Two splice variants of the bovine lactoferrin gene identified in Staphylococcus aureus isolated from mastitis in dairy cattle.

PubMed

Huang, J M; Wang, Z Y; Ju, Z H; Wang, C F; Li, Q L; Sun, T; Hou, Q L; Hang, S Q; Hou, M H; Zhong, J F

2011-12-21

Bovine lactoferrin (bLF) is a member of the transferrin family; it plays an important role in the innate immune response. We identified novel splice variants of the bLF gene in mastitis-infected and healthy cows. Reverse transcription-polymerase chain reaction (RT-PCR) and clone sequencing analysis were used to screen the splice variants of the bLF gene in the mammary gland, spleen and liver tissues. One main transcript corresponding to the bLF reference sequence was found in three tissues in both healthy and mastitis-infected cows. Quantitative real-time PCR analysis showed that the expression levels of the LF gene's main transcript were not significantly different in tissues from healthy versus mastitis-infected cows. However, the new splice variant, LF-AS2, which has the exon-skipping alternative splicing pattern, was only identified in mammary glands infected with Staphylococcus aureus. Sequencing analysis showed that the new splice variant was 251 bp in length, including exon 1, part of exon 2, part of exon 16, and exon 17. We conclude that bLF may play a role in resistance to mastitis through alternative splicing mechanisms.

Alternative splice variants of AID are not stoichiometrically present at the protein level in chronic lymphocytic leukemia

PubMed Central

Rebhandl, Stefan; Huemer, Michael; Zaborsky, Nadja; Gassner, Franz Josef; Catakovic, Kemal; Felder, Thomas Klaus; Greil, Richard; Geisberger, Roland

2014-01-01

Activation-induced deaminase (AID) is a DNA-mutating enzyme that mediates class-switch recombination as well as somatic hypermutation of antibody genes in B cells. Due to off-target activity, AID is implicated in lymphoma development by introducing genome-wide DNA damage and initiating chromosomal translocations such as c-myc/IgH. Several alternative splice transcripts of AID have been reported in activated B cells as well as malignant B cells such as chronic lymphocytic leukemia (CLL). As most commercially available antibodies fail to recognize alternative splice variants, their abundance in vivo, and hence their biological significance, has not been determined. In this study, we assessed the protein levels of AID splice isoforms by introducing an AID splice reporter construct into cell lines and primary CLL cells from patients as well as from WT and TCL1tg C57BL/6 mice (where TCL1 is T-cell leukemia/lymphoma 1). The splice construct is 5′-fused to a GFP-tag, which is preserved in all splice isoforms and allows detection of translated protein. Summarizing, we show a thorough quantification of alternatively spliced AID transcripts and demonstrate that the corresponding protein abundances, especially those of splice variants AID-ivs3 and AID-ΔE4, are not stoichiometrically equivalent. Our data suggest that enhanced proteasomal degradation of low-abundance proteins might be causative for this discrepancy. PMID:24668151

Alternative splicing and nonsense-mediated decay of circadian clock genes under environmental stress conditions in Arabidopsis

PubMed Central

2014-01-01

Background The circadian clock enables living organisms to anticipate recurring daily and seasonal fluctuations in their growth habitats and synchronize their biology to the environmental cycle. The plant circadian clock consists of multiple transcription-translation feedback loops that are entrained by environmental signals, such as light and temperature. In recent years, alternative splicing emerges as an important molecular mechanism that modulates the clock function in plants. Several clock genes are known to undergo alternative splicing in response to changes in environmental conditions, suggesting that the clock function is intimately associated with environmental responses via the alternative splicing of the clock genes. However, the alternative splicing events of the clock genes have not been studied at the molecular level. Results We systematically examined whether major clock genes undergo alternative splicing under various environmental conditions in Arabidopsis. We also investigated the fates of the RNA splice variants of the clock genes. It was found that the clock genes, including EARLY FLOWERING 3 (ELF3) and ZEITLUPE (ZTL) that have not been studied in terms of alternative splicing, undergo extensive alternative splicing through diverse modes of splicing events, such as intron retention, exon skipping, and selection of alternative 5′ splice site. Their alternative splicing patterns were differentially influenced by changes in photoperiod, temperature extremes, and salt stress. Notably, the RNA splice variants of TIMING OF CAB EXPRESSION 1 (TOC1) and ELF3 were degraded through the nonsense-mediated decay (NMD) pathway, whereas those of other clock genes were insensitive to NMD. Conclusion Taken together, our observations demonstrate that the major clock genes examined undergo extensive alternative splicing under various environmental conditions, suggesting that alternative splicing is a molecular scheme that underlies the linkage between the clock and environmental stress adaptation in plants. It is also envisioned that alternative splicing of the clock genes plays more complex roles than previously expected. PMID:24885185

