untranslated region (UTR). Mammalian models of CDKL5 disorder are currently limited to mouse, and little is known about Cdkl5 in other organisms used to model neurodevelopmental disorders, such as rat. In this study we characterise, both bioinformatically and experimentally, the rat Cdkl5 gene structure and its associated transcript isoforms. New exonic regions, splice sites and UTRs are described, confirming the presence of four distinct transcript isoforms. The predominant isoform in the brain, which we name rCdkl5_1, is orthologous to the human hCDKL5_1 and mouse mCdkl5_1 isoforms and is the most highly expressed isoform across all brain regions tested. This updated gene model of Cdkl5 in rat provides a framework for studies into its protein products and provides a reference for the development of molecular therapies for testing in rat models of CDKL5 disorder. Copyright © 2016 Elsevier B.V. All rights reserved.

Characterization of fusion genes and the significantly expressed fusion isoforms in breast cancer by hybrid sequencing

PubMed Central

Weirather, Jason L.; Afshar, Pegah Tootoonchi; Clark, Tyson A.; Tseng, Elizabeth; Powers, Linda S.; Underwood, Jason G.; Zabner, Joseph; Korlach, Jonas; Wong, Wing Hung; Au, Kin Fai

2015-01-01

We developed an innovative hybrid sequencing approach, IDP-fusion, to detect fusion genes, determine fusion sites and identify and quantify fusion isoforms. IDP-fusion is the first method to study gene fusion events by integrating Third Generation Sequencing long reads and Second Generation Sequencing short reads. We applied IDP-fusion to PacBio data and Illumina data from the MCF-7 breast cancer cells. Compared with the existing tools, IDP-fusion detects fusion genes at higher precision and a very low false positive rate. The results show that IDP-fusion will be useful for unraveling the complexity of multiple fusion splices and fusion isoforms within tumorigenesis-relevant fusion genes. PMID:26040699

Evolutionary origins and functions of the carotenoid biosynthetic pathway in marine diatoms.

PubMed

Coesel, Sacha; Oborník, Miroslav; Varela, Joao; Falciatore, Angela; Bowler, Chris

2008-08-06

Carotenoids are produced by all photosynthetic organisms, where they play essential roles in light harvesting and photoprotection. The carotenoid biosynthetic pathway of diatoms is largely unstudied, but is of particular interest because these organisms have a very different evolutionary history with respect to the Plantae and are thought to be derived from an ancient secondary endosymbiosis between heterotrophic and autotrophic eukaryotes. Furthermore, diatoms have an additional xanthophyll-based cycle for dissipating excess light energy with respect to green algae and higher plants. To explore the origins and functions of the carotenoid pathway in diatoms we searched for genes encoding pathway components in the recently completed genome sequences of two marine diatoms. Consistent with the supplemental xanthophyll cycle in diatoms, we found more copies of the genes encoding violaxanthin de-epoxidase (VDE) and zeaxanthin epoxidase (ZEP) enzymes compared with other photosynthetic eukaryotes. However, the similarity of these enzymes with those of higher plants indicates that they had very probably diversified before the secondary endosymbiosis had occurred, implying that VDE and ZEP represent early eukaryotic innovations in the Plantae. Consequently, the diatom chromist lineage likely obtained all paralogues of ZEP and VDE genes during the process of secondary endosymbiosis by gene transfer from the nucleus of the algal endosymbiont to the host nucleus. Furthermore, the presence of a ZEP gene in Tetrahymena thermophila provides the first evidence for a secondary plastid gene encoded in a heterotrophic ciliate, providing support for the chromalveolate hypothesis. Protein domain structures and expression analyses in the pennate diatom Phaeodactylum tricornutum indicate diverse roles for the different ZEP and VDE isoforms and demonstrate that they are differentially regulated by light. These studies therefore reveal the ancient origins of several components of the carotenoid biosynthesis pathway in photosynthetic eukaryotes and provide information about how they have diversified and acquired new functions in the diatoms.

Modulation of NF-κB and Nrf2 control of inflammatory responses in FHs 74 Int cell line is tocopherol isoform-specific

PubMed Central

Elisia, Ingrid

2013-01-01

The present study investigates the relative ability of α-, γ-, and δ-tocopherol (Toc) to modulate cell signaling events that are associated with inflammatory responses in fetal-derived intestinal (FHs 74 Int) cells. Secretion of the proinflammatory cytokine IL-8 in FHs 74 Int cells was stimulated in the following order: α-Toc < γ-Toc < δ-Toc. A similar proinflammatory response was observed when inflammation was induced in FHs 74 Int cells. Modulation of IL-8 expression by Toc corresponded to an isoform-specific modulation of NF-κB and nuclear factor-erythroid 2-related factor 2 (Nrf2) cell signaling pathways involved in expression of proinflammatory cytokines and antioxidant enzymes, respectively. δ-Toc and, to a lesser extent, γ-Toc activated NF-κB and Nrf2 signaling, as indicated by the greater nuclear translocation of transcription factors. Activation of NF-κB signaling by γ- and δ-Toc was accompanied by upregulation of NF-κB target genes, such as IL-8 and prostaglandin-endoperoxide synthase 2, with and without a prior IFNγ-PMA challenge. Nevertheless, γ- and δ-Toc, particularly δ-Toc, concurrently downregulated glutamate-cysteine ligase, a Nrf2 target gene that encodes for glutathione biosynthesis. This observation was substantiated by confirmation that γ- and δ-Toc were effective at decreasing glutamate-cysteine ligase protein expression and cellular glutathione content. Downregulation of glutathione content in fetal intestinal cells corresponded to induction of apoptosis-mediated cytotoxicity. In conclusion, γ- and δ-Toc are biologically active isoforms of vitamin E and show superior bioactivity to α-Toc in modulating cell signaling events that contribute to a proinflammatory response in fetal-derived intestinal cells. PMID:24136788

Gibberellic Acid, Synthetic Auxins, and Ethylene Differentially Modulate α-l-Arabinofuranosidase Activities in Antisense 1-Aminocyclopropane-1-Carboxylic Acid Synthase Tomato Pericarp Discs1

PubMed Central

Sozzi, Gabriel O.; Greve, L. Carl; Prody, Gerry A.; Labavitch, John M.

2002-01-01

α-l-Arabinofuranosidases (α-Afs) are plant enzymes capable of releasing terminal arabinofuranosyl residues from cell wall matrix polymers, as well as from different glycoconjugates. Three different α-Af isoforms were distinguished by size exclusion chromatography of protein extracts from control tomatoes (Lycopersicon esculentum) and an ethylene synthesis-suppressed (ESS) line expressing an antisense 1-aminocyclopropane-1-carboxylic synthase transgene. α-Af I and II are active throughout fruit ontogeny. α-Af I is the first Zn-dependent cell wall enzyme isolated from tomato pericarp tissues, thus suggesting the involvement of zinc in fruit cell wall metabolism. This isoform is inhibited by 1,10-phenanthroline, but remains stable in the presence of NaCl and sucrose. α-Af II activity accounts for over 80% of the total α-Af activity in 10-d-old fruit, but activity drops during ripening. In contrast, α-Af III is ethylene dependent and specifically active during ripening. α-Af I released monosaccharide arabinose from KOH-soluble polysaccharides from tomato cell walls, whereas α-Af II and III acted on Na2CO3-soluble pectins. Different α-Af isoform responses to gibberellic acid, synthetic auxins, and ethylene were followed by using a novel ESS mature-green tomato pericarp disc system. α-Af I and II activity increased when gibberellic acid or 2,4-dichlorophenoxyacetic acid was applied, whereas ethylene treatment enhanced only α-Af III activity. Results suggest that tomato α-Afs are encoded by a gene family under differential hormonal controls, and probably have different in vivo functions. The ESS pericarp explant system allows comprehensive studies involving effects of physiological levels of different growth regulators on gene expression and enzyme activity with negligible wound-induced ethylene production. PMID:12114586

Gene dosage-dependent rescue of HSP neurite defects in SPG4 patients’ neurons

PubMed Central

Havlicek, Steven; Kohl, Zacharias; Mishra, Himanshu K.; Prots, Iryna; Eberhardt, Esther; Denguir, Naime; Wend, Holger; Plötz, Sonja; Boyer, Leah; Marchetto, Maria C.N.; Aigner, Stefan; Sticht, Heinrich; Groemer, Teja W.; Hehr, Ute; Lampert, Angelika; Schlötzer-Schrehardt, Ursula; Winkler, Jürgen; Gage, Fred H.; Winner, Beate

2014-01-01

The hereditary spastic paraplegias (HSPs) are a heterogeneous group of motorneuron diseases characterized by progressive spasticity and paresis of the lower limbs. Mutations in Spastic Gait 4 (SPG4), encoding spastin, are the most frequent cause of HSP. To understand how mutations in SPG4 affect human neurons, we generated human induced pluripotent stem cells (hiPSCs) from fibroblasts of two patients carrying a c.1684C>T nonsense mutation and from two controls. These SPG4 and control hiPSCs were able to differentiate into neurons and glia at comparable efficiency. All known spastin isoforms were reduced in SPG4 neuronal cells. The complexity of SPG4 neurites was decreased, which was paralleled by an imbalance of axonal transport with less retrograde movement. Prominent neurite swellings with disrupted microtubules were present in SPG4 neurons at an ultrastructural level. While some of these swellings contain acetylated and detyrosinated tubulin, these tubulin modifications were unchanged in total cell lysates of SPG4 neurons. Upregulation of another microtubule-severing protein, p60 katanin, may partially compensate for microtubuli dynamics in SPG4 neurons. Overexpression of the M1 or M87 spastin isoforms restored neurite length, branching, numbers of primary neurites and reduced swellings in SPG4 neuronal cells. We conclude that neurite complexity and maintenance in HSP patient-derived neurons are critically sensitive to spastin gene dosage. Our data show that elevation of single spastin isoform levels is sufficient to restore neurite complexity and reduce neurite swellings in patient cells. Furthermore, our human model offers an ideal platform for pharmacological screenings with the goal to restore physiological spastin levels in SPG4 patients. PMID:24381312

Characterization of Ovine A3Z1 Restriction Properties against Small Ruminant Lentiviruses (SRLVs)

PubMed Central

de Pablo-Maiso, Lorena; Glaria, Idoia; Crespo, Helena; Nistal-Villán, Estanislao; Andrésdóttir, Valgerdur; de Andrés, Damián; Amorena, Beatriz

2017-01-01

Intrinsic factors of the innate immune system include the apolipoprotein B editing enzyme catalytic polypeptide-like 3 (APOBEC3) protein family. APOBEC3 inhibits replication of different virus families by cytosine deamination of viral DNA and a not fully characterized cytosine deamination-independent mechanism. Sheep are susceptible to small ruminant lentivirus (SRLVs) infection and contain three APOBEC3 genes encoding four proteins (A3Z1, Z2, Z3 and Z2-Z3) with yet not deeply described antiviral properties. Using sheep blood monocytes and in vitro-derived macrophages, we found that A3Z1 expression is associated with lower viral replication in this cellular type. A3Z1 transcripts may also contain spliced variants (A3Z1Tr) lacking the cytidine deaminase motif. A3Z1 exogenous expression in fully permissive fibroblast-like cells restricted SRLVs infection while A3Z1Tr allowed infection. A3Z1Tr was induced after SRLVs infection or stimulation of blood-derived macrophages with interferon gamma (IFN-γ). Interaction between truncated isoform and native A3Z1 protein was detected as well as incorporation of both proteins into virions. A3Z1 and A3Z1Tr interacted with SRLVs Vif, but this interaction was not associated with degradative properties. Similar A3Z1 truncated isoforms were also present in human and monkey cells suggesting a conserved alternative splicing regulation in primates. A3Z1-mediated retroviral restriction could be constrained by different means, including gene expression and specific alternative splicing regulation, leading to truncated protein isoforms lacking a cytidine-deaminase motif. PMID:29149056

Alternative splicing for members of human mosaic domain superfamilies. I. The CH and LIM domains containing group of proteins.

PubMed

Friedberg, Felix

2009-05-01

In this paper we examine (restricted to homo sapiens) the products resulting from gene duplication and the subsequent alternative splicing for the members of a multidomain group of proteins which possess the evolutionary conserved calponin homology CH domain, i.e. an "actin binding domain", as a singlet and which, in addition, contain the conserved cysteine rich double Zn finger possessing Lim domain, also as a singlet. Seven genes, resulting from gene duplications, were identified that code for seven group members for which pre-mRNAs appear to have undergone multiple alternative splicing: Mical 1, 2 and 3 are located on chromosomes 6q21, 11p15 and 22q11, respectively. The LMO7 gene is present on chromosome 13q22 and the LIMCH1 gene on chromosome 4p13. Micall1 is mapped to chromosome 22q13 and Micall2 to chromosome 7p22. Translated Gen/Bank ESTs suggest the existence of multiple products alternatively spliced from the pre-mRNAs encoded by these genes. Characteristic indicators of such splicing among the proteins derived from one gene must include containment of some common extensive 100% identical regions. In some instances only one exon might be partly or completely eliminated. Sometimes alternative splicing is also associated with an increased frequency of creation of an exon or part of an exon from an intron. Not only coding regions for the body of the protein but also for its N- or -C ends could be affected by the splicing. If created forms are merely beginning at different starting points but remain identical in sequence thereafter, their existence as products of alternate splicing must be questioned. In the splicings, described in this paper, multiple isoforms rather than a single isoform appear as products during the gene expression.

Sorting of tropomyosin isoforms in synchronised NIH 3T3 fibroblasts: evidence for distinct microfilament populations.

PubMed

Percival, J M; Thomas, G; Cock, T A; Gardiner, E M; Jeffrey, P L; Lin, J J; Weinberger, R P; Gunning, P

2000-11-01

The nonmuscle actin cytoskeleton consists of multiple networks of actin microfilaments. Many of these filament systems are bound by the actin-binding protein tropomyosin (Tm). We investigated whether Tm isoforms could be cell cycle regulated during G0 and G1 phases of the cell cycle in synchronised NIH 3T3 fibroblasts. Using Tm isoform-specific antibodies, we investigated protein expression levels of specific Tms in G0 and G1 phases and whether co-expressed isoforms could be sorted into different compartments. Protein levels of Tms 1, 2, 5a, 6, from the alpha Tm(fast) and beta-Tm genes increased approximately 2-fold during mid-late G1. Tm 3 levels did not change appreciably during G1 progression. In contrast, Tm 5NM gene isoform levels (Tm 5NM-1-11) increased 2-fold at 5 h into G1 and this increase was maintained for the following 3 h. However, Tm 5NM-1 and -2 levels decreased by a factor of three during this time. Comparison of the staining of the antibodies CG3 (detects all Tm 5NM gene products), WS5/9d (detects only two Tms from the Tm 5NM gene, Tm 5NM-1 and -2) and alpha(f)9d (detects specific Tms from the alpha Tm(fast) and beta-Tm genes) antibodies revealed 3 spatially distinct microfilament systems. Tm isoforms detected by alpha(f)9d were dramatically sorted from isoforms from the Tm 5NM gene detected by CG3. Tm 5NM-1 and Tm 5NM-2 were not incorporated into stress fibres, unlike other Tm 5NM isoforms, and marked a discrete, punctate, and highly polarised compartment in NIH 3T3 fibroblasts. All microfilament systems, excluding that detected by the WS5/9d antibody, were observed to coalign into parallel stress fibres at 8 h into G1. However, Tms detected by the CG3 and alpha(f)9d antibodies were incorporated into filaments at different times indicating distinct temporal control mechanisms. Microfilaments in NIH 3T3 cells containing Tm 5NM isoforms were more resistant to cytochalasin D-mediated actin depolymerisation than filaments containing isoforms from the alpha Tm(fast) and beta-Tm genes. This suggests that Tm 5NM isoforms may be in different microfilaments to alpha Tm(fast) and beta-Tm isoforms even when present in the same stress fibre. Staining of primary mouse fibroblasts showed identical Tm sorting patterns to those seen in cultured NIH 3T3 cells. Furthermore, we demonstrate that sorting of Tms is not restricted to cultured cells and can be observed in human columnar epithelial cells in vivo. We conclude that the expression and localisation of Tm isoforms are differentially regulated in G0 and G1 phase of the cell cycle. Tms mark multiple microfilament compartments with restricted tropomyosin composition. The creation of distinct microfilament compartments by differential sorting of Tm isoforms is observable in primary fibroblasts, cultured 3T3 cells and epithelial cells in vivo. Copyright 2000 Wiley-Liss, Inc.

Lack of variation in voltage-gated sodium channels of common bottlenose dolphins (Tursiops truncatus) exposed to neurotoxic algal blooms.

PubMed

Cammen, Kristina M; Rosel, Patricia E; Wells, Randall S; Read, Andrew J

2014-12-01

In coastal marine ecosystems, neurotoxins produced by harmful algal blooms (HABs) often result in large-scale mortality events of many marine species. Historical and frequent exposure to HABs therefore may provide a strong selective pressure for adaptations that result in toxin resistance. Neurotoxin resistance has independently evolved in a variety of terrestrial and marine species via mutations in genes encoding the toxin binding sites within the voltage-gated sodium channel gene complex. Accordingly, we tested the hypothesis that genetic variation in the putative binding site of brevetoxins in common bottlenose dolphins (Tursiops truncatus) explains differences among individuals or populations in resistance to harmful Karenia brevis blooms in the Gulf of Mexico. We found very little variation in the sodium channel exons encoding the putative brevetoxin binding site among bottlenose dolphins from central-west Florida and the Florida Panhandle. Our study included samples from several bottlenose dolphin mortality events associated with HABs, but we found no association between genetic variation and survival. We observed a significant effect of geographic region on genetic variation for some sodium channel isoforms, but this can be primarily explained by rare private alleles and is more likely a reflection of regional genetic differentiation than the cause of different levels of HAB resistance between regions. In contrast to many other previously studied neurotoxin-resistant species, we conclude that bottlenose dolphins have not evolved resistance to HABs via mutations in genes encoding the brevetoxin binding site on the voltage-gated sodium channels. Copyright © 2014 Elsevier B.V. All rights reserved.

Activity Augmentation of Amphioxus Peptidoglycan Recognition Protein BbtPGRP3 via Fusion with a Chitin Binding Domain

PubMed Central

Wang, Wen-Jie; Cheng, Wang; Luo, Ming; Yan, Qingyu; Yu, Hong-Mei; Li, Qiong; Cao, Dong-Dong; Huang, Shengfeng; Xu, Anlong; Mariuzza, Roy A.; Chen, Yuxing; Zhou, Cong-Zhao

2015-01-01

Peptidoglycan recognition proteins (PGRPs), which have been identified in most animals, are pattern recognition molecules that involve antimicrobial defense. Resulting from extraordinary expansion of innate immune genes, the amphioxus encodes many PGRPs of diverse functions. For instance, three isoforms of PGRP encoded by Branchiostoma belcheri tsingtauense, termed BbtPGRP1~3, are fused with a chitin binding domain (CBD) at the N-terminus. Here we report the 2.7 Å crystal structure of BbtPGRP3, revealing an overall structure of an N-terminal hevein-like CBD followed by a catalytic PGRP domain. Activity assays combined with site-directed mutagenesis indicated that the individual PGRP domain exhibits amidase activity towards both DAP-type and Lys-type peptidoglycans (PGNs), the former of which is favored. The N-terminal CBD not only has the chitin-binding activity, but also enables BbtPGRP3 to gain a five-fold increase of amidase activity towards the Lys-type PGNs, leading to a significantly broadened substrate spectrum. Together, we propose that modular evolution via domain shuffling combined with gene horizontal transfer makes BbtPGRP1~3 novel PGRPs of augmented catalytic activity and broad recognition spectrum. PMID:26479246

RNA Splicing: Regulation and Dysregulation in the Heart.

PubMed

van den Hoogenhof, Maarten M G; Pinto, Yigal M; Creemers, Esther E

2016-02-05

RNA splicing represents a post-transcriptional mechanism to generate multiple functional RNAs or proteins from a single transcript. The evolution of RNA splicing is a prime example of the Darwinian function follows form concept. A mutation that leads to a new mRNA (form) that encodes for a new functional protein (function) is likely to be retained, and this way, the genome has gradually evolved to encode for genes with multiple isoforms, thereby creating an enormously diverse transcriptome. Advances in technologies to characterize RNA populations have led to a better understanding of RNA processing in health and disease. In the heart, alternative splicing is increasingly being recognized as an important layer of post-transcriptional gene regulation. Moreover, the recent identification of several cardiac splice factors, such as RNA-binding motif protein 20 and SF3B1, not only provided important insight into the mechanisms underlying alternative splicing but also revealed how these splicing factors impact functional properties of the heart. Here, we review our current knowledge of alternative splicing in the heart, with a particular focus on the major and minor spliceosome, the factors controlling RNA splicing, and the role of alternative splicing in cardiac development and disease. © 2016 American Heart Association, Inc.

Structural analysis of the RH-like blood group gene products in nonhuman primates

DOE Office of Scientific and Technical Information (OSTI.GOV)

Salvignol, I.; Calvas, P.; Blancher, A.

1995-03-01

Rh-related transcripts present in bone marrow samples from several species of nonhuman primates (chimpanzee, gorilla, gibbon, crab-eating macaque) have been amplified by RT-polymerase chain reaction using primers deduced from the sequence of human RH genes. Nucleotide sequence analysis of the nonhuman transcripts revealed a high degree of similarity to human blood group Rh sequences, suggesting a great conservation of the RH genes throughout evolution. Full-length transcripts, potentially encoding 417 amino acid long proteins homologous to Rh polypeptides, were characterized, as well as mRNA isoforms which harbored nucleotide deletions or insertions and potentially encode truncated proteins. Proteins of 30-40,000 M{sub r},more » immunologically related to human Rh proteins, were detected by western blot analysis with antipeptide antibodies, indicating that Rh-like transcripts are translated into membrane proteins. Comparison of human and nonhuman protein sequences was pivotal in clarifying the molecular basis of the blood group C/c polymorphism, showing that only the Pro103Ser substitution was correlated with C/c polymorphism. In addition, it was shown that a proline residue at position 102 was critical in the expression of C and c epitopes, most likely by providing an appropriate conformation of Rh polypeptides. From these data a phylogenetic reconstruction of the RH locus evolution has been calculated from which an unrooted phylogenetic tree could be proposed, indicating that African ape Rh-like genes would be closer to the human RhD gene than to the human RhCE gene. 55 refs., 4 figs., 1 tab.« less

20-Hydroxyecdysone (20E) Primary Response Gene E75 Isoforms Mediate Steroidogenesis Autoregulation and Regulate Developmental Timing in Bombyx*

PubMed Central

Li, Kang; Tian, Ling; Guo, Zhongjian; Guo, Sanyou; Zhang, Jianzhen; Gu, Shi-Hong; Palli, Subba R.; Cao, Yang; Li, Sheng

2016-01-01

The temporal control mechanisms that precisely control animal development remain largely elusive. The timing of major developmental transitions in insects, including molting and metamorphosis, is coordinated by the steroid hormone 20-hydroxyecdysone (20E). 20E involves feedback loops to maintain pulses of ecdysteroid biosynthesis leading to its upsurge, whereas the underpinning molecular mechanisms are not well understood. Using the silkworm Bombyx mori as a model, we demonstrated that E75, the 20E primary response gene, mediates a regulatory loop between ecdysteroid biosynthesis and 20E signaling. E75 isoforms A and C directly bind to retinoic acid receptor-related response elements in Halloween gene promoter regions to induce gene expression thus promoting ecdysteroid biosynthesis and developmental transition, whereas isoform B antagonizes the transcriptional activity of isoform A/C through physical interaction. As the expression of E75 isoforms is differentially induced by 20E, the E75-mediated regulatory loop represents a fine autoregulation of steroidogenesis, which contributes to the precise control of developmental timing. PMID:27365399

A structured sparse regression method for estimating isoform expression level from multi-sample RNA-seq data.

PubMed

Zhang, L; Liu, X J

2016-06-03

With the rapid development of next-generation high-throughput sequencing technology, RNA-seq has become a standard and important technique for transcriptome analysis. For multi-sample RNA-seq data, the existing expression estimation methods usually deal with each single-RNA-seq sample, and ignore that the read distributions are consistent across multiple samples. In the current study, we propose a structured sparse regression method, SSRSeq, to estimate isoform expression using multi-sample RNA-seq data. SSRSeq uses a non-parameter model to capture the general tendency of non-uniformity read distribution for all genes across multiple samples. Additionally, our method adds a structured sparse regularization, which not only incorporates the sparse specificity between a gene and its corresponding isoform expression levels, but also reduces the effects of noisy reads, especially for lowly expressed genes and isoforms. Four real datasets were used to evaluate our method on isoform expression estimation. Compared with other popular methods, SSRSeq reduced the variance between multiple samples, and produced more accurate isoform expression estimations, and thus more meaningful biological interpretations.

Characterization of the heterooligomeric red-type rubisco activase from red algae

PubMed Central

Loganathan, Nitin; Tsai, Yi-Chin Candace; Mueller-Cajar, Oliver

2016-01-01

The photosynthetic CO2-fixing enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (rubisco) is inhibited by nonproductive binding of its substrate ribulose-1,5-bisphosphate (RuBP) and other sugar phosphates. Reactivation requires ATP-hydrolysis–powered remodeling of the inhibited complexes by diverse molecular chaperones known as rubisco activases (Rcas). Eukaryotic phytoplankton of the red plastid lineage contain so-called red-type rubiscos, some of which have been shown to possess superior kinetic properties to green-type rubiscos found in higher plants. These organisms are known to encode multiple homologs of CbbX, the α-proteobacterial red-type activase. Here we show that the gene products of two cbbX genes encoded by the nuclear and plastid genomes of the red algae Cyanidioschyzon merolae are nonfunctional in isolation, but together form a thermostable heterooligomeric Rca that can use both α-proteobacterial and red algal-inhibited rubisco complexes as a substrate. The mechanism of rubisco activation appears conserved between the bacterial and the algal systems and involves threading of the rubisco large subunit C terminus. Whereas binding of the allosteric regulator RuBP induces oligomeric transitions to the bacterial activase, it merely enhances the kinetics of ATP hydrolysis in the algal enzyme. Mutational analysis of nuclear and plastid isoforms demonstrates strong coordination between the subunits and implicates the nuclear-encoded subunit as being functionally dominant. The plastid-encoded subunit may be catalytically inert. Efforts to enhance crop photosynthesis by transplanting red algal rubiscos with enhanced kinetics will need to take into account the requirement for a compatible Rca. PMID:27872295

Characterization of the heterooligomeric red-type rubisco activase from red algae.

PubMed

Loganathan, Nitin; Tsai, Yi-Chin Candace; Mueller-Cajar, Oliver

2016-12-06

The photosynthetic CO 2 -fixing enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (rubisco) is inhibited by nonproductive binding of its substrate ribulose-1,5-bisphosphate (RuBP) and other sugar phosphates. Reactivation requires ATP-hydrolysis-powered remodeling of the inhibited complexes by diverse molecular chaperones known as rubisco activases (Rcas). Eukaryotic phytoplankton of the red plastid lineage contain so-called red-type rubiscos, some of which have been shown to possess superior kinetic properties to green-type rubiscos found in higher plants. These organisms are known to encode multiple homologs of CbbX, the α-proteobacterial red-type activase. Here we show that the gene products of two cbbX genes encoded by the nuclear and plastid genomes of the red algae Cyanidioschyzon merolae are nonfunctional in isolation, but together form a thermostable heterooligomeric Rca that can use both α-proteobacterial and red algal-inhibited rubisco complexes as a substrate. The mechanism of rubisco activation appears conserved between the bacterial and the algal systems and involves threading of the rubisco large subunit C terminus. Whereas binding of the allosteric regulator RuBP induces oligomeric transitions to the bacterial activase, it merely enhances the kinetics of ATP hydrolysis in the algal enzyme. Mutational analysis of nuclear and plastid isoforms demonstrates strong coordination between the subunits and implicates the nuclear-encoded subunit as being functionally dominant. The plastid-encoded subunit may be catalytically inert. Efforts to enhance crop photosynthesis by transplanting red algal rubiscos with enhanced kinetics will need to take into account the requirement for a compatible Rca.

MicroRNA-281 regulates the expression of ecdysone receptor (EcR) isoform B in the silkworm, Bombyx mori

USDA-ARS?s Scientific Manuscript database

Hundreds of Bombyx mori miRNAs had been identified in recent years, but their function in vivo remains poorly understood. The silkworm EcR gene (BmEcR) has three transcriptional isoforms, A, B1 and B2. Isoform sequences are different in the 3’UTR region of the gene, which is the case only in insects...

Functional redundancy and nonredundancy between two Troponin C isoforms in Drosophila adult muscles

PubMed Central

Chechenova, Maria B.; Maes, Sara; Oas, Sandy T.; Nelson, Cloyce; Kiani, Kaveh G.; Bryantsev, Anton L.; Cripps, Richard M.

2017-01-01

We investigated the functional overlap of two muscle Troponin C (TpnC) genes that are expressed in the adult fruit fly, Drosophila melanogaster: TpnC4 is predominantly expressed in the indirect flight muscles (IFMs), whereas TpnC41C is the main isoform in the tergal depressor of the trochanter muscle (TDT; jump muscle). Using CRISPR/Cas9, we created a transgenic line with a homozygous deletion of TpnC41C and compared its phenotype to a line lacking functional TpnC4. We found that the removal of either of these genes leads to expression of the other isoform in both muscle types. The switching between isoforms occurs at the transcriptional level and involves minimal enhancers located upstream of the transcription start points of each gene. Functionally, the two TpnC isoforms were not equal. Although ectopic TpnC4 in TDT muscles was able to maintain jumping ability, TpnC41C in IFMs could not effectively support flying. Simultaneous functional disruption of both TpnC genes resulted in jump-defective and flightless phenotypes of the survivors, as well as abnormal sarcomere organization. These results indicated that TpnC is required for myofibril assembly, and that there is functional specialization among TpnC isoforms in Drosophila. PMID:28077621

Divergent functional isoforms drive niche specialisation for nutrient acquisition and use in rumen microbiome

PubMed Central

Rubino, Francesco; Carberry, Ciara; M Waters, Sinéad; Kenny, David; McCabe, Matthew S; Creevey, Christopher J

2017-01-01

Many microbes in complex competitive environments share genes for acquiring and utilising nutrients, questioning whether niche specialisation exists and if so, how it is maintained. We investigated the genomic signatures of niche specialisation in the rumen microbiome, a highly competitive, anaerobic environment, with limited nutrient availability determined by the biomass consumed by the host. We generated individual metagenomic libraries from 14 cows fed an ad libitum diet of grass silage and calculated functional isoform diversity for each microbial gene identified. The animal replicates were used to calculate confidence intervals to test for differences in diversity of functional isoforms between microbes that may drive niche specialisation. We identified 153 genes with significant differences in functional isoform diversity between the two most abundant bacterial genera in the rumen (Prevotella and Clostridium). We found Prevotella possesses a more diverse range of isoforms capable of degrading hemicellulose, whereas Clostridium for cellulose. Furthermore, significant differences were observed in key metabolic processes indicating that isoform diversity plays an important role in maintaining their niche specialisation. The methods presented represent a novel approach for untangling complex interactions between microorganisms in natural environments and have resulted in an expanded catalogue of gene targets central to rumen cellulosic biomass degradation. PMID:28085156

Role of JNK isoforms in the kainic acid experimental model of epilepsy and neurodegeneration.

PubMed

Auladell, Carme; de Lemos, Luisa; Verdaguer, Ester; Ettcheto, Miren; Busquets, Oriol; Lazarowski, Alberto; Beas-Zarate, Carlos; Olloquequi, Jordi; Folch, Jaume; Camins, Antoni

2017-01-01

Chemoconvulsants that induce status epilepticus in rodents have been widely used over the past decades due to their capacity to reproduce with high similarity neuropathological and electroencephalographic features observed in patients with temporal lobe epilepsy (TLE). Kainic acid  is one of the most used chemoconvulsants in experimental models. KA administration mainly induces neuronal loss in the hippocampus. We focused the present review inthe c-Jun N-terminal kinase-signaling pathway (JNK), since it has been shown to play a key role in the process of neuronal death following KA activation. Among the three isoforms of JNK (JNK1, JNK2, JNK3), JNK3 is widely localized in the majority of areas of the hippocampus, whereas JNK1 levels are located exclusively in the CA3 and CA4 areas and in dentate gyrus. Disruption of the gene encoding JNK3 in mice renders neuroprotection to KA, since these animals showed a reduction in seizure activity and a diminution in hippocampal neuronal apoptosis. In light of this, JNK3 could be a promising subcellular target for future therapeutic interventions in epilepsy.

Cloning and expression of sheep DNA methyltransferase 1 and its development-specific isoform.

PubMed

Taylor, Jane; Moore, Hannah; Beaujean, Nathalie; Gardner, John; Wilmut, Ian; Meehan, Richard; Young, Lorraine

2009-05-01

Unlike the mouse embryo, where loss of DNA methylation in the embryonic nucleus leaves cleavage stage embryos globally hypomethylated, sheep preimplantation embryos retain high levels of methylation until the blastocyst stage. We have cloned and sequenced sheep Dnmt1 and found it to be highly conserved with both the human and mouse homologues. Furthermore, we observed that the transcript normally expressed in adult somatic tissues is highly abundant in sheep oocytes. Throughout sheep preimplantation development the protein is retained in the cytoplasm whereas Dnmt1 transcript production declines after the embryonic genome activation at the 8-16 cell stage. Attempts to clone oocyte-specific 5' regions of Dnmt1, known to be present in the mouse and human gene, were unsuccessful. However, a novel ovine Dnmt1 exon, theoretically encoding 13 amino acids, was found to be expressed in sheep oocytes, preimplantation embryos and early fetal lineages, but not in the adult tissue. RNAi-mediated knockdown of this novel transcript resulted in embryonic developmental arrest at the late morula stage, suggesting an essential role for this isoform in sheep blastocyst formation. (c) 2008 Wiley-Liss, Inc.

NOVA2-mediated RNA regulation is required for axonal pathfinding during development.

PubMed

Saito, Yuhki; Miranda-Rottmann, Soledad; Ruggiu, Matteo; Park, Christopher Y; Fak, John J; Zhong, Ru; Duncan, Jeremy S; Fabella, Brian A; Junge, Harald J; Chen, Zhe; Araya, Roberto; Fritzsch, Bernd; Hudspeth, A J; Darnell, Robert B

2016-05-25

The neuron specific RNA-binding proteins NOVA1 and NOVA2 are highly homologous alternative splicing regulators. NOVA proteins regulate at least 700 alternative splicing events in vivo, yet relatively little is known about the biologic consequences of NOVA action and in particular about functional differences between NOVA1 and NOVA2. Transcriptome-wide searches for isoform-specific functions, using NOVA1 and NOVA2 specific HITS-CLIP and RNA-seq data from mouse cortex lacking either NOVA isoform, reveals that NOVA2 uniquely regulates alternative splicing events of a series of axon guidance related genes during cortical development. Corresponding axonal pathfinding defects were specific to NOVA2 deficiency: Nova2-/- but not Nova1-/- mice had agenesis of the corpus callosum, and axonal outgrowth defects specific to ventral motoneuron axons and efferent innervation of the cochlea. Thus we have discovered that NOVA2 uniquely regulates alternative splicing of a coordinate set of transcripts encoding key components in cortical, brainstem and spinal axon guidance/outgrowth pathways during neural differentiation, with severe functional consequences in vivo.

A mitochondrial alkaline/neutral invertase isoform (A/N-InvC) functions in developmental energy-demanding processes in Arabidopsis.

PubMed

Martín, Mariana L; Lechner, Leandra; Zabaleta, Eduardo J; Salerno, Graciela L

2013-03-01

Recent findings demonstrate that alkaline/neutral invertases (A/N-Invs), enzymes that catalyze the breakdown of sucrose into glucose and fructose, are essential proteins in plant life. The fact that different isoforms are present in multiple locations makes them candidates for the coordination of metabolic processes. In the present study, we functionally characterized the encoding gene of a novel A/N-Inv (named A/N-InvC) from Arabidopsis, which localizes in mitochondria. A/N-InvC is expressed in roots, in aerial parts (shoots and leaves) and flowers. A detailed phenotypic analysis of knockout mutant plants (invc) reveals an impaired growth phenotype. Shoot growth was severely reduced, but root development was not affected as reported for A/N-InvA mutant (inva) plants. Remarkably, germination and flowering, two energy demanding processes, were the most affected stages. The effect of exogenous growth regulators led us to suggest that A/N-InvC may be modulating hormone balance in relation to the radicle emergence. We also show that oxygen consumption is reduced in inva and invc in comparison with wild-type plants, indicating that both organelle isoenzymes may play a fundamental role in mitochondrion functionality. Taken together, our results emphasize the involvement of mitochondrial A/N-Invs in developmental processes and uncover the possibility of playing different roles for the two isoforms located in the organelle.