Integrative genome-wide analysis of the determinants of RNA splicing in kidney renal clear cell carcinoma.

PubMed

Lehmann, Kjong-Van; Kahles, André; Kandoth, Cyriac; Lee, William; Schultz, Nikolaus; Stegle, Oliver; Rätsch, Gunnar

2015-01-01

We present a genome-wide analysis of splicing patterns of 282 kidney renal clear cell carcinoma patients in which we integrate data from whole-exome sequencing of tumor and normal samples, RNA-seq and copy number variation. We proposed a scoring mechanism to compare splicing patterns in tumor samples to normal samples in order to rank and detect tumor-specific isoforms that have a potential for new biomarkers. We identified a subset of genes that show introns only observable in tumor but not in normal samples, ENCODE and GEUVADIS samples. In order to improve our understanding of the underlying genetic mechanisms of splicing variation we performed a large-scale association analysis to find links between somatic or germline variants with alternative splicing events. We identified 915 cis- and trans-splicing quantitative trait loci (sQTL) associated with changes in splicing patterns. Some of these sQTL have previously been associated with being susceptibility loci for cancer and other diseases. Our analysis also allowed us to identify the function of several COSMIC variants showing significant association with changes in alternative splicing. This demonstrates the potential significance of variants affecting alternative splicing events and yields insights into the mechanisms related to an array of disease phenotypes.

A family of splice variants of CstF-64 expressed in vertebrate nervous systems

PubMed Central

Shankarling, Ganesh S; Coates, Penelope W; Dass, Brinda; MacDonald, Clinton C

2009-01-01

Background Alternative splicing and polyadenylation are important mechanisms for creating the proteomic diversity necessary for the nervous system to fulfill its specialized functions. The contribution of alternative splicing to proteomic diversity in the nervous system has been well documented, whereas the role of alternative polyadenylation in this process is less well understood. Since the CstF-64 polyadenylation protein is known to be an important regulator of tissue-specific polyadenylation, we examined its expression in brain and other organs. Results We discovered several closely related splice variants of CstF-64 – collectively called βCstF-64 – that could potentially contribute to proteomic diversity in the nervous system. The βCstF-64 splice variants are found predominantly in the brains of several vertebrate species including mice and humans. The major βCstF-64 variant mRNA is generated by inclusion of two alternate exons (that we call exons 8.1 and 8.2) found between exons 8 and 9 of the CstF-64 gene, and contains an additional 147 nucleotides, encoding 49 additional amino acids. Some variants of βCstF-64 contain only the first alternate exon (exon 8.1) while other variants contain both alternate exons (8.1 and 8.2). In mice, the predominant form of βCstF-64 also contains a deletion of 78 nucleotides from exon 9, although that variant is not seen in any other species examined, including rats. Immunoblot and 2D-PAGE analyses of mouse nuclear extracts indicate that a protein corresponding to βCstF-64 is expressed in brain at approximately equal levels to CstF-64. Since βCstF-64 splice variant family members were found in the brains of all vertebrate species examined (including turtles and fish), this suggests that βCstF-64 has an evolutionarily conserved function in these animals. βCstF-64 was present in both pre- and post-natal mice and in different regions of the nervous system, suggesting an important role for βCstF-64 in neural gene expression throughout development. Finally, experiments in representative cell lines suggest that βCstF-64 is expressed in neurons but not glia. Conclusion This is the first report of a family of splice variants encoding a key polyadenylation protein that is expressed in a nervous system-specific manner. We propose that βCstF-64 contributes to proteomic diversity by regulating alternative polyadenylation of neural mRNAs. PMID:19284619