Heterologous expression of a rice metallothionein isoform (OsMTI-1b) in Saccharomyces cerevisiae enhances cadmium, hydrogen peroxide and ethanol tolerance.

PubMed

Ansarypour, Zahra; Shahpiri, Azar

Metallothioneins are a superfamily of low-molecular-weight, cysteine (Cys)-rich proteins that are believed to play important roles in protection against metal toxicity and oxidative stress. The main purpose of this study was to investigate the effect of heterologous expression of a rice metallothionein isoform (OsMTI-1b) on the tolerance of Saccharomyces cerevisiae to Cd 2+ , H 2 O 2 and ethanol stress. The gene encoding OsMTI-1b was cloned into p426GPD as a yeast expression vector. The new construct was transformed to competent cells of S. cerevisiae. After verification of heterologous expression of OsMTI-1b, the new strain and control were grown under stress conditions. In comparison to control strain, the transformed S. cerevisiae cells expressing OsMTI-1b showed more tolerance to Cd 2+ and accumulated more Cd 2+ ions when they were grown in the medium containing CdCl 2 . In addition, the heterologous expression of GST-OsMTI-1b conferred H 2 O 2 and ethanol tolerance to S. cerevisiae cells. The results indicate that heterologous expression of plant MT isoforms can enhance the tolerance of S. cerevisiae to multiple stresses. Copyright © 2017 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

Enoyl-Acyl Carrier Protein Reductase I (FabI) Is Essential for the Intracellular Growth of Listeria monocytogenes

PubMed Central

Ericson, Megan E.; Frank, Matthew W.

2016-01-01

Enoyl-acyl carrier protein reductase catalyzes the last step in each elongation cycle of type II bacterial fatty acid synthesis and is a key regulatory protein in bacterial fatty acid synthesis. Genes of the facultative intracellular pathogen Listeria monocytogenes encode two functional enoyl-acyl carrier protein isoforms based on their ability to complement the temperature-sensitive growth phenotype of Escherichia coli strain JP1111 [fabI(Ts)]. The FabI isoform was inactivated by the FabI selective inhibitor AFN-1252, but the FabK isoform was not affected by the drug, as expected. Inhibition of FabI by AFN-1252 decreased endogenous fatty acid synthesis by 80% and lowered the growth rate of L. monocytogenes in laboratory medium. Robust exogenous fatty acid incorporation was not detected in L. monocytogenes unless the pathway was partially inactivated by AFN-1252 treatment. However, supplementation with exogenous fatty acids did not restore normal growth in the presence of AFN-1252. FabI inactivation prevented the intracellular growth of L. monocytogenes, showing that neither FabK nor the incorporation of host cellular fatty acids was sufficient to support the intracellular growth of L. monocytogenes. Our results show that FabI is the primary enoyl-acyl carrier protein reductase of type II bacterial fatty acid synthesis and is essential for the intracellular growth of L. monocytogenes. PMID:27736774

Molecular structure and differential function of choline kinases CHKα and CHKβ in musculoskeletal system and cancer.

PubMed

Chen, Xi; Qiu, Heng; Wang, Chao; Yuan, Yu; Tickner, Jennifer; Xu, Jiake; Zou, Jun

2017-02-01

Choline, a hydrophilic cation, has versatile physiological roles throughout the body, including cholinergic neurotransmission, memory consolidation and membrane biosynthesis and metabolism. Choline kinases possess enzyme activity that catalyses the conversion of choline to phosphocholine, which is further converted to cytidine diphosphate-coline (CDP-choline) in the biosynthesis of phosphatidylcholine (PC). PC is a major constituent of the phospholipid bilayer which constitutes the eukaryotic cell membrane, and regulates cell signal transduction. Choline Kinase consists of three isoforms, CHKα1, CHKα2 and CHKβ, encoded by two separate genes (CHKA(Human)/Chka(Mouse) and CHKB(Human)/Chkb(Mouse)). Both isoforms have similar structures and enzyme activity, but display some distinct molecular structural domains and differential tissue expression patterns. Whilst Choline Kinase was discovered in early 1950, its pivotal role in the development of muscular dystrophy, bone deformities, and cancer has only recently been identified. CHKα has been proposed as a cancer biomarker and its inhibition as an anti-cancer therapy. In contrast, restoration of CHKβ deficiency through CDP-choline supplements like citicoline may be beneficial for the treatment of muscular dystrophy, bone metabolic diseases, and cognitive conditions. The molecular structure and expression pattern of Choline Kinase, the differential roles of Choline Kinase isoforms and their potential as novel therapeutic targets for muscular dystrophy, bone deformities, cognitive conditions and cancer are discussed. Copyright © 2016. Published by Elsevier Ltd.

mRNA Quantification of NIPBL Isoforms A and B in Adult and Fetal Human Tissues, and a Potentially Pathological Variant Affecting Only Isoform A in Two Patients with Cornelia de Lange Syndrome

PubMed Central

Puisac, Beatriz; Teresa-Rodrigo, María-Esperanza; Hernández-Marcos, María; Baquero-Montoya, Carolina; Gil-Rodríguez, María-Concepción; Visnes, Torkild; Bot, Christopher; Gómez-Puertas, Paulino; Kaiser, Frank J.; Ramos, Feliciano J.; Ström, Lena; Pié, Juan

2017-01-01

Cornelia de Lange syndrome (CdLS) is a congenital developmental disorder characterized by craniofacial dysmorphia, growth retardation, limb malformations, and intellectual disability. Approximately 60% of patients with CdLS carry a recognizable pathological variant in the NIPBL gene, of which two isoforms, A and B, have been identified, and which only differ in the C-terminal segment. In this work, we describe the distribution pattern of the isoforms A and B mRNAs in tissues of adult and fetal origin, by qPCR (quantitative polymerase chain reaction). Our results show a higher gene expression of the isoform A, even though both seem to have the same tissue distribution. Interestingly, the expression in fetal tissues is higher than that of adults, especially in brain and skeletal muscle. Curiously, the study of fibroblasts of two siblings with a mild CdLS phenotype and a pathological variant specific of the isoform A of NIPBL (c.8387A > G; P.Tyr2796Cys), showed a similar reduction in both isoforms, and a normal sensitivity to DNA damage. Overall, these results suggest that the position of the pathological variant at the 3´ end of the NIPBL gene affecting only isoform A, is likely to be the cause of the atypical mild phenotype of the two brothers. PMID:28241484

Steroid receptor RNA activator (SRA1): unusual bifaceted gene products with suspected relevance to breast cancer

PubMed Central

Leygue, Etienne

2007-01-01

The steroid receptor RNA activator (SRA) is a unique modulator of steroid receptor transcriptional activity, as it is able to mediate its coregulatory effects as a RNA molecule. Recent findings, however, have painted a more complex picture of the SRA gene (SRA1) products. Indeed, even though SRA was initially thought to be noncoding, several RNA isoforms have now been found to encode an endogenous protein (SRAP), which is well conserved among Chordata. Although the function of SRAP remains largely unknown, it has been proposed that, much like its corresponding RNA, the protein itself might regulate estrogen and androgen receptor signaling pathways. As such, data suggest that both SRA and SRAP might participate in the mechanisms underlying breast, as well as prostate tumorigenesis. This review summarizes the published literature dealing with these two faces of the SRA gene products and underscores the relevance of this bifaceted system to breast cancer development. PMID:17710122

Short- and long-term memory are modulated by multiple isoforms of the fragile X mental retardation protein.

PubMed

Banerjee, Paromita; Schoenfeld, Brian P; Bell, Aaron J; Choi, Catherine H; Bradley, Michael P; Hinchey, Paul; Kollaros, Maria; Park, Jae H; McBride, Sean M J; Dockendorff, Thomas C

2010-05-12

The diversity of protein isoforms arising from alternative splicing is thought to modulate fine-tuning of synaptic plasticity. Fragile X mental retardation protein (FMRP), a neuronal RNA binding protein, exists in isoforms as a result of alternative splicing, but the contribution of these isoforms to neural plasticity are not well understood. We show that two isoforms of Drosophila melanogaster FMRP (dFMR1) have differential roles in mediating neural development and behavior functions conferred by the dfmr1 gene. These isoforms differ in the presence of a protein interaction module that is related to prion domains and is functionally conserved between FMRPs. Expression of both isoforms is necessary for optimal performance in tests of short- and long-term memory of courtship training. The presence or absence of the protein interaction domain may govern the types of ribonucleoprotein (RNP) complexes dFMR1 assembles into, with different RNPs regulating gene expression in a manner necessary for establishing distinct phases of memory formation.

Transcription patterns of genes encoding four metallothionein homologs in Daphnia pulex exposed to copper and cadmium are time- and homolog- dependent

PubMed Central

Asselman, Jana; Shaw, Joseph R.; Glaholt, Stephen P.; Colbourne, John K.; De Schamphelaere, Karel AC.

2013-01-01

Metallothioneins are proteins that play an essential role in metal homeostasis and detoxification in nearly all organisms studied to date. Yet discrepancies between outcomes of chronic and acute exposure experiments hamper the understanding of the regulatory mechanisms of their isoforms following metal exposure. Here, we investigated transcriptional differences among four identified homologs (mt1–mt4) in Daphnia pulex exposed across time to copper and cadmium relative to a control. Transcriptional upregulation of mt1 and mt3 was detected on day four following exposure to cadmium, whereas that of mt2 and mt4 was detected on day two and day eight following exposure to copper. These results confirm temporal and metal-specific differences in the transcriptional induction of genes encoding metallothionein homologs upon metal exposure which should be considered in ecotoxicological monitoring programs of metal-contaminated water bodies. Indeed, the mRNA expression patterns observed here illustrate the complex regulatory system associated with metallothioneins, as these patterns are not only dependent on the metal, but also on exposure time and the homolog studied. Further phylogenetic analysis and analysis of regulatory elements in upstream promoter regions revealed a high degree of similarity between metallothionein genes of Daphnia pulex and Daphnia magna, a species belonging to the same genus. These findings, combined with a limited amount of available expression data for D. magna metallothionein genes, tentatively suggest a potential generalization of the metallothionein response system between these Daphnia species. PMID:24113165

Insights into rubber biosynthesis from transcriptome analysis of Hevea brasiliensis latex.

PubMed

Chow, Keng-See; Wan, Kiew-Lian; Isa, Mohd Noor Mat; Bahari, Azlina; Tan, Siang-Hee; Harikrishna, K; Yeang, Hoong-Yeet

2007-01-01

Hevea brasiliensis is the most widely cultivated species for commercial production of natural rubber (cis-polyisoprene). In this study, 10,040 expressed sequence tags (ESTs) were generated from the latex of the rubber tree, which represents the cytoplasmic content of a single cell type, in order to analyse the latex transcription profile with emphasis on rubber biosynthesis-related genes. A total of 3,441 unique transcripts (UTs) were obtained after quality editing and assembly of EST sequences. Functional classification of UTs according to the Gene Ontology convention showed that 73.8% were related to genes of unknown function. Among highly expressed ESTs, a significant proportion encoded proteins related to rubber biosynthesis and stress or defence responses. Sequences encoding rubber particle membrane proteins (RPMPs) belonging to three protein families accounted for 12% of the ESTs. Characterization of these ESTs revealed nine RPMP variants (7.9-27 kDa) including the 14 kDa REF (rubber elongation factor) and 22 kDa SRPP (small rubber particle protein). The expression of multiple RPMP isoforms in latex was shown using antibodies against REF and SRPP. Both EST and quantitative reverse transcription-PCR (QRT-PCR) analyses demonstrated REF and SRPP to be the most abundant transcripts in latex. Besides rubber biosynthesis, comparative sequence analysis showed that the RPMPs are highly similar to sequences in the plant kingdom having stress-related functions. Implications of the RPMP function in cis-polyisoprene biosynthesis in the context of transcript abundance and differential gene expression are discussed.

Molecular and biochemical characterization of two tungsten- and selenium-containing formate dehydrogenases from Eubacterium acidaminophilum that are associated with components of an iron-only hydrogenase.

PubMed

Graentzdoerffer, Andrea; Rauh, David; Pich, Andreas; Andreesen, Jan R

2003-01-01

Two gene clusters encoding similar formate dehydrogenases (FDH) were identified in Eubacterium acidaminophilum. Each cluster is composed of one gene coding for a catalytic subunit ( fdhA-I, fdhA-II) and one for an electron-transferring subunit ( fdhB-I, fdhB-II). Both fdhA genes contain a TGA codon for selenocysteine incorporation and the encoded proteins harbor five putative iron-sulfur clusters in their N-terminal region. Both FdhB subunits resemble the N-terminal region of FdhA on the amino acid level and contain five putative iron-sulfur clusters. Four genes thought to encode the subunits of an iron-only hydrogenase are located upstream of the FDH gene cluster I. By sequence comparison, HymA and HymB are predicted to contain one and four iron-sulfur clusters, respectively, the latter protein also binding sites for FMN and NAD(P). Thus, HymA and HymB seem to represent electron-transferring subunits, and HymC the putative catalytic subunit containing motifs for four iron-sulfur clusters and one H-cluster specific for Fe-only hydrogenases. HymD has six predicted transmembrane helices and might be an integral membrane protein. Viologen-dependent FDH activity was purified from serine-grown cells of E. acidaminophilum and the purified protein complex contained four subunits, FdhA and FdhB, encoded by FDH gene cluster II, and HymA and HymB, identified after determination of their N-terminal sequences. Thus, this complex might represent the most simple type of a formate hydrogen lyase. The purified formate dehydrogenase fraction contained iron, tungsten, a pterin cofactor, and zinc, but no molybdenum. FDH-II had a two-fold higher K(m) for formate (0.37 mM) than FDH-I and also catalyzed CO(2) reduction to formate. Reverse transcription (RT)-PCR pointed to increased expression of FDH-II in serine-grown cells, supporting the isolation of this FDH isoform. The fdhA-I gene was expressed as inactive protein in Escherichia coli. The in-frame UGA codon for selenocysteine incorporation was read in the heterologous system only as stop codon, although its potential SECIS element exhibited a quite high similarity to that of E. coli FDH.

Functional divergences of GAPDH isoforms during early development in two perciform fish species.

PubMed

Sarropoulou, Elena; Nousdili, Dimitra; Kotoulas, Georgios; Magoulas, Antonios

2011-12-01

Glyceraldehyde-3-phospate dehydrogenase (GAPDH) is involved in basic cell catabolic processes and, as it is thought to be continuously expressed, belongs to the group of housekeeping genes. Thus, it is frequently used as an internal control in quantitative gene expression studies. However, the evidence of different expression patterns in a broad range of organisms and tissues, as well as the occurrence of different isoforms, shows that GAPDH has to be reevaluated as an internal control in qPCR studies, and its annotation has to be enriched. GAPDH has been shown to be involved in the pathway of energy and carbon molecule supply as well as in transcription and apoptosis. In the present study, we isolated the two isoforms, GAPDH-1 and GAPDH-2, of the gilthead sea bream (Sparus aurata) and the European sea bass (Dicentrarchus labrax). We inferred the phylogenetic relationships to ten other fish species and gave the gene structure of both genes. We further investigated gene expression analysis in both species for different developmental stages showing divergent gene expression of the two isoforms and the possible function of GAPDH-1 as a maternal gene.

APPRIS: annotation of principal and alternative splice isoforms

PubMed Central

Rodriguez, Jose Manuel; Maietta, Paolo; Ezkurdia, Iakes; Pietrelli, Alessandro; Wesselink, Jan-Jaap; Lopez, Gonzalo; Valencia, Alfonso; Tress, Michael L.

2013-01-01

Here, we present APPRIS (http://appris.bioinfo.cnio.es), a database that houses annotations of human splice isoforms. APPRIS has been designed to provide value to manual annotations of the human genome by adding reliable protein structural and functional data and information from cross-species conservation. The visual representation of the annotations provided by APPRIS for each gene allows annotators and researchers alike to easily identify functional changes brought about by splicing events. In addition to collecting, integrating and analyzing reliable predictions of the effect of splicing events, APPRIS also selects a single reference sequence for each gene, here termed the principal isoform, based on the annotations of structure, function and conservation for each transcript. APPRIS identifies a principal isoform for 85% of the protein-coding genes in the GENCODE 7 release for ENSEMBL. Analysis of the APPRIS data shows that at least 70% of the alternative (non-principal) variants would lose important functional or structural information relative to the principal isoform. PMID:23161672

A novel single nucleotide splice site mutation in FHL1 confirms an Emery-Dreifuss plus phenotype with pulmonary artery hypoplasia and facial dysmorphology.

PubMed

Pen, Anja E; Nyegaard, Mette; Fang, Mingyan; Jiang, Hui; Christensen, Rikke; Mølgaard, Henning; Andersen, Henning; Ulhøi, Benedicte Parm; Østergaard, John R; Væth, Signe; Sommerlund, Mette; de Brouwer, Arjan P M; Zhang, Xiuqing; Jensen, Uffe B

2015-04-01

We describe a Danish family with an, until recently, unknown X-linked disease with muscular dystrophy (MD), facial dysmorphology and pulmonary artery hypoplasia. One patient died suddenly before age 20 and another was resuscitated from cardiac arrest at the age of 28. Linkage analysis pointed to a region of 25 Mb from 123.6 Mb to 148.4 Mb on chromosome X containing over 100 genes. Exome sequencing identified a single nucleotide splice site mutation c.502-2A > T, which is located 5' to exon 6 in the gene encoding four and a half LIM domain 1 (FHL1) protein. FHL1 expresses three main splice variants, known as FHL1A, FHL1B and FHL1C. In healthy individuals, FHL1A is the predominant splice variant and is mainly found in skeletal and cardiac muscle. The FHL1 transcript profiles from two affected individuals were investigated in skin fibroblasts with quantitative real-time PCR. This demonstrated loss of isoform A and B, and an almost 200-fold overexpression of isoform C confirming that lack of FHL1A and overexpression of FHL1C results in an extended phenotype of EDMD as recently shown by Tiffin et al. [2013]. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Exonic Deletions in AUTS2 Cause a Syndromic Form of Intellectual Disability and Suggest a Critical Role for the C Terminus

PubMed Central

Beunders, Gea; Voorhoeve, Els; Golzio, Christelle; Pardo, Luba M.; Rosenfeld, Jill A.; Talkowski, Michael E.; Simonic, Ingrid; Lionel, Anath C.; Vergult, Sarah; Pyatt, Robert E.; van de Kamp, Jiddeke; Nieuwint, Aggie; Weiss, Marjan M.; Rizzu, Patrizia; Verwer, Lucilla E.N.I.; van Spaendonk, Rosalina M.L.; Shen, Yiping; Wu, Bai-lin; Yu, Tingting; Yu, Yongguo; Chiang, Colby; Gusella, James F.; Lindgren, Amelia M.; Morton, Cynthia C.; van Binsbergen, Ellen; Bulk, Saskia; van Rossem, Els; Vanakker, Olivier; Armstrong, Ruth; Park, Soo-Mi; Greenhalgh, Lynn; Maye, Una; Neill, Nicholas J.; Abbott, Kristin M.; Sell, Susan; Ladda, Roger; Farber, Darren M.; Bader, Patricia I.; Cushing, Tom; Drautz, Joanne M.; Konczal, Laura; Nash, Patricia; de Los Reyes, Emily; Carter, Melissa T.; Hopkins, Elizabeth; Marshall, Christian R.; Osborne, Lucy R.; Gripp, Karen W.; Thrush, Devon Lamb; Hashimoto, Sayaka; Gastier-Foster, Julie M.; Astbury, Caroline; Ylstra, Bauke; Meijers-Heijboer, Hanne; Posthuma, Danielle; Menten, Björn; Mortier, Geert; Scherer, Stephen W.; Eichler, Evan E.; Girirajan, Santhosh; Katsanis, Nicholas; Groffen, Alexander J.; Sistermans, Erik A.

2013-01-01

Genomic rearrangements involving AUTS2 (7q11.22) are associated with autism and intellectual disability (ID), although evidence for causality is limited. By combining the results of diagnostic testing of 49,684 individuals, we identified 24 microdeletions that affect at least one exon of AUTS2, as well as one translocation and one inversion each with a breakpoint within the AUTS2 locus. Comparison of 17 well-characterized individuals enabled identification of a variable syndromic phenotype including ID, autism, short stature, microcephaly, cerebral palsy, and facial dysmorphisms. The dysmorphic features were more pronounced in persons with 3′ AUTS2 deletions. This part of the gene is shown to encode a C-terminal isoform (with an alternative transcription start site) expressed in the human brain. Consistent with our genetic data, suppression of auts2 in zebrafish embryos caused microcephaly that could be rescued by either the full-length or the C-terminal isoform of AUTS2. Our observations demonstrate a causal role of AUTS2 in neurocognitive disorders, establish a hitherto unappreciated syndromic phenotype at this locus, and show how transcriptional complexity can underpin human pathology. The zebrafish model provides a valuable tool for investigating the etiology of AUTS2 syndrome and facilitating gene-function analysis in the future. PMID:23332918

Functional analysis of the Hikeshi-like protein and its interaction with HSP70 in Arabidopsis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Koizumi, Shinya; Ohama, Naohiko; Mizoi, Junya

2014-07-18

Highlights: • HKL, a Hikeshi homologous gene is identified in Arabidopsis. • HKL interacts with two HSP70 isoforms and regulates the subcellular localization of HSC70-1. • The two HSP70 translocate into nucleus in response to heat stress. • Overexpression of HKL confers thermotolerance in transgenic plants. - Abstract: Heat shock proteins (HSPs) refold damaged proteins and are an essential component of the heat shock response. Previously, the 70 kDa heat shock protein (HSP70) has been reported to translocate into the nucleus in a heat-dependent manner in many organisms. In humans, the heat-induced translocation of HSP70 requires the nuclear carrier proteinmore » Hikeshi. In the Arabidopsis genome, only one gene encodes a protein with high homology to Hikeshi, and we named this homolog Hikeshi-like (HKL) protein. In this study, we show that two Arabidopsis HSP70 isoforms accumulate in the nucleus in response to heat shock and that HKL interacts with these HSP70s. Our histochemical analysis revealed that HKL is predominantly expressed in meristematic tissues, suggesting the potential importance of HKL during cell division in Arabidopsis. In addition, we show that HKL regulates HSP70 localization, and HKL overexpression conferred thermotolerance to transgenic Arabidopsis plants. Our results suggest that HKL plays a positive role in the thermotolerance of Arabidopsis plants and cooperatively interacts with HSP70.« less

Presynaptic congenital myasthenic syndrome with a homozygous sequence variant in LAMA5 combines myopia, facial tics, and failure of neuromuscular transmission.

PubMed

Maselli, Ricardo A; Arredondo, Juan; Vázquez, Jessica; Chong, Jessica X; Bamshad, Michael J; Nickerson, Deborah A; Lara, Marian; Ng, Fiona; Lo, Victoria L; Pytel, Peter; McDonald, Craig M

2017-08-01

Defects in genes encoding the isoforms of the laminin alpha subunit have been linked to various phenotypic manifestations, including brain malformations, muscular dystrophy, ocular defects, cardiomyopathy, and skin abnormalities. We report here a severe defect of neuromuscular transmission in a consanguineous patient with a homozygous variant in the laminin alpha-5 subunit gene (LAMA5). The variant c.8046C>T (p.Arg2659Trp) is rare and has a predicted deleterious effect. The affected individual, who also carries a rare homozygous sequence variant in LAMA1, had muscle weakness, myopia, and facial tics. Magnetic resonance imaging of brain showed mild volume loss and periventricular T2 prolongation. Repetitive nerve stimulation revealed 50% decrement of compound muscle action potential amplitudes and 250% facilitation immediately after exercise, Endplate studies identified a profound reduction of the endplate potential quantal content and endplates with normal postsynaptic folding that were denuded or partially occupied by small nerve terminals. Expression studies revealed that p.Arg2659Trp caused decreased binding of laminin alpha-5 to SV2A and impaired laminin-521 cell-adhesion and cell projection support in primary neuronal cultures. In summary, this report describing severe neuromuscular transmission failure in a patient with a LAMA5 mutation expands the list of phenotypes associated with defects in genes encoding alpha-laminins. © 2017 Wiley Periodicals, Inc.

TFIIH subunit alterations causing xeroderma pigmentosum and trichothiodystrophy specifically disturb several steps during transcription.

PubMed

Singh, Amita; Compe, Emanuel; Le May, Nicolas; Egly, Jean-Marc

2015-02-05

Mutations in genes encoding the ERCC3 (XPB), ERCC2 (XPD), and GTF2H5 (p8 or TTD-A) subunits of the transcription and DNA-repair factor TFIIH lead to three autosomal-recessive disorders: xeroderma pigmentosum (XP), XP associated with Cockayne syndrome (XP/CS), and trichothiodystrophy (TTD). Although these diseases were originally associated with defects in DNA repair, transcription deficiencies might be also implicated. By using retinoic acid receptor beta isoform 2 (RARB2) as a model in several cells bearing mutations in genes encoding TFIIH subunits, we observed that (1) the recruitment of the TFIIH complex was altered at the activated RARB2 promoter, (2) TFIIH participated in the recruitment of nucleotide excision repair (NER) factors during transcription in a manner different from that observed during NER, and (3) the different TFIIH variants disturbed transcription by having distinct consequences on post-translational modifications of histones, DNA-break induction, DNA demethylation, and gene-loop formation. The transition from heterochromatin to euchromatin was disrupted depending on the variant, illustrating the fact that TFIIH, by contributing to NER factor recruitment, orchestrates chromatin remodeling. The subtle transcriptional differences found between various TFIIH variants thus participate in the phenotypic variability observed among XP, XP/CS, and TTD individuals. Copyright © 2015 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

Transgenic Expression of the piRNA-Resistant Masculinizer Gene Induces Female-Specific Lethality and Partial Female-to-Male Sex Reversal in the Silkworm, Bombyx mori.

PubMed

Sakai, Hiroki; Sumitani, Megumi; Chikami, Yasuhiko; Yahata, Kensuke; Uchino, Keiro; Kiuchi, Takashi; Katsuma, Susumu; Aoki, Fugaku; Sezutsu, Hideki; Suzuki, Masataka G

2016-08-01

In Bombyx mori (B. mori), Fem piRNA originates from the W chromosome and is responsible for femaleness. The Fem piRNA-PIWI complex targets and cleaves mRNAs transcribed from the Masc gene. Masc encodes a novel CCCH type zinc-finger protein and is required for male-specific splicing of B. mori doublesex (Bmdsx) transcripts. In the present study, several silkworm strains carrying a transgene, which encodes a Fem piRNA-resistant Masc mRNA (Masc-R), were generated. Forced expression of the Masc-R transgene caused female-specific lethality during the larval stages. One of the Masc-R strains weakly expressed Masc-R in various tissues. Females heterozygous for the transgene expressed male-specific isoform of the Bombyx homolog of insulin-like growth factor II mRNA-binding protein (ImpM) and Bmdsx. All examined females showed a lower inducibility of vitellogenin synthesis and exhibited abnormalities in the ovaries. Testis-like tissues were observed in abnormal ovaries and, notably, the tissues contained considerable numbers of sperm bundles. Homozygous expression of the transgene resulted in formation of the male-specific abdominal segment in adult females and caused partial male differentiation in female genitalia. These results strongly suggest that Masc is an important regulatory gene of maleness in B. mori.

Z-disc-associated, Alternatively Spliced, PDZ Motif-containing Protein (ZASP) Mutations in the Actin-binding Domain Cause Disruption of Skeletal Muscle Actin Filaments in Myofibrillar Myopathy*

PubMed Central

Lin, Xiaoyan; Ruiz, Janelle; Bajraktari, Ilda; Ohman, Rachel; Banerjee, Soojay; Gribble, Katherine; Kaufman, Joshua D.; Wingfield, Paul T.; Griggs, Robert C.; Fischbeck, Kenneth H.; Mankodi, Ami

2014-01-01

The core of skeletal muscle Z-discs consists of actin filaments from adjacent sarcomeres that are cross-linked by α-actinin homodimers. Z-disc-associated, alternatively spliced, PDZ motif-containing protein (ZASP)/Cypher interacts with α-actinin, myotilin, and other Z-disc proteins via the PDZ domain. However, these interactions are not sufficient to maintain the Z-disc structure. We show that ZASP directly interacts with skeletal actin filaments. The actin-binding domain is between the modular PDZ and LIM domains. This ZASP region is alternatively spliced so that each isoform has unique actin-binding domains. All ZASP isoforms contain the exon 6-encoded ZASP-like motif that is mutated in zaspopathy, a myofibrillar myopathy (MFM), whereas the exon 8–11 junction-encoded peptide is exclusive to the postnatal long ZASP isoform (ZASP-LΔex10). MFM is characterized by disruption of skeletal muscle Z-discs and accumulation of myofibrillar degradation products. Wild-type and mutant ZASP interact with α-actin, α-actinin, and myotilin. Expression of mutant, but not wild-type, ZASP leads to Z-disc disruption and F-actin accumulation in mouse skeletal muscle, as in MFM. Mutations in the actin-binding domain of ZASP-LΔex10, but not other isoforms, cause disruption of the actin cytoskeleton in muscle cells. These isoform-specific mutation effects highlight the essential role of the ZASP-LΔex10 isoform in F-actin organization. Our results show that MFM-associated ZASP mutations in the actin-binding domain have deleterious effects on the core structure of the Z-discs in skeletal muscle. PMID:24668811

Arabidopsis thaliana RALF1 opposes brassinosteroid effects on root cell elongation and lateral root formation

PubMed Central

Moura, Daniel S.

2014-01-01

Rapid alkalinization factor (RALF) is a peptide signal that plays a basic role in cell biology and most likely regulates cell expansion. In this study, transgenic Arabidopsis thaliana lines with high and low levels of AtRALF1 transcripts were used to investigate this peptide’s mechanism of action. Overexpression of the root-specific isoform AtRALF1 resulted in reduced cell size. Conversely, AtRALF1 silencing increased root length by increasing the size of root cells. AtRALF1-silenced plants also showed an increase in the number of lateral roots, whereas AtRALF1 overexpression produced the opposite effect. In addition, four AtRALF1-inducible genes were identified: two genes encoding proline-rich proteins (AtPRP1 and AtPRP3), one encoding a hydroxyproline-rich glycoprotein (AtHRPG2), and one encoding a xyloglucan endotransglucosylase (TCH4). These genes were expressed in roots and involved in cell-wall rearrangement, and their induction was concentration dependent. Furthermore, AtRALF1-overexpressing plants were less sensitive to exogenous brassinolide (BL); upon BL treatment, the plants showed no increase in root length and a compromised increase in hypocotyl elongation. In addition, the treatment had no effect on the number of emerged lateral roots. AtRALF1 also induces two brassinosteroid (BR)-downregulated genes involved in the BR biosynthetic pathway: the cytochrome P450 monooxygenases CONSTITUTIVE PHOTOMORPHISM AND DWARFISM (CPD) and DWARF4 (DWF4). Simultaneous treatment with both AtRALF1 and BL caused a reduction in AtRALF1-inducible gene expression levels, suggesting that these signals may compete for components shared by both pathways. Taken together, these results indicate an opposing effect of AtRALF1 and BL, and suggest that RALF’s mechanism of action could be to interfere with the BR signalling pathway. PMID:24620000

TP53INP1 is a novel p73 target gene that induces cell cycle arrest and cell death by modulating p73 transcriptional activity.

PubMed

Tomasini, Richard; Seux, Mylène; Nowak, Jonathan; Bontemps, Caroline; Carrier, Alice; Dagorn, Jean-Charles; Pébusque, Marie-Josèphe; Iovanna, Juan L; Dusetti, Nelson J

2005-12-08

TP53INP1 is an alternatively spliced gene encoding two nuclear protein isoforms (TP53INP1alpha and TP53INP1beta), whose transcription is activated by p53. When overexpressed, both isoforms induce cell cycle arrest in G1 and enhance p53-mediated apoptosis. TP53INP1s also interact with the p53 gene and regulate p53 transcriptional activity. We report here that TP53INP1 expression is induced during experimental acute pancreatitis in p53-/- mice and in cisplatin-treated p53-/- mouse embryo fibroblasts (MEFs). We demonstrate that ectopic expression of p73, a p53 homologue, leads to TP53INP1 induction in p53-deficient cells. In turn, TP53INP1s alters the transactivation capacity of p73 on several p53-target genes, including TP53INP1 itself, demonstrating a functional association between p73 and TP53INP1s. Also, when overexpressed in p53-deficient cells, TP53INP1s inhibit cell growth and promote cell death as assessed by cell cycle analysis and colony formation assays. Finally, we show that TP53INP1s potentiate the capacity of p73 to inhibit cell growth, that effect being prevented when the p53 mutant R175H is expressed or when p73 expression is blocked by a siRNA. These results suggest that TP53INP1s are functionally associated with p73 to regulate cell cycle progression and apoptosis, independently from p53.

Molecular codes for neuronal individuality and cell assembly in the brain

PubMed Central

Yagi, Takeshi

2012-01-01

The brain contains an enormous, but finite, number of neurons. The ability of this limited number of neurons to produce nearly limitless neural information over a lifetime is typically explained by combinatorial explosion; that is, by the exponential amplification of each neuron's contribution through its incorporation into “cell assemblies” and neural networks. In development, each neuron expresses diverse cellular recognition molecules that permit the formation of the appropriate neural cell assemblies to elicit various brain functions. The mechanism for generating neuronal assemblies and networks must involve molecular codes that give neurons individuality and allow them to recognize one another and join appropriate networks. The extensive molecular diversity of cell-surface proteins on neurons is likely to contribute to their individual identities. The clustered protocadherins (Pcdh) is a large subfamily within the diverse cadherin superfamily. The clustered Pcdh genes are encoded in tandem by three gene clusters, and are present in all known vertebrate genomes. The set of clustered Pcdh genes is expressed in a random and combinatorial manner in each neuron. In addition, cis-tetramers composed of heteromultimeric clustered Pcdh isoforms represent selective binding units for cell-cell interactions. Here I present the mathematical probabilities for neuronal individuality based on the random and combinatorial expression of clustered Pcdh isoforms and their formation of cis-tetramers in each neuron. Notably, clustered Pcdh gene products are known to play crucial roles in correct axonal projections, synaptic formation, and neuronal survival. Their molecular and biological features induce a hypothesis that the diverse clustered Pcdh molecules provide the molecular code by which neuronal individuality and cell assembly permit the combinatorial explosion of networks that supports enormous processing capability and plasticity of the brain. PMID:22518100

Characterization of the canine mda-7 gene, transcripts and expression patterns

PubMed Central

Sandey, Maninder; Bird, R. Curtis; Das, Swadesh K.; Sarkar, Devanand; Curiel, David T.; Fisher, Paul B.; Smith, Bruce F.

2014-01-01

Human melanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24) displays potent growth suppressing and cell killing activity against a wide variety of human and rodent cancer cells. In this study, we identified a canine ortholog of the human mda-7/IL-24 gene located within a cluster of IL-10 family members on chromosome 7. The full-length mRNA sequence of canine mda-7 was determined, which encodes a 186-amino acid protein that has 66% similarity to human MDA-7/IL-24. Canine MDA-7 is constitutively expressed in cultured normal canine epidermal keratinocytes (NCEKs), and its expression levels are increased after lipopolysaccharide stimulation. In cultured NCEKs, the canine mda-7 pre-mRNA is differentially spliced, via exon skipping and alternate 5′-splice donor sites, to yield five splice variants (canine mda-7sv1, canine mda-7sv2, canine mda-7sv3, canine mda-7sv4 and canine mda-7sv5) that encode four protein isoforms of the canine MDA-7 protein. These protein isoforms have a conserved N-terminus (signal peptide sequence) and are dissimilar in amino acid sequences at their C-terminus. Canine MDA-7 is not expressed in primary canine tumor samples, and most tumor derived cancer cell lines tested, like its human counterpart. Unlike human MDA-7/IL-24, canine mda-7 mRNA is not expressed in unstimulated or lipopolysaccharide (LPS), concanavalin A (ConA) or phytohemagglutinin (PHA) stimulated canine peripheral blood mononuclear cells (PBMCs). Furthermore, in-silico analysis revealed that canonical canine MDA-7 has a potential 28 amino acid signal peptide sequence that can target it for active secretion. This data suggests that canine mda-7 is indeed an ortholog of human mda-7/IL-24, its protein product has high amino acid similarity to human MDA-7/IL-24 protein and it may possess similar biological properties to human MDA-7/IL-24, but its expression pattern is more restricted than its human ortholog. PMID:24865935

Differential expression of growth factors at the cellular level in virus-infected brain

PubMed Central

Prosniak, Mikhail; Zborek, Anna; Scott, Gwen S.; Roy, Anirban; Phares, Timothy W.; Koprowski, Hilary; Hooper, D. Craig

2003-01-01

The contribution of host factors to rabies virus (RV) transcription/replication and axonal/transsynaptic spread is largely unknown. We previously identified several host genes that are up-regulated in the mouse brain during RV infection, including neuroleukin, which is involved in neuronal growth and survival, cell motility, and differentiation, and fibroblast growth factor homologous factor 4 (FHF4), which has been implicated in limb and nervous system development. In this study, we used real-time quantitative RT-PCR to assess the expression of mRNAs specific for neuroleukin, the two isoforms of FHF4 (FHF4-1a and -1b) encoded by the FHF4 gene, and N protein of RV in neurons and astrocytes isolated by laser capture microdissection from mouse brains infected with the laboratory-adapted RV strain CVS-N2c or with a street RV of silver-haired bat origin. Differences in the gene expression patterns suggest that the capacity of RV strains to infect nonneuronal cells and differentially modulate host gene expression may be important in virus replication and spread in the CNS. PMID:12736376

Characterization of fusion genes and the significantly expressed fusion isoforms in breast cancer by hybrid sequencing.

PubMed

Weirather, Jason L; Afshar, Pegah Tootoonchi; Clark, Tyson A; Tseng, Elizabeth; Powers, Linda S; Underwood, Jason G; Zabner, Joseph; Korlach, Jonas; Wong, Wing Hung; Au, Kin Fai

2015-10-15

We developed an innovative hybrid sequencing approach, IDP-fusion, to detect fusion genes, determine fusion sites and identify and quantify fusion isoforms. IDP-fusion is the first method to study gene fusion events by integrating Third Generation Sequencing long reads and Second Generation Sequencing short reads. We applied IDP-fusion to PacBio data and Illumina data from the MCF-7 breast cancer cells. Compared with the existing tools, IDP-fusion detects fusion genes at higher precision and a very low false positive rate. The results show that IDP-fusion will be useful for unraveling the complexity of multiple fusion splices and fusion isoforms within tumorigenesis-relevant fusion genes. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Active FOXO1 is a Key Determinant of Isoform-Specific Progesterone Receptor Transactivation and Senescence Programming

PubMed Central

Diep, Caroline H.; Knutson, Todd P.; Lange, Carol A.

2015-01-01

Progesterone promotes differentiation coupled to proliferation and pro-survival in the breast, but inhibits estrogen-driven growth in the reproductive tract and ovaries. Herein, it is demonstrated, using progesterone receptor (PR) isoform-specific ovarian cancer model systems, that PR-A and PR-B promote distinct gene expression profiles that differ from PR-driven genes in breast cancer cells. In ovarian cancer models, PR-A primarily regulates genes independently of progestin, while PR-B is the dominant ligand-dependent isoform. Notably, FOXO1 and the PR/FOXO1 target-gene p21 (CDKN1A) are repressed by PR-A, but induced by PR-B. In the presence of progestin, PR-B, but not PR-A, robustly induced cellular senescence via FOXO1-dependent induction of p21 and p15 (CDKN2B). Chromatin immunoprecipitation (ChIP) assays performed on PR-isoform specific cells demonstrated that while each isoform is recruited to the same PRE-containing region of the p21 promoter in response to progestin, only PR-B elicits active chromatin marks. Overexpression of constitutively active FOXO1 in PR-A-expressing cells conferred robust ligand-dependent upregulation of the PR-B target genes GZMA, IGFBP1, and p21, and induced cellular senescence. In the presence of endogenous active FOXO1, PR-A was phosphorylated on Ser294 and transactivated PR-B at PR-B target genes; these events were blocked by the FOXO1 inhibitor (AS1842856). PR isoform-specific regulation of the FOXO1/p21 axis recapitulated in human primary ovarian tumor explants treated with progestin; loss of progestin sensitivity correlated with high AKT activity. PMID:26577046

Casein Kinase I Isoform Hrr25 Is a Negative Regulator of Haa1 in the Weak Acid Stress Response Pathway in Saccharomyces cerevisiae.

PubMed

Collins, Morgan E; Black, Joshua J; Liu, Zhengchang

2017-07-01

Haa1 is a transcription factor that adapts Saccharomyces cerevisiae cells to weak organic acid stresses by activating the expression of various genes. Many of these genes encode membrane proteins, such as TPO2 and YRO2 How Haa1 is activated by weak acids is not clear. Here, we show that casein kinase I isoform Hrr25 is an important negative regulator of Haa1. Haa1 is known to be multiply phosphorylated. We found that mutations in HRR25 lead to reduced Haa1 phosphorylation and increased expression of Haa1 target genes and that Hrr25 interacts with Haa1. The other three casein kinase I isoforms, Yck1, Yck2, and Yck3, do not seem to play critical roles in Haa1 regulation. Hrr25 has a 200-residue C-terminal region, including a proline- and glutamine-rich domain. Our data suggest that the C-terminal region of Hrr25 is required for normal inhibition of expression of Haa1 target genes TPO2 and YRO2 and is important for cell growth but is not required for cell morphogenesis. We propose that Hrr25 is an important regulator of cellular adaptation to weak acid stress by inhibiting Haa1 through phosphorylation. IMPORTANCE Our study has revealed the casein kinase I protein Hrr25 to be a negative regulator of Haa1, a transcription factor mediating the cellular response to stresses caused by weak acids. Many studies have focused on the target genes of Haa1 and their roles in weak acid stress responses, but little has been reported on the regulatory mechanism of Haa1. Weak acids, such as acetic acid, have long been used for food preservation by slowing down the growth of fungal species, including S. cerevisiae In the biofuel industry, acetic acid in the lignocellulosic hydrolysates limits the production of ethanol, which is undesirable. By understanding how Haa1 is regulated, we can make advances in the field of food sciences to better preserve food and engineer acetic acid-resistant strains that will increase productivity in the biofuel industry. Copyright © 2017 American Society for Microbiology.

Isoform-level gene expression patterns in single-cell RNA-sequencing data.

PubMed

Vu, Trung Nghia; Wills, Quin F; Kalari, Krishna R; Niu, Nifang; Wang, Liewei; Pawitan, Yudi; Rantalainen, Mattias

2018-02-27

RNA sequencing of single cells enables characterization of transcriptional heterogeneity in seemingly homogeneous cell populations. Single-cell sequencing has been applied in a wide range of researches fields. However, few studies have focus on characterization of isoform-level expression patterns at the single-cell level. In this study we propose and apply a novel method, ISOform-Patterns (ISOP), based on mixture modeling, to characterize the expression patterns of isoform pairs from the same gene in single-cell isoform-level expression data. We define six principal patterns of isoform expression relationships and describe a method for differential-pattern analysis. We demonstrate ISOP through analysis of single-cell RNA-sequencing data from a breast cancer cell line, with replication in three independent datasets. We assigned the pattern types to each of 16,562 isoform-pairs from 4,929 genes. Among those, 26% of the discovered patterns were significant (p<0.05), while remaining patterns are possibly effects of transcriptional bursting, drop-out and stochastic biological heterogeneity. Furthermore, 32% of genes discovered through differential-pattern analysis were not detected by differential-expression analysis. The effect of drop-out events, mean expression level, and properties of the expression distribution on the performances of ISOP were also investigated through simulated datasets. To conclude, ISOP provides a novel approach for characterization of isoformlevel preference, commitment and heterogeneity in single-cell RNA-sequencing data. The ISOP method has been implemented as a R package and is available at https://github.com/nghiavtr/ISOP under a GPL-3 license. mattias.rantalainen@ki.se. Supplementary data are available at Bioinformatics online.

Brassicaceae Express Multiple Isoforms of Biotin Carboxyl Carrier Protein in a Tissue-Specific Manner1

PubMed Central

Thelen, Jay J.; Mekhedov, Sergei; Ohlrogge, John B.

2001-01-01

Plastidial acetyl-coenzyme A carboxylase from most plants is a multi-enzyme complex comprised of four different subunits. One of these subunits, the biotin carboxyl carrier protein (BCCP), was previously proposed to be encoded by a single gene in Arabidopsis. We report and characterize here a second Arabidopsis BCCP (AtBCCP2) cDNA with 42% amino acid identity to AtBCCP1 and 75% identity to a class of oilseed rape (Brassica napus) BCCPs. Both Arabidopsis BCCP isoforms were expressed in Escherichia coli and found to be biotinylated and supported carboxylation activity when reconstituted with purified, recombinant Arabidopsis biotin carboxylase. In vitro translated AtBCCP2 was competent for import into pea (Pisum sativum) chloroplasts and processed to a 25-kD polypeptide. Extracts of Arabidopsis seeds contained biotinylated polypeptides of 35 and 25 kD, in agreement with the masses of recombinant AtBCCP1 and 2, respectively. AtBCCP1 protein was present in developing tissues from roots, leaves, flowers, siliques, and seeds, whereas AtBCCP2 protein was primarily expressed in 7 to 10 d-after-flowering seeds at levels approximately 2-fold less abundant than AtBCCP1. AtBCCP1 transcript reflected these protein expression profiles present in all developing organs and highest in 14-d leaves and siliques, whereas AtBCCP2 transcript was present in flowers and siliques. In protein blots, four different BCCP isoforms were detected in developing seeds from oilseed rape. Of these, a 35-kD BCCP was detected in immature leaves and developing seeds, whereas developing seeds also contained 22-, 25-, and 37-kD isoforms highly expressed 21 d after flowering. These data indicate that oilseed plants in the family Brassicaceae contain at least one to three seed-up-regulated BCCP isoforms, depending upon genome complexity. PMID:11299381

High resolution structure of cleaved Serpin 42 Da from Drosophila melanogaster.

PubMed

Ellisdon, Andrew M; Zhang, Qingwei; Henstridge, Michelle A; Johnson, Travis K; Warr, Coral G; Law, Ruby Hp; Whisstock, James C

2014-04-24

The Drosophila melanogaster Serpin 42 Da gene (previously Serpin 4) encodes a serine protease inhibitor that is capable of remarkable functional diversity through the alternative splicing of four different reactive centre loop exons. Eight protein isoforms of Serpin 42 Da have been identified to date, targeting the protease inhibitor to both different proteases and cellular locations. Biochemical and genetic studies suggest that Serpin 42 Da inhibits target proteases through the classical serpin 'suicide' inhibition mechanism, however the crystal structure of a representative Serpin 42 Da isoform remains to be determined. We report two high-resolution crystal structures of Serpin 42 Da representing the A/B isoforms in the cleaved conformation, belonging to two different space-groups and diffracting to 1.7 Å and 1.8 Å. Structural analysis reveals the archetypal serpin fold, with the major elements of secondary structure displaying significant homology to the vertebrate serpin, neuroserpin. Key residues known to have central roles in the serpin inhibitory mechanism are conserved in both the hinge and shutter regions of Serpin 42 Da. Furthermore, these structures identify important conserved interactions that appear to be of crucial importance in allowing the Serpin 42 Da fold to act as a versatile template for multiple reactive centre loops that have different sequences and protease specificities. In combination with previous biochemical and genetic studies, these structures confirm for the first time that the Serpin 42 Da isoforms are typical inhibitory serpin family members with the conserved serpin fold and inhibitory mechanism. Additionally, these data reveal the remarkable structural plasticity of serpins, whereby the basic fold is harnessed as a template for inhibition of a large spectrum of proteases by reactive centre loop exon 'switching'. This is the first structure of a Drosophila serpin reported to date, and will provide a platform for future mutational studies in Drosophila to ascertain the functional role of each of the Serpin 42 Da isoforms.

Reassimilation of Photorespiratory Ammonium in Lotus japonicus Plants Deficient in Plastidic Glutamine Synthetase

PubMed Central

Pérez-Delgado, Carmen M.; García-Calderón, Margarita; Márquez, Antonio J.; Betti, Marco

2015-01-01

It is well established that the plastidic isoform of glutamine synthetase (GS2) is the enzyme in charge of photorespiratory ammonium reassimilation in plants. The metabolic events associated to photorespiratory NH4 + accumulation were analyzed in a Lotus japonicus photorespiratory mutant lacking GS2. The mutant plants accumulated high levels of NH4 + when photorespiration was active, followed by a sudden drop in the levels of this compound. In this paper it was examined the possible existence of enzymatic pathways alternative to GS2 that could account for this decline in the photorespiratory ammonium. Induction of genes encoding for cytosolic glutamine synthetase (GS1), glutamate dehydrogenase (GDH) and asparagine synthetase (ASN) was observed in the mutant in correspondence with the diminishment of NH4 +. Measurements of gene expression, polypeptide levels, enzyme activity and metabolite levels were carried out in leaf samples from WT and mutant plants after different periods of time under active photorespiratory conditions. In the case of asparagine synthetase it was not possible to determine enzyme activity and polypeptide content; however, an increased asparagine content in parallel with the induction of ASN gene expression was detected in the mutant plants. This increase in asparagine levels took place concomitantly with an increase in glutamine due to the induction of cytosolic GS1 in the mutant, thus revealing a major role of cytosolic GS1 in the reassimilation and detoxification of photorespiratory NH4 + when the plastidic GS2 isoform is lacking. Moreover, a diminishment in glutamate levels was observed, that may be explained by the induction of NAD(H)-dependent GDH activity. PMID:26091523

Cloning of apg-2 encoding a novel member of heat shock protein 110 family.

PubMed

Kaneko, Y; Kimura, T; Kishishita, M; Noda, Y; Fujita, J

1997-04-11

Chinese hamster heat shock protein 110-encoding gene (hsp110), mouse apg-1 and human hsp70RY are structurally related genes, with the first two encoding about 110-kDa HSPs [Yoon et al. (1995) J. Biol. Chem. 270, 15725-15733; Kaneko et al. (1997) J. Biol. Chem., in press; Fathallah et al. (1993) J. Immunol. 151, 810-813]. Using apg-1 cDNA as a probe, we isolated a novel cDNA, apg-2 from a mouse testis cDNA library, which was highly homologous to human hsp70RY. However, the predicted amino acid (aa) sequence of APG-2 was longer (841 aa) than that of HSP70RY (701 aa) and comparable to those of HSP110 and APG-1. Northern blot analysis revealed that the expression of apg-2 transcripts was ubiquitous in various mouse tissues, and most abundant in the testis and ovary. While induction of hsp70 transcripts was observed in mouse TAMA26 Sertoli cells and NIH/3T3 fibroblasts on temperature shift from 37 degrees C to 42 degrees C (traditional heat shock) or from 32 degrees C to 39 degrees C, apg-2 transcripts were not induced under either condition. These results suggest that apg-2 is an isoform of mouse homolog of hsp70RY, but that it belongs to the hsp110 family instead of hsp70 family, and that it plays a role under non-stress conditions.

The origin and evolution of human glutaminases and their atypical C-terminal ankyrin repeats

DOE PAGES

Pasquali, Camila Cristina; Islam, Zeyaul; Adamoski, Douglas; ...

2017-05-19

On the basis of tissue-specific enzyme activity and inhibition by catalytic products, Hans Krebs first demonstrated the existence of multiple glutaminases in mammals. Currently, two human genes are known to encode at least four glutaminase isoforms. But, the phylogeny of these medically relevant enzymes remains unclear, prompting us to investigate their origin and evolution. Using prokaryotic and eukaryotic glutaminase sequences, we built a phylogenetic tree whose topology suggested that the multidomain architecture was inherited from bacterial ancestors, probably simultaneously with the hosting of the proto-mitochondrion endosymbiont. We propose an evolutionary model wherein the appearance of the most active enzyme isoform,more » glutaminase C (GAC), which is expressed in many cancers, was a late retrotransposition event that occurred in fishes from the Chondrichthyes class. The ankyrin (ANK) repeats in the glutaminases were acquired early in their evolution. In order to obtain information on ANK folding, we solved two high-resolution structures of the ANK repeat-containing C termini of both kidney-type glutaminase (KGA) and GLS2 isoforms (glutaminase B and liver-type glutaminase). We also found that the glutaminase ANK repeats form unique intramolecular contacts through two highly conserved motifs; curiously, this arrangement occludes a region usually involved in ANK-mediated protein-protein interactions. We also solved the crystal structure of full-length KGA and present a small-angle X-ray scattering model for full-length GLS2. These structures explain these proteins' compromised ability to assemble into catalytically active supra-tetrameric filaments, as previously shown for GAC. Collectively, these results provide information about glutaminases that may aid in the design of isoform-specific glutaminase inhibitors.« less

The novel Solanum tuberosum calcium dependent protein kinase, StCDPK3, is expressed in actively growing organs.

PubMed

Grandellis, Carolina; Giammaria, Verónica; Bialer, Magalí; Santin, Franco; Lin, Tian; Hannapel, David J; Ulloa, Rita M

2012-12-01

Calcium-dependent protein kinases (CDPKs) are key components of calcium regulated signaling cascades in plants. In this work, isoform StCDPK3 from Solanum tuberosum was studied and fully described. StCDPK3 encodes a 63 kDa protein with an N-terminal variable domain (NTV), rich in prolines and glutamines, which presents myristoylation and palmitoylation consensus sites and a PEST sequence indicative of rapid protein degradation. StCDPK3 gene (circa 11 kb) is localized in chromosome 3, shares the eight exons and seven introns structure with other isoforms from subgroup IIa and contains an additional intron in the 5'UTR region. StCDPK3 expression is ubiquitous being transcripts more abundant in early elongating stolons (ES), leaves and roots, however isoform specific antibodies only detected the protein in leaf particulate extracts. The recombinant 6xHis-StCDPK3 is an active kinase that differs in its kinetic parameters and calcium requirements from StCDPK1 and 2 isoforms. In vitro, StCDPK3 undergoes autophosphorylation regardless of the addition of calcium. The StCDPK3 promoter region (circa 1,800 bp) was subcloned by genome walking and fused to GUS. Light and ABRE responsive elements were identified in the promoter region as well as elements associated to expression in roots. StCDPK3 expression was enhanced by ABA while GA decreased it. Potato transgenic lines harboring StCDPK3 promoter∷GUS construct were generated by Agrobacterium tumefaciens mediated plant transformation. Promoter activity was detected in leaves, root tips and branching points, early ES, tuber eyes and developing sprouts indicating that StCDPK3 is expressed in actively growing organs.

The origin and evolution of human glutaminases and their atypical C-terminal ankyrin repeats

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pasquali, Camila Cristina; Islam, Zeyaul; Adamoski, Douglas

On the basis of tissue-specific enzyme activity and inhibition by catalytic products, Hans Krebs first demonstrated the existence of multiple glutaminases in mammals. Currently, two human genes are known to encode at least four glutaminase isoforms. But, the phylogeny of these medically relevant enzymes remains unclear, prompting us to investigate their origin and evolution. Using prokaryotic and eukaryotic glutaminase sequences, we built a phylogenetic tree whose topology suggested that the multidomain architecture was inherited from bacterial ancestors, probably simultaneously with the hosting of the proto-mitochondrion endosymbiont. We propose an evolutionary model wherein the appearance of the most active enzyme isoform,more » glutaminase C (GAC), which is expressed in many cancers, was a late retrotransposition event that occurred in fishes from the Chondrichthyes class. The ankyrin (ANK) repeats in the glutaminases were acquired early in their evolution. In order to obtain information on ANK folding, we solved two high-resolution structures of the ANK repeat-containing C termini of both kidney-type glutaminase (KGA) and GLS2 isoforms (glutaminase B and liver-type glutaminase). We also found that the glutaminase ANK repeats form unique intramolecular contacts through two highly conserved motifs; curiously, this arrangement occludes a region usually involved in ANK-mediated protein-protein interactions. We also solved the crystal structure of full-length KGA and present a small-angle X-ray scattering model for full-length GLS2. These structures explain these proteins' compromised ability to assemble into catalytically active supra-tetrameric filaments, as previously shown for GAC. Collectively, these results provide information about glutaminases that may aid in the design of isoform-specific glutaminase inhibitors.« less

Novel causative mutations in patients with Nance-Horan syndrome and altered localization of the mutant NHS-A protein isoform

PubMed Central

Burdon, Kathryn P.; Dave, Alpana; Jamieson, Robyn V.; Yaron, Yuval; Billson, Frank; Van Maldergem, Lionel; Lorenz, Birgit; Gécz, Jozef; Craig, Jamie E.

2008-01-01

Purpose Nance-Horan syndrome is typically characterized by severe bilateral congenital cataracts and dental abnormalities. Truncating mutations in the Nance-Horan syndrome (NHS) gene cause this X-linked genetic disorder. NHS encodes two isoforms, NHS-A and NHS-1A. The ocular lens expresses NHS-A, the epithelial and neuronal cell specific isoform. The NHS-A protein localizes in the lens epithelium at the cellular periphery. The data to date suggest a role for this isoform at cell-cell junctions in epithelial cells. This study aimed to identify the causative mutations in new patients diagnosed with Nance-Horan syndrome and to investigate the effect of mutations on subcellular localization of the NHS-A protein. Methods All coding exons of NHS were screened for mutations by polymerase chain reaction (PCR) and sequencing. PCR-based mutagenesis was performed to introduce three independent mutations in the NHS-A cDNA. Expression and localization of the mutant proteins was determined in mammalian epithelial cells. Results Truncating mutations were found in 6 out of 10 unrelated patients from four countries. Each of four patients carried a novel mutation (R248X, P264fs, K1198fs, and I1302fs), and each of the two other patients carried two previously reported mutations (R373X and R879X). No mutation was found in the gene in four patients. Two disease-causing mutations (R134fs and R901X) and an artificial mutation (T1357fs) resulted in premature truncation of the NHS-A protein. All three mutant proteins failed to localize to the cellular periphery in epithelial cells and instead were found in the cytoplasm. Conclusions This study brings the total number of mutations identified in NHS to 18. The mislocalization of the mutant NHS-A protein, revealed by mutation analysis, is expected to adversely affect cell-cell junctions in epithelial cells such as the lens epithelium, which may explain cataractogenesis in Nance-Horan syndrome patients. Mutation analysis also shed light on the significance of NHS-A regions for its localization and, hence, its function at epithelial cell junctions. PMID:18949062

Novel causative mutations in patients with Nance-Horan syndrome and altered localization of the mutant NHS-A protein isoform.

PubMed

Sharma, Shiwani; Burdon, Kathryn P; Dave, Alpana; Jamieson, Robyn V; Yaron, Yuval; Billson, Frank; Van Maldergem, Lionel; Lorenz, Birgit; Gécz, Jozef; Craig, Jamie E

2008-01-01

Nance-Horan syndrome is typically characterized by severe bilateral congenital cataracts and dental abnormalities. Truncating mutations in the Nance-Horan syndrome (NHS) gene cause this X-linked genetic disorder. NHS encodes two isoforms, NHS-A and NHS-1A. The ocular lens expresses NHS-A, the epithelial and neuronal cell specific isoform. The NHS-A protein localizes in the lens epithelium at the cellular periphery. The data to date suggest a role for this isoform at cell-cell junctions in epithelial cells. This study aimed to identify the causative mutations in new patients diagnosed with Nance-Horan syndrome and to investigate the effect of mutations on subcellular localization of the NHS-A protein. All coding exons of NHS were screened for mutations by polymerase chain reaction (PCR) and sequencing. PCR-based mutagenesis was performed to introduce three independent mutations in the NHS-A cDNA. Expression and localization of the mutant proteins was determined in mammalian epithelial cells. Truncating mutations were found in 6 out of 10 unrelated patients from four countries. Each of four patients carried a novel mutation (R248X, P264fs, K1198fs, and I1302fs), and each of the two other patients carried two previously reported mutations (R373X and R879X). No mutation was found in the gene in four patients. Two disease-causing mutations (R134fs and R901X) and an artificial mutation (T1357fs) resulted in premature truncation of the NHS-A protein. All three mutant proteins failed to localize to the cellular periphery in epithelial cells and instead were found in the cytoplasm. This study brings the total number of mutations identified in NHS to 18. The mislocalization of the mutant NHS-A protein, revealed by mutation analysis, is expected to adversely affect cell-cell junctions in epithelial cells such as the lens epithelium, which may explain cataractogenesis in Nance-Horan syndrome patients. Mutation analysis also shed light on the significance of NHS-A regions for its localization and, hence, its function at epithelial cell junctions.

Isolation and characterization of a novel calmodulin-binding protein from potato

NASA Technical Reports Server (NTRS)

Reddy, Anireddy S N.; Day, Irene S.; Narasimhulu, S. B.; Safadi, Farida; Reddy, Vaka S.; Golovkin, Maxim; Harnly, Melissa J.

2002-01-01

Tuberization in potato is controlled by hormonal and environmental signals. Ca(2+), an important intracellular messenger, and calmodulin (CaM), one of the primary Ca(2+) sensors, have been implicated in controlling diverse cellular processes in plants including tuberization. The regulation of cellular processes by CaM involves its interaction with other proteins. To understand the role of Ca(2+)/CaM in tuberization, we have screened an expression library prepared from developing tubers with biotinylated CaM. This screening resulted in isolation of a cDNA encoding a novel CaM-binding protein (potato calmodulin-binding protein (PCBP)). Ca(2+)-dependent binding of the cDNA-encoded protein to CaM is confirmed by (35)S-labeled CaM. The full-length cDNA is 5 kb long and encodes a protein of 1309 amino acids. The deduced amino acid sequence showed significant similarity with a hypothetical protein from another plant, Arabidopsis. However, no homologs of PCBP are found in nonplant systems, suggesting that it is likely to be specific to plants. Using truncated versions of the protein and a synthetic peptide in CaM binding assays we mapped the CaM-binding region to a 20-amino acid stretch (residues 1216-1237). The bacterially expressed protein containing the CaM-binding domain interacted with three CaM isoforms (CaM2, CaM4, and CaM6). PCBP is encoded by a single gene and is expressed differentially in the tissues tested. The expression of CaM, PCBP, and another CaM-binding protein is similar in different tissues and organs. The predicted protein contained seven putative nuclear localization signals and several strong PEST motifs. Fusion of the N-terminal region of the protein containing six of the seven nuclear localization signals to the reporter gene beta-glucuronidase targeted the reporter gene to the nucleus, suggesting a nuclear role for PCBP.

Complementation for an essential ancillary nonstructural protein function across parvovirus genera

PubMed Central

Mihaylov, Ivailo S.; Cotmore, Susan F.; Tattersall, Peter

2014-01-01

Parvoviruses encode a small number of ancillary proteins that differ substantially between genera. Within the genus Protoparvovirus, minute virus of mice (MVM) encodes three isoforms of its ancillary protein NS2, while human bocavirus 1 (HBoV1), in the genus Bocaparvovirus, encodes an NP1 protein that is unrelated in primary sequence to MVM NS2. To search for functional overlap between NS2 and NP1, we generated murine A9 cell populations that inducibly express HBoV1 NP1. These were used to test whether NP1 expression could complement specific defects resulting from depletion of MVM NS2 isoforms. NP1 induction had little impact on cell viability or cell cycle progression in uninfected cells, and was unable to complement late defects in MVM virion production associated with low NS2 levels. However, NP1 did relocate to MVM replication centers, and supports both the normal expansion of these foci and overcomes the early paralysis of DNA replication in NS2-null infections. PMID:25194919

Pairwise comparisons of ten porcine tissues identify differential transcriptional regulation at the gene, isoform, promoter and transcription start site level

DOE Office of Scientific and Technical Information (OSTI.GOV)

Farajzadeh, Leila; Hornshøj, Henrik; Momeni, Jamal

Highlights: •Transcriptome sequencing yielded 223 mill porcine RNA-seq reads, and 59,000 transcribed locations. •Establishment of unique transcription profiles for ten porcine tissues including four brain tissues. •Comparison of transcription profiles at gene, isoform, promoter and transcription start site level. •Highlights a high level of regulation of neuro-related genes at both gene, isoform, and TSS level. •Our results emphasize the pig as a valuable animal model with respect to human biological issues. -- Abstract: The transcriptome is the absolute set of transcripts in a tissue or cell at the time of sampling. In this study RNA-Seq is employed to enable themore » differential analysis of the transcriptome profile for ten porcine tissues in order to evaluate differences between the tissues at the gene and isoform expression level, together with an analysis of variation in transcription start sites, promoter usage, and splicing. Totally, 223 million RNA fragments were sequenced leading to the identification of 59,930 transcribed gene locations and 290,936 transcript variants using Cufflinks with similarity to approximately 13,899 annotated human genes. Pairwise analysis of tissues for differential expression at the gene level showed that the smallest differences were between tissues originating from the porcine brain. Interestingly, the relative level of differential expression at the isoform level did generally not vary between tissue contrasts. Furthermore, analysis of differential promoter usage between tissues, revealed a proportionally higher variation between cerebellum (CBE) versus frontal cortex and cerebellum versus hypothalamus (HYP) than in the remaining comparisons. In addition, the comparison of differential transcription start sites showed that the number of these sites is generally increased in comparisons including hypothalamus in contrast to other pairwise assessments. A comprehensive analysis of one of the tissue contrasts, i.e. cerebellum versus heart for differential variation at the gene, isoform, and transcription start site (TSS), and promoter level showed that several of the genes differed at all four levels. Interestingly, these genes were mainly annotated to the “electron transport chain” and neuronal differentiation, emphasizing that “tissue important” genes are regulated at several levels. Furthermore, our analysis shows that the “across tissue approach” has a promising potential when screening for possible explanations for variations, such as those observed at the gene expression levels.« less

Oligomeric properties and DNA binding specificities of repressor isoforms from the Streptomyces bacteriophage phiC31.

PubMed

Wilson, S E; Smith, M C

1998-05-15

Three protein isoforms (74, 54 and 42 kDa) are expressed from repressor gene c in the Streptomyces temperate bacteriophage phiC31. Because expression of the two smaller isoforms, 54 and 42 kDa, is sufficient for superinfection immunity, the interaction between these isoforms was studied. The native 42 kDa repressor (Nat42) and an N-terminally 6x histidine-tagged 54 kDa isoform (His54) were shown by co-purification on a Ni-NTA column to interact in Streptomyces lividans . In vitro three repressor preparations, containing Nat42, His54 and the native 54 and 42 kDa isoforms expressed together (Nat54&42), were subjected to chemical crosslinking and gel filtration analysis. Homo- and hetero-tetramers were observed. Previous work showed that the smallest isoform bound to 17 bp operators containing aconservedinvertedrepeat (CIR) and that the CIRs were located at 16 loci throughout the phiC31 genome. One of the CIRs (CIR6) is believed to be critical for regulating the lytic pathway. The DNA binding activities of the three repressor preparations were studied using fragments containing CIRs (CIR3-CIR6) from the essential early region as templates for DNase I footprinting. Whereas Nat42 bound to CIR6, poorly to CIR5 but undetectably to CIR3 or CIR4, the Nat54&42 preparation could bind to all CIRs tested, albeit poorly to CIR3 and CIR4. The His54 isoform bound all CIRs tested. Isoforms expressed from the phiC31 repressor gene, like those which are expressed from many eukaryotic transcription factor genes, apparently have different binding specificities.

A global analysis of the complex landscape of isoforms and regulatory networks of p63 in human cells and tissues.

PubMed

Sethi, Isha; Romano, Rose-Anne; Gluck, Christian; Smalley, Kirsten; Vojtesek, Borivoj; Buck, Michael J; Sinha, Satrajit

2015-08-07

The transcription factor p63 belongs to the p53/p63/p73 family and plays key functional roles during normal epithelial development and differentiation and in pathological states such as squamous cell carcinomas. The human TP63 gene, located on chromosome 3q28 is driven by two promoters that generate the full-length transactivating (TA) and N-terminal truncated (ΔN) isoforms. Furthermore alternative splicing at the C-terminus gives rise to additional α, β, γ and likely several other minor variants. Teasing out the expression and biological function of each p63 variant has been both the focus of, and a cause for contention in the p63 field. Here we have taken advantage of a burgeoning RNA-Seq based genomic data-sets to examine the global expression profiles of p63 isoforms across commonly utilized human cell-lines and major tissues and organs. Consistent with earlier studies, we find ΔNp63 transcripts, primarily that of the ΔNp63α isoforms, to be expressed in most cells of epithelial origin such as those of skin and oral tissues, mammary glands and squamous cell carcinomas. In contrast, TAp63 is not expressed in the majority of normal cell-types and tissues; rather it is selectively expressed at moderate to high levels in a subset of Burkitt's and diffuse large B-cell lymphoma cell lines. We verify this differential expression pattern of p63 isoforms by Western blot analysis, using newly developed ΔN and TA specific antibodies. Furthermore using unsupervised clustering of human cell lines, tissues and organs, we show that ΔNp63 and TAp63 driven transcriptional networks involve very distinct sets of molecular players, which may underlie their different biological functions. In this study we report comprehensive and global expression profiles of p63 isoforms and their relationship to p53/p73 and other potential transcriptional co-regulators. We curate publicly available data generated in part by consortiums such as ENCODE, FANTOM and Human Protein Atlas to delineate the vastly different transcriptomic landscapes of ΔNp63 and TAp63. Our studies help not only in dispelling prevailing myths and controversies on p63 expression in commonly used human cell lines but also augur new isoform- and cell type-specific activities of p63.

Ultraconserved elements are associated with homeostatic control of splicing regulators by alternative splicing and nonsense-mediated decay

PubMed Central

Ni, Julie Z.; Grate, Leslie; Donohue, John Paul; Preston, Christine; Nobida, Naomi; O’Brien, Georgeann; Shiue, Lily; Clark, Tyson A.; Blume, John E.; Ares, Manuel

2007-01-01

Many alternative splicing events create RNAs with premature stop codons, suggesting that alternative splicing coupled with nonsense-mediated decay (AS-NMD) may regulate gene expression post-transcriptionally. We tested this idea in mice by blocking NMD and measuring changes in isoform representation using splicing-sensitive microarrays. We found a striking class of highly conserved stop codon-containing exons whose inclusion renders the transcript sensitive to NMD. A genomic search for additional examples identified >50 such exons in genes with a variety of functions. These exons are unusually frequent in genes that encode splicing activators and are unexpectedly enriched in the so-called “ultraconserved” elements in the mammalian lineage. Further analysis show that NMD of mRNAs for splicing activators such as SR proteins is triggered by splicing activation events, whereas NMD of the mRNAs for negatively acting hnRNP proteins is triggered by splicing repression, a polarity consistent with widespread homeostatic control of splicing regulator gene expression. We suggest that the extreme genomic conservation surrounding these regulatory splicing events within splicing factor genes demonstrates the evolutionary importance of maintaining tightly tuned homeostasis of RNA-binding protein levels in the vertebrate cell. PMID:17369403

A truncated, activin-induced Smad3 isoform acts as a transcriptional repressor of FSHβ expression in mouse pituitary

PubMed Central

Kim, So-Youn; Zhu, Jie; Woodruff, Teresa K.

2011-01-01

The receptor-regulated protein Smad3 is key player in the signaling cascade stimulated by the binding of activin to its cell surface receptor. Upon phosphorylation, Smad3 forms a heterocomplex with Smad2 and Smad4, translocates to the nucleus and acts as a transcriptional co-activator. We have identified a unique isoform of Smad3 that is expressed in mature pituitary gonadotropes. 5' RACE revealed that this truncated Smad3 isoform is transcribed from an ATG site within exon 4 and consists of 7 exons encoding half of the linker region and the MH2 region. In pituitary cells, the truncated Smad3 isoform was phosphorylated upon activin treatment, in a manner that was temporally distinct from the phosphorylation of full-length Smad3. Activin-induced phosphorylation of Smad3 and the truncated Smad3 isoform was blocked by both follistatin and siRNA-mediated knockdown of Smad3. The truncated Smad3 isoform antagonized Smad3-mediated, activin-responsive promoter activity. We propose that the pituitary gonadotrope contains an ultra-short, activin-responsive feedback loop utilizing two different isoforms of Smad3, one which acts as an agonist (Smad3) and another that acts as an intracrine antagonist (truncated Smad3 isoform) to regulate FSHβ production. PMID:21664424

Arsenic trioxide (ATO) influences the gene expression of metallothioneins in human glioblastoma cells.