Comparison of Two Methods for Detecting Alternative Splice Variants Using GeneChip® Exon Arrays

PubMed Central

Fan, Wenhong; Stirewalt, Derek L.; Radich, Jerald P.; Zhao, Lueping

2011-01-01

The Affymetrix GeneChip Exon Array can be used to detect alternative splice variants. Microarray Detection of Alternative Splicing (MIDAS) and Partek® Genomics Suite (Partek® GS) are among the most popular analytical methods used to analyze exon array data. While both methods utilize statistical significance for testing, MIDAS and Partek® GS could produce somewhat different results due to different underlying assumptions. Comparing MIDAS and Partek® GS is quite difficult due to their substantially different mathematical formulations and assumptions regarding alternative splice variants. For meaningful comparison, we have used the previously published generalized probe model (GPM) which encompasses both MIDAS and Partek® GS under different assumptions. We analyzed a colon cancer exon array data set using MIDAS, Partek® GS and GPM. MIDAS and Partek® GS produced quite different sets of genes that are considered to have alternative splice variants. Further, we found that GPM produced results similar to MIDAS as well as to Partek® GS under their respective assumptions. Within the GPM, we show how discoveries relating to alternative variants can be quite different due to different assumptions. MIDAS focuses on relative changes in expression values across different exons within genes and tends to be robust but less efficient. Partek® GS, however, uses absolute expression values of individual exons within genes and tends to be more efficient but more sensitive to the presence of outliers. From our observations, we conclude that MIDAS and Partek® GS produce complementary results, and discoveries from both analyses should be considered. PMID:23675234

The roles played by highly truncated splice variants of G protein-coupled receptors

PubMed Central

2012-01-01

Alternative splicing of G protein-coupled receptor (GPCR) genes greatly increases the total number of receptor isoforms which may be expressed in a cell-dependent and time-dependent manner. This increased diversity of cell signaling options caused by the generation of splice variants is further enhanced by receptor dimerization. When alternative splicing generates highly truncated GPCRs with less than seven transmembrane (TM) domains, the predominant effect in vitro is that of a dominant-negative mutation associated with the retention of the wild-type receptor in the endoplasmic reticulum (ER). For constitutively active (agonist-independent) GPCRs, their attenuated expression on the cell surface, and consequent decreased basal activity due to the dominant-negative effect of truncated splice variants, has pathological consequences. Truncated splice variants may conversely offer protection from disease when expression of co-receptors for binding of infectious agents to cells is attenuated due to ER retention of the wild-type co-receptor. In this review, we will see that GPCRs retained in the ER can still be functionally active but also that highly truncated GPCRs may also be functionally active. Although rare, some truncated splice variants still bind ligand and activate cell signaling responses. More importantly, by forming heterodimers with full-length GPCRs, some truncated splice variants also provide opportunities to generate receptor complexes with unique pharmacological properties. So, instead of assuming that highly truncated GPCRs are associated with faulty transcription processes, it is time to reassess their potential benefit to the host organism. PMID:22938630

Alcoholism and alternative splicing of candidate genes.

PubMed

Sasabe, Toshikazu; Ishiura, Shoichi

2010-04-01

Gene expression studies have shown that expression patterns of several genes have changed during the development of alcoholism. Gene expression is regulated not only at the level of transcription but also through alternative splicing of pre-mRNA. In this review, we discuss some of the evidence suggesting that alternative splicing of candidate genes such as DRD2 (encoding dopamine D2 receptor) may form the basis of the mechanisms underlying the pathophysiology of alcoholism. These reports suggest that aberrant expression of splice variants affects alcohol sensitivities, and alcohol consumption also regulates alternative splicing. Thus, investigations of alternative splicing are essential for understanding the molecular events underlying the development of alcoholism.

Identification of interleukin-26 in the dromedary camel (Camelus dromedarius): Evidence of alternative splicing and isolation of novel splice variants.