PubMed

Falnoga, Ingrid; Zelenik Pevec, Andreja; Šlejkovec, Zdenka; Žnidarič, Magda Tušek; Zajc, Irena; Mlakar, Simona Jurković; Marc, Janja

2012-12-01

Arsenic trioxide (As(2)O(3); ATO, TRISENOX®) is used to treat patients with refractory or relapsed acute promyelocytic leukaemia while its application for treatment of solid cancers like glioblastoma is still under evaluation. In the present study, we investigated the interaction of arsenic trioxide with metallothionein (MT) isoforms as a possible (protective response) resistance of glioblastoma cells to arsenic-induced cytotoxicity. Special attention was focused on MT3, the isoform expressed mainly in the brain. MT3 has low metal inducibility, fast metal binding/releasing properties and outstanding neuronal inhibitory activity. The human astrocytoma (glioblastoma) cell line U87 MG was treated with 0.6, 2 and 6-7 μM arsenic (equivalent to 0.3, 1 and 3-3.5 μM As(2)O(3)) for 12, 24 or 48 h and gene expression for different MT isoforms, namely MT2A, MT1A, MT1F, MT1X, MT1E and MT3, was measured by real time qPCR using SYBR Green I and Taqman® gene expression assays. TfR, 18S rRNA, GAPDH and AB were tested as reference genes, and the last two evaluated to be appropriate in conditions of low (GAPDH) and high (AB) arsenic exposure. The gene expression of MT3 gene was additionally tested and confirmed by restriction enzyme analysis with PvuII. In the given conditions the mRNAs of six MT isoforms were identified in human glioblastoma cell line U87 MG. Depending on arsenic exposure conditions, an increase or decrease of MT gene expression was observed for each isoform, with the highest increase for isoforms MT1X, MT1F and MT2A mRNA (up to 13-fold) and more persistent decreases for MT1A, MT1E and MT3 mRNA. Despite the common assumption of the noninducibility of MT3, the evident MT3 mRNA increase was observed during high As exposure (up to 4-fold). In conclusion, our results clearly demonstrate the influence of As on MT isoform gene expression. The MT1X, MT1F and MT2A increase could represent brain tumour acquired resistance to As cytotoxicity while the MT3 increase is more enigmatic, with its possible involvement in arsenic-related induction of type II cell death.

Embryonic Lethality Due to Arrested Cardiac Development in Psip1/Hdgfrp2 Double-Deficient Mice.

PubMed

Wang, Hao; Shun, Ming-Chieh; Dickson, Amy K; Engelman, Alan N

2015-01-01

Hepatoma-derived growth factor (HDGF) related protein 2 (HRP2) and lens epithelium-derived growth factor (LEDGF)/p75 are closely related members of the HRP2 protein family. LEDGF/p75 has been implicated in numerous human pathologies including cancer, autoimmunity, and infectious disease. Knockout of the Psip1 gene, which encodes for LEDGF/p75 and the shorter LEDGF/p52 isoform, was previously shown to cause perinatal lethality in mice. The function of HRP2 was by contrast largely unknown. To learn about the role of HRP2 in development, we knocked out the Hdgfrp2 gene, which encodes for HRP2, in both normal and Psip1 knockout mice. Hdgfrp2 knockout mice developed normally and were fertile. By contrast, the double deficient mice died at approximate embryonic day (E) 13.5. Histological examination revealed ventricular septal defect (VSD) associated with E14.5 double knockout embryos. To investigate the underlying molecular mechanism(s), RNA recovered from ventricular tissue was subjected to RNA-sequencing on the Illumina platform. Bioinformatic analysis revealed several genes and biological pathways that were significantly deregulated by the Psip1 knockout and/or Psip1/Hdgfrp2 double knockout. Among the dozen genes known to encode for LEDGF/p75 binding factors, only the expression of Nova1, which encodes an RNA splicing factor, was significantly deregulated by the knockouts. However the expression of other RNA splicing factors, including the LEDGF/p52-interacting protein ASF/SF2, was not significantly altered, indicating that deregulation of global RNA splicing was not a driving factor in the pathology of the VSD. Tumor growth factor (Tgf) β-signaling, which plays a key role in cardiac morphogenesis during development, was the only pathway significantly deregulated by the double knockout as compared to control and Psip1 knockout samples. We accordingly speculate that deregulated Tgf-β signaling was a contributing factor to the VSD and prenatal lethality of Psip1/Hdgfrp2 double-deficient mice.

Three rare diseases in one Sib pair: RAI1, PCK1, GRIN2B mutations associated with Smith-Magenis Syndrome, cytosolic PEPCK deficiency and NMDA receptor glutamate insensitivity.

PubMed

Adams, David R; Yuan, Hongjie; Holyoak, Todd; Arajs, Katrina H; Hakimi, Parvin; Markello, Thomas C; Wolfe, Lynne A; Vilboux, Thierry; Burton, Barbara K; Fajardo, Karin Fuentes; Grahame, George; Holloman, Conisha; Sincan, Murat; Smith, Ann C M; Wells, Gordon A; Huang, Yan; Vega, Hugo; Snyder, James P; Golas, Gretchen A; Tifft, Cynthia J; Boerkoel, Cornelius F; Hanson, Richard W; Traynelis, Stephen F; Kerr, Douglas S; Gahl, William A

2014-11-01

The National Institutes of Health Undiagnosed Diseases Program evaluates patients for whom no diagnosis has been discovered despite a comprehensive diagnostic workup. Failure to diagnose a condition may arise from the mutation of genes previously unassociated with disease. However, we hypothesized that this could also co-occur with multiple genetic disorders. Demonstrating a complex syndrome caused by multiple disorders, we report two siblings manifesting both similar and disparate signs and symptoms. They shared a history of episodes of hypoglycemia and lactic acidosis, but had differing exam findings and developmental courses. Clinical acumen and exome sequencing combined with biochemical and functional studies identified three genetic conditions. One sibling had Smith-Magenis Syndrome and a nonsense mutation in the RAI1 gene. The second sibling had a de novo mutation in GRIN2B, which resulted in markedly reduced glutamate potency of the encoded receptor. Both siblings had a protein-destabilizing homozygous mutation in PCK1, which encodes the cytosolic isoform of phosphoenolpyruvate carboxykinase (PEPCK-C). In summary, we present the first clinically-characterized mutation of PCK1 and demonstrate that complex medical disorders can represent the co-occurrence of multiple diseases. Copyright © 2014 Elsevier Inc. All rights reserved.

Minimally invasive transcriptome profiling in salmon: detection of biological response in rainbow trout caudal fin following exposure to environmental chemical contaminants.

PubMed

Veldhoen, Nik; Stevenson, Mitchel R; Skirrow, Rachel C; Rieberger, Kevin J; van Aggelen, Graham; Meays, Cynthia L; Helbing, Caren C

2013-10-15

An increasing number of anthropogenic chemicals have demonstrated potential for disruption of biological processes critical to normal growth and development of wildlife species. Both anadromous and freshwater salmon species are at risk of exposure to environmental chemical contaminants that may affect migratory behavior, environmental fitness, and reproductive success. A sensitive metric in determination of the presence and impact of such environmental chemical contaminants is through detection of changes in the status of gene transcript levels using a targeted quantitative real-time polymerase chain reaction assay. Ideally, the wildlife assessment strategy would incorporate conservation-centered non-lethal practices. Herein, we describe the development of such an assay for rainbow trout, Oncorhynchus mykiss, following an acute 96 h exposure to increasing concentrations of either 17α-ethinyl estradiol or cadmium. The estrogenic screen included measurement of mRNA encoding estrogen receptor α and β isoforms, vitellogenin, vitelline envelope protein γ, cytochrome p450 family 19 subfamily A, aryl hydrocarbon receptor, and the stress indicator, catalase. The metal exposure screen included evaluation of the latter two mRNA transcripts along with those encoding the metallothionein A and B isoforms. Exposure-dependent transcript abundance profiles were detected in both liver and caudal fin supporting the use of the caudal fin as a non-lethally obtained tissue source. The potential for both transcriptome profiling and genotypic sex determination from fin biopsy was extended, in principle, to field-captured Chinook salmon (Oncorhynchus tshawytscha). Copyright © 2013 Elsevier B.V. All rights reserved.

The 20-hydroxyecdysone-induced signalling pathway in G2/M arrest of Plodia interpunctella imaginal wing cells.

PubMed

Siaussat, David; Bozzolan, Françoise; Porcheron, Patrick; Debernard, Stéphane

2008-05-01

The mechanisms involved in the control of cellular proliferation by the steroid hormone 20-hydroxyecdysone (20E) in insects are not known. We dissected the 20E signalling pathway responsible for G2/M arrest of imaginal cells from the IAL-PID2 cells of the Indian meal moth Plodia interpunctella. We first used a 5'-3' RACE-based strategy to clone a 4479bp cDNA encoding a putative P. interpunctella HR3 transcription factor named PiHR3. The deduced amino acid sequence of PiHR3 was highly similar to those of HR3 proteins from other lepidopterans, e.g. Manduca sexta and Bombyx mori. Using double-stranded RNA-mediated interference (dsRNAi), we then succeeded in blocking the ability of 20E to induce the expression of PiEcR-B1, PiUSP-2 and PiHR3 genes that encode the P. interpunctella ecdysone receptor B1-isoform, Ultraspiracle-2 isoform, the insect homologue of the vertebrate retinoid X receptor, and the HR3 transcription factor. We showed that inhibiting the 20E induction of PiEcR-B1, PiUSP-2 and PiHR3 mRNAs prevented the decreased expression of B cyclin and consequently the G2/M arrest of IAL-PID2 cells. Using this functional approach, we revealed the participation of EcR, USP and HR3 in a 20E signalling pathway that controls the proliferation of imaginal cells by regulating the expression of B cyclin.

Quantitative evaluation of alternatively spliced mRNA isoforms by label-free real-time plasmonic sensing.

PubMed

Huertas, César S; Carrascosa, L G; Bonnal, S; Valcárcel, J; Lechuga, L M

2016-04-15

Alternative splicing of mRNA precursors enables cells to generate different protein outputs from the same gene depending on their developmental or homeostatic status. Its deregulation is strongly linked to disease onset and progression. Current methodologies for monitoring alternative splicing demand elaborate procedures and often present difficulties in discerning between closely related isoforms, e.g. due to cross-hybridization during their detection. Herein, we report a general methodology using a Surface Plasmon Resonance (SPR) biosensor for label-free monitoring of alternative splicing events in real-time, without any cDNA synthesis or PCR amplification requirements. We applied this methodology to RNA isolated from HeLa cells for the quantification of alternatively spliced isoforms of the Fas gene, involved in cancer progression through regulation of programmed cell death. We demonstrate that our methodology is isoform-specific, with virtually no cross-hybridization, achieving limits of detection (LODs) in the picoMolar (pM) range. Similar results were obtained for the detection of the BCL-X gene mRNA isoforms. The results were independently validated by RT-qPCR, with excellent concordance in the determination of isoform ratios. The simplicity and robustness of this biosensor technology can greatly facilitate the exploration of alternative splicing biomarkers in disease diagnosis and therapy. Copyright © 2015 Elsevier B.V. All rights reserved.

Enoyl-Acyl Carrier Protein Reductase I (FabI) Is Essential for the Intracellular Growth of Listeria monocytogenes.

PubMed

Yao, Jiangwei; Ericson, Megan E; Frank, Matthew W; Rock, Charles O

2016-12-01

Enoyl-acyl carrier protein reductase catalyzes the last step in each elongation cycle of type II bacterial fatty acid synthesis and is a key regulatory protein in bacterial fatty acid synthesis. Genes of the facultative intracellular pathogen Listeria monocytogenes encode two functional enoyl-acyl carrier protein isoforms based on their ability to complement the temperature-sensitive growth phenotype of Escherichia coli strain JP1111 [fabI(Ts)]. The FabI isoform was inactivated by the FabI selective inhibitor AFN-1252, but the FabK isoform was not affected by the drug, as expected. Inhibition of FabI by AFN-1252 decreased endogenous fatty acid synthesis by 80% and lowered the growth rate of L. monocytogenes in laboratory medium. Robust exogenous fatty acid incorporation was not detected in L. monocytogenes unless the pathway was partially inactivated by AFN-1252 treatment. However, supplementation with exogenous fatty acids did not restore normal growth in the presence of AFN-1252. FabI inactivation prevented the intracellular growth of L. monocytogenes, showing that neither FabK nor the incorporation of host cellular fatty acids was sufficient to support the intracellular growth of L. monocytogenes Our results show that FabI is the primary enoyl-acyl carrier protein reductase of type II bacterial fatty acid synthesis and is essential for the intracellular growth of L. monocytogenes. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

The β subunit of yeast AMP-activated protein kinase directs substrate specificity in response to alkaline stress.

PubMed

Chandrashekarappa, Dakshayini G; McCartney, Rhonda R; O'Donnell, Allyson F; Schmidt, Martin C

2016-12-01

Saccharomyces cerevisiae express three isoforms of Snf1 kinase that differ by which β subunit is present, Gal83, Sip1 or Sip2. Here we investigate the abundance, activation, localization and signaling specificity of the three Snf1 isoforms. The relative abundance of these isoforms was assessed by quantitative immunoblotting using two different protein extraction methods and by fluorescence microscopy. The Gal83 containing isoform is the most abundant in all assays while the abundance of the Sip1 and Sip2 isoforms is typically underestimated especially in glass-bead extractions. Earlier studies to assess Snf1 isoform function utilized gene deletions as a means to inactivate specific isoforms. Here we use point mutations in Gal83 and Sip2 and a 17 amino acid C-terminal truncation of Sip1 to inactivate specific isoforms without affecting their abundance or association with the other subunits. The effect of low glucose and alkaline stresses was examined for two Snf1 phosphorylation substrates, the Mig1 and Mig2 proteins. Any of the three isoforms was capable of phosphorylating Mig1 in response to glucose stress. In contrast, the Gal83 isoform of Snf1 was both necessary and sufficient for the phosphorylation of the Mig2 protein in response to alkaline stress. Alkaline stress led to the activation of all three isoforms yet only the Gal83 isoform translocates to the nucleus and phosphorylates Mig2. Deletion of the SAK1 gene blocked nuclear translocation of Gal83 and signaling to Mig2. These data strongly support the idea that Snf1 signaling specificity is mediated by localization of the different Snf1 isoforms. Copyright © 2016 Elsevier Inc. All rights reserved.

The β subunit of yeast AMP-activated protein kinase directs substrate specificity in response to alkaline stress

PubMed Central

Chandrashekarappa, Dakshayini G.; McCartney, Rhonda R.; O’Donnell, Allyson F.; Schmidt, Martin C.

2016-01-01

Saccharomyces cerevisiae express three isoforms of Snf1 kinase that differ by which β subunit is present, Gal83, Sip1 or Sip2. Here we investigate the abundance, activation, localization and signaling specificity of the three Snf1 isoforms. The relative abundance of these isoforms was assessed by quantitative immunoblotting using two different protein extraction methods and by fluorescence microscopy. The Gal83 containing isoform is the most abundant in all assays while the abundance of the Sip1 and Sip2 isoforms is typically underestimated especially in glass-bead extractions. Earlier studies to assess Snf1 isoform function utilized gene deletions as a means to inactivate specific isoforms. Here we use point mutations in Gal83 and Sip2 and a 17 amino acid C-terminal truncation of Sip1 to inactivate specific isoforms without affecting their abundance or association with the other subunits. The effect of low glucose and alkaline stresses was examined for two Snf1 phosphorylation substrates, the Mig1 and Mig2 proteins. Any of the three isoforms was capable of phosphorylating Mig1 in response to glucose stress. In contrast, the Gal83 isoform of Snf 1 was both necessary and sufficient for the phosphorylation of the Mig2 protein in response to alkaline stress. Alkaline stress led to the activation of all three isoforms yet only the Gal83 isoform translocates to the nucleus and phosphorylates Mig2. Deletion of the SAK1 gene blocked nuclear translocation of Gal83 and signaling to Mig2. These data strongly support the idea that Snf1 signaling specificity is mediated by localization of the different Snf1 isoforms. PMID:27592031

MEF2 Transcription Factors Regulate Distinct Gene Programs in Mammalian Skeletal Muscle Differentiation*

PubMed Central

Estrella, Nelsa L.; Desjardins, Cody A.; Nocco, Sarah E.; Clark, Amanda L.; Maksimenko, Yevgeniy; Naya, Francisco J.

2015-01-01

Skeletal muscle differentiation requires precisely coordinated transcriptional regulation of diverse gene programs that ultimately give rise to the specialized properties of this cell type. In Drosophila, this process is controlled, in part, by MEF2, the sole member of an evolutionarily conserved transcription factor family. By contrast, vertebrate MEF2 is encoded by four distinct genes, Mef2a, -b, -c, and -d, making it far more challenging to link this transcription factor to the regulation of specific muscle gene programs. Here, we have taken the first step in molecularly dissecting vertebrate MEF2 transcriptional function in skeletal muscle differentiation by depleting individual MEF2 proteins in myoblasts. Whereas MEF2A is absolutely required for proper myoblast differentiation, MEF2B, -C, and -D were found to be dispensable for this process. Furthermore, despite the extensive redundancy, we show that mammalian MEF2 proteins regulate a significant subset of nonoverlapping gene programs. These results suggest that individual MEF2 family members are able to recognize specific targets among the entire cohort of MEF2-regulated genes in the muscle genome. These findings provide opportunities to modulate the activity of MEF2 isoforms and their respective gene programs in skeletal muscle homeostasis and disease. PMID:25416778

eQTL Mapping Using RNA-seq Data

PubMed Central

Hu, Yijuan

2012-01-01

As RNA-seq is replacing gene expression microarrays to assess genome-wide transcription abundance, gene expression Quantitative Trait Locus (eQTL) studies using RNA-seq have emerged. RNA-seq delivers two novel features that are important for eQTL studies. First, it provides information on allele-specific expression (ASE), which is not available from gene expression microarrays. Second, it generates unprecedentedly rich data to study RNA-isoform expression. In this paper, we review current methods for eQTL mapping using ASE and discuss some future directions. We also review existing works that use RNA-seq data to study RNA-isoform expression and we discuss the gaps between these works and isoform-specific eQTL mapping. PMID:23667399

Regulation of xylose metabolism in recombinant Saccharomyces cerevisiae

PubMed Central

Salusjärvi, Laura; Kankainen, Matti; Soliymani, Rabah; Pitkänen, Juha-Pekka; Penttilä, Merja; Ruohonen, Laura

2008-01-01

Background Considerable interest in the bioconversion of lignocellulosic biomass into ethanol has led to metabolic engineering of Saccharomyces cerevisiae for fermentation of xylose. In the present study, the transcriptome and proteome of recombinant, xylose-utilising S. cerevisiae grown in aerobic batch cultures on xylose were compared with those of glucose-grown cells both in glucose repressed and derepressed states. The aim was to study at the genome-wide level how signalling and carbon catabolite repression differ in cells grown on either glucose or xylose. The more detailed knowledge whether xylose is sensed as a fermentable carbon source, capable of catabolite repression like glucose, or is rather recognised as a non-fermentable carbon source is important for further engineering this yeast for more efficient anaerobic fermentation of xylose. Results Genes encoding respiratory proteins, proteins of the tricarboxylic acid and glyoxylate cycles, and gluconeogenesis were only partially repressed by xylose, similar to the genes encoding their transcriptional regulators HAP4, CAT8 and SIP1-2 and 4. Several genes that are repressed via the Snf1p/Mig1p-pathway during growth on glucose had higher expression in the cells grown on xylose than in the glucose repressed cells but lower than in the glucose derepressed cells. The observed expression profiles of the transcription repressor RGT1 and its target genes HXT2-3, encoding hexose transporters suggested that extracellular xylose was sensed by the glucose sensors Rgt2p and Snf3p. Proteome analyses revealed distinct patterns in phosphorylation of hexokinase 2, glucokinase and enolase isoenzymes in the xylose- and glucose-grown cells. Conclusion The results indicate that the metabolism of yeast growing on xylose corresponds neither to that of fully glucose repressed cells nor that of derepressed cells. This may be one of the major reasons for the suboptimal fermentation of xylose by recombinant S. cerevisiae strains. Phosphorylation of different isoforms of glycolytic enzymes suggests that regulation of glycolysis also occurred at a post-translational level, supporting prior findings. PMID:18533012

SMCHD1 regulates a limited set of gene clusters on autosomal chromosomes.

PubMed

Mason, Amanda G; Slieker, Roderick C; Balog, Judit; Lemmers, Richard J L F; Wong, Chao-Jen; Yao, Zizhen; Lim, Jong-Won; Filippova, Galina N; Ne, Enrico; Tawil, Rabi; Heijmans, Bas T; Tapscott, Stephen J; van der Maarel, Silvère M

2017-06-06

Facioscapulohumeral muscular dystrophy (FSHD) is in most cases caused by a contraction of the D4Z4 macrosatellite repeat on chromosome 4 (FSHD1) or by mutations in the SMCHD1 or DNMT3B gene (FSHD2). Both situations result in the incomplete epigenetic repression of the D4Z4-encoded retrogene DUX4 in somatic cells, leading to the aberrant expression of DUX4 in the skeletal muscle. In mice, Smchd1 regulates chromatin repression at different loci, having a role in CpG methylation establishment and/or maintenance. To investigate the global effects of harboring heterozygous SMCHD1 mutations on DNA methylation in humans, we combined 450k methylation analysis on mononuclear monocytes from female heterozygous SMCHD1 mutation carriers and unaffected controls with reduced representation bisulfite sequencing (RRBS) on FSHD2 and control myoblast cell lines. Candidate loci were then evaluated for SMCHD1 binding using ChIP-qPCR and expression was evaluated using RT-qPCR. We identified a limited number of clustered autosomal loci with CpG hypomethylation in SMCHD1 mutation carriers: the protocadherin (PCDH) cluster on chromosome 5, the transfer RNA (tRNA) and 5S rRNA clusters on chromosome 1, the HOXB and HOXD clusters on chromosomes 17 and 2, respectively, and the D4Z4 repeats on chromosomes 4 and 10. Furthermore, minor increases in RNA expression were seen in FSHD2 myoblasts for some of the PCDHβ cluster isoforms, tRNA isoforms, and a HOXB isoform in comparison to controls, in addition to the previously reported effects on DUX4 expression. SMCHD1 was bound at DNAseI hypersensitivity sites known to regulate the PCDHβ cluster and at the chromosome 1 tRNA cluster, with decreased binding in SMCHD1 mutation carriers at the PCDHβ cluster sites. Our study is the first to investigate the global methylation effects in humans resulting from heterozygous mutations in SMCHD1. Our results suggest that SMCHD1 acts as a repressor on a limited set of autosomal gene clusters, as an observed reduction in methylation associates with a loss of SMCHD1 binding and increased expression for some of the loci.

Molecular characterization and transcriptional regulation of the Na +/K+ ATPase α subunit isoforms during development and salinity challenge in a teleost fish, the Senegalese sole (Solea senegalensis).

PubMed

Armesto, Paula; Campinho, Marco A; Rodríguez-Rúa, Ana; Cousin, Xavier; Power, Deborah M; Manchado, Manuel; Infante, Carlos

2014-09-01

In the present work, five genes encoding different Na(+),K(+) ATPase (NKA) α-isoforms in the teleost Solea senegalensis are described for the first time. Sequence analysis of predicted polypeptides revealed a high degree of conservation across teleosts and mammals. Phylogenetic analysis clustered the five genes into three main clades: α1 (designated atp1a1a and atp1a1b), α2 (designated atp1a2) and α3 (designated atp1a3a and atp1a3b) isoforms. Transcriptional analysis in larvae showed distinct expression profiles during development. In juvenile tissues, the atp1a1a gene was highly expressed in osmoregulatory organs, atp1a2 in skeletal muscle, atp1a1b in brain and heart and atp1a3a and atp1a3b mainly in brain. Quantification of mRNA abundance after a salinity challenge showed that atp1a1a transcript levels increased significantly in the gill of soles transferred to high salinity water (60 ppt). In contrast, atp1a3a transcripts increased at low salinity (5 ppt). In situ hybridization (ISH) analysis revealed that the number of ionocytes expressing atp1a1a transcripts in the primary gill filaments was higher at 35 and 60 ppt than at 5 ppt and remained undetectable or at very low levels in the lamellae at 5 and 35 ppt but increased at 60 ppt. Immunohistochemistry showed a higher number of positive cells in the lamellae. Whole-mount analysis of atp1a1a mRNA in young sole larvae revealed that it was localized in gut, pronephric tubule, gill, otic vesicle, yolk sac ionocytes and chordacentrum. Moreover, atp1a1a mRNAs increased at mouth opening (3 DPH) in larvae incubated at 36 ppt with a greater signal in gills. Copyright © 2014 Elsevier Inc. All rights reserved.

Identification and characterization of vp7 gene in Bombyx mori cytoplasmic polyhedrosis virus.

PubMed

He, Lei; Hu, Xiaolong; Zhu, Min; Liang, Zi; Chen, Fei; Zhu, Liyuan; Kuang, Sulan; Cao, Guangli; Xue, Renyu; Gong, Chengliang

2017-09-05

The genome of Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) contains 10 double stranded RNA segments (S1-S10). The segment 7 (S7) encodes 50kDa protein which is considered as a structural protein. The expression pattern and function of p50 in the virus life cycle are still unclear. In this study, the viral structural protein 7 (VP7) polyclonal antibody was prepared with immunized mouse to explore the presence of small VP7 gene-encoded proteins in Bombyx mori cytoplasmic polyhedrosis virus. The expression pattern of vp7 gene was investigated by its overexpression in BmN cells. In addition to VP7, supplementary band was identified with western blotting technique. The virion, BmCPV infected cells and midguts were also examined using western blotting technique. 4, 2 and 5 bands were detected in the corresponding samples, respectively. The replication of BmCPV genome in the cultured cells and midgut of silkworm was decreased by reducing the expression level of vp7 gene using RNA interference. In immunoprecipitation experiments, using a polyclonal antiserum directed against the VP7, one additional shorter band in BmCPV infected midguts was detected, and then the band was analyzed with mass spectrum (MS), the MS results showed thatone candidate interacted protein (VP7 voltage-dependent anion-selective channel-like isoform, VDAC) was identified from silkworm. We concluded that the novel viral product was generated with a leaky scanning mechanism and the VDAC may be an interacted protein with VP7. Copyright © 2017 Elsevier B.V. All rights reserved.

Dom34 Links Translation to Protein O-mannosylation

PubMed Central

van Wijlick, Lasse; Geissen, René; Hilbig, Jessica S.; Lagadec, Quentin; Cantero, Pilar D.; Juchimiuk, Mateusz; Kluge, Sven; Wickert, Stephan; Alepuz, Paula; Ernst, Joachim F.

2016-01-01

In eukaryotes, Dom34 upregulates translation by securing levels of activatable ribosomal subunits. We found that in the yeast Saccharomyces cerevisiae and the human fungal pathogen Candida albicans, Dom34 interacts genetically with Pmt1, a major isoform of protein O-mannosyltransferase. In C. albicans, lack of Dom34 exacerbated defective phenotypes of pmt1 mutants, while they were ameliorated by Dom34 overproduction that enhanced Pmt1 protein but not PMT1 transcript levels. Translational effects of Dom34 required the 5′-UTR of the PMT1 transcript, which bound recombinant Dom34 directly at a CA/AC-rich sequence and regulated in vitro translation. Polysomal profiling revealed that Dom34 stimulates general translation moderately, but that it is especially required for translation of transcripts encoding Pmt isoforms 1, 4 and 6. Because defective protein N- or O-glycosylation upregulates transcription of PMT genes, it appears that Dom34-mediated specific translational upregulation of the PMT transcripts optimizes cellular responses to glycostress. Its translational function as an RNA binding protein acting at the 5′-UTR of specific transcripts adds another facet to the known ribosome-releasing functions of Dom34 at the 3′-UTR of transcripts. PMID:27768707

Dom34 Links Translation to Protein O-mannosylation.

PubMed

van Wijlick, Lasse; Geissen, René; Hilbig, Jessica S; Lagadec, Quentin; Cantero, Pilar D; Pfeifer, Eugen; Juchimiuk, Mateusz; Kluge, Sven; Wickert, Stephan; Alepuz, Paula; Ernst, Joachim F

2016-10-01

In eukaryotes, Dom34 upregulates translation by securing levels of activatable ribosomal subunits. We found that in the yeast Saccharomyces cerevisiae and the human fungal pathogen Candida albicans, Dom34 interacts genetically with Pmt1, a major isoform of protein O-mannosyltransferase. In C. albicans, lack of Dom34 exacerbated defective phenotypes of pmt1 mutants, while they were ameliorated by Dom34 overproduction that enhanced Pmt1 protein but not PMT1 transcript levels. Translational effects of Dom34 required the 5'-UTR of the PMT1 transcript, which bound recombinant Dom34 directly at a CA/AC-rich sequence and regulated in vitro translation. Polysomal profiling revealed that Dom34 stimulates general translation moderately, but that it is especially required for translation of transcripts encoding Pmt isoforms 1, 4 and 6. Because defective protein N- or O-glycosylation upregulates transcription of PMT genes, it appears that Dom34-mediated specific translational upregulation of the PMT transcripts optimizes cellular responses to glycostress. Its translational function as an RNA binding protein acting at the 5'-UTR of specific transcripts adds another facet to the known ribosome-releasing functions of Dom34 at the 3'-UTR of transcripts.

Structure and function of splice variants of the cardiac voltage-gated sodium channel Na(v)1.5.

PubMed

Schroeter, Annett; Walzik, Stefan; Blechschmidt, Steve; Haufe, Volker; Benndorf, Klaus; Zimmer, Thomas

2010-07-01

Voltage-gated sodium channels mediate the rapid upstroke of the action potential in excitable tissues. The tetrodotoxin (TTX) resistant isoform Na(v)1.5, encoded by the SCN5A gene, is the predominant isoform in the heart. This channel plays a key role for excitability of atrial and ventricular cardiomyocytes and for rapid impulse propagation through the specific conduction system. During recent years, strong evidence has been accumulated in support of the expression of several Na(v)1.5 splice variants in the heart, and in various other tissues and cell lines including brain, dorsal root ganglia, breast cancer cells and neuronal stem cell lines. This review summarizes our knowledge on the structure and putative function of nine Na(v)1.5 splice variants detected so far. Attention will be paid to the distinct biophysical properties of the four functional splice variants, to the pronounced tissue- and species-specific expression, and to the developmental regulation of Na(v)1.5 splicing. The implications of alternative splicing for SCN5A channelopathies, and for a better understanding of genotype-phenotype correlations, are discussed. Copyright 2010 Elsevier Ltd. All rights reserved.

A small RNA activates CFA synthase by isoform-specific mRNA stabilization

PubMed Central

Fröhlich, Kathrin Sophie; Papenfort, Kai; Fekete, Agnes; Vogel, Jörg

2013-01-01

Small RNAs use a diversity of well-characterized mechanisms to repress mRNAs, but how they activate gene expression at the mRNA level remains not well understood. The predominant activation mechanism of Hfq-associated small RNAs has been translational control whereby base pairing with the target prevents the formation of an intrinsic inhibitory structure in the mRNA and promotes translation initiation. Here, we report a translation-independent mechanism whereby the small RNA RydC selectively activates the longer of two isoforms of cfa mRNA (encoding cyclopropane fatty acid synthase) in Salmonella enterica. Target activation is achieved through seed pairing of the pseudoknot-exposed, conserved 5′ end of RydC to an upstream region of the cfa mRNA. The seed pairing stabilizes the messenger, likely by interfering directly with RNase E-mediated decay in the 5′ untranslated region. Intriguingly, this mechanism is generic such that the activation is equally achieved by seed pairing of unrelated small RNAs, suggesting that this mechanism may be utilized in the design of RNA-controlled synthetic circuits. Physiologically, RydC is the first small RNA known to regulate membrane stability. PMID:24141880

A small RNA activates CFA synthase by isoform-specific mRNA stabilization.

PubMed

Fröhlich, Kathrin Sophie; Papenfort, Kai; Fekete, Agnes; Vogel, Jörg

2013-11-13

Small RNAs use a diversity of well-characterized mechanisms to repress mRNAs, but how they activate gene expression at the mRNA level remains not well understood. The predominant activation mechanism of Hfq-associated small RNAs has been translational control whereby base pairing with the target prevents the formation of an intrinsic inhibitory structure in the mRNA and promotes translation initiation. Here, we report a translation-independent mechanism whereby the small RNA RydC selectively activates the longer of two isoforms of cfa mRNA (encoding cyclopropane fatty acid synthase) in Salmonella enterica. Target activation is achieved through seed pairing of the pseudoknot-exposed, conserved 5' end of RydC to an upstream region of the cfa mRNA. The seed pairing stabilizes the messenger, likely by interfering directly with RNase E-mediated decay in the 5' untranslated region. Intriguingly, this mechanism is generic such that the activation is equally achieved by seed pairing of unrelated small RNAs, suggesting that this mechanism may be utilized in the design of RNA-controlled synthetic circuits. Physiologically, RydC is the first small RNA known to regulate membrane stability.

NOVA2-mediated RNA regulation is required for axonal pathfinding during development

PubMed Central

Saito, Yuhki; Miranda-Rottmann, Soledad; Ruggiu, Matteo; Park, Christopher Y; Fak, John J; Zhong, Ru; Duncan, Jeremy S; Fabella, Brian A; Junge, Harald J; Chen, Zhe; Araya, Roberto; Fritzsch, Bernd; Hudspeth, A J; Darnell, Robert B

2016-01-01

The neuron specific RNA-binding proteins NOVA1 and NOVA2 are highly homologous alternative splicing regulators. NOVA proteins regulate at least 700 alternative splicing events in vivo, yet relatively little is known about the biologic consequences of NOVA action and in particular about functional differences between NOVA1 and NOVA2. Transcriptome-wide searches for isoform-specific functions, using NOVA1 and NOVA2 specific HITS-CLIP and RNA-seq data from mouse cortex lacking either NOVA isoform, reveals that NOVA2 uniquely regulates alternative splicing events of a series of axon guidance related genes during cortical development. Corresponding axonal pathfinding defects were specific to NOVA2 deficiency: Nova2-/- but not Nova1-/- mice had agenesis of the corpus callosum, and axonal outgrowth defects specific to ventral motoneuron axons and efferent innervation of the cochlea. Thus we have discovered that NOVA2 uniquely regulates alternative splicing of a coordinate set of transcripts encoding key components in cortical, brainstem and spinal axon guidance/outgrowth pathways during neural differentiation, with severe functional consequences in vivo. DOI: http://dx.doi.org/10.7554/eLife.14371.001 PMID:27223325

Tissue specificity and regulation of the N-terminal diversity of reticulon 3

PubMed Central

Di Scala, Franck; Dupuis, Luc; Gaiddon, Christian; De Tapia, Marc; Jokic, Natasa; Gonzalez De Aguilar, Jose-Luis; Raul, Jean-Sébastien; Ludes, Bertrand; Loeffler, Jean-Philippe

2004-01-01

Over the last few years, the widely distributed family of reticulons (RTNs) is receiving renewed interest because of the implication of RTN4/Nogo in neurite regeneration. Four genes were identified in mammals and are referred to as RTN1, 2, 3 and the neurite outgrowth inhibitor RTN4/Nogo. In the present paper, we describe the existence of five new isoforms of RTN3 that differ in their N-termini, and analysed their tissue distribution and expression in neurons. We redefined the structure of human and murine rtn3 genes, and identified two supplementary exons that may generate up to seven putative isoforms arising by alternative splicing or differential promoter usage. We confirmed the presence of five of these isoforms at the mRNA and protein levels, and showed their preferential expression in the central nervous system. We analysed rtn3 expression in the cerebellum further, and observed increased levels of several of the RTN3 isoforms during cerebellum development and during in vitro maturation of cerebellar granule cells. This pattern of expression paralleled that shown by RTN4/Nogo isoforms. Specifically, RTN3A1 expression was down-regulated upon cell death of cerebellar granule neurons triggered by potassium deprivation. Altogether, our results demonstrate that the rtn3 gene generates multiple isoforms varying in their N-termini, and that their expression is tightly regulated in neurons. These findings suggest that RTN3 isoforms may contribute, by as yet unknown mechanisms, to neuronal survival and plasticity. PMID:15350194

Bicistronic CACNA1A Gene Expression in Neurons Derived from Spinocerebellar Ataxia Type 6 Patient-Induced Pluripotent Stem Cells

PubMed Central

Bavassano, Carlo; Eigentler, Andreas; Stanika, Ruslan; Obermair, Gerald J.; Boesch, Sylvia; Dechant, Georg

2017-01-01

Spinocerebellar ataxia type 6 (SCA6) is an autosomal-dominant neurodegenerative disorder that is caused by a CAG trinucleotide repeat expansion in the CACNA1A gene. As one of the few bicistronic genes discovered in the human genome, CACNA1A encodes not only the α1A subunit of the P/Q type voltage-gated Ca2+ channel CaV2.1 but also the α1ACT protein, a 75 kDa transcription factor sharing the sequence of the cytoplasmic C-terminal tail of the α1A subunit. Isoforms of both proteins contain the polyglutamine (polyQ) domain that is expanded in SCA6 patients. Although certain SCA6 phenotypes appear to be specific for Purkinje neurons, other pathogenic effects of the SCA6 polyQ mutation can affect a broad spectrum of central nervous system (CNS) neuronal subtypes. We investigated the expression and function of CACNA1A gene products in human neurons derived from induced pluripotent stem cells from two SCA6 patients. Expression levels of CACNA1A encoding α1A subunit were similar between SCA6 and control neurons, and no differences were found in the subcellular distribution of CaV2.1 channel protein. The α1ACT immunoreactivity was detected in the majority of cell nuclei of SCA6 and control neurons. Although no SCA6 genotype-dependent differences in CaV2.1 channel function were observed, they were found in the expression levels of the α1ACT target gene Granulin (GRN) and in glutamate-induced cell vulnerability. PMID:28946818

Intron retention regulates the expression of pectin methyl esterase inhibitor (Pmei) genes during wheat growth and development.

PubMed

Rocchi, V; Janni, M; Bellincampi, D; Giardina, T; D'Ovidio, R

2012-03-01

Pectin is an important component of the plant cell wall and its remodelling occurs during normal plant growth or following stress responses. Pectin is secreted into the cell wall in a highly methyl-esterified form and subsequently de-methyl-esterified by pectin methyl esterase (PME), whose activity is controlled by the pectin methyl esterase inhibitor protein (PMEI). Cereal cell wall contains a low amount of pectin; nonetheless the level and pattern of pectin methyl esterification play a primary role during development or pathogen infection. Since few data are available on the role of PMEI in plant development and defence of cereal species, we isolated and characterised three Pmei genes (Tdpmei2.1, Tdpmei2.2 and Tdpmei3) and their encoded products in wheat. Sequence comparisons showed a low level of intra- and inter-specific sequence conservation of PMEIs. Tdpmei2.1 and Tdpmei2.2 share 94% identity at protein level, but only 20% identity with the product of Tdpmei3. All three Tdpmei genes code for functional inhibitors of plant PMEs and do not inhibit microbial PMEs or a plant invertase. RT-PCR analyses demonstrated, for the first time to our knowledge, that Pmei genes are regulated by intron retention. Processed and unprocessed transcripts of Tdpmei2.1 and Tdpmei2.2 accumulated in several organs, but anthers contained only mature transcripts. Tdpmei3 lacks introns and its transcript accumulated mainly in stem internodes. These findings suggest that products encoded by these Tdpmei genes control organ- or tissue-specific activity of specific PME isoforms in wheat. © 2011 German Botanical Society and The Royal Botanical Society of the Netherlands.

Functions and regulation of the Nox family in the filamentous fungus Podospora anserina: a new role in cellulose degradation.

PubMed

Brun, Sylvain; Malagnac, Fabienne; Bidard, Frédérique; Lalucque, Hervé; Silar, Philippe

2009-10-01

NADPH oxidases are enzymes that produce reactive oxygen species. Studies in mammals, plants and fungi have shown that they play important roles in differentiation, defence, host/pathogen interaction and mutualistic symbiosis. In this paper, we have identified a Podospora anserina mutant strain impaired for processes controlled by PaNox1 and PaNox2, the two Nox isoforms characterized in this model ascomycete. We show that the gene mutated is PaNoxR, the homologue of the gene encoding the regulatory subunit p67(phox), conserved in mammals and fungi, and that PaNoxR regulates both PaNox1 and PaNox2. Genome sequence analysis of P. anserina reveals that this fungus posses a third Nox isoform, PaNox3, related to human Nox5/Duox and plant Rboh. We have generated a knock-out mutant of PaNox3 and report that PaNox3 plays a minor role in P. anserina, if any. We show that PaNox1 and PaNox2 play antagonist roles in cellulose degradation. Finally, we report for the first time that a saprobic fungus, P. anserina, develops special cell structures dedicated to breach and to exploit a solid cellulosic substrate, cellophane. Importantly, as for similar structures present in some plant pathogens, their proper differentiation requires PaNox1, PaNox2, PaNoxR and the tetraspanin PaPls1.

Mutations in STT3A and STT3B cause two congenital disorders of glycosylation

PubMed Central

Shrimal, Shiteshu; Ng, Bobby G.; Losfeld, Marie-Estelle; Gilmore, Reid; Freeze, Hudson H.

2013-01-01

We describe two unreported types of congenital disorders of glycosylation (CDG) which are caused by mutations in different isoforms of the catalytic subunit of the oligosaccharyltransferase (OST). Each isoform is encoded by a different gene (STT3A or STT3B), resides in a different OST complex and has distinct donor and acceptor substrate specificities with partially overlapping functions in N-glycosylation. The two cases from unrelated consanguineous families both show neurologic abnormalities, hypotonia, intellectual disability, failure to thrive and feeding problems. A homozygous mutation (c.1877T > C) in STT3A causes a p.Val626Ala change and a homozygous intronic mutation (c.1539 + 20G > T) in STT3B causes the other disorder. Both mutations impair glycosylation of a GFP biomarker and are rescued with the corresponding cDNA. Glycosylation of STT3A- and STT3B-specific acceptors is decreased in fibroblasts carrying the corresponding mutated gene and expression of the STT3A (p.Val626Ala) allele in STT3A-deficient HeLa cells does not rescue glycosylation. No additional cases were found in our collection or in reviewing various databases. The STT3A mutation significantly impairs glycosylation of the biomarker transferrin, but the STT3B mutation only slightly affects its glycosylation. Additional cases of STT3B-CDG may be missed by transferrin analysis and will require exome or genome sequencing. PMID:23842455

Aldo-Keto Reductases 1B in Adrenal Cortex Physiology

PubMed Central

Pastel, Emilie; Pointud, Jean-Christophe; Martinez, Antoine; Lefrançois-Martinez, A. Marie

2016-01-01

Aldose reductase (AKR1B) proteins are monomeric enzymes, belonging to the aldo-keto reductase (AKR) superfamily. They perform oxidoreduction of carbonyl groups from a wide variety of substrates, such as aliphatic and aromatic aldehydes or ketones. Due to the involvement of human aldose reductases in pathologies, such as diabetic complications and cancer, AKR1B subgroup enzymatic properties have been extensively characterized. However, the issue of AKR1B function in non-pathologic conditions remains poorly resolved. Adrenal activities generated large amount of harmful aldehydes from lipid peroxidation and steroidogenesis, including 4-hydroxynonenal (4-HNE) and isocaproaldehyde (4-methylpentanal), which can both be reduced by AKR1B proteins. More recently, some AKR1B isoforms have been shown to be endowed with prostaglandin F synthase (PGFS) activity, suggesting that, in addition to possible scavenger function, they could instigate paracrine signals. Interestingly, the adrenal gland is one of the major sites for human and murine AKR1B expression, suggesting that their detoxifying/signaling activity could be specifically required for the correct handling of adrenal function. Moreover, chronic effects of ACTH result in a coordinated regulation of genes encoding the steroidogenic enzymes and some AKR1B isoforms. This review presents the molecular mechanisms accounting for the adrenal-specific expression of some AKR1B genes. Using data from recent mouse genetic models, we will try to connect their enzymatic properties and regulation with adrenal functions. PMID:27499746

NFATc1 releases BCL6-dependent repression of CCR2 agonist expression in peritoneal macrophages from Saccharomyces cerevisiae infected mice.

PubMed

Busch, Rhoda; Murti, Krisna; Liu, Jiming; Patra, Amiya K; Muhammad, Khalid; Knobeloch, Klaus-Peter; Lichtinger, Monika; Bonifer, Constanze; Wörtge, Simone; Waisman, Ari; Reifenberg, Kurt; Ellenrieder, Volker; Serfling, Edgar; Avots, Andris

2016-03-01

The link between the extensive usage of calcineurin (CN) inhibitors cyclosporin A and tacrolimus (FK506) in transplantation medicine and the increasing rate of opportunistic infections within this segment of patients is alarming. Currently, how peritoneal infections are favored by these drugs, which impair the activity of several signaling pathways including the Ca(++) /CN/NFAT, Ca(++) /CN/cofilin, Ca(++) /CN/BAD, and NF-κB networks, is unknown. Here, we show that Saccharomyces cerevisiae infection of peritoneal resident macrophages triggers the transient nuclear translocation of NFATc1β isoforms, resulting in a coordinated, CN-dependent induction of the Ccl2, Ccl7, and Ccl12 genes, all encoding CCR2 agonists. CN inhibitors block the CCR2-dependent recruitment of inflammatory monocytes (IM) to the peritoneal cavities of S. cerevisiae infected mice. In myeloid cells, NFATc1/β proteins represent the most prominent NFATc1 isoforms. NFATc1/β ablation leads to a decrease of CCR2 chemokines, impaired mobilization of IMs, and delayed clearance of infection. We show that, upon binding to a composite NFAT/BCL6 regulatory element within the Ccl2 promoter, NFATc1/β proteins release the BCL6-dependent repression of Ccl2 gene in macrophages. These findings suggest a novel CN-dependent cross-talk between NFAT and BCL6 transcription factors, which may affect the outcome of opportunistic fungal infections in immunocompromised patients. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The RPN5 subunit of the 26s proteasome is essential for gametogenesis, sporophyte development, and complex assembly in Arabidopsis.

PubMed

Book, Adam J; Smalle, Jan; Lee, Kwang-Hee; Yang, Peizhen; Walker, Joseph M; Casper, Sarah; Holmes, James H; Russo, Laura A; Buzzinotti, Zachri W; Jenik, Pablo D; Vierstra, Richard D

2009-02-01

The 26S proteasome is an essential multicatalytic protease complex that degrades a wide range of intracellular proteins, especially those modified with ubiquitin. Arabidopsis thaliana and other plants use pairs of genes to encode most of the core subunits, with both of the isoforms often incorporated into the mature complex. Here, we show that the gene pair encoding the regulatory particle non-ATPase subunit (RPN5) has a unique role in proteasome function and Arabidopsis development. Homozygous rpn5a rpn5b mutants could not be generated due to a defect in male gametogenesis. While single rpn5b mutants appear wild-type, single rpn5a mutants display a host of morphogenic defects, including abnormal embryogenesis, partially deetiolated development in the dark, a severely dwarfed phenotype when grown in the light, and infertility. Proteasome complexes missing RPN5a are less stable in vitro, suggesting that some of the rpn5a defects are caused by altered complex integrity. The rpn5a phenotype could be rescued by expression of either RPN5a or RPN5b, indicating functional redundancy. However, abnormal phenotypes generated by overexpression implied that paralog-specific functions also exist. Collectively, the data point to a specific role for RPN5 in the plant 26S proteasome and suggest that its two paralogous genes in Arabidopsis have both redundant and unique roles in development.

Circadian Clock Regulates Response to Pesticides in Drosophila via Conserved Pdp1 Pathway

PubMed Central

Beaver, Laura Michelle; Hooven, Louisa Ada; Butcher, Shawn Michael; Krishnan, Natraj; Sherman, Katherine Alice; Chow, Eileen Shin-Yeu; Giebultowicz, Jadwiga Maria

2010-01-01

Daily rhythms generated by the circadian clock regulate many life functions, including responses to xenobiotic compounds. In Drosophila melanogaster, the circadian clock consists of positive elements encoded by cycle (cyc) and Clock (Clk) and negative elements encoded by period (per) and timeless (tim) genes. The ϵ-isoform of the PAR-domain protein 1 (Pdp1ε) transcription factor is controlled by positive clock elements and regulates daily locomotor activity rhythms. Pdp1 target genes have not been identified, and its involvement in other clock output pathways is not known. Mammalian orthologs of Pdp1 have been implicated in the regulation of xenobiotic metabolism; therefore, we asked whether Pdp1 has a similar role in the fly. Using pesticides as model toxicants, we determined that disruption of Pdp1ε increased pesticide-induced mortality in flies. Flies deficient for cyc also showed increased mortality, while disruption of per and tim had no effect. Day/night and Pdp1-dependent differences in the expression of xenobiotic-metabolizing enzymes Cyp6a2, Cyp6g1, and α-Esterase-7 were observed and likely contribute to impaired detoxification. DHR96, a homolog of constitutive androstane receptor and pregnane X receptor, is involved in pesticide response, and DHR96 expression decreased when Pdp1 was suppressed. Taken together, our data uncover a pathway from the positive arm of the circadian clock through Pdp1 to detoxification effector genes, demonstrating a conserved role of the circadian system in modulating xenobiotic toxicity. PMID:20348229

A specialized flavone biosynthetic pathway has evolved in the medicinal plant, Scutellaria baicalensis

PubMed Central

Zhao, Qing; Zhang, Yang; Wang, Gang; Hill, Lionel; Weng, Jing-Ke; Chen, Xiao-Ya; Xue, Hongwei; Martin, Cathie

2016-01-01

Wogonin and baicalein are bioactive flavones in the popular Chinese herbal remedy Huang-Qin (Scutellaria baicalensis Georgi). These specialized flavones lack a 4′-hydroxyl group on the B ring (4′-deoxyflavones) and induce apoptosis in a wide spectrum of human tumor cells in vitro and inhibit tumor growth in vivo in different mouse tumor models. Root-specific flavones (RSFs) from Scutellaria have a variety of reported additional beneficial effects including antioxidant and antiviral properties. We describe the characterization of a new pathway for the synthesis of these compounds, in which pinocembrin (a 4′-deoxyflavanone) serves as a key intermediate. Although two genes encoding flavone synthase II (FNSII) are expressed in the roots of S. baicalensis, FNSII-1 has broad specificity for flavanones as substrates, whereas FNSII-2 is specific for pinocembrin. FNSII-2 is responsible for the synthesis of 4′-deoxyRSFs, such as chrysin and wogonin, wogonoside, baicalein, and baicalin, which are synthesized from chrysin. A gene encoding a cinnamic acid–specific coenzyme A ligase (SbCLL-7), which is highly expressed in roots, is required for the synthesis of RSFs by FNSII-2, as demonstrated by gene silencing. A specific isoform of chalcone synthase (SbCHS-2) that is highly expressed in roots producing RSFs is also required for the synthesis of chrysin. Our studies reveal a recently evolved pathway for biosynthesis of specific, bioactive 4′-deoxyflavones in the roots of S. baicalensis. PMID:27152350

Structure of the human myelin/oligodendrocyte glycoprotein gene and multiple alternative spliced isoforms

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pham-Dinh, D.; Gaspera, D.B.; Dautigny, A.

1995-09-20

Myelin/oligodendrocyte glycoprotein (MOG), a special component of the central nervous system localization on the outermost lamellae of mature myelin, is a member of the immunoglobulin superfamily. We report here the organization of the human MOG gene, which spans approximately 17 kb, and the characterization of six MOG mRNA splicing variants. The intron/exon structure of the human MOG gene confirmed the splicing pattern, supporting the hypothesis that mRNA isoforms could arise by alternative splicing of a single gene. In addition to the eight exons coding for the major MOG isoform, the human MOG gene also contains 3` region, a previously unknownmore » alternatively spliced coding exon, VIA. Alternative utilization of two acceptor splicing sites for exon VIII could produce two different C-termini. The nucleotide sequences presented here may be a useful tool to study further possible involvement if the MOG gene in hereditary neurological disorders. 23 refs., 5 figs.« less

Oligophrenin-1 (OPHN1), a Gene Involved in X-Linked Intellectual Disability, Undergoes RNA Editing and Alternative Splicing during Human Brain Development

PubMed Central

Athanasiadis, Alekos; Galeano, Federica; Locatelli, Franco; Bertini, Enrico; Zanni, Ginevra; Gallo, Angela

2014-01-01

Oligophrenin-1 (OPHN1) encodes for a Rho-GTPase-activating protein, important for dendritic morphogenesis and synaptic function. Mutations in this gene have been identified in patients with X-linked intellectual disability associated with cerebellar hypoplasia. ADAR enzymes are responsible for A-to-I RNA editing, an essential post-transcriptional RNA modification contributing to transcriptome and proteome diversification. Specifically, ADAR2 activity is essential for brain development and function. Herein, we show that the OPHN1 transcript undergoes post-transcriptional modifications such as A-to-I RNA editing and alternative splicing in human brain and other tissues. We found that OPHN1 editing is detectable already at the 18th week of gestation in human brain with a boost of editing at weeks 20 to 33, concomitantly with OPHN1 expression increase and the appearance of a novel OPHN1 splicing isoform. Our results demonstrate that multiple post-transcriptional events occur on OPHN1, a gene playing an important role in brain function and development. PMID:24637888

Oligophrenin-1 (OPHN1), a gene involved in X-linked intellectual disability, undergoes RNA editing and alternative splicing during human brain development.

PubMed

Barresi, Sabina; Tomaselli, Sara; Athanasiadis, Alekos; Galeano, Federica; Locatelli, Franco; Bertini, Enrico; Zanni, Ginevra; Gallo, Angela

2014-01-01

Oligophrenin-1 (OPHN1) encodes for a Rho-GTPase-activating protein, important for dendritic morphogenesis and synaptic function. Mutations in this gene have been identified in patients with X-linked intellectual disability associated with cerebellar hypoplasia. ADAR enzymes are responsible for A-to-I RNA editing, an essential post-transcriptional RNA modification contributing to transcriptome and proteome diversification. Specifically, ADAR2 activity is essential for brain development and function. Herein, we show that the OPHN1 transcript undergoes post-transcriptional modifications such as A-to-I RNA editing and alternative splicing in human brain and other tissues. We found that OPHN1 editing is detectable already at the 18th week of gestation in human brain with a boost of editing at weeks 20 to 33, concomitantly with OPHN1 expression increase and the appearance of a novel OPHN1 splicing isoform. Our results demonstrate that multiple post-transcriptional events occur on OPHN1, a gene playing an important role in brain function and development.

α-Synuclein-induced dopaminergic neurodegeneration in a rat model of Parkinson's disease occurs independent of ATP13A2 (PARK9).

PubMed

Daniel, Guillaume; Musso, Alessandra; Tsika, Elpida; Fiser, Aris; Glauser, Liliane; Pletnikova, Olga; Schneider, Bernard L; Moore, Darren J

2015-01-01

Mutations in the ATP13A2 (PARK9) gene cause early-onset, autosomal recessive Parkinson's disease (PD) and Kufor-Rakeb syndrome. ATP13A2 mRNA is spliced into three distinct isoforms encoding a P5-type ATPase involved in regulating heavy metal transport across vesicular membranes. Here, we demonstrate that three ATP13A2 mRNA isoforms are expressed in the normal human brain and are modestly increased in the cingulate cortex of PD cases. ATP13A2 can mediate protection toward a number of stressors in mammalian cells and can protect against α-synuclein-induced toxicity in cellular and invertebrate models of PD. Using a primary cortical neuronal model combined with lentiviral-mediated gene transfer, we demonstrate that human ATP13A2 isoforms 1 and 2 display selective neuroprotective effects toward toxicity induced by manganese and hydrogen peroxide exposure through an ATPase-independent mechanism. The familial PD mutations, F182L and G504R, abolish the neuroprotective effects of ATP13A2 consistent with a loss-of-function mechanism. We further demonstrate that the AAV-mediated overexpression of human ATP13A2 is not sufficient to attenuate dopaminergic neurodegeneration, neuropathology, and striatal dopamine and motoric deficits induced by human α-synuclein expression in a rat model of PD. Intriguingly, the delivery of an ATPase-deficient form of ATP13A2 (D513N) to the substantia nigra is sufficient to induce dopaminergic neuronal degeneration and motor deficits in rats, potentially suggesting a dominant-negative mechanism of action. Collectively, our data demonstrate a distinct lack of ATP13A2-mediated protection against α-synuclein-induced neurotoxicity in the rat nigrostriatal dopaminergic pathway, and limited neuroprotective capacity overall, and raise doubts about the potential of ATP13A2 as a therapeutic target for PD. Copyright © 2015 Elsevier Inc. All rights reserved.

A Mutation of the Prdm9 Mouse Hybrid Sterility Gene Carried by a Transgene.

PubMed

Mihola, O; Trachtulec, Z

2017-01-01

PRDM9 is a protein with histone-3-methyltransferase activity, which specifies the sites of meiotic recombination in mammals. Deficiency of the Prdm9 gene in the laboratory mouse results in complete arrest of the meiotic prophase of both sexes. Moreover, the combination of certain PRDM9 alleles from different mouse subspecies causes hybrid sterility, e.g., the male-specific meiotic arrest found in the (PWD/Ph × C57BL/6J)F1 animals. The fertility of all these mice can be rescued using a Prdm9-containing transgene. Here we characterized a transgene made from the clone RP24-346I22 that was expected to encompass the entire Prdm9 gene. Both (PWD/Ph × C57BL/6J)F1 intersubspecific hybrid males and Prdm9-deficient laboratory mice of both sexes carrying this transgene remained sterile, suggesting that Prdm9 inactivation occurred in the Tg(RP24-346I22) transgenics. Indeed, comparative qRT-PCR analysis of testicular RNAs from transgene-positive versus negative animals revealed similar expression levels of Prdm9 mRNAs from the exons encoding the C-terminal part of the protein but elevated expression from the regions coding for the N-terminus of PRDM9, indicating that the transgenic carries a new null Prdm9 allele. Two naturally occurring alternative Prdm9 mRNA isoforms were overexpressed in Tg(RP24-346I22), one formed via splicing to a 3'-terminal exon consisting of short interspersed element B2 and one isoform including an alternative internal exon of 28 base pairs. However, the overexpression of these alternative transcripts was apparently insufficient for Prdm9 function or for increasing the fertility of the hybrid males.

Regulation of an antisense RNA with the transition of neonatal to IIb myosin heavy chain during postnatal development and hypothyroidism in rat skeletal muscle

PubMed Central

Jiang, Weihua; Qin, Anqi X.; Bodell, Paul W.; Baldwin, Kenneth M.; Haddad, Fadia

2012-01-01

Postnatal development of fast skeletal muscle is characterized by a transition in expression of myosin heavy chain (MHC) isoforms, from primarily neonatal MHC at birth to primarily IIb MHC in adults, in a tightly coordinated manner. These isoforms are encoded by distinct genes, which are separated by ∼17 kb on rat chromosome 10. The neonatal-to-IIb MHC transition is inhibited by a hypothyroid state. We examined RNA products [mRNA, pre-mRNA, and natural antisense transcript (NAT)] of developmental and adult-expressed MHC genes (embryonic, neonatal, I, IIa, IIx, and IIb) at 2, 10, 20, and 40 days after birth in normal and thyroid-deficient rat neonates treated with propylthiouracil. We found that a long noncoding antisense-oriented RNA transcript, termed bII NAT, is transcribed from a site within the IIb-Neo intergenic region and across most of the IIb MHC gene. NATs have previously been shown to mediate transcriptional repression of sense-oriented counterparts. The bII NAT is transcriptionally regulated during postnatal development and in response to hypothyroidism. Evidence for a regulatory mechanism is suggested by an inverse relationship between IIb MHC and bII NAT in normal and hypothyroid-treated muscle. Neonatal MHC transcription is coordinately expressed with bII NAT. A comparative phylogenetic analysis also suggests that bII NAT-mediated regulation has been a conserved trait of placental mammals for most of the eutherian evolutionary history. The evidence in support of the regulatory model implicates long noncoding antisense RNA as a mechanism to coordinate the transition between neonatal and IIb MHC during postnatal development. PMID:22262309

Regulation of an antisense RNA with the transition of neonatal to IIb myosin heavy chain during postnatal development and hypothyroidism in rat skeletal muscle.

PubMed

Pandorf, Clay E; Jiang, Weihua; Qin, Anqi X; Bodell, Paul W; Baldwin, Kenneth M; Haddad, Fadia

2012-04-01

Postnatal development of fast skeletal muscle is characterized by a transition in expression of myosin heavy chain (MHC) isoforms, from primarily neonatal MHC at birth to primarily IIb MHC in adults, in a tightly coordinated manner. These isoforms are encoded by distinct genes, which are separated by ∼17 kb on rat chromosome 10. The neonatal-to-IIb MHC transition is inhibited by a hypothyroid state. We examined RNA products [mRNA, pre-mRNA, and natural antisense transcript (NAT)] of developmental and adult-expressed MHC genes (embryonic, neonatal, I, IIa, IIx, and IIb) at 2, 10, 20, and 40 days after birth in normal and thyroid-deficient rat neonates treated with propylthiouracil. We found that a long noncoding antisense-oriented RNA transcript, termed bII NAT, is transcribed from a site within the IIb-Neo intergenic region and across most of the IIb MHC gene. NATs have previously been shown to mediate transcriptional repression of sense-oriented counterparts. The bII NAT is transcriptionally regulated during postnatal development and in response to hypothyroidism. Evidence for a regulatory mechanism is suggested by an inverse relationship between IIb MHC and bII NAT in normal and hypothyroid-treated muscle. Neonatal MHC transcription is coordinately expressed with bII NAT. A comparative phylogenetic analysis also suggests that bII NAT-mediated regulation has been a conserved trait of placental mammals for most of the eutherian evolutionary history. The evidence in support of the regulatory model implicates long noncoding antisense RNA as a mechanism to coordinate the transition between neonatal and IIb MHC during postnatal development.

Fatal mitochondrial encephalopathy caused by fumarase deficiency: A molecular-genetic study

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gellera, C.; Cavadini, P.; Baratta, S.

Fumarase deficiency is a rare autosomal recessive disorder of the citric acid cycle resulting in severe organic aciduria and encephalopathy. Mammalian cells contain two fumarase isoenzymes, one mitochondrial and one cytosolic. In rat, the two proteins are encoded by the same gene and are synthesized by alternative initiation of translation at two in-phase AUG codons. One single fumarase gene locus has been identified on human chromosome 1. In most of the patients so far described, the activities of both isozymes are severely affected, suggesting that mutations within a single gene may underlie the disease. Here, we report the molecular studymore » of fumarase deficiency in a patient exhibiting compound heterozygosity for two different allelic mutations affecting the amino acid composition of both isoforms. The proband, an Italian boy of nonconsanguineous parents, died at 7 months of age of a progressive encephalopathy. Immunoblot demonstrated absence of cross-reacting material in both cytosolic and mitochondrial fraction of all tissues examined. Molecular analysis of the patient`s fumarase cDNA amplified by RT-PCR showed the presence of two mutations affecting the amino acid composition of both isoforms, a missense mutation resulting in the nonconservative amino acid substitution at codon 190 (Arg190Cys) and an amino acid in-frame insertion at codon 434 (Lys434ins). SSCP analysis of genomic PCR fragments encompassing the mutations demonstrated that the patient was heterozygous for both mutations, having inherited the Arg-to-Cys substitution from the father and the in-frame insertion from the mother. Finally, the effects of the mutations on enzyme function were investigated by expressing both normal and mutated fumarase cDNAs in a fumarase-deficient ({delta}FUM1) S. cerevisiae strain.« less

Peroxisome proliferator-activated receptors for hypertension

PubMed Central

Usuda, Daisuke; Kanda, Tsugiyasu

2014-01-01

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors belonging to the nuclear receptor superfamily, which is composed of four members encoded by distinct genes (α, β, γ, and δ). The genes undergo transactivation or transrepression under specific mechanisms that lead to the induction or repression of target gene expression. As is the case with other nuclear receptors, all four PPAR isoforms contain five or six structural regions in four functional domains; namely, A/B, C, D, and E/F. PPARs have many functions, particularly functions involving control of vascular tone, inflammation, and energy homeostasis, and are, therefore, important targets for hypertension, obesity, obesity-induced inflammation, and metabolic syndrome in general. Hence, PPARs also represent drug targets, and PPARα and PPARγ agonists are used clinically in the treatment of dyslipidemia and type 2 diabetes mellitus, respectively. Because of their pleiotropic effects, they have been identified as active in a number of diseases and are targets for the development of a broad range of therapies for a variety of diseases. It is likely that the range of PPARγ agonist therapeutic actions will result in novel approaches to lifestyle and other diseases. The combination of PPARs with reagents or with other cardiovascular drugs, such as diuretics and angiotensin II receptor blockers, should be studied. This article provides a review of PPAR isoform characteristics, a discussion of progress in our understanding of the biological actions of PPARs, and a summary of PPAR agonist development for patient management. We also include a summary of the experimental and clinical evidence obtained from animal studies and clinical trials conducted to evaluate the usefulness and effectiveness of PPAR agonists in the treatment of lifestyle-related diseases. PMID:25228953

Mg chelatase in chlorophyll synthesis and retrograde signaling in Chlamydomonas reinhardtii : CHLI2 cannot substitute for CHLI1

DOE PAGES

Brzezowski, Pawel; Sharifi, Marina N.; Dent, Rachel M.; ...

2016-01-25

The oligomeric Mg chelatase (MgCh), consisting of the subunits CHLH, CHLI, and CHLD, is located at the central site of chlorophyll synthesis, but is also thought to have an additional function in regulatory feedback control of the tetrapyrrole biosynthesis pathway and in chloroplast retrograde signaling. In Arabidopsis thaliana and Chlamydomonas reinhardtii, two genes have been proposed to encode the CHLI subunit of MgCh. While the role of CHLI1 in A. thaliana MgCh has been substantially elucidated, different reports provide inconsistent results with regard to the function of CHLI2 in Mg chelation and retrograde signaling. In the present report, the possiblemore » functions of both isoforms were analyzed in C. reinhardtii. Knockout of the CHLI1 gene resulted in complete loss of MgCh activity, absence of chlorophyll, acute light sensitivity, and, as a consequence, down-regulation of tetrapyrrole biosynthesis and photosynthesis-associated nuclear genes. These observations indicate a phenotypical resemblance of chli1 to the chlh and chld C. reinhardtii mutants previously reported. The key role of CHLI1 for MgCh reaction in comparison with the second isoform was confirmed by the rescue of chli1 with genomic CHLI1. Because CHLI2 in C. reinhardtii shows lower expression than CHLI1, strains overexpressing CHLI2 were produced in the chli1 background. However, no complementation of the chli1 phenotype was observed. Silencing of CHLI2 in the wild-type background did not result in any changes in the accumulation of tetrapyrrole intermediates or of chlorophyll. The results suggest that, unlike in A. thaliana, changes in CHLI2 content observed in the present studies do not affect formation and activity of MgCh in C. reinhardtii.« less

Retinoic acid-induced pancreatic stellate cell quiescence reduces paracrine Wnt-β-catenin signaling to slow tumor progression.

PubMed

Froeling, Fieke E M; Feig, Christine; Chelala, Claude; Dobson, Richard; Mein, Charles E; Tuveson, David A; Clevers, Hans; Hart, Ian R; Kocher, Hemant M

2011-10-01

Patients with pancreatic ductal adenocarcinoma are deficient in vitamin A, resulting in activation of pancreatic stellate cells (PSCs). We investigated whether restoration of retinol to PSCs restores their quiescence and affects adjacent cancer cells. PSCs and cancer cell lines (AsPc1 and Capan1) were exposed to doses and isoforms of retinoic acid (RA) in 2-dimensional and 3-dimensional culture conditions (physiomimetic organotypic culture). The effects of all-trans retinoic acid (ATRA) were studied in LSL-KrasG12D/+;LSL-Trp53R172H/+;Pdx-1-Cre mice, a model of human pancreatic ductal adenocarcinoma. After incubation with ATRA, PSCs were quiescent and had altered expression of genes that regulate proliferation, morphology, and motility; genes that encode cytoskeletal proteins and cytokines; and genes that control other functions, irrespective of culture conditions or dosage. In the organotypic model, and in mice, ATRA induced quiescence of PSCs and thereby reduced cancer cell proliferation and translocation of β-catenin to the nucleus, increased cancer cell apoptosis, and altered tumor morphology. ATRA reduced the motility of PSCs, so these cells created a "wall" at the junction between the tumor and the matrix that prevented cancer cell invasion. Restoring secreted frizzled-related protein 4 (sFRP4) secretion to quiescent PSCs reduced Wnt-β-catenin signaling in cancer cells and their invasive ability. Human primary and metastatic pancreatic tumor tissues stained strongly for cancer cell nuclear β-catenin but had low levels of sFRP4 (in cancer cells and PSCs). RA induces quiescence and reduces motility of PSCs, leading to reduced proliferation and increased apoptosis of surrounding pancreatic cancer cells. RA isoforms might be developed as therapeutic reagents for pancreatic cancer. Copyright © 2011 AGA Institute. Published by Elsevier Inc. All rights reserved.

Characterization and expression of cyp19a gene in the Chinese giant salamander Andrias davidianus.

PubMed

Hu, Qiaomu; Xiao, Hanbing; Tian, HaiFeng; Meng, Yan

2016-02-01

We cloned the full length cyp19a of Chinese giant salamander Andrias davidianus, determined its distribution in tissues and developing gonads, and analyzed the CpG methylation pattern of the cyp19a promoter. The results revealed isoforms of 1706 bp (G arom) and 1698 bp (B arom) in length, differing in the 5' flanking region, both encoding 502 amino acids. The G arom gene was observed mainly in the ovary and kidney, with little in other investigated tissues, while B arom expression was high in the brain, ovary, testis, and pituitary, with low or undetected expression in other examined tissues. Total aromatase expression was high in the ovary; moderate in the kidney, brain, testis, and pituitary; and low in the remaining tissues. G arom expression was significantly higher in the ovary than in the testis and gradually decreased with maturation of the salamander. A single injection of methyltestosterone or letrozole resulted in ovarian G arom expression decreasing over a 12-96 h period. A 1366 bp sequence of the cyp19a promoter was cloned and shown to be conserved in selected species. CpG methylation level was negatively correlated with cyp19a expression in the examined tissues and developing ovaries. Five and three CpG methylation sites positively correlated with DNA methylation levels in tissues and developing ovary, suggesting that they play an important role in regulating cyp19a expression. The aromatase gene showed two isoforms with distinct expression patterns, and the promoter methylation level at specific CpG sites was associated with variation in expression profiles of tissues and developing ovaries. Copyright © 2015 Elsevier Inc. All rights reserved.

Phenotypic variability in patients with osteogenesis imperfecta caused by BMP1 mutations.

PubMed

Pollitt, Rebecca C; Saraff, Vrinda; Dalton, Ann; Webb, Emma A; Shaw, Nick J; Sobey, Glenda J; Mughal, M Zulf; Hobson, Emma; Ali, Farhan; Bishop, Nicholas J; Arundel, Paul; Högler, Wolfgang; Balasubramanian, Meena

2016-12-01

Osteogenesis Imperfecta (OI) is an inherited bone fragility disorder most commonly associated with autosomal dominant mutations in the type I collagen genes. Autosomal recessive mutations in a number of genes have also been described, including the BMP1 gene that encodes the mammalian Tolloid (mTLD) and its shorter isoform bone morphogenic protein-1 (BMP1). To date, less than 20 individuals with OI have been identified with BMP1 mutations, with skeletal phenotypes ranging from mild to severe and progressively deforming. In the majority of patients, bone fragility was associated with increased bone mineral density (BMD); however, the full range of phenotypes associated with BMP1 remains unclear. Here, we describe three children with mutations in BMP1 associated with a highly variable phenotype: a sibship homozygous for the c.2188delC mutation that affects only the shorter BMP1 isoform and a further patient who is compound heterozygous for a c.1293C>G nonsense mutation and a c.1148G>A missense mutation in the CUB1 domain. These individuals had recurrent fractures from early childhood, are hypermobile and have no evidence of dentinogenesis imperfecta. The homozygous siblings with OI had normal areal BMD by dual energy X-ray absorptiometry whereas the third patient presented with a high bone mass phenotype. Intravenous bisphosphonate therapy was started in all patients, but discontinued in two patients and reduced in another due to concerns about increasing bone stiffness leading to chalk-stick fractures. Given the association of BMP1-related OI with very high bone material density, concerns remain whether anti-resorptive therapy is indicated in this ultra-rare form of OI.© 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Alternative Splicing Profile and Sex-Preferential Gene Expression in the Female and Male Pacific Abalone Haliotis discus hannai.

PubMed

Kim, Mi Ae; Rhee, Jae-Sung; Kim, Tae Ha; Lee, Jung Sick; Choi, Ah-Young; Choi, Beom-Soon; Choi, Ik-Young; Sohn, Young Chang

2017-03-09

In order to characterize the female or male transcriptome of the Pacific abalone and further increase genomic resources, we sequenced the mRNA of full-length complementary DNA (cDNA) libraries derived from pooled tissues of female and male Haliotis discus hannai by employing the Iso-Seq protocol of the PacBio RSII platform. We successfully assembled whole full-length cDNA sequences and constructed a transcriptome database that included isoform information. After clustering, a total of 15,110 and 12,145 genes that coded for proteins were identified in female and male abalones, respectively. A total of 13,057 putative orthologs were retained from each transcriptome in abalones. Overall Gene Ontology terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways analyzed in each database showed a similar composition between sexes. In addition, a total of 519 and 391 isoforms were genome-widely identified with at least two isoforms from female and male transcriptome databases. We found that the number of isoforms and their alternatively spliced patterns are variable and sex-dependent. This information represents the first significant contribution to sex-preferential genomic resources of the Pacific abalone. The availability of whole female and male transcriptome database and their isoform information will be useful to improve our understanding of molecular responses and also for the analysis of population dynamics in the Pacific abalone.