PubMed

Premraj, Avinash; Nautiyal, Binita; Aleyas, Abi G; Rasool, Thaha Jamal

2015-10-01

Interleukin-26 (IL-26) is a member of the IL-10 family of cytokines. Though conserved across vertebrates, the IL-26 gene is functionally inactivated in a few mammals like rat, mouse and horse. We report here the identification, isolation and cloning of the cDNA of IL-26 from the dromedary camel. The camel cDNA contains a 516 bp open reading frame encoding a 171 amino acid precursor protein, including a 21 amino acid signal peptide. Sequence analysis revealed high similarity with other mammalian IL-26 homologs and the conservation of IL-10 cytokine family domain structure including key amino acid residues. We also report the identification and cloning of four novel transcript variants produced by alternative splicing at the Exon 3-Exon 4 regions of the gene. Three of the alternative splice variants had premature termination codons and are predicted to code for truncated proteins. The transcript variant 4 (Tv4) having an insertion of an extra 120 bp nucleotides in the ORF was predicted to encode a full length protein product with 40 extra amino acid residues. The mRNA transcripts of all the variants were identified in lymph node, where as fewer variants were observed in other tissues like blood, liver and kidney. The expression of Tv2 and Tv3 were found to be up regulated in mitogen induced camel peripheral blood mononuclear cells. IL-26-Tv2 expression was also induced in camel fibroblast cells infected with Camel pox virus in-vitro. The identification of the transcript variants of IL-26 from the dromedary camel is the first report of alternative splicing for IL-26 in a species in which the gene has not been inactivated. Copyright © 2015 Elsevier Ltd. All rights reserved.

The fitness cost of mis-splicing is the main determinant of alternative splicing patterns.

PubMed

Saudemont, Baptiste; Popa, Alexandra; Parmley, Joanna L; Rocher, Vincent; Blugeon, Corinne; Necsulea, Anamaria; Meyer, Eric; Duret, Laurent

2017-10-30

Most eukaryotic genes are subject to alternative splicing (AS), which may contribute to the production of protein variants or to the regulation of gene expression via nonsense-mediated messenger RNA (mRNA) decay (NMD). However, a fraction of splice variants might correspond to spurious transcripts and the question of the relative proportion of splicing errors to functional splice variants remains highly debated. We propose a test to quantify the fraction of AS events corresponding to errors. This test is based on the fact that the fitness cost of splicing errors increases with the number of introns in a gene and with expression level. We analyzed the transcriptome of the intron-rich eukaryote Paramecium tetraurelia. We show that in both normal and in NMD-deficient cells, AS rates strongly decrease with increasing expression level and with increasing number of introns. This relationship is observed for AS events that are detectable by NMD as well as for those that are not, which invalidates the hypothesis of a link with the regulation of gene expression. Our results show that in genes with a median expression level, 92-98% of observed splice variants correspond to errors. We observed the same patterns in human transcriptomes and we further show that AS rates correlate with the fitness cost of splicing errors. These observations indicate that genes under weaker selective pressure accumulate more maladaptive substitutions and are more prone to splicing errors. Thus, to a large extent, patterns of gene expression variants simply reflect the balance between selection, mutation, and drift.

Alternative Splice Variants in TIM Barrel Proteins from Human Genome Correlate with the Structural and Evolutionary Modularity of this Versatile Protein Fold

PubMed Central

Ochoa-Leyva, Adrián; Montero-Morán, Gabriela; Saab-Rincón, Gloria; Brieba, Luis G.; Soberón, Xavier