Alternative Splicing Profile and Sex-Preferential Gene Expression in the Female and Male Pacific Abalone Haliotis discus hannai

PubMed Central

Kim, Mi Ae; Rhee, Jae-Sung; Kim, Tae Ha; Lee, Jung Sick; Choi, Ah-Young; Choi, Beom-Soon; Choi, Ik-Young; Sohn, Young Chang

2017-01-01

In order to characterize the female or male transcriptome of the Pacific abalone and further increase genomic resources, we sequenced the mRNA of full-length complementary DNA (cDNA) libraries derived from pooled tissues of female and male Haliotis discus hannai by employing the Iso-Seq protocol of the PacBio RSII platform. We successfully assembled whole full-length cDNA sequences and constructed a transcriptome database that included isoform information. After clustering, a total of 15,110 and 12,145 genes that coded for proteins were identified in female and male abalones, respectively. A total of 13,057 putative orthologs were retained from each transcriptome in abalones. Overall Gene Ontology terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways analyzed in each database showed a similar composition between sexes. In addition, a total of 519 and 391 isoforms were genome-widely identified with at least two isoforms from female and male transcriptome databases. We found that the number of isoforms and their alternatively spliced patterns are variable and sex-dependent. This information represents the first significant contribution to sex-preferential genomic resources of the Pacific abalone. The availability of whole female and male transcriptome database and their isoform information will be useful to improve our understanding of molecular responses and also for the analysis of population dynamics in the Pacific abalone. PMID:28282934

The developmental transcriptome of Drosophila melanogaster

DOE Office of Scientific and Technical Information (OSTI.GOV)

University of Connecticut; Graveley, Brenton R.; Brooks, Angela N.

Drosophila melanogaster is one of the most well studied genetic model organisms; nonetheless, its genome still contains unannotated coding and non-coding genes, transcripts, exons and RNA editing sites. Full discovery and annotation are pre-requisites for understanding how the regulation of transcription, splicing and RNA editing directs the development of this complex organism. Here we used RNA-Seq, tiling microarrays and cDNA sequencing to explore the transcriptome in 30 distinct developmental stages. We identified 111,195 new elements, including thousands of genes, coding and non-coding transcripts, exons, splicing and editing events, and inferred protein isoforms that previously eluded discovery using established experimental, predictionmore » and conservation-based approaches. These data substantially expand the number of known transcribed elements in the Drosophila genome and provide a high-resolution view of transcriptome dynamics throughout development. Drosophila melanogaster is an important non-mammalian model system that has had a critical role in basic biological discoveries, such as identifying chromosomes as the carriers of genetic information and uncovering the role of genes in development. Because it shares a substantial genic content with humans, Drosophila is increasingly used as a translational model for human development, homeostasis and disease. High-quality maps are needed for all functional genomic elements. Previous studies demonstrated that a rich collection of genes is deployed during the life cycle of the fly. Although expression profiling using microarrays has revealed the expression of, 13,000 annotated genes, it is difficult to map splice junctions and individual base modifications generated by RNA editing using such approaches. Single-base resolution is essential to define precisely the elements that comprise the Drosophila transcriptome. Estimates of the number of transcript isoforms are less accurate than estimates of the number of genes. Whereas, 20% of Drosophila genes are annotated as encoding alternatively spliced premRNAs, splice-junction microarray experiments indicate that this number is at least 40% (ref. 7). Determining the diversity of mRNAs generated by alternative promoters, alternative splicing and RNA editing will substantially increase the inferred protein repertoire. Non-coding RNA genes (ncRNAs) including short interfering RNAs (siRNAs) and microRNAS (miRNAs) (reviewed in ref. 10), and longer ncRNAs such as bxd (ref. 11) and rox (ref. 12), have important roles in gene regulation, whereas others such as small nucleolar RNAs (snoRNAs)and small nuclear RNAs (snRNAs) are important components of macromolecular machines such as the ribosome and spliceosome. The transcription and processing of these ncRNAs must also be fully documented and mapped. As part of the modENCODE project to annotate the functional elements of the D. melanogaster and Caenorhabditis elegans genomes, we used RNA-Seq and tiling microarrays to sample the Drosophila transcriptome at unprecedented depth throughout development from early embryo to ageing male and female adults. We report on a high-resolution view of the discovery, structure and dynamic expression of the D. melanogaster transcriptome.« less

A truncated, activin-induced Smad3 isoform acts as a transcriptional repressor of FSHβ expression in mouse pituitary.

PubMed

Kim, So-Youn; Zhu, Jie; Woodruff, Teresa K

2011-08-06

The receptor-regulated protein Smad3 is key player in the signaling cascade stimulated by the binding of activin to its cell surface receptor. Upon phosphorylation, Smad3 forms a heterocomplex with Smad2 and Smad4, translocates to the nucleus and acts as a transcriptional co-activator. We have identified a unique isoform of Smad3 that is expressed in mature pituitary gonadotropes. 5' RACE revealed that this truncated Smad3 isoform is transcribed from an ATG site within exon 4 and consists of 7 exons encoding half of the linker region and the MH2 region. In pituitary cells, the truncated Smad3 isoform was phosphorylated upon activin treatment, in a manner that was temporally distinct from the phosphorylation of full-length Smad3. Activin-induced phosphorylation of Smad3 and the truncated Smad3 isoform was blocked by both follistatin and siRNA-mediated knockdown of Smad3. The truncated Smad3 isoform antagonized Smad3-mediated, activin-responsive promoter activity. We propose that the pituitary gonadotrope contains an ultra-short, activin-responsive feedback loop utilizing two different isoforms of Smad3, one which acts as an agonist (Smad3) and another that acts as an intracrine antagonist (truncated Smad3 isoform) to regulate FSHβ production. Copyright © 2011 Elsevier Ltd. All rights reserved.

Alternative Splicing of Four Trafficking Genes Regulates Myofiber Structure and Skeletal Muscle Physiology.

PubMed

Giudice, Jimena; Loehr, James A; Rodney, George G; Cooper, Thomas A

2016-11-15

During development, transcriptional and post-transcriptional networks are coordinately regulated to drive organ maturation. Alternative splicing contributes by producing temporal-specific protein isoforms. We previously found that genes undergoing splicing transitions during mouse postnatal heart development are enriched for vesicular trafficking and membrane dynamics functions. Here, we show that adult trafficking isoforms are also expressed in adult skeletal muscle and hypothesize that striated muscle utilizes alternative splicing to generate specific isoforms required for function of adult tissue. We deliver morpholinos into flexor digitorum brevis muscles in adult mice to redirect splicing of four trafficking genes to the fetal isoforms. The splicing switch results in multiple structural and functional defects, including transverse tubule (T-tubule) disruption and dihydropyridine receptor alpha (DHPR) and Ryr1 mislocalization, impairing excitation-contraction coupling, calcium handling, and force generation. The results demonstrate a previously unrecognized role for trafficking functions in adult muscle tissue homeostasis and a specific requirement for the adult splice variants. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

Functional characterization of EZH2β reveals the increased complexity of EZH2 isoforms involved in the regulation of mammalian gene expression

PubMed Central

2013-01-01

Background Histone methyltransferase enhancer of zeste homologue 2 (EZH2) forms an obligate repressive complex with suppressor of zeste 12 and embryonic ectoderm development, which is thought, along with EZH1, to be primarily responsible for mediating Polycomb-dependent gene silencing. Polycomb-mediated repression influences gene expression across the entire gamut of biological processes, including development, differentiation and cellular proliferation. Deregulation of EZH2 expression is implicated in numerous complex human diseases. To date, most EZH2-mediated function has been primarily ascribed to a single protein product of the EZH2 locus. Results We report that the EZH2 locus undergoes alternative splicing to yield at least two structurally and functionally distinct EZH2 methyltransferases. The longest protein encoded by this locus is the conventional enzyme, which we refer to as EZH2α, whereas EZH2β, characterized here, represents a novel isoform. We find that EZH2β localizes to the cell nucleus, complexes with embryonic ectoderm development and suppressor of zeste 12, trimethylates histone 3 at lysine 27, and mediates silencing of target promoters. At the cell biological level, we find that increased EZH2β induces cell proliferation, demonstrating that this protein is functional in the regulation of processes previously attributed to EZH2α. Biochemically, through the use of genome-wide expression profiling, we demonstrate that EZH2β governs a pattern of gene repression that is often ontologically redundant from that of EZH2α, but also divergent for a wide variety of specific target genes. Conclusions Combined, these results demonstrate that an expanded repertoire of EZH2 writers can modulate histone code instruction during histone 3 lysine 27-mediated gene silencing. These data support the notion that the regulation of EZH2-mediated gene silencing is more complex than previously anticipated and should guide the design and interpretation of future studies aimed at understanding the biochemical and biological roles of this important family of epigenomic regulators. PMID:23448518

Complementation for an essential ancillary non-structural protein function across parvovirus genera.

PubMed

Mihaylov, Ivailo S; Cotmore, Susan F; Tattersall, Peter

2014-11-01

Parvoviruses encode a small number of ancillary proteins that differ substantially between genera. Within the genus Protoparvovirus, minute virus of mice (MVM) encodes three isoforms of its ancillary protein NS2, while human bocavirus 1 (HBoV1), in the genus Bocaparvovirus, encodes an NP1 protein that is unrelated in primary sequence to MVM NS2. To search for functional overlap between NS2 and NP1, we generated murine A9 cell populations that inducibly express HBoV1 NP1. These were used to test whether NP1 expression could complement specific defects resulting from depletion of MVM NS2 isoforms. NP1 induction had little impact on cell viability or cell cycle progression in uninfected cells, and was unable to complement late defects in MVM virion production associated with low NS2 levels. However, NP1 did relocate to MVM replication centers, and supports both the normal expansion of these foci and overcomes the early paralysis of DNA replication in NS2-null infections. Copyright © 2014 Elsevier Inc. All rights reserved.

Convergence of the Insulin and Serotonin Programs in the Pancreatic β-Cell

PubMed Central

Ohta, Yasuharu; Kosaka, Yasuhiro; Kishimoto, Nina; Wang, Juehu; Smith, Stuart B.; Honig, Gerard; Kim, Hail; Gasa, Rosa M.; Neubauer, Nicole; Liou, Angela; Tecott, Laurence H.; Deneris, Evan S.; German, Michael S.

2011-01-01

OBJECTIVE Despite their origins in different germ layers, pancreatic islet cells share many common developmental features with neurons, especially serotonin-producing neurons in the hindbrain. Therefore, we tested whether these developmental parallels have functional consequences. RESEARCH DESIGN AND METHODS We used transcriptional profiling, immunohistochemistry, DNA-binding analyses, and mouse genetic models to assess the expression and function of key serotonergic genes in the pancreas. RESULTS We found that islet cells expressed the genes encoding all of the products necessary for synthesizing, packaging, and secreting serotonin, including both isoforms of the serotonin synthetic enzyme tryptophan hydroxylase and the archetypal serotonergic transcription factor Pet1. As in serotonergic neurons, Pet1 expression in islets required homeodomain transcription factor Nkx2.2 but not Nkx6.1. In β-cells, Pet1 bound to the serotonergic genes but also to a conserved insulin gene regulatory element. Mice lacking Pet1 displayed reduced insulin production and secretion and impaired glucose tolerance. CONCLUSIONS These studies demonstrate that a common transcriptional cascade drives the differentiation of β-cells and serotonergic neurons and imparts the shared ability to produce serotonin. The interrelated biology of these two cell types has important implications for the pathology and treatment of diabetes. PMID:22013016

Convergence of the insulin and serotonin programs in the pancreatic β-cell.

PubMed

Ohta, Yasuharu; Kosaka, Yasuhiro; Kishimoto, Nina; Wang, Juehu; Smith, Stuart B; Honig, Gerard; Kim, Hail; Gasa, Rosa M; Neubauer, Nicole; Liou, Angela; Tecott, Laurence H; Deneris, Evan S; German, Michael S

2011-12-01

Despite their origins in different germ layers, pancreatic islet cells share many common developmental features with neurons, especially serotonin-producing neurons in the hindbrain. Therefore, we tested whether these developmental parallels have functional consequences. We used transcriptional profiling, immunohistochemistry, DNA-binding analyses, and mouse genetic models to assess the expression and function of key serotonergic genes in the pancreas. We found that islet cells expressed the genes encoding all of the products necessary for synthesizing, packaging, and secreting serotonin, including both isoforms of the serotonin synthetic enzyme tryptophan hydroxylase and the archetypal serotonergic transcription factor Pet1. As in serotonergic neurons, Pet1 expression in islets required homeodomain transcription factor Nkx2.2 but not Nkx6.1. In β-cells, Pet1 bound to the serotonergic genes but also to a conserved insulin gene regulatory element. Mice lacking Pet1 displayed reduced insulin production and secretion and impaired glucose tolerance. These studies demonstrate that a common transcriptional cascade drives the differentiation of β-cells and serotonergic neurons and imparts the shared ability to produce serotonin. The interrelated biology of these two cell types has important implications for the pathology and treatment of diabetes.

The Peculiar Characteristics of Fish Type I Interferons

PubMed Central

Boudinot, Pierre; Langevin, Christelle; Secombes, Christopher J.; Levraud, Jean-Pierre

2016-01-01

Antiviral type I interferons (IFNs) have been discovered in fish. Genomic studies revealed their considerable number in many species; some genes encode secreted and non-secreted isoforms. Based on cysteine motifs, fish type I IFNs fall in two subgroups, which use two different receptors. Mammalian type I IFN genes are intronless while type III have introns; in fish, all have introns, but structurally, both subgroups belong to type I. Type I IFNs likely appeared early in vertebrates as intron containing genes, and evolved in parallel in tetrapods and fishes. The diversity of their repertoires in fish and mammals is likely a convergent feature, selected as a response to the variety of viral strategies. Several alternative nomenclatures have been established for different taxonomic fish groups, calling for a unified system. The specific functions of each type I gene remains poorly understood, as well as their interactions in antiviral responses. However, distinct induction pathways, kinetics of response, and tissue specificity indicate that fish type I likely are highly specialized, especially in groups where they are numerous such as salmonids or cyprinids. Unravelling their functional integration constitutes the next challenge to understand how these cytokines evolved to orchestrate antiviral innate immunity in vertebrates. PMID:27827855

Distinct Interactions of EBP1 Isoforms with FBXW7 Elicits Different Functions in Cancer

DOE PAGES

Wang, Yuli; Zhang, Pengju; Wang, Yunshan; ...

2017-02-16

The ErbB3 receptor–binding protein EBP1 encodes two alternatively spliced isoforms P48 and P42. While there is evidence of differential roles for these isoforms in tumorigenesis, little is known about their underlying mechanisms. In this paper, we demonstrate that EBP1 isoforms interact with the SCF-type ubiquitin ligase FBXW7 in distinct ways to exert opposing roles in tumorigenesis. EBP1 P48 bound to the WD domain of FBXW7 as an oncogenic substrate of FBXW7. EBP1 P48 binding sequestered FBXW7α to the cytosol, modulating its role in protein degradation and attenuating its tumor suppressor function. In contrast, EBP1 P42 bound to both the F-boxmore » domain of FBXW7 as well as FBXW7 substrates. This adapter function of EBP1 P42 stabilized the interaction of FBXW7 with its substrates and promoted FBXW7-mediated degradation of oncogenic targets, enhancing its overall tumor-suppressing function. Finally and overall, our results establish distinct physical and functional interactions between FBXW7 and EBP1 isoforms, which yield their mechanistically unique isoform-specific functions of EBP1 in cancer.« less

The alternatively-included 11a sequence modifies the effects of Mena on actin cytoskeletal organization and cell behavior

PubMed Central

Balsamo, Michele; Mondal, Chandrani; Carmona, Guillaume; McClain, Leslie M.; Riquelme, Daisy N.; Tadros, Jenny; Ma, Duan; Vasile, Eliza; Condeelis, John S.; Lauffenburger, Douglas A.; Gertler, Frank B.

2016-01-01

During tumor progression, alternative splicing gives rise to different Mena protein isoforms. We analyzed how Mena11a, an isoform enriched in epithelia and epithelial-like cells, affects Mena-dependent regulation of actin dynamics and cell behavior. While other Mena isoforms promote actin polymerization and drive membrane protrusion, we find that Mena11a decreases actin polymerization and growth factor-stimulated membrane protrusion at lamellipodia. Ectopic Mena11a expression slows mesenchymal-like cell motility, while isoform-specific depletion of endogenous Mena11a in epithelial-like tumor cells perturbs cell:cell junctions and increases membrane protrusion and overall cell motility. Mena11a can dampen membrane protrusion and reduce actin polymerization in the absence of other Mena isoforms, indicating that it is not simply an inactive Mena isoform. We identify a phosphorylation site within 11a that is required for some Mena11a-specific functions. RNA-seq data analysis from patient cohorts demonstrates that the difference between mRNAs encoding constitutive Mena sequences and those containing the 11a exon correlates with metastasis in colorectal cancer, suggesting that 11a exon exclusion contributes to invasive phenotypes and leads to poor clinical outcomes. PMID:27748415

The alternatively-included 11a sequence modifies the effects of Mena on actin cytoskeletal organization and cell behavior.

PubMed

Balsamo, Michele; Mondal, Chandrani; Carmona, Guillaume; McClain, Leslie M; Riquelme, Daisy N; Tadros, Jenny; Ma, Duan; Vasile, Eliza; Condeelis, John S; Lauffenburger, Douglas A; Gertler, Frank B

2016-10-17

During tumor progression, alternative splicing gives rise to different Mena protein isoforms. We analyzed how Mena11a, an isoform enriched in epithelia and epithelial-like cells, affects Mena-dependent regulation of actin dynamics and cell behavior. While other Mena isoforms promote actin polymerization and drive membrane protrusion, we find that Mena11a decreases actin polymerization and growth factor-stimulated membrane protrusion at lamellipodia. Ectopic Mena11a expression slows mesenchymal-like cell motility, while isoform-specific depletion of endogenous Mena11a in epithelial-like tumor cells perturbs cell:cell junctions and increases membrane protrusion and overall cell motility. Mena11a can dampen membrane protrusion and reduce actin polymerization in the absence of other Mena isoforms, indicating that it is not simply an inactive Mena isoform. We identify a phosphorylation site within 11a that is required for some Mena11a-specific functions. RNA-seq data analysis from patient cohorts demonstrates that the difference between mRNAs encoding constitutive Mena sequences and those containing the 11a exon correlates with metastasis in colorectal cancer, suggesting that 11a exon exclusion contributes to invasive phenotypes and leads to poor clinical outcomes.

Characterization of rtSH3p13 gene encoding a development protein involved in vesicular traffic in spermiogenesis.

PubMed

Qiao, Yuan; Yang, Ju Xiang; Zhang, Xiao Dong L; Liu, Yu; Zhang, Jian Chao; Zong, Shu Dong; Miao, Shi Ying; Wang, Lin Fang; Koide, Samuel S

2004-06-01

A cDNA, designated as rtSH3p13, was isolated from a rat testis cDNA library. It consists of 1463 bp nuclear acids, which encodes a protein of 312 amino acids and was assigned the GenBank accession number AF227439. The deduced rtSH3p13 protein is a truncated isoform of SH3p13 as a result of mRNA alternative splicing. It is mainly expressed in the rat testis, detected in spermatids at the steps 8-19 of spermiogenesis, and found around the acrosome. During postnatal development, rtSH3p13 appears on day 18 and reaches maximum on day 60. Further experimental results suggested that rtSH3p13 forms a complex with activated epidermal growth factor receptor (EGFR) and interacts with synaptojanin I. Surprisingly, similar to SH3 domain, the V region of rtSH3p13 also inhibits endocytosis in CHO cells. Our results reveal a link between an rtSH3p13-synaptojanin-clathrin complex-mediated formation of pits and the process of spermiogenesis.

Genome-wide characterization of differential transcript usage in Arabidopsis thaliana.

PubMed

Vaneechoutte, Dries; Estrada, April R; Lin, Ying-Chen; Loraine, Ann E; Vandepoele, Klaas

2017-12-01

Alternative splicing and the usage of alternate transcription start- or stop sites allows a single gene to produce multiple transcript isoforms. Most plant genes express certain isoforms at a significantly higher level than others, but under specific conditions this expression dominance can change, resulting in a different set of dominant isoforms. These events of differential transcript usage (DTU) have been observed for thousands of Arabidopsis thaliana, Zea mays and Vitis vinifera genes, and have been linked to development and stress response. However, neither the characteristics of these genes, nor the implications of DTU on their protein coding sequences or functions, are currently well understood. Here we present a dataset of isoform dominance and DTU for all genes in the AtRTD2 reference transcriptome based on a protocol that was benchmarked on simulated data and validated through comparison with a published reverse transciptase-polymerase chain reaction panel. We report DTU events for 8148 genes across 206 public RNA-Seq samples, and find that protein sequences are affected in 22% of the cases. The observed DTU events show high consistency across replicates, and reveal reproducible patterns in response to treatment and development. We also demonstrate that genes with different evolutionary ages, expression breadths and functions show large differences in the frequency at which they undergo DTU, and in the effect that these events have on their protein sequences. Finally, we showcase how the generated dataset can be used to explore DTU events for genes of interest or to find genes with specific DTU in samples of interest. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

Characterisation of CDKL5 Transcript Isoforms in Human and Mouse

PubMed Central

Dando, Owen; Landsberger, Nicoletta; Kilstrup-Nielsen, Charlotte; Kind, Peter C.; Bailey, Mark E. S.; Cobb, Stuart R.

2016-01-01

Mutations in the X-linked Cyclin-Dependent Kinase-Like 5 gene (CDKL5) cause early onset infantile spasms and subsequent severe developmental delay in affected children. Deleterious mutations have been reported to occur throughout the CDKL5 coding region. Several studies point to a complex CDKL5 gene structure in terms of exon usage and transcript expression. Improvements in molecular diagnosis and more extensive research into the neurobiology of CDKL5 and pathophysiology of CDKL5 disorders necessitate an updated analysis of the gene. In this study, we have analysed human and mouse CDKL5 transcript patterns both bioinformatically and experimentally. We have characterised the predominant brain isoform of CDKL5, a 9.7 kb transcript comprised of 18 exons with a large 6.6 kb 3’-untranslated region (UTR), which we name hCDKL5_1. In addition we describe new exonic regions and a range of novel splice and UTR isoforms. This has enabled the description of an updated gene model in both species and a standardised nomenclature system for CDKL5 transcripts. Profiling revealed tissue- and brain development stage-specific differences in expression between transcript isoforms. These findings provide an essential backdrop for the diagnosis of CDKL5-related disorders, for investigations into the basic biology of this gene and its protein products, and for the rational design of gene-based and molecular therapies for these disorders. PMID:27315173

Characterisation of CDKL5 Transcript Isoforms in Human and Mouse.

PubMed

Hector, Ralph D; Dando, Owen; Landsberger, Nicoletta; Kilstrup-Nielsen, Charlotte; Kind, Peter C; Bailey, Mark E S; Cobb, Stuart R

2016-01-01

Mutations in the X-linked Cyclin-Dependent Kinase-Like 5 gene (CDKL5) cause early onset infantile spasms and subsequent severe developmental delay in affected children. Deleterious mutations have been reported to occur throughout the CDKL5 coding region. Several studies point to a complex CDKL5 gene structure in terms of exon usage and transcript expression. Improvements in molecular diagnosis and more extensive research into the neurobiology of CDKL5 and pathophysiology of CDKL5 disorders necessitate an updated analysis of the gene. In this study, we have analysed human and mouse CDKL5 transcript patterns both bioinformatically and experimentally. We have characterised the predominant brain isoform of CDKL5, a 9.7 kb transcript comprised of 18 exons with a large 6.6 kb 3'-untranslated region (UTR), which we name hCDKL5_1. In addition we describe new exonic regions and a range of novel splice and UTR isoforms. This has enabled the description of an updated gene model in both species and a standardised nomenclature system for CDKL5 transcripts. Profiling revealed tissue- and brain development stage-specific differences in expression between transcript isoforms. These findings provide an essential backdrop for the diagnosis of CDKL5-related disorders, for investigations into the basic biology of this gene and its protein products, and for the rational design of gene-based and molecular therapies for these disorders.

SlgA, encoded by the homolog of the human schizophrenia-associated gene PRODH, acts in clock neurons to regulate Drosophila aggression

PubMed Central

Zwarts, Liesbeth; Vulsteke, Veerle; Buhl, Edgar; Hodge, James J. L.

2017-01-01

ABSTRACT Mutations in the proline dehydrogenase gene PRODH are linked to behavioral alterations in schizophrenia and as part of DiGeorge and velo-cardio-facial syndromes, but the role of PRODH in their etiology remains unclear. Here, we establish a Drosophila model to study the role of PRODH in behavioral disorders. We determine the distribution of the Drosophila PRODH homolog slgA in the brain and show that knockdown and overexpression of human PRODH and slgA in the lateral neurons ventral (LNv) lead to altered aggressive behavior. SlgA acts in an isoform-specific manner and is regulated by casein kinase II (CkII). Our data suggest that these effects are, at least partially, due to effects on mitochondrial function. We thus show that precise regulation of proline metabolism is essential to drive normal behavior and we identify Drosophila aggression as a model behavior relevant for the study of the mechanisms that are impaired in neuropsychiatric disorders. PMID:28331058

Differential Properties of Cytomegalovirus pUL97 Kinase Isoforms Affect Viral Replication and Maribavir Susceptibility

PubMed Central

Webel, Rike; Hakki, Morgan; Prichard, Mark N.; Rawlinson, William D.; Marschall, Manfred

2014-01-01

ABSTRACT The human cytomegalovirus (HCMV)-encoded kinase pUL97 is required for efficient viral replication. Previous studies described two isoforms of pUL97, the full-length isoform (M1) and a smaller isoform likely resulting from translation initiation at codon 74 (M74). Here, we report the detection of a third pUL97 isoform during viral infection resulting from translation initiation at codon 157 (isoform M157). The consistent expression of isoform M157 as a minor component of pUL97 during infection with clinical and laboratory-adapted HCMV strains was suppressed when codon 157 was mutagenized. Viral mutants expressing specific isoforms were generated to compare their growth and drug susceptibility phenotypes, as well as pUL97 intracellular localization patterns and kinase activities. The exclusive expression of isoform M157 resulted in substantially reduced viral growth and resistance to the pUL97 inhibitor maribavir while retaining susceptibility to ganciclovir. Confocal imaging demonstrated reduced nuclear import of amino-terminal deletion isoforms compared to isoform M1. Isoform M157 showed reduced efficiency of various substrate protein interactions and autophosphorylation, whereas Rb phosphorylation was preserved. These results reveal differential properties of pUL97 isoforms that affect viral replication, with implications for the antiviral efficacy of maribavir. IMPORTANCE The HCMV UL97 kinase performs important functions in viral replication that are targeted by the antiviral drug maribavir. Here, we describe a naturally occurring short isoform of the kinase that when expressed by itself in a recombinant virus results in altered intracellular localization, impaired growth, and high-level resistance to maribavir compared to those of the predominant full-length counterpart. This is another factor to consider in explaining why maribavir appears to have variable antiviral activity in cell culture and in vivo. PMID:24522923

Discovery of a Potent Class of PI3Kα Inhibitors with Unique Binding Mode via Encoded Library Technology (ELT).

PubMed

Yang, Hongfang; Medeiros, Patricia F; Raha, Kaushik; Elkins, Patricia; Lind, Kenneth E; Lehr, Ruth; Adams, Nicholas D; Burgess, Joelle L; Schmidt, Stanley J; Knight, Steven D; Auger, Kurt R; Schaber, Michael D; Franklin, G Joseph; Ding, Yun; DeLorey, Jennifer L; Centrella, Paolo A; Mataruse, Sibongile; Skinner, Steven R; Clark, Matthew A; Cuozzo, John W; Evindar, Ghotas

2015-05-14

In the search of PI3K p110α wild type and H1047R mutant selective small molecule leads, an encoded library technology (ELT) campaign against the desired target proteins was performed which led to the discovery of a selective chemotype for PI3K isoforms from a three-cycle DNA encoded library. An X-ray crystal structure of a representative inhibitor from this chemotype demonstrated a unique binding mode in the p110α protein.

Discovery of a Potent Class of PI3Kα Inhibitors with Unique Binding Mode via Encoded Library Technology (ELT)

PubMed Central

2015-01-01

In the search of PI3K p110α wild type and H1047R mutant selective small molecule leads, an encoded library technology (ELT) campaign against the desired target proteins was performed which led to the discovery of a selective chemotype for PI3K isoforms from a three-cycle DNA encoded library. An X-ray crystal structure of a representative inhibitor from this chemotype demonstrated a unique binding mode in the p110α protein. PMID:26005528

Septin functions in organ system physiology and pathology

PubMed Central

Dolat, Lee; Hu, Qicong

2015-01-01

Human septins comprise a family of 13 genes that encode for >30 protein isoforms with ubiquitous and tissue-specific expressions. Septins are GTP-binding proteins that assemble into higher-order oligomers and filamentous polymers, which associate with cell membranes and the cytoskeleton. In the last decade, much progress has been made in understanding the biochemical properties and cell biological functions of septins. In parallel, a growing number of studies show that septins play important roles for the development and physiology of specific tissues and organs. Here, we review the expression and function of septins in the cardiovascular, immune, nervous, urinary, digestive, respiratory, endocrine, reproductive, and integumentary organ systems. Furthermore, we discuss how the tissue-specific functions of septins relate to the pathology of human diseases that arise from aberrations in septin expression. PMID:24114910

Importin-α7 Is Involved in the Formation of Ebola Virus Inclusion Bodies but Is Not Essential for Pathogenicity in Mice.

PubMed

Gabriel, Gülsah; Feldmann, Friederike; Reimer, Rudolph; Thiele, Swantje; Fischer, Meike; Hartmann, Enno; Bader, Michael; Ebihara, Hideki; Hoenen, Thomas; Feldmann, Heinz

2015-10-01

Ebola virus (EBOV) protein 24 antagonizes the host interferon (IFN) response by hijacking select nuclear importin-α isoforms. Thereby, it blocks STAT1-mediated IFN-α/β and IFN-γ synthesis. However, owing to the lack of importin-α knockout animal models in the past, their role in EBOV pathogenesis remained largely unknown. Here, we demonstrate that importin-α7 is involved in the formation of EBOV inclusion bodies and replication. However, deletion of the gene encoding importin-α7 was not sufficient to increase survival rates among mice infected with EBOV. © The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Comparative Proteomics Reveals a Significant Bias Toward Alternative Protein Isoforms with Conserved Structure and Function

PubMed Central

Ezkurdia, Iakes; del Pozo, Angela; Frankish, Adam; Rodriguez, Jose Manuel; Harrow, Jennifer; Ashman, Keith; Valencia, Alfonso; Tress, Michael L.

2012-01-01

Advances in high-throughput mass spectrometry are making proteomics an increasingly important tool in genome annotation projects. Peptides detected in mass spectrometry experiments can be used to validate gene models and verify the translation of putative coding sequences (CDSs). Here, we have identified peptides that cover 35% of the genes annotated by the GENCODE consortium for the human genome as part of a comprehensive analysis of experimental spectra from two large publicly available mass spectrometry databases. We detected the translation to protein of “novel” and “putative” protein-coding transcripts as well as transcripts annotated as pseudogenes and nonsense-mediated decay targets. We provide a detailed overview of the population of alternatively spliced protein isoforms that are detectable by peptide identification methods. We found that 150 genes expressed multiple alternative protein isoforms. This constitutes the largest set of reliably confirmed alternatively spliced proteins yet discovered. Three groups of genes were highly overrepresented. We detected alternative isoforms for 10 of the 25 possible heterogeneous nuclear ribonucleoproteins, proteins with a key role in the splicing process. Alternative isoforms generated from interchangeable homologous exons and from short indels were also significantly enriched, both in human experiments and in parallel analyses of mouse and Drosophila proteomics experiments. Our results show that a surprisingly high proportion (almost 25%) of the detected alternative isoforms are only subtly different from their constitutive counterparts. Many of the alternative splicing events that give rise to these alternative isoforms are conserved in mouse. It was striking that very few of these conserved splicing events broke Pfam functional domains or would damage globular protein structures. This evidence of a strong bias toward subtle differences in CDS and likely conserved cellular function and structure is remarkable and strongly suggests that the translation of alternative transcripts may be subject to selective constraints. PMID:22446687

Identification, gene expression and immune function of the novel Bm-STAT gene in virus-infected Bombyx mori.

PubMed

Zhang, Xiaoli; Guo, Rui; Kumar, Dhiraj; Ma, Huanyan; Liu, Jiabin; Hu, Xiaolong; Cao, Guangli; Xue, Renyu; Gong, Chengliang

2016-02-10

Genes in the signal transducer and activator of transcription (STAT) family are vital for activities including gene expression and immune response. To investigate the functions of the silkworm Bombyx mori STAT (Bm-STAT) gene in antiviral immunity, two Bm-STAT gene isoforms, Bm-STAT-L for long form and Bm-STAT-S for short form, were cloned. Sequencing showed that the open reading frames were 2313 bp encoding 770 amino acid residues for Bm-STAT-L and 2202 bp encoding 734 amino acid residues for Bm-STAT-S. The C-terminal 42 amino acid residues of Bm-STAT-L were different from the last 7 amino acid residues of Bm-STAT-S. Immunofluorescence showed that Bm-STAT was primarily distributed in the nucleus. Transcription levels of Bm-STAT in different tissues were determined by quantitative PCR, and the results revealed Bm-STAT was mainly expressed in testes. Western blots showed two bands with molecular weights of 70 kDa and 130 kDa in testes, but no bands were detected in ovaries by using anti-Bm-STAT antibody as the primary antibody. Expression of Bm-STAT in hemolymph at 48 h post infection with B. mori macula-like virus (BmMLV) was slightly enhanced compared with controls, suggesting a weak response induced by infection with BmMLV. Hemocyte immunofluorescence showed that Bm-STAT expression was elevated in B. mori nucleopolyhedrovirus (BmNPV)-infected cells. Moreover, resistance of BmN cells to BmNPV was reduced by downregulation of Bm-STAT expression and increased by upregulation. Resistance of BmN cells to BmCPV was not significantly improved by upregulating Bm-STAT expression. Therefore, we concluded that Bm-STAT is a newly identified insect gene of the STAT family. The JAK-STAT pathway has a more specialized role in antiviral defense in silkworms, but JAK-STAT pathway is not triggered in response to all viruses. Copyright © 2015 Elsevier B.V. All rights reserved.

Myocardium expression of connexin 43, SERCA2a, and myosin heavy chain isoforms are preserved in nitrofen-induced congenital diaphragmatic hernia rat model.

PubMed

Baptista, Maria João; Recamán, Mónica; Melo-Rocha, Gustavo; Nogueira-Silva, Cristina; Roriz, José-Mário; Soares-Fernandes, João; Gonzaga, Silvia; Santos, Marta; Leite-Moreira, Adelino; Areias, José Carlos; Correia-Pinto, Jorge

2006-09-01

Previous morphological studies had produced controversial results with regard to heart development in congenital diaphragmatic hernia (CDH), whereas a few publications investigated cardiac function and myocardial maturation. Myocardium maturation is associated with age-dependent increasing of gene expression of gap junction protein connexin 43 (Cx43), adenosine triphosphatase of the sarcoplasmic reticulum (SERCA2a), as well as switching of myosin heavy chains (MHCs) from beta to alpha isoforms. Our aim was to evaluate myocardium maturity in nitrofen-induced CDH rat model. Fetuses from dated pregnant Sprague-Dawley rats were assigned to 3 experimental groups: control, nitrofen (exposed to nitrofen, without CDH), and CDH (exposed to nitrofen, with CDH). Myocardial samples collected from left ventricle free wall were processed to (i) quantification of messenger RNA (mRNA) of Cx43, SERCA2a, alpha and beta MHC isoforms, as well as beta-actin (housekeeping gene); and (ii) separation of MHC isoforms (alpha and beta isoforms) by sodium dodecyl sulfate polyacrylamide gel electrophoresis silver stained. We demonstrated that there is no difference in myocardial gene expression of Cx43 (control, 1.0 +/- 0.1; nitrofen, 1.1 +/- 0.2; CDH, 1.3 +/- 0.2) and SERCA2a (control, 1.0 +/- 0.1; nitrofen, 0.9 +/- 0.1; CDH, 1.0 +/- 0.2). Myocardial gene expressions of alpha and beta mRNA of MHC isoforms were slightly decreased both in nitrofen and CDH fetuses when compared with control fetuses, but evaluation of the alpha-to-beta ratios of MHC isoforms at protein level revealed no significant differences between CDH and control (control, 16.9 +/- 2.5; CDH, 17.0 +/- 2.0). Myocardial quantification of Cx43 and SERCA2a mRNA, as well as the expression pattern of MHC isoforms at protein levels, was similar in all studied groups. We predict, therefore, that acute heart failure commonly observed in CDH infants might be attributed predominantly to cardiac overload secondary to severe pulmonary hypertension rather than to myocardial immaturity.

Genome-wide mapping of alternative splicing in Arabidopsis thaliana

PubMed Central

Filichkin, Sergei A.; Priest, Henry D.; Givan, Scott A.; Shen, Rongkun; Bryant, Douglas W.; Fox, Samuel E.; Wong, Weng-Keen; Mockler, Todd C.

2010-01-01

Alternative splicing can enhance transcriptome plasticity and proteome diversity. In plants, alternative splicing can be manifested at different developmental stages, and is frequently associated with specific tissue types or environmental conditions such as abiotic stress. We mapped the Arabidopsis transcriptome at single-base resolution using the Illumina platform for ultrahigh-throughput RNA sequencing (RNA-seq). Deep transcriptome sequencing confirmed a majority of annotated introns and identified thousands of novel alternatively spliced mRNA isoforms. Our analysis suggests that at least ∼42% of intron-containing genes in Arabidopsis are alternatively spliced; this is significantly higher than previous estimates based on cDNA/expressed sequence tag sequencing. Random validation confirmed that novel splice isoforms empirically predicted by RNA-seq can be detected in vivo. Novel introns detected by RNA-seq were substantially enriched in nonconsensus terminal dinucleotide splice signals. Alternative isoforms with premature termination codons (PTCs) comprised the majority of alternatively spliced transcripts. Using an example of an essential circadian clock gene, we show that intron retention can generate relatively abundant PTC+ isoforms and that this specific event is highly conserved among diverse plant species. Alternatively spliced PTC+ isoforms can be potentially targeted for degradation by the nonsense mediated mRNA decay (NMD) surveillance machinery or regulate the level of functional transcripts by the mechanism of regulated unproductive splicing and translation (RUST). We demonstrate that the relative ratios of the PTC+ and reference isoforms for several key regulatory genes can be considerably shifted under abiotic stress treatments. Taken together, our results suggest that like in animals, NMD and RUST may be widespread in plants and may play important roles in regulating gene expression. PMID:19858364

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Yuli; Zhang, Pengju; Wang, Yunshan

The ErbB3 receptor–binding protein EBP1 encodes two alternatively spliced isoforms P48 and P42. While there is evidence of differential roles for these isoforms in tumorigenesis, little is known about their underlying mechanisms. In this paper, we demonstrate that EBP1 isoforms interact with the SCF-type ubiquitin ligase FBXW7 in distinct ways to exert opposing roles in tumorigenesis. EBP1 P48 bound to the WD domain of FBXW7 as an oncogenic substrate of FBXW7. EBP1 P48 binding sequestered FBXW7α to the cytosol, modulating its role in protein degradation and attenuating its tumor suppressor function. In contrast, EBP1 P42 bound to both the F-boxmore » domain of FBXW7 as well as FBXW7 substrates. This adapter function of EBP1 P42 stabilized the interaction of FBXW7 with its substrates and promoted FBXW7-mediated degradation of oncogenic targets, enhancing its overall tumor-suppressing function. Finally and overall, our results establish distinct physical and functional interactions between FBXW7 and EBP1 isoforms, which yield their mechanistically unique isoform-specific functions of EBP1 in cancer.« less

SASD: the Synthetic Alternative Splicing Database for identifying novel isoform from proteomics

PubMed Central

2013-01-01

Background Alternative splicing is an important and widespread mechanism for generating protein diversity and regulating protein expression. High-throughput identification and analysis of alternative splicing in the protein level has more advantages than in the mRNA level. The combination of alternative splicing database and tandem mass spectrometry provides a powerful technique for identification, analysis and characterization of potential novel alternative splicing protein isoforms from proteomics. Therefore, based on the peptidomic database of human protein isoforms for proteomics experiments, our objective is to design a new alternative splicing database to 1) provide more coverage of genes, transcripts and alternative splicing, 2) exclusively focus on the alternative splicing, and 3) perform context-specific alternative splicing analysis. Results We used a three-step pipeline to create a synthetic alternative splicing database (SASD) to identify novel alternative splicing isoforms and interpret them at the context of pathway, disease, drug and organ specificity or custom gene set with maximum coverage and exclusive focus on alternative splicing. First, we extracted information on gene structures of all genes in the Ensembl Genes 71 database and incorporated the Integrated Pathway Analysis Database. Then, we compiled artificial splicing transcripts. Lastly, we translated the artificial transcripts into alternative splicing peptides. The SASD is a comprehensive database containing 56,630 genes (Ensembl gene IDs), 95,260 transcripts (Ensembl transcript IDs), and 11,919,779 Alternative Splicing peptides, and also covering about 1,956 pathways, 6,704 diseases, 5,615 drugs, and 52 organs. The database has a web-based user interface that allows users to search, display and download a single gene/transcript/protein, custom gene set, pathway, disease, drug, organ related alternative splicing. Moreover, the quality of the database was validated with comparison to other known databases and two case studies: 1) in liver cancer and 2) in breast cancer. Conclusions The SASD provides the scientific community with an efficient means to identify, analyze, and characterize novel Exon Skipping and Intron Retention protein isoforms from mass spectrometry and interpret them at the context of pathway, disease, drug and organ specificity or custom gene set with maximum coverage and exclusive focus on alternative splicing. PMID:24267658

Significant differentiation in the apolipoprotein(a)/lipoprotein(a) trait between chimpanzees from Western and Central Africa.

PubMed

Noureen, Asma; Ronke, Claudius; Khalifa, Mahmoud; Halbwax, Michel; Fischer, Anne; André, Claudine; Atencia, Rebeca; Garriga, Rosa; Mugisha, Lawrence; Ceglarek, Uta; Thiery, Joachim; Utermann, Gerd; Schmidt, Konrad

2017-09-01

Elevated Lipoprotein(a) (Lp(a)) plasma concentrations are a risk factor for cardiovascular disease in humans, largely controlled by the LPA gene encoding apolipoprotein(a) (apo(a)). Lp(a) is composed of low-density lipoprotein (LDL) and apo(a) and restricted to Catarrhini. A variable number of kringle IV (KIV) domains in LPA lead to a size polymorphism of apo(a) that is inversely correlated with Lp(a) concentrations. Smaller apo(a) isoforms and higher Lp(a) levels in central chimpanzees (Pan troglodytes troglodytes [PTT]) compared to humans from Europe had been reported. We studied apo(a) isoforms and Lp(a) concentrations in 75 western (Pan troglodytes verus [PTV]) and 112 central chimpanzees, and 12 bonobos (Pan paniscus [PPA]), all wild born and living in sanctuaries in Sierra Leone, Republic of the Congo, and DR Congo, respectively, and 116 humans from Gabon. Lp(a) levels were severalfold higher in western than in central chimpanzees (181.0 ± 6.7 mg/dl vs. 56.5 ± 4.3 mg/dl), whereas bonobos showed intermediate levels (134.8 ± 33.4 mg/dl). Apo(a) isoform sizes differed significantly between subspecies (means 20.9 ± 2.2, 22.9 ± 4.4, and 23.8 ± 3.8 KIV repeats in PTV, PTT, and PPA, respectively). However, far higher isoform-associated Lp(a) concentrations for all isoform sizes in western chimpanzees offered the main explanation for the higher overall Lp(a) levels in this subspecies. Human Lp(a) concentrations (mean 47.9 ± 2.8 mg/dl) were similar to those in central chimpanzees despite larger isoforms (mean 27.1 ± 4.9 KIV). Lp(a) and LDL, apoB-100, and total cholesterol levels only correlated in PTV. This remarkable differentiation between chimpanzees from different African habitats and the trait's similarity in humans and chimpanzees from Central Africa poses the question of a possible impact of an environmental factor that has shaped the genetic architecture of LPA. Overall, studies on the cholesterol-containing particles of Lp(a) and LDL in chimpanzees should consider differentiation between subspecies. © 2017 Wiley Periodicals, Inc.

Genome-Wide Posttranscriptional Dysregulation by MicroRNAs in Human Asthma as Revealed by Frac-seq.

PubMed

Martinez-Nunez, Rocio T; Rupani, Hitasha; Platé, Manuela; Niranjan, Mahesan; Chambers, Rachel C; Howarth, Peter H; Sanchez-Elsner, Tilman

2018-05-16

MicroRNAs are small noncoding RNAs that inhibit gene expression posttranscriptionally, implicated in virtually all biological processes. Although the effect of individual microRNAs is generally studied, the genome-wide role of multiple microRNAs is less investigated. We assessed paired genome-wide expression of microRNAs with total (cytoplasmic) and translational (polyribosome-bound) mRNA levels employing subcellular fractionation and RNA sequencing (Frac-seq) in human primary bronchoepithelium from healthy controls and severe asthmatics. Severe asthma is a chronic inflammatory disease of the airways characterized by poor response to therapy. We found genes (i.e., isoforms of a gene) and mRNA isoforms differentially expressed in asthma, with novel inflammatory and structural pathophysiological mechanisms related to bronchoepithelium disclosed solely by polyribosome-bound mRNAs (e.g., IL1A and LTB genes or ITGA6 and ITGA2 alternatively spliced isoforms). Gene expression (i.e., isoforms of a gene) and mRNA expression analysis revealed different molecular candidates and biological pathways, with differentially expressed polyribosome-bound and total mRNAs also showing little overlap. We reveal a hub of six dysregulated microRNAs accounting for ∼90% of all microRNA targeting, displaying preference for polyribosome-bound mRNAs. Transfection of this hub in bronchial epithelial cells from healthy donors mimicked asthma characteristics. Our work demonstrates extensive posttranscriptional gene dysregulation in human asthma, in which microRNAs play a central role, illustrating the feasibility and importance of assessing posttranscriptional gene expression when investigating human disease. Copyright © 2018 by The American Association of Immunologists, Inc.

Aspergillus Oryzae S2 α-Amylase Domain C Involvement in Activity and Specificity: In Vivo Proteolysis, Molecular and Docking Studies

PubMed Central

Sahnoun, Mouna; Jemli, Sonia; Trabelsi, Sahar; Ayadi, Leila; Bejar, Samir

2016-01-01

We previously reported that Aspergillus oryzae strain S2 had produced two α-amylase isoforms named AmyA and AmyB. The apparent molecular masses revealed by SDS-PAGE were 50 and 42 kDa, respectively. Yet AmyB has a higher catalytic efficiency. Based on a monitoring study of the α-amylase production in both the presence and absence of different protease inhibitors, a chymotrypsin proteolysis process was detected in vivo generating AmyB. A. oryzae S2 α-amylase gene was amplified, cloned and sequenced. The sequence analysis revealed nine exons, eight introns and an encoding open reading frame of 1500 bp corresponding to AmyA isoform. The amino-acid sequence analysis revealed aY371 potential chymotrypsin cleaving site, likely to be the AmyB C-Terminal end and two other potential sites at Y359, and F379. A zymogram with a high acrylamide concentration was used. It highlighted two other closed apparent molecular mass α-amylases termed AmyB1 and AmyB2 reaching40 kDa and 43 kDa. These isoforms could be possibly generated fromY359, and F379secondary cut, respectively. The molecular modeling study showed that AmyB preserved the (β/α)8 barrel domain and the domain B but lacked the C-terminal domain C. The contact map analysis and the docking studies strongly suggested a higher activity and substrate binding affinity for AmyB than AmyA which was previously experimentally exhibited. This could be explained by the easy catalytic cleft accessibility. PMID:27101008

Glycerol-3-phosphate acyltransferase (GPAT)-1, but not GPAT4, incorporates newly synthesized fatty acids into triacylglycerol and diminishes fatty acid oxidation.

PubMed

Wendel, Angela A; Cooper, Daniel E; Ilkayeva, Olga R; Muoio, Deborah M; Coleman, Rosalind A

2013-09-20

Four glycerol-3-phosphate acyltransferase (GPAT) isoforms, each encoded by a separate gene, catalyze the initial step in glycerolipid synthesis; in liver, the major isoforms are GPAT1 and GPAT4. To determine whether each of these hepatic isoforms performs a unique function in the metabolism of fatty acid, we measured the incorporation of de novo synthesized fatty acid or exogenous fatty acid into complex lipids in primary mouse hepatocytes from control, Gpat1(-/-), and Gpat4(-/-) mice. Although hepatocytes from each genotype incorporated a similar amount of exogenous fatty acid into triacylglycerol (TAG), only control and Gpat4(-/-) hepatocytes were able to incorporate de novo synthesized fatty acid into TAG. When compared with controls, Gpat1(-/-) hepatocytes oxidized twice as much exogenous fatty acid. To confirm these findings and to assess hepatic β-oxidation metabolites, we measured acylcarnitines in liver from mice after a 24-h fast and after a 24-h fast followed by 48 h of refeeding with a high sucrose diet to promote lipogenesis. Confirming the in vitro findings, the hepatic content of long-chain acylcarnitine in fasted Gpat1(-/-) mice was 3-fold higher than in controls. When compared with control and Gpat4(-/-) mice, after the fasting-refeeding protocol, Gpat1(-/-) hepatic TAG was depleted, and long-chain acylcarnitine content was 3.5-fold higher. Taken together, these data demonstrate that GPAT1, but not GPAT4, is required to incorporate de novo synthesized fatty acids into TAG and to divert them away from oxidation.

Identification and comparative analysis of the epidermal differentiation complex in snakes

PubMed Central

Brigit Holthaus, Karin; Mlitz, Veronika; Strasser, Bettina; Tschachler, Erwin; Alibardi, Lorenzo; Eckhart, Leopold

2017-01-01

The epidermis of snakes efficiently protects against dehydration and mechanical stress. However, only few proteins of the epidermal barrier to the environment have so far been identified in snakes. Here, we determined the organization of the Epidermal Differentiation Complex (EDC), a cluster of genes encoding protein constituents of cornified epidermal structures, in snakes and compared it to the EDCs of other squamates and non-squamate reptiles. The EDC of snakes displays shared synteny with that of the green anole lizard, including the presence of a cluster of corneous beta-protein (CBP)/beta-keratin genes. We found that a unique CBP comprising 4 putative beta-sheets and multiple cysteine-rich EDC proteins are conserved in all snakes and other squamates investigated. Comparative genomics of squamates suggests that the evolution of snakes was associated with a gene duplication generating two isoforms of the S100 fused-type protein, scaffoldin, the origin of distinct snake-specific EDC genes, and the loss of other genes that were present in the EDC of the last common ancestor of snakes and lizards. Taken together, our results provide new insights into the evolution of the skin in squamates and a basis for the characterization of the molecular composition of the epidermis in snakes. PMID:28345630

Characterization of the Sucrose Phosphate Phosphatase (SPP) Isoforms from Arabidopsis thaliana and Role of the S6PPc Domain in Dimerization.

PubMed

Albi, Tomás; Ruiz, M Teresa; de Los Reyes, Pedro; Valverde, Federico; Romero, José M

2016-01-01

Sucrose-phosphate phosphatase (SPP) catalyses the final step in the sucrose biosynthesis pathway. Arabidopsis thaliana genome codifies four SPP isoforms. In this study, the four Arabidopsis thaliana genes coding for SPP isoforms have been cloned, expressed in Escherichia coli and the kinetic and regulatory properties of the purified enzymes analysed. SPP2 is the isoform showing the highest activity, with SPP3b and SPP3a showing lower activity levels. No activity was detected for SPP1. We propose that this lack of activity is probably due to the absence of an essential amino acid participating in catalysis and/or in the binding of the substrate, sucrose-6-phosphate (Suc6P). The expression patterns of Arabidopsis SPP genes indicate that SPP2 and SPP3b are the main isoforms expressed in different tissues and organs, although the non-catalytic SPP1 is the main isoform expressed in roots. Thus, SPP1 could have acquired new unknown functions. We also show that the three catalytically active SPPs from Arabidopsis are dimers. By generating a chimeric SPP composed of the monomeric cyanobacterial SPP fused to the higher plant non-catalytic S6PPc domain (from SPP2), we show that the S6PPc domain is responsible for SPP dimerization. This is the first experimental study on the functionality and gene expression pattern of all the SPPs from a single plant species.

Adaptive selection of an incretin gene in Eurasian populations

PubMed Central

Chang, Chia Lin; Cai, James J.; Lo, Chiening; Amigo, Jorge; Park, Jae-Il; Hsu, Sheau Yu Teddy

2011-01-01

Diversities in human physiology have been partially shaped by adaptation to natural environments and changing cultures. Recent genomic analyses have revealed single nucleotide polymorphisms (SNPs) that are associated with adaptations in immune responses, obvious changes in human body forms, or adaptations to extreme climates in select human populations. Here, we report that the human GIP locus was differentially selected among human populations based on the analysis of a nonsynonymous SNP (rs2291725). Comparative and functional analyses showed that the human GIP gene encodes a cryptic glucose-dependent insulinotropic polypeptide (GIP) isoform (GIP55S or GIP55G) that encompasses the SNP and is resistant to serum degradation relative to the known mature GIP peptide. Importantly, we found that GIP55G, which is encoded by the derived allele, exhibits a higher bioactivity compared with GIP55S, which is derived from the ancestral allele. Haplotype structure analysis suggests that the derived allele at rs2291725 arose to dominance in East Asians ∼8100 yr ago due to positive selection. The combined results suggested that rs2291725 represents a functional mutation and may contribute to the population genetics observation. Given that GIP signaling plays a critical role in homeostasis regulation at both the enteroinsular and enteroadipocyte axes, our study highlights the importance of understanding adaptations in energy-balance regulation in the face of the emerging diabetes and obesity epidemics. PMID:20978139

Progesterone signalling in broiler skeletal muscle is associated with divergent feed efficiency.

PubMed

Bottje, Walter; Kong, Byung-Whi; Reverter, Antonio; Waardenberg, Ashley J; Lassiter, Kentu; Hudson, Nicholas J

2017-02-24

We contrast the pectoralis muscle transcriptomes of broilers selected from within a single genetic line expressing divergent feed efficiency (FE) in an effort to improve our understanding of the mechanistic basis of FE. Application of a virtual muscle model to gene expression data pointed to a coordinated reduction in slow twitch muscle isoforms of the contractile apparatus (MYH15, TPM3, MYOZ2, TNNI1, MYL2, MYOM3, CSRP3, TNNT2), consistent with diminishment in associated slow machinery (myoglobin and phospholamban) in the high FE animals. These data are in line with the repeated transition from red slow to white fast muscle fibres observed in agricultural species selected on mass and FE. Surprisingly, we found that the expression of 699 genes encoding the broiler mitoproteome is modestly-but significantly-biased towards the high FE group, suggesting a slightly elevated mitochondrial content. This is contrary to expectation based on the slow muscle isoform data and theoretical physiological capacity arguments. Reassuringly, the extreme 40 most DE genes can successfully cluster the 12 individuals into the appropriate FE treatment group. Functional groups contained in this DE gene list include metabolic proteins (including opposing patterns of CA3 and CA4), mitochondrial proteins (CKMT1A), oxidative status (SEPP1, HIG2A) and cholesterol homeostasis (APOA1, INSIG1). We applied a differential network method (Regulatory Impact Factors) whose aim is to use patterns of differential co-expression to detect regulatory molecules transcriptionally rewired between the groups. This analysis clearly points to alterations in progesterone signalling (via the receptor PGR) as the major driver. We show the progesterone receptor localises to the mitochondria in a quail muscle cell line. Progesterone is sometimes used in the cattle industry in exogenous hormone mixes that lead to a ~20% increase in FE. Because the progesterone receptor can localise to avian mitochondria, our data continue to point to muscle mitochondrial metabolism as an important component of the phenotypic expression of variation in broiler FE.

Functional analysis of the isoforms of an ABI3-like factor of Pisum sativum generated by alternative splicing.

PubMed

Gagete, Andrés P; Riera, Marta; Franco, Luis; Rodrigo, M Isabel

2009-01-01

At least seven isoforms (PsABI3-1 to PsABI3-7) of a putative, pea ABI3-like factor, originated by alternative splicing, have been identified after cDNA cloning. A similar variability had previously only been described for monocot genes. The full-length isoform, PsABI3-1, contains the typical N-terminal acidic domains and C-terminal basic subdomains, B1 to B3. Reverse transcriptase-PCR analysis revealed that the gene is expressed just in seeds, starting at middle embryogenesis; no gene products are observed in embryo axes after 18 h post-imbibition although they are more persistent in cotyledons. The activity of the isoforms was studied by yeast one-hybrid assays. When yeast was transformed with the isoforms fused to the DNA binding domain of Gal4p, only the polypeptides PsABI3-2 and PsABI3-7 failed to complement the activity of Gal4p. Acidic domains A1 and A2 exhibit transactivating activity, but the former requires a small C-terminal extension to be active. Yeast two-hybrid analysis showed that PsABI3 is able to heterodimerize with Arabidopsis thaliana ABI5, thus proving that PsABI3 is functionally active. The minimum requirement for the interaction PsABI3-AtABI5 is the presence of the subdomain B1 with an extension, 81 amino acids long, at their C-terminal side. Finally, a transient onion transformation assay showed that both the active PsABI3-1 and the inactive PsABI3-2 isoforms are localized to nuclei. Considering that the major isoforms remain approximately constant in developing seeds although their relative proportion varied, the possible role of splicing in the regulatory network of ABA signalling is discussed.

Tissue- and cell-specific expression of metallothionein genes in cadmium- and copper-exposed mussels analyzed by in situ hybridization and RT-PCR

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zorita, I.; Bilbao, E.; Schad, A.

2007-04-15

Metallothioneins (MTs) are metal-inducible proteins that can be used as biomarkers of metal exposure. In mussels two families of MT isoforms (MT10 and MT20) have been characterized. In this study, mussels (Mytilus galloprovincialis) were exposed to 200 ppb Cd and 40 ppb Cu for 2 and 9 days to characterize the tissue and isoform specificity of metal-induced MT expression. Non-radioactive in situ hybridization demonstrated that both MT isoforms were mainly transcribed in digestive tubule epithelial cells, especially in basophilic cells. Weaker MT expression was detected in non-ciliated duct cells, stomach and gill epithelial cells, haemocytes, adipogranular cells, spermatic follicles andmore » oocytes. RT-PCR resulted in cloning of a novel M. galloprovincialis isoform homologous to recently cloned Mytilus edulis intron-less MT10B isoform. In gills, Cd only affected MT10 gene expression after 2 days of exposure while increases in MT protein levels occurred at day 9. In the digestive gland, a marked increase of both isoforms, but especially of MT20, was accompanied by increased levels of MT proteins and basophilic cell volume density (Vv{sub BAS}) after 2 and 9 days and of intralysosomal metal accumulation in digestive cells after 9 days. Conversely, although metal was accumulated in digestive cells lysosomes and the Vv{sub BAS} increased in Cu-exposed mussels, Cu exposure did not produce an increase of MT gene expression or MT protein levels. These data suggest that MTs are expressed in a tissue-, cell- and isoform-specific way in response to different metals.« less

m6A level and isoform characterization sequencing (m6A-LAIC-seq) reveals the census and complexity of the m6A epitranscriptome

PubMed Central

Molinie, Benoit; Wang, Jinkai; Lim, Kok-Seong; Hillebrand, Roman; Lu, Zhi-xiang; Van Wittenberghe, Nicholas; Howard, Benjamin D.; Daneshvar, Kaveh; Mullen, Alan C.; Dedon, Peter

2017-01-01

N6-Methyladenosine (m6A) is a widespread, reversible chemical modification of RNA molecules, implicated in many aspects of RNA metabolism. Little quantitative information exists as to either how many transcript copies of particular genes are m6A modified (‘m6A levels’) or the relationship of m6A modification(s) to alternative RNA isoforms. To deconvolute the m6A epitranscriptome, we developed m6A-level and isoform-characterization sequencing (m6A-LAIC-seq). We found that cells exhibit a broad range of nonstoichiometric m6A levels with cell-type specificity. At the level of isoform characterization, we discovered widespread differences in the use of tandem alternative polyadenylation (APA) sites by methylated and nonmethylated transcript isoforms of individual genes. Strikingly, there is a strong bias for methylated transcripts to be coupled with proximal APA sites, resulting in shortened 3′ untranslated regions, while nonmethylated transcript isoforms tend to use distal APA sites. m6A-LAIC-seq yields a new perspective on transcriptome complexity and links APA usage to m6A modifications. PMID:27376769

DEIsoM: a hierarchical Bayesian model for identifying differentially expressed isoforms using biological replicates

PubMed Central

Peng, Hao; Yang, Yifan; Zhe, Shandian; Wang, Jian; Gribskov, Michael; Qi, Yuan

2017-01-01

Abstract Motivation High-throughput mRNA sequencing (RNA-Seq) is a powerful tool for quantifying gene expression. Identification of transcript isoforms that are differentially expressed in different conditions, such as in patients and healthy subjects, can provide insights into the molecular basis of diseases. Current transcript quantification approaches, however, do not take advantage of the shared information in the biological replicates, potentially decreasing sensitivity and accuracy. Results We present a novel hierarchical Bayesian model called Differentially Expressed Isoform detection from Multiple biological replicates (DEIsoM) for identifying differentially expressed (DE) isoforms from multiple biological replicates representing two conditions, e.g. multiple samples from healthy and diseased subjects. DEIsoM first estimates isoform expression within each condition by (1) capturing common patterns from sample replicates while allowing individual differences, and (2) modeling the uncertainty introduced by ambiguous read mapping in each replicate. Specifically, we introduce a Dirichlet prior distribution to capture the common expression pattern of replicates from the same condition, and treat the isoform expression of individual replicates as samples from this distribution. Ambiguous read mapping is modeled as a multinomial distribution, and ambiguous reads are assigned to the most probable isoform in each replicate. Additionally, DEIsoM couples an efficient variational inference and a post-analysis method to improve the accuracy and speed of identification of DE isoforms over alternative methods. Application of DEIsoM to an hepatocellular carcinoma (HCC) dataset identifies biologically relevant DE isoforms. The relevance of these genes/isoforms to HCC are supported by principal component analysis (PCA), read coverage visualization, and the biological literature. Availability and implementation The software is available at https://github.com/hao-peng/DEIsoM Contact pengh@alumni.purdue.edu Supplementary information Supplementary data are available at Bioinformatics online. PMID:28595376

Highly specific targeting of the TMPRSS2/ERG fusion gene using liposomal nanovectors

PubMed Central

Shao, Longjiang; Tekedereli, Ibrahim; Wang, Jianghua; Yuca, Erkan; Tsang, Susan; Sood, Anil; Lopez-Berestein, Gabriel; Ozpolat, Bulent; Ittmann, Michael

2012-01-01

Purpose The TMPRSS2/ERG (T/E) fusion gene is present in half of all prostate cancer (PCa) tumors. Fusion of the oncogenic ERG gene with the androgen-regulated TMPRSS2 gene promoter results in expression of fusion mRNAs in PCa cells. The junction of theTMPRSS2 and ERG derived portions of the fusion mRNA constitutes a cancer specific target in cells containing the T/E fusion gene. Targeting the most common alternatively spliced fusion gene mRNA junctional isoforms in vivo using siRNAs in liposomal nanovectors may potentially be a novel, low toxicity treatment for PCa. Experimental Design We designed and optimized siRNAs targeting the two most common T/E fusion gene mRNA junctional isoforms (Type III or Type VI). Specificity of siRNAs was assessed by transient co-transfection in vitro. To test their ability to inhibit growth of PCa cells expressing these fusion gene isoforms in vivo, specific siRNAs in liposomal nanovectors were used to treat mice bearing orthotopic or subcutaneous xenograft tumors expressing the targeted fusion isoforms. Results The targeting siRNAs were both potent and highly specific in vitro. In vivo they significantly inhibited tumor growth. The degree of growth inhibition was variable and was correlated with the extent of fusion gene knockdown. The growth inhibition was associated with marked inhibition of angiogenesis and, to a lesser degree, proliferation and a marked increase in apoptosis of tumor cells. No toxicity was observed. Conclusions Targeting the T/E fusion junction in vivo with specific siRNAs delivered via liposomal nanovectors is a promising therapy for men with PCa. PMID:23052253

Highly specific targeting of the TMPRSS2/ERG fusion gene using liposomal nanovectors.

PubMed

Shao, Longjiang; Tekedereli, Ibrahim; Wang, Jianghua; Yuca, Erkan; Tsang, Susan; Sood, Anil; Lopez-Berestein, Gabriel; Ozpolat, Bulent; Ittmann, Michael

2012-12-15

The TMPRSS2/ERG (T/E) fusion gene is present in half of all prostate cancer tumors. Fusion of the oncogenic ERG gene with the androgen-regulated TMPRSS2 gene promoter results in expression of fusion mRNAs in prostate cancer cells. The junction of theTMPRSS2- and ERG-derived portions of the fusion mRNA constitutes a cancer-specific target in cells containing the T/E fusion gene. Targeting the most common alternatively spliced fusion gene mRNA junctional isoforms in vivo using siRNAs in liposomal nanovectors may potentially be a novel, low-toxicity treatment for prostate cancer. We designed and optimized siRNAs targeting the two most common T/E fusion gene mRNA junctional isoforms (type III or type VI). Specificity of siRNAs was assessed by transient co-transfection in vitro. To test their ability to inhibit growth of prostate cancer cells expressing these fusion gene isoforms in vivo, specific siRNAs in liposomal nanovectors were used to treat mice bearing orthotopic or subcutaneous xenograft tumors expressing the targeted fusion isoforms. The targeting siRNAs were both potent and highly specific in vitro. In vivo they significantly inhibited tumor growth. The degree of growth inhibition was variable and was correlated with the extent of fusion gene knockdown. The growth inhibition was associated with marked inhibition of angiogenesis and, to a lesser degree, proliferation and a marked increase in apoptosis of tumor cells. No toxicity was observed. Targeting the T/E fusion junction in vivo with specific siRNAs delivered via liposomal nanovectors is a promising therapy for men with prostate cancer. ©2012 AACR.

L-Ilf3 and L-NF90 Traffic to the Nucleolus Granular Component: Alternatively-Spliced Exon 3 Encodes a Nucleolar Localization Motif

PubMed Central

Viranaicken, Wildriss; Gasmi, Laila; Chaumet, Alexandre; Durieux, Christiane; Georget, Virginie; Denoulet, Philippe; Larcher, Jean-Christophe

2011-01-01

Ilf3 and NF90, two proteins containing double-stranded RNA-binding domains, are generated by alternative splicing and involved in several functions. Their heterogeneity results from posttranscriptional and posttranslational modifications. Alternative splicing of exon 3, coding for a 13 aa N-terminal motif, generates for each protein a long and short isoforms. Subcellular fractionation and localization of recombinant proteins showed that this motif acts as a nucleolar localization signal. Deletion and substitution mutants identified four arginines, essential for nucleolar targeting, and three histidines to stabilize the proteins within the nucleolus. The short isoforms are never found in the nucleoli, whereas the long isoforms are present in the nucleoplasm and the nucleoli. For Ilf3, only the posttranslationally-unmodified long isoform is nucleolar, suggesting that this nucleolar targeting is abrogated by posttranslational modifications. Confocal microscopy and FRAP experiments have shown that the long Ilf3 isoform localizes to the granular component of the nucleolus, and that L-Ilf3 and L-NF90 exchange rapidly between nucleoli. The presence of this 13 aminoacid motif, combined with posttranslational modifications, is responsible for the differences in Ilf3 and NF90 isoforms subcellular localizations. The protein polymorphism of Ilf3/NF90 and the various subcellular localizations of their isoforms may partially explain the various functions previously reported for these proteins. PMID:21811582

Lipid metabolism in Rhodnius prolixus: Lessons from the genome.

PubMed

Majerowicz, David; Calderón-Fernández, Gustavo M; Alves-Bezerra, Michele; De Paula, Iron F; Cardoso, Lívia S; Juárez, M Patricia; Atella, Georgia C; Gondim, Katia C

2017-01-05

The kissing bug Rhodnius prolixus is both an important vector of Chagas' disease and an interesting model for investigation into the field of physiology, including lipid metabolism. The publication of this insect genome will bring a huge amount of new molecular biology data to be used in future experiments. Although this work represents a promising scenario, a preliminary analysis of the sequence data is necessary to identify and annotate the genes involved in lipid metabolism. Here, we used bioinformatics tools and gene expression analysis to explore genes from different genes families and pathways, including genes for fat breakdown, as lipases and phospholipases, and enzymes from β-oxidation, fatty acid metabolism, and acyl-CoA and glycerolipid synthesis. The R. prolixus genome encodes 31 putative lipase genes, including 21 neutral lipases and 5 acid lipases. The expression profiles of some of these genes were analyzed. We were able to identify nine phospholipase A2 genes. A variety of gene families that participate in fatty acid synthesis and modification were studied, including fatty acid synthase, elongase, desaturase and reductase. Concerning the synthesis of glycerolipids, we found a second isoform of glycerol-3-phosphate acyltransferase that was ubiquitously expressed throughout the organs. Finally, all genes involved in fatty acid β-oxidation were identified, but not a long-chain acyl-CoA dehydrogenase. These results provide fundamental data to be used in future research on insect lipid metabolism and its possible relevance to Chagas' disease transmission. Copyright © 2016 Elsevier B.V. All rights reserved.

Identification of U2AF(35)-dependent exons by RNA-Seq reveals a link between 3′ splice-site organization and activity of U2AF-related proteins

PubMed Central

Kralovicova, Jana; Knut, Marcin; Cross, Nicholas C. P.; Vorechovsky, Igor

2015-01-01

The auxiliary factor of U2 small nuclear RNA (U2AF) is a heterodimer consisting of 65- and 35-kD proteins that bind the polypyrimidine tract (PPT) and AG dinucleotides at the 3′ splice site (3′ss). The gene encoding U2AF35 (U2AF1) is alternatively spliced, giving rise to two isoforms U2AF35a and U2AF35b. Here, we knocked down U2AF35 and each isoform and characterized transcriptomes of HEK293 cells with varying U2AF35/U2AF65 and U2AF35a/b ratios. Depletion of both isoforms preferentially modified alternative RNA processing events without widespread failure to recognize 3′ss or constitutive exons. Over a third of differentially used exons were terminal, resulting largely from the use of known alternative polyadenylation (APA) sites. Intronic APA sites activated in depleted cultures were mostly proximal whereas tandem 3′UTR APA was biased toward distal sites. Exons upregulated in depleted cells were preceded by longer AG exclusion zones and PPTs than downregulated or control exons and were largely activated by PUF60 and repressed by CAPERα. The U2AF(35) repression and activation was associated with a significant interchange in the average probabilities to form single-stranded RNA in the optimal PPT and branch site locations and sequences further upstream. Although most differentially used exons were responsive to both U2AF subunits and their inclusion correlated with U2AF levels, a small number of transcripts exhibited distinct responses to U2AF35a and U2AF35b, supporting the existence of isoform-specific interactions. These results provide new insights into function of U2AF and U2AF35 in alternative RNA processing. PMID:25779042

Recommendations for Accurate Resolution of Gene and Isoform Allele-Specific Expression in RNA-Seq Data

PubMed Central

Wood, David L. A.; Nones, Katia; Steptoe, Anita; Christ, Angelika; Harliwong, Ivon; Newell, Felicity; Bruxner, Timothy J. C.; Miller, David; Cloonan, Nicole; Grimmond, Sean M.

2015-01-01

Genetic variation modulates gene expression transcriptionally or post-transcriptionally, and can profoundly alter an individual’s phenotype. Measuring allelic differential expression at heterozygous loci within an individual, a phenomenon called allele-specific expression (ASE), can assist in identifying such factors. Massively parallel DNA and RNA sequencing and advances in bioinformatic methodologies provide an outstanding opportunity to measure ASE genome-wide. In this study, matched DNA and RNA sequencing, genotyping arrays and computationally phased haplotypes were integrated to comprehensively and conservatively quantify ASE in a single human brain and liver tissue sample. We describe a methodological evaluation and assessment of common bioinformatic steps for ASE quantification, and recommend a robust approach to accurately measure SNP, gene and isoform ASE through the use of personalized haplotype genome alignment, strict alignment quality control and intragenic SNP aggregation. Our results indicate that accurate ASE quantification requires careful bioinformatic analyses and is adversely affected by sample specific alignment confounders and random sampling even at moderate sequence depths. We identified multiple known and several novel ASE genes in liver, including WDR72, DSP and UBD, as well as genes that contained ASE SNPs with imbalance direction discordant with haplotype phase, explainable by annotated transcript structure, suggesting isoform derived ASE. The methods evaluated in this study will be of use to researchers performing highly conservative quantification of ASE, and the genes and isoforms identified as ASE of interest to researchers studying those loci. PMID:25965996

Bacterial Production, Characterization and Protein Modeling of a Novel Monofuctional Isoform of FAD Synthase in Humans: An Emergency Protein?