2013-01-01

After the surprisingly low number of genes identified in the human genome, alternative splicing emerged as a major mechanism to generate protein diversity in higher eukaryotes. However, it is still not known if its prevalence along the genome evolution has contributed to the overall functional protein diversity or if it simply reflects splicing noise. The (βα)8 barrel or TIM barrel is one of the most frequent, versatile, and ancient fold encountered among enzymes. Here, we analyze the structural modifications present in TIM barrel proteins from the human genome product of alternative splicing events. We found that 87% of all splicing events involved deletions; most of these events resulted in protein fragments that corresponded to the (βα)2, (βα)4, (βα)5, (βα)6, and (βα)7 subdomains of TIM barrels. Because approximately 7% of all the splicing events involved internal β-strand substitutions, we decided, based on the genomic data, to design β-strand and α-helix substitutions in a well-studied TIM barrel enzyme. The biochemical characterization of one of the chimeric variants suggests that some of the splice variants in the human genome with β-strand substitutions may be evolving novel functions via either the oligomeric state or substrate specificity. We provide results of how the splice variants represent subdomains that correlate with the independently folding and evolving structural units previously reported. This work is the first to observe a link between the structural features of the barrel and a recurrent genetic mechanism. Our results suggest that it is reasonable to expect that a sizeable fraction of splice variants found in the human genome represent structurally viable functional proteins. Our data provide additional support for the hypothesis of the origin of the TIM barrel fold through the assembly of smaller subdomains. We suggest a model of how nature explores new proteins through alternative splicing as a mechanism to diversify the proteins encoded in the human genome. PMID:23950966

P52 Activation and Enzalutamide Therapy in Prostate Cancer

DTIC Science & Technology

2017-10-01

c-Myc:hnRNPA1 pathway regulates expression of androgen receptor splice variants and enzalutamide sensitivity in prostate cancer . Castration resistant... prostate cancer (CRPC) remains dependent on androgen receptor (AR) signaling. Alternative splicing of the AR to generate constitutively active... receptor splice variants and enzalutamide sensitivity in prostate cancer . • We discovered that quercetin, a naturally occurring polyphenolic compound

Heterozygous KIDINS220/ARMS nonsense variants cause spastic paraplegia, intellectual disability, nystagmus, and obesity.

PubMed

Josifova, Dragana J; Monroe, Glen R; Tessadori, Federico; de Graaff, Esther; van der Zwaag, Bert; Mehta, Sarju G; Harakalova, Magdalena; Duran, Karen J; Savelberg, Sanne M C; Nijman, Isaäc J; Jungbluth, Heinz; Hoogenraad, Casper C; Bakkers, Jeroen; Knoers, Nine V; Firth, Helen V; Beales, Philip L; van Haaften, Gijs; van Haelst, Mieke M

2016-06-01

We identified de novo nonsense variants in KIDINS220/ARMS in three unrelated patients with spastic paraplegia, intellectual disability, nystagmus, and obesity (SINO). KIDINS220 is an essential scaffold protein coordinating neurotrophin signal pathways in neurites and is spatially and temporally regulated in the brain. Molecular analysis of patients' variants confirmed expression and translation of truncated transcripts similar to recently characterized alternative terminal exon splice isoforms of KIDINS220 KIDINS220 undergoes extensive alternative splicing in specific neuronal populations and developmental time points, reflecting its complex role in neuronal maturation. In mice and humans, KIDINS220 is alternative spliced in the middle region as well as in the last exon. These full-length and KIDINS220 splice variants occur at precise moments in cortical, hippocampal, and motor neuron development, with splice variants similar to the variants seen in our patients and lacking the last exon of KIDINS220 occurring in adult rather than in embryonic brain. We conducted tissue-specific expression studies in zebrafish that resulted in spasms, confirming a functional link with disruption of the KIDINS220 levels in developing neurites. This work reveals a crucial physiological role of KIDINS220 in development and provides insight into how perturbation of the complex interplay of KIDINS220 isoforms and their relative expression can affect neuron control and human metabolism. Altogether, we here show that de novo protein-truncating KIDINS220 variants cause a new syndrome, SINO. This is the first report of KIDINS220 variants causing a human disease. © The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Alternative Splicing Governs Cone Cyclic Nucleotide-gated (CNG) Channel Sensitivity to Regulation by Phosphoinositides*

PubMed Central

Dai, Gucan; Sherpa, Tshering; Varnum, Michael D.