PubMed

Leone, Piero; Galluccio, Michele; Barbiroli, Alberto; Eberini, Ivano; Tolomeo, Maria; Vrenna, Flavia; Gianazza, Elisabetta; Iametti, Stefania; Bonomi, Francesco; Indiveri, Cesare; Barile, Maria

2018-01-06

FAD synthase (FADS, EC 2.7.7.2) is the last essential enzyme involved in the pathway of biosynthesis of Flavin cofactors starting from Riboflavin (Rf). Alternative splicing of the human FLAD1 gene generates different isoforms of the enzyme FAD synthase. Besides the well characterized isoform 1 and 2, other FADS isoforms with different catalytic domains have been detected, which are splice variants. We report the characterization of one of these novel isoforms, a 320 amino acid protein, consisting of the sole C-terminal 3'-phosphoadenosine 5'-phosphosulfate (PAPS) reductase domain (named FADS6). This isoform has been previously detected in Riboflavin-Responsive (RR-MADD) and Non-responsive Multiple Acyl-CoA Dehydrogenase Deficiency (MADD) patients with frameshift mutations of FLAD1 gene. To functionally characterize the hFADS6, it has been over-expressed in Escherichia coli and purified with a yield of 25 mg·L -1 of cell culture. The protein has a monomeric form, it binds FAD and is able to catalyze FAD synthesis (k cat about 2.8 min -1 ), as well as FAD pyrophosphorolysis in a strictly Mg 2+ -dependent manner. The synthesis of FAD is inhibited by HgCl₂. The enzyme lacks the ability to hydrolyze FAD. It behaves similarly to PAPS. Combining threading and ab-initio strategy a 3D structural model for such isoform has been built. The relevance to human physio-pathology of this FADS isoform is discussed.

Gene organization and alternative splicing of human prohormone convertase PC8.

PubMed Central

Goodge, K A; Thomas, R J; Martin, T J; Gillespie, M T

1998-01-01

The mammalian Ca2+-dependent serine protease prohormone convertase PC8 is expressed ubiquitously, being transcribed as 3.5, 4.3 and 6.0 kb mRNA isoforms in various tissues. To determine the origin of these various mRNA isoforms we report the characterization of the human PC8 gene, which has been previously localized to chromosome 11q23-24. Consisting of 16 exons, the human PC8 gene spans approx. 27 kb. A comparison of the position of intron-exon junctions of the human PC8 gene with the gene structures of previously reported prohormone convertase genes demonstrated a divergence of the human PC8 from the highly conserved nature of the gene organization of this enzyme family. The nucleotide sequence of the 5'-flanking region of the human PC8 is reported and possesses putative promoter elements characteristic of a GC-rich promoter. Further supporting the potential role of a GC-rich promoter element, multiple transcriptional initiation sites within a 200 bp region were demonstrated. We propose that the various mRNA isoforms of PC8 result from the inclusion of intronic sequences within transcripts. PMID:9820811

Functional characterization of transmembrane adenylyl cyclases from the honeybee brain.

PubMed

Balfanz, Sabine; Ehling, Petra; Wachten, Sebastian; Jordan, Nadine; Erber, Joachim; Mujagic, Samir; Baumann, Arnd

2012-06-01

The second messenger cAMP has a pivotal role in animals' physiology and behavior. Intracellular concentrations of cAMP are balanced by cAMP-synthesizing adenylyl cyclases (ACs) and cAMP-cleaving phosphodiesterases. Knowledge about ACs in the honeybee (Apis mellifera) is rather limited and only an ortholog of the vertebrate AC3 isoform has been functionally characterized, so far. Employing bioinformatics and functional expression we characterized two additional honeybee genes encoding membrane-bound (tm)ACs. The proteins were designated AmAC2t and AmAC8. Unlike the common structure of tmACs, AmAC2t lacks the first transmembrane domain. Despite this unusual topography, AmAC2t-activity could be stimulated by norepinephrine and NKH477 with EC(50s) of 0.07 μM and 3 μM. Both ligands stimulated AmAC8 with EC(50s) of 0.24 μM and 3.1 μM. In brain cryosections, intensive staining of mushroom bodies was observed with specific antibodies against AmAC8, an expression pattern highly reminiscent of the Drosophila rutabaga AC. In a current release of the honeybee genome database we identified three additional tmAC- and one soluble AC-encoding gene. These results suggest that (1) the AC-gene family in honeybees is comparably large as in other species, and (2) based on the restricted expression of AmAC8 in mushroom bodies, this enzyme might serve important functions in honeybee behavior. Copyright © 2012 Elsevier Ltd. All rights reserved.

Alternative splicing: a novel mechanism of regulation identified in the chorismate mutase gene of the potato cyst nematode Globodera rostochiensis.

PubMed

Lu, Shun-Wen; Tian, Duanhua; Borchardt-Wier, Harmony B; Wang, Xiaohong

2008-11-01

Chorismate mutase (CM) secreted from the stylet of plant-parasitic nematodes plays an important role in plant parasitism. We isolated and characterized a new nematode CM gene (Gr-cm-1) from the potato cyst nematode, Globodera rostochiensis. The Gr-cm-1 gene was found to exist in the nematode genome as a single-copy gene that has two different alleles, Gr-cm-1A and Gr-cm-1B, both of which could give rise to two different mRNA transcripts of Gr-cm-1 and Gr-cm-1-IRII. In situ mRNA hybridization showed that the Gr-cm-1 gene was exclusively expressed within the subventral oesophageal gland cells of the nematode. Gr-cm-1 was demonstrated to encode a functional CM (GR-CM-1) potentially having a dimeric structure as the secreted bacterial *AroQ CMs. Gr-cm-1-IRII, generated by retention of intron 2 of the Gr-cm-1 pre-mRNA through alternative splicing (AS), would encode a truncated protein (GR-CM-1t) lacking the CM domain with no CM activity. The quantitative real-time reverse transcription-PCR assay revealed that splicing of the Gr-cm-1 gene was developmentally regulated; Gr-cm-1 was up-regulated whereas Gr-cm-1-IRII was down-regulated in early nematode parasitic stages compared to the preparasitic juvenile stage. Low-temperature SDS-PAGE analysis revealed that GR-CM-1 could form homodimers when expressed in Escherichia coli and the dimerization domain was retained in the truncated GR-CM-1t protein. The specific interaction between the two proteins was demonstrated in yeast. Our data suggested that the novel splice variant might function as a dominant negative isoform through heterodimerization with the full-length GR-CM-1 protein and that AS may represent an important mechanism for regulating CM activity during nematode parasitism.

Ectromelia virus upregulates the expression of heat shock protein 70 to promote viral replication.

PubMed

Cheng, Wenyu; Jia, Huaijie; Wang, Xiaoxia; He, Xiaobing; Jin, Qiwang; Cao, Jingxin; Jing, Zhizhong

2018-08-01

The ectromelia virus (ECTV) is a mouse specific Orthopoxvirus that causes lethal infection in some mouse strains. ECTV infection of these mouse strains has been used as a valuable model for understanding the interplay between Orthopoxvirus species and their hosts, including variola virus in humans. Although poxviruses encode numerous proteins required for DNA and RNA synthesis, and are less dependent on host functions than other DNA viruses, a detailed understanding of the host factors required for the replication of poxviruses is lacking. Heat shock protein 70 (Hsp70) isoforms have been reported to serve various roles in the replication cycle of numerous viruses. In the present study, microarray and reverse transcription‑quantitative polymerase chain reaction analysis were conducted to investigate the host gene expression profiles following ECTV infection in mice and cell cultures. The results indicated that one Hsp70 isoform, Hsp70 member 1B (Hspa1b), was highly upregulated during ECTV infection in vitro and in vivo. Subsequently, overexpression of Hspa1b protein and small interfering RNA‑mediated gene silencing of Hspa1b revealed that Hspa1b is required for efficient replication of ECTV. Furthermore, the results demonstrated that ECTV replication may be significantly suppressed by two chemical Hspa1b inhibitors: Quercetin and VER155008. In conclusion, the present study clearly demonstrated that ECTV infection upregulates the expression of Hspa1b in order to promote its replication. The dependence on Hsp70 may be used as a novel therapeutic target for the treatment of Orthopoxvirus infection.

The Nance-Horan syndrome protein encodes a functional WAVE homology domain (WHD) and is important for co-ordinating actin remodelling and maintaining cell morphology.

PubMed

Brooks, Simon P; Coccia, Margherita; Tang, Hao R; Kanuga, Naheed; Machesky, Laura M; Bailly, Maryse; Cheetham, Michael E; Hardcastle, Alison J

2010-06-15

Nance-Horan syndrome (NHS) is an X-linked developmental disorder, characterized by bilateral congenital cataracts, dental anomalies, facial dysmorphism and mental retardation. Null mutations in a novel gene, NHS, cause the syndrome. The NHS gene appears to have multiple isoforms as a result of alternative transcription, but a cellular function for the NHS protein has yet to be defined. We describe NHS as a founder member of a new protein family (NHS, NHSL1 and NHSL2). Here, we demonstrate that NHS is a novel regulator of actin remodelling and cell morphology. NHS localizes to sites of cell-cell contact, the leading edge of lamellipodia and focal adhesions. The N-terminus of isoforms NHS-A and NHS-1A, implicated in the pathogenesis of NHS, have a functional WAVE homology domain that interacts with the Abi protein family, haematopoietic stem/progenitor cell protein 300 (HSPC300), Nap1 and Sra1. NHS knockdown resulted in the disruption of the actin cytoskeleton. We show that NHS controls cell morphology by maintaining the integrity of the circumferential actin ring and controlling lamellipod formation. NHS knockdown led to a striking increase in cell spreading. Conversely, ectopic overexpression of NHS inhibited lamellipod formation. Remodelling of the actin cytoskeleton and localized actin polymerization into branched actin filaments at the plasma membrane are essential for mediating changes in cell shape, migration and cell contact. Our data identify NHS as a new regulator of actin remodelling. We suggest that NHS orchestrates actin regulatory protein function in response to signalling events during development.

The PI3K Isoforms p110α and p110δ are Essential for Pre-B Cell Receptor Signaling and B Cell Development

PubMed Central

Ramadani, Faruk; Bolland, Daniel J.; Garcon, Fabien; Emery, Juliet L.; Vanhaesebroeck, Bart; Corcoran, Anne E.; Okkenhaug, Klaus

2013-01-01

B cell development is controlled by a series of checkpoints that ensure that the immunoglobulin (Ig)-encoding genes are assembled in frame to produce a functional B cell receptor (BCR) and antibodies. The BCR consists of Ig proteins in complex with the immunoreceptor tyrosine-based activation motif (ITAM)-containing Igα and Igβ chains. Whereas the activation of Src and Syk tyrosine kinases is essential for BCR signaling, the pathways that act downstream of these kinases are incompletely defined. Previous work has revealed a key role for the p110δ isoform of phosphoinositide 3-kinase (PI3K) in agonist-induced BCR signaling; however, early B cell development and mature B cell survival, which depend on tonic BCR signaling, are not substantially affected by a deficiency in p110δ. Here, we show that in the absence of p110δ, p110α, but not p110β, can compensate to promote early B cell development in the bone marrow and B cell survival in the spleen. In the absence of both p110α and p110δ activities, pre-BCR signaling fails to suppress the production of recombination-activating gene (Rag) protein and to promote developmental progression of B cell progenitors. By contrast, p110α does not contribute to agonist-induced BCR signaling. These studies indicate that either p110α or p110δ can mediate tonic signaling from the BCR, but that only p110δ can contribute to antigen-dependent activation of B cells. PMID:20699475

Extraction, integration and analysis of alternative splicing and protein structure distributed information

PubMed Central

D'Antonio, Matteo; Masseroli, Marco

2009-01-01

Background Alternative splicing has been demonstrated to affect most of human genes; different isoforms from the same gene encode for proteins which differ for a limited number of residues, thus yielding similar structures. This suggests possible correlations between alternative splicing and protein structure. In order to support the investigation of such relationships, we have developed the Alternative Splicing and Protein Structure Scrutinizer (PASS), a Web application to automatically extract, integrate and analyze human alternative splicing and protein structure data sparsely available in the Alternative Splicing Database, Ensembl databank and Protein Data Bank. Primary data from these databases have been integrated and analyzed using the Protein Identifier Cross-Reference, BLAST, CLUSTALW and FeatureMap3D software tools. Results A database has been developed to store the considered primary data and the results from their analysis; a system of Perl scripts has been implemented to automatically create and update the database and analyze the integrated data; a Web interface has been implemented to make the analyses easily accessible; a database has been created to manage user accesses to the PASS Web application and store user's data and searches. Conclusion PASS automatically integrates data from the Alternative Splicing Database with protein structure data from the Protein Data Bank. Additionally, it comprehensively analyzes the integrated data with publicly available well-known bioinformatics tools in order to generate structural information of isoform pairs. Further analysis of such valuable information might reveal interesting relationships between alternative splicing and protein structure differences, which may be significantly associated with different functions. PMID:19828075

Expression of phosphoinositide-specific phospholipase C isoforms in native endothelial cells.

PubMed

Béziau, Delphine M; Toussaint, Fanny; Blanchette, Alexandre; Dayeh, Nour R; Charbel, Chimène; Tardif, Jean-Claude; Dupuis, Jocelyn; Ledoux, Jonathan

2015-01-01

Phospholipase C (PLC) comprises a superfamily of enzymes that play a key role in a wide array of intracellular signalling pathways, including protein kinase C and intracellular calcium. Thirteen different mammalian PLC isoforms have been identified and classified into 6 families (PLC-β, γ, δ, ε, ζ and η) based on their biochemical properties. Although the expression of PLC isoforms is tissue-specific, concomitant expression of different PLC has been reported, suggesting that PLC family is involved in multiple cellular functions. Despite their critical role, the PLC isoforms expressed in native endothelial cells (ECs) remains undetermined. A conventional PCR approach was initially used to elucidate the mRNA expression pattern of PLC isoforms in 3 distinct murine vascular beds: mesenteric (MA), pulmonary (PA) and middle cerebral arteries (MCA). mRNA encoding for most PLC isoforms was detected in MA, MCA and PA with the exception of η2 and β2 (only expressed in PA), δ4 (only expressed in MCA), η1 (expressed in all but MA) and ζ (not detected in any vascular beds tested). The endothelial-specific PLC expression was then sought in freshly isolated ECs. Interestingly, the PLC expression profile appears to differ across the investigated arterial beds. While mRNA for 8 of the 13 PLC isoforms was detected in ECs from MA, two additional PLC isoforms were detected in ECs from PA and MCA. Co-expression of multiple PLC isoforms in ECs suggests an elaborate network of signalling pathways: PLC isoforms may contribute to the complexity or diversity of signalling by their selective localization in cellular microdomains. However in situ immunofluorescence revealed a homogeneous distribution for all PLC isoforms probed (β3, γ2 and δ1) in intact endothelium. Although PLC isoforms play a crucial role in endothelial signal transduction, subcellular localization alone does not appear to be sufficient to determine the role of PLC in the signalling microdomains found in the native endothelium.

Characterization of cDNAs encoding the chick retinoic acid receptor gamma 2 and preferential distribution of retinoic acid receptor gamma transcripts during chick skin development.

PubMed

Michaille, J J; Blanchet, S; Kanzler, B; Garnier, J M; Dhouailly, D

1994-12-01

Retinoic acid receptors alpha, beta and gamma (RAR alpha, beta and gamma) are ligand-inductible transcriptional activators which belong to the steroid/thyroid hormone receptor superfamily. At least two major isoforms (1 and 2) of each RAR arise by differential use of two promoters and alternative splicing. In mouse, the three RAR genes are expressed in stage- and tissue-specific patterns during embryonic development. In order to understand the role of the different RARs in chick, RAR gamma 2 cDNAs were isolated from an 8.5-day (stage 35 of Hamburger and Hamilton) chick embryo skin library. The deduced chick RAR gamma 2 amino acid sequence displays uncommon features such as 21 specific amino acid replacements, 12 of them being clustered in the amino-terminal region (domains A2 and B), and a truncated acidic carboxy-terminal region (F domain). However, the pattern of RAR gamma expression in chick embryo resembles that reported in mouse, particularly in skin where RAR gamma expression occurs in both the dermal and epidermal layers at the beginning of feather formation, and is subsequently restricted to the differentiating epidermal cells. Northern blot analysis suggests that different RAR gamma isoforms could be successively required during chick development.

Class I HDACs Regulate Angiotensin II-Dependent Cardiac Fibrosis via Fibroblasts and Circulating Fibrocytes

PubMed Central

Williams, Sarah M.; Golden-Mason, Lucy; Ferguson, Bradley S.; Douglas, Katherine B.; Cavasin, Maria A.; Demos-Davies, Kim; Yeager, Michael E.; Stenmark, Kurt R.; McKinsey, Timothy A.

2014-01-01

Fibrosis, which is defined as excessive accumulation of fibrous connective tissue, contributes to the pathogenesis of numerous diseases involving diverse organ systems. Cardiac fibrosis predisposes individuals to myocardial ischemia, arrhythmias and sudden death, and is commonly associated with diastolic dysfunction. Histone deacetylase (HDAC) inhibitors block cardiac fibrosis in pre-clinical models of heart failure. However, which HDAC isoforms govern cardiac fibrosis, and the mechanisms by which they do so, remains unclear. Here, we show that selective inhibition of class I HDACs potently suppresses angiotensin II (Ang II)-mediated cardiac fibrosis by targeting two key effector cell populations, cardiac fibroblasts and bone marrow-derived fibrocytes. Class I HDAC inhibition blocks cardiac fibroblast cell cycle progression through derepression of the genes encoding the cyclin-dependent kinase (CDK) inhibitors, p15 and p57. In contrast, class I HDAC inhibitors block agonist-dependent differentiation of fibrocytes through a mechanism involving repression of ERK1/2 signaling. These findings define novel roles for class I HDACs in the control of pathological cardiac fibrosis. Furthermore, since fibrocytes have been implicated in the pathogenesis of a variety of human diseases, including heart, lung and kidney failure, our results suggest broad utility for isoform-selective HDAC inhibitors as anti-fibrotic agents that function, in part, by targeting these circulating mesenchymal cells. PMID:24374140

The Mitochondrial Protein Import Component, TRANSLOCASE OF THE INNER MEMBRANE17-1, Plays a Role in Defining the Timing of Germination in Arabidopsis1[W][OPEN

PubMed Central

Wang, Yan; Law, Simon R.; Ivanova, Aneta; van Aken, Olivier; Kubiszewski-Jakubiak, Szymon; Uggalla, Vindya; van der Merwe, Margaretha; Duncan, Owen; Narsai, Reena; Whelan, James; Murcha, Monika W.

2014-01-01

In Arabidopsis (Arabidopsis thaliana), small gene families encode multiple isoforms for many of the components of the mitochondrial protein import apparatus. There are three isoforms of the TRANSLOCASE OF THE INNER MEMBRANE17 (Tim17). Transcriptome analysis indicates that AtTim17-1 is only detectable in dry seed. In this study, two independent transfer DNA insertional mutant lines of tim17-1 exhibited a germination-specific phenotype, showing a significant increase in the rate of germination. Microarray analyses revealed that Attim17-1 displayed alterations in the temporal sequence of transcriptomic events during germination, peaking earlier compared with the wild type. Promoter analysis of AtTim17-1 further identified an abscisic acid (ABA)-responsive element, which binds ABA-responsive transcription factors, acting to repress the expression of AtTim17-1. Attim17-1 dry seeds contained significantly increased levels of ABA and gibberellin, 2- and 5-fold, respectively. These results support the model that mitochondrial biogenesis is regulated in a tight temporal sequence of events during germination and that altering mitochondrial biogenesis feeds back to alter the germination rate, as evidenced by the altered levels of the master regulatory hormones that define germination. PMID:25253887

Cloning of a newly identified heart-specific troponin I isoform, which lacks the troponin T binding portion, using the yeast hybrid system.

PubMed

Suzuki, Hideaki; Arakawa, Yasuhiro; Ito, Masaki; Yamada, Hisashi; Horiguchi-Yamada, Junko

2006-01-01

To elucidate the molecular pathogenesis behind increased levels of laminin in cardiac muscle cells in cardiomyopathy by using a yeast hybrid screen. The present study reports the cloning of a newly identified heart-specific troponin I isoform, which is putatively linked to laminin. Future studies will explore the functional significance of this connection. Yeast two-hybrid screen analysis was performed using MLF1-interacting protein (amino acids 1 to 318) as bait. The human heart complementary DNA library was screened by using the yeast-mating method for overnight culture. Two final positive clones from the heart library were isolated. These two clones encoded the same protein, a short isoform of human cardiac troponin I (TnI) that lacked TnI exons 5 and 6. The TnI isoform has a heart-specific expression pattern and it shares several sequence features with human cardiac TnI; however, it lacks the troponin T binding portion. The heart-specific segment of the human cardiac TnI isoform shares several sequence features with human cardiac TnI, but it lacks the troponin T binding portion. These results suggest that the heart-specific TnI isoform may be involved in cardiac development and disease.

Cloning of a newly identified heart-specific troponin I isoform, which lacks the troponin T binding portion, using the yeast hybrid system

PubMed Central

Suzuki, Hideaki; Arakawa, Yasuhiro; Ito, Masaki; Yamada, Hisashi; Horiguchi-Yamada, Junko

2006-01-01

OBJECTIVE To elucidate the molecular pathogenesis behind increased levels of laminin in cardiac muscle cells in cardiomyopathy by using a yeast hybrid screen. The present study reports the cloning of a newly identified heart-specific troponin I isoform, which is putatively linked to laminin. Future studies will explore the functional significance of this connection. METHODS Yeast two-hybrid screen analysis was performed using MLF1-interacting protein (amino acids 1 to 318) as bait. The human heart complementary DNA library was screened by using the yeast-mating method for overnight culture. RESULTS Two final positive clones from the heart library were isolated. These two clones encoded the same protein, a short isoform of human cardiac troponin I (TnI) that lacked TnI exons 5 and 6. The TnI isoform has a heart-specific expression pattern and it shares several sequence features with human cardiac TnI; however, it lacks the troponin T binding portion. CONCLUSION The heart-specific segment of the human cardiac TnI isoform shares several sequence features with human cardiac TnI, but it lacks the troponin T binding portion. These results suggest that the heart-specific TnI isoform may be involved in cardiac development and disease. PMID:18651010

First de novo ANK3 nonsense mutation in a boy with intellectual disability, speech impairment and autistic features.

PubMed

Kloth, Katja; Denecke, Jonas; Hempel, Maja; Johannsen, Jessika; Strom, Tim M; Kubisch, Christian; Lessel, Davor

2017-09-01

Ankyrin-G, encoded by ANK3, plays an important role in neurodevelopment and neuronal function. There are multiple isoforms of Ankyrin-G resulting in differential tissue expression and function. Heterozygous missense mutations in ANK3 have been associated with autism spectrum disorder. Further, in three siblings a homozygous frameshift mutation affecting only the longest isoform and a patient with a balanced translocation disrupting all isoforms were documented. The latter four patients were affected by a variable degree of intellectual disability, attention deficit hyperactivity disorder and autism. Here, we report on a boy with speech impairment, intellectual disability, autistic features, macrocephaly, macrosomia, chronic hunger and an altered sleeping pattern. By trio-whole-exome sequencing, we identified the first de novo nonsense mutation affecting all ANK3 transcripts. Thus, our data expand the phenotype of ANK3-associated diseases and suggest an isoform-based, phenotypic continuum between dominant and recessive ANK3-associated pathologies. Copyright © 2017. Published by Elsevier Masson SAS.

The venom gland transcriptome of the Desert Massasauga Rattlesnake (Sistrurus catenatus edwardsii): towards an understanding of venom composition among advanced snakes (Superfamily Colubroidea)

PubMed Central

Pahari, Susanta; Mackessy, Stephen P; Kini, R Manjunatha

2007-01-01

Background Snake venoms are complex mixtures of pharmacologically active proteins and peptides which belong to a small number of superfamilies. Global cataloguing of the venom transcriptome facilitates the identification of new families of toxins as well as helps in understanding the evolution of venom proteomes. Results We have constructed a cDNA library of the venom gland of a threatened rattlesnake (a pitviper), Sistrurus catenatus edwardsii (Desert Massasauga), and sequenced 576 ESTs. Our results demonstrate a high abundance of serine proteinase and metalloproteinase transcripts, indicating that the disruption of hemostasis is a principle mechanism of action of the venom. In addition to the transcripts encoding common venom proteins, we detected two varieties of low abundance unique transcripts in the library; these encode for three-finger toxins and a novel toxin possibly generated from the fusion of two genes. We also observed polyadenylated ribosomal RNAs in the venom gland library, an interesting preliminary obsevation of this unusual phenomenon in a reptilian system. Conclusion The three-finger toxins are characteristic of most elapid venoms but are rare in viperid venoms. We detected several ESTs encoding this group of toxins in this study. We also observed the presence of a transcript encoding a fused protein of two well-characterized toxins (Kunitz/BPTI and Waprins), and this is the first report of this kind of fusion in a snake toxin transcriptome. We propose that these new venom proteins may have ancillary functions for envenomation. The presence of a fused toxin indicates that in addition to gene duplication and accelerated evolution, exon shuffling or transcriptional splicing may also contribute to generating the diversity of toxins and toxin isoforms observed among snake venoms. The detection of low abundance toxins, as observed in this and other studies, indicates a greater compositional similarity of venoms (though potency will differ) among advanced snakes than has been previously recognized. PMID:18096037

Statistical modeling of isoform splicing dynamics from RNA-seq time series data.

PubMed

Huang, Yuanhua; Sanguinetti, Guido

2016-10-01

Isoform quantification is an important goal of RNA-seq experiments, yet it remains problematic for genes with low expression or several isoforms. These difficulties may in principle be ameliorated by exploiting correlated experimental designs, such as time series or dosage response experiments. Time series RNA-seq experiments, in particular, are becoming increasingly popular, yet there are no methods that explicitly leverage the experimental design to improve isoform quantification. Here, we present DICEseq, the first isoform quantification method tailored to correlated RNA-seq experiments. DICEseq explicitly models the correlations between different RNA-seq experiments to aid the quantification of isoforms across experiments. Numerical experiments on simulated datasets show that DICEseq yields more accurate results than state-of-the-art methods, an advantage that can become considerable at low coverage levels. On real datasets, our results show that DICEseq provides substantially more reproducible and robust quantifications, increasing the correlation of estimates from replicate datasets by up to 10% on genes with low or moderate expression levels (bottom third of all genes). Furthermore, DICEseq permits to quantify the trade-off between temporal sampling of RNA and depth of sequencing, frequently an important choice when planning experiments. Our results have strong implications for the design of RNA-seq experiments, and offer a novel tool for improved analysis of such datasets. Python code is freely available at http://diceseq.sf.net G.Sanguinetti@ed.ac.uk Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Analysis of transcriptional isoforms of collagen types IX, II, and I in the developing avian cornea by competitive polymerase chain reaction.

PubMed

Fitch, J M; Gordon, M K; Gibney, E P; Linsenmayer, T F

1995-01-01

The genes for the alpha 1(IX), alpha 1(II), and alpha 2(I) collagen chains can give rise to different isoforms of mRNA, generated by alternative promotor usage [for alpha 1(IX) and alpha 2(I)] or alternative splicing [for alpha 1(II)]. In this study, we employed competitive reverse transcriptase PCR to quantitate the amounts of transcriptional isoforms for these genes in the embryonic avian cornea from its inception (about 3 1/2 days of development) to 11 days. In order to compare values at different time points, the results were normalized to those obtained for the "housekeeping" enzyme, glycerol-3-phosphate dehydrogenase (G3PDH). These values were compared to those obtained from other tissues (anterior optic cup and cartilage) that synthesize different combinations of the collagen isoforms. We found that, in the cornea, transcripts from the upstream promotor of alpha 1(IX) collagen (termed "long IX") were predominant at stage 18-20 (about 3 1/2 days), but then fell rapidly, and remained at a low level. By 5 days (just before stromal swelling) the major mRNA isoform of alpha 1(IX) was from the downstream promoter (termed "short IX"). The relative amount of transcript for the short form of type IX collagen rose to a peak at about 6 days of development, and then declined. Throughout this period, the predominant transcriptional isoform of the collagen type II gene was IIA (i.e., containing the alternatively spliced exon 2). This indicates that the molecules of type II collagen that are assembled into heterotypic fibrils with type I collagen possess, at least transiently, an amino-terminal globular domain similar to that found in collagen types I, III, and V. For type I, the "bone/tendon" mRNA isoform of the alpha 2(I) collagen gene was predominant; transcripts from the downstream promotor were at basal levels. In other tissues expressing collagen types IX and II, long IX was expressed predominantly with the IIA form in the anterior optic cup at stage 22/23; in 14 1/2 day cartilage, long IX was expressed predominantly along with the IIB form of alpha 1(II). The downstream transcript of the alpha 2(I) gene (Icart) was found at high levels only in cartilage.

A Comprehensive Analysis of Alternative Splicing in Paleopolyploid Maize.

PubMed

Mei, Wenbin; Liu, Sanzhen; Schnable, James C; Yeh, Cheng-Ting; Springer, Nathan M; Schnable, Patrick S; Barbazuk, William B

2017-01-01

Identifying and characterizing alternative splicing (AS) enables our understanding of the biological role of transcript isoform diversity. This study describes the use of publicly available RNA-Seq data to identify and characterize the global diversity of AS isoforms in maize using the inbred lines B73 and Mo17, and a related species, sorghum. Identification and characterization of AS within maize tissues revealed that genes expressed in seed exhibit the largest differential AS relative to other tissues examined. Additionally, differences in AS between the two genotypes B73 and Mo17 are greatest within genes expressed in seed. We demonstrate that changes in the level of alternatively spliced transcripts (intron retention and exon skipping) do not solely reflect differences in total transcript abundance, and we present evidence that intron retention may act to fine-tune gene expression across seed development stages. Furthermore, we have identified temperature sensitive AS in maize and demonstrate that drought-induced changes in AS involve distinct sets of genes in reproductive and vegetative tissues. Examining our identified AS isoforms within B73 × Mo17 recombinant inbred lines (RILs) identified splicing QTL (sQTL). The 43.3% of cis- sQTL regulated junctions are actually identified as alternatively spliced junctions in our analysis, while 10 Mb windows on each side of 48.2% of trans -sQTLs overlap with splicing related genes. Using sorghum as an out-group enabled direct examination of loss or conservation of AS between homeologous genes representing the two subgenomes of maize. We identify several instances where AS isoforms that are conserved between one maize homeolog and its sorghum ortholog are absent from the second maize homeolog, suggesting that these AS isoforms may have been lost after the maize whole genome duplication event. This comprehensive analysis provides new insights into the complexity of AS in maize.

The multidrug resistance 1 gene Abcb1 in brain and placenta: comparative analysis in human and guinea pig.

PubMed

Pappas, Jane J; Petropoulos, Sophie; Suderman, Matthew; Iqbal, Majid; Moisiadis, Vasilis; Turecki, Gustavo; Matthews, Stephen G; Szyf, Moshe

2014-01-01

The Multidrug Resistance 1 (MDR1; alternatively ABCB1) gene product P-glycoprotein (P-gp), an ATP binding cassette transporter, extrudes multiple endogenous and exogenous substrates from the cell, playing an important role in normal physiology and xenobiotic distribution and bioavailability. To date, the predominant animal models used to investigate the role of P-gp have been the mouse and rat, which have two distinct genes, Abcb1a and Abcb1b. In contrast, the human has a single gene, ABCB1, for which only a single isoform has been validated. We and others have previously shown important differences between Abcb1a and Abcb1b, limiting the extrapolation from rodent findings to the human. Since the guinea pig has a relatively long gestation, hemomonochorial placentation and neuroanatomically mature offspring, it is more similar to the human, and may provide a more comparable model for investigating the regulation of P-gp in the brain and placenta, however, to date, the Abcb1 gene in the guinea pig remains to be characterized. The placenta and fetal brain are barrier sites that express P-gp and that play a critical role of protection of the fetus and the fetal brain from maternally administered drugs and other xenobiotics. Using RNA sequencing (RNA-seq), reverse transcription-polymerase chain reaction (RT-PCR) and quantitative PCR (QPCR) to sequence the expressed isoforms of guinea pig Abcb1, we demonstrate that like the human, the guinea pig genome contains one gene for Abcb1 but that it is expressed as at least three different isoforms via alternative splicing and alternate exon usage. Further, we demonstrate that these isoforms are more closely related to human than to rat or mouse isoforms. This striking, overall similarity and evolutionary relatedness between guinea pig Abcb1 and human ABCB1 indicate that the guinea pig represents a relevant animal model for investigating the function and regulation of P-gp in the placenta and brain.

Comparative Genomics and Reverse Genetics Analysis Reveal Indispensable Functions of the Serine Acetyltransferase Gene Family in Arabidopsis[W][OA

PubMed Central

Watanabe, Mutsumi; Mochida, Keiichi; Kato, Tomohiko; Tabata, Satoshi; Yoshimoto, Naoko; Noji, Masaaki; Saito, Kazuki

2008-01-01

Ser acetyltransferase (SERAT), which catalyzes O-acetyl-Ser (OAS) formation, plays a key role in sulfur assimilation and Cys synthesis. Despite several studies on SERATs from various plant species, the in vivo function of multiple SERAT genes in plant cells remains unaddressed. Comparative genomics studies with the five genes of the SERAT gene family in Arabidopsis thaliana indicated that all three Arabidopsis SERAT subfamilies are conserved across five plant species with available genome sequences. Single and multiple knockout mutants of all Arabidopsis SERAT gene family members were analyzed. All five quadruple mutants with a single gene survived, with three mutants showing dwarfism. However, the quintuple mutant lacking all SERAT genes was embryo-lethal. Thus, all five isoforms show functional redundancy in vivo. The developmental and compartment-specific roles of each SERAT isoform were also demonstrated. Mitochondrial SERAT2;2 plays a predominant role in cellular OAS formation, while plastidic SERAT2;1 contributes less to OAS formation and subsequent Cys synthesis. Three cytosolic isoforms, SERAT1;1, SERAT3;1, and SERAT3;2, may play a major role during seed development. Thus, the evolutionally conserved SERAT gene family is essential in cellular processes, and the substrates and products of SERAT must be exchangeable between the cytosol and organelles. PMID:18776059

A little sugar goes a long way: The cell biology of O-GlcNAc

PubMed Central

2015-01-01

Unlike the complex glycans decorating the cell surface, the O-linked β-N-acetyl glucosamine (O-GlcNAc) modification is a simple intracellular Ser/Thr-linked monosaccharide that is important for disease-relevant signaling and enzyme regulation. O-GlcNAcylation requires uridine diphosphate–GlcNAc, a precursor responsive to nutrient status and other environmental cues. Alternative splicing of the genes encoding the O-GlcNAc cycling enzymes O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) yields isoforms targeted to discrete sites in the nucleus, cytoplasm, and mitochondria. OGT and OGA also partner with cellular effectors and act in tandem with other posttranslational modifications. The enzymes of O-GlcNAc cycling act preferentially on intrinsically disordered domains of target proteins impacting transcription, metabolism, apoptosis, organelle biogenesis, and transport. PMID:25825515

NURD: an implementation of a new method to estimate isoform expression from non-uniform RNA-seq data

PubMed Central

2013-01-01

Background RNA-Seq technology has been used widely in transcriptome study, and one of the most important applications is to estimate the expression level of genes and their alternative splicing isoforms. There have been several algorithms published to estimate the expression based on different models. Recently Wu et al. published a method that can accurately estimate isoform level expression by considering position-related sequencing biases using nonparametric models. The method has advantages in handling different read distributions, but there hasn’t been an efficient program to implement this algorithm. Results We developed an efficient implementation of the algorithm in the program NURD. It uses a binary interval search algorithm. The program can correct both the global tendency of sequencing bias in the data and local sequencing bias specific to each gene. The correction makes the isoform expression estimation more reliable under various read distributions. And the implementation is computationally efficient in both the memory cost and running time and can be readily scaled up for huge datasets. Conclusion NURD is an efficient and reliable tool for estimating the isoform expression level. Given the reads mapping result and gene annotation file, NURD will output the expression estimation result. The package is freely available for academic use at http://bioinfo.au.tsinghua.edu.cn/software/NURD/. PMID:23837734