2014-01-01

Precursor mRNA encoding CNGA3 subunits of cone photoreceptor cyclic nucleotide-gated (CNG) channels undergoes alternative splicing, generating isoforms differing in the N-terminal cytoplasmic region of the protein. In humans, four variants arise from alternative splicing, but the functional significance of these changes has been a persistent mystery. Heterologous expression of the four possible CNGA3 isoforms alone or with CNGB3 subunits did not reveal significant differences in basic channel properties. However, inclusion of optional exon 3, with or without optional exon 5, produced heteromeric CNGA3 + CNGB3 channels exhibiting an ∼2-fold greater shift in K1/2,cGMP after phosphatidylinositol 4,5-biphosphate or phosphatidylinositol 3,4,5-trisphosphate application compared with channels lacking the sequence encoded by exon 3. We have previously identified two structural features within CNGA3 that support phosphoinositides (PIPn) regulation of cone CNG channels: N- and C-terminal regulatory modules. Specific mutations within these regions eliminated PIPn sensitivity of CNGA3 + CNGB3 channels. The exon 3 variant enhanced the component of PIPn regulation that depends on the C-terminal region rather than the nearby N-terminal region, consistent with an allosteric effect on PIPn sensitivity because of altered N-C coupling. Alternative splicing of CNGA3 occurs in multiple species, although the exact variants are not conserved across CNGA3 orthologs. Optional exon 3 appears to be unique to humans, even compared with other primates. In parallel, we found that a specific splice variant of canine CNGA3 removes a region of the protein that is necessary for high sensitivity to PIPn. CNGA3 alternative splicing may have evolved, in part, to tune the interactions between cone CNG channels and membrane-bound phosphoinositides. PMID:24675082

Alternative splicing governs cone cyclic nucleotide-gated (CNG) channel sensitivity to regulation by phosphoinositides.

PubMed

Dai, Gucan; Sherpa, Tshering; Varnum, Michael D

2014-05-09

Precursor mRNA encoding CNGA3 subunits of cone photoreceptor cyclic nucleotide-gated (CNG) channels undergoes alternative splicing, generating isoforms differing in the N-terminal cytoplasmic region of the protein. In humans, four variants arise from alternative splicing, but the functional significance of these changes has been a persistent mystery. Heterologous expression of the four possible CNGA3 isoforms alone or with CNGB3 subunits did not reveal significant differences in basic channel properties. However, inclusion of optional exon 3, with or without optional exon 5, produced heteromeric CNGA3 + CNGB3 channels exhibiting an ∼2-fold greater shift in K1/2,cGMP after phosphatidylinositol 4,5-biphosphate or phosphatidylinositol 3,4,5-trisphosphate application compared with channels lacking the sequence encoded by exon 3. We have previously identified two structural features within CNGA3 that support phosphoinositides (PIPn) regulation of cone CNG channels: N- and C-terminal regulatory modules. Specific mutations within these regions eliminated PIPn sensitivity of CNGA3 + CNGB3 channels. The exon 3 variant enhanced the component of PIPn regulation that depends on the C-terminal region rather than the nearby N-terminal region, consistent with an allosteric effect on PIPn sensitivity because of altered N-C coupling. Alternative splicing of CNGA3 occurs in multiple species, although the exact variants are not conserved across CNGA3 orthologs. Optional exon 3 appears to be unique to humans, even compared with other primates. In parallel, we found that a specific splice variant of canine CNGA3 removes a region of the protein that is necessary for high sensitivity to PIPn. CNGA3 alternative splicing may have evolved, in part, to tune the interactions between cone CNG channels and membrane-bound phosphoinositides.

Alternative splicing and the progesterone receptor in breast cancer

PubMed Central

Cork, David MW; Lennard, Thomas WJ; Tyson-Capper, Alison J

2008-01-01

Progesterone receptor status is a marker for hormone responsiveness and disease prognosis in breast cancer. Progesterone receptor negative tumours have generally been shown to have a poorer prognosis than progesterone receptor positive tumours. The observed loss of progesterone receptor could be through a range of mechanisms, including the generation of alternatively spliced progesterone receptor variants that are not detectable by current screening methods. Many progesterone receptor mRNA variants have been described with deletions of various whole, multiple or partial exons that encode differing protein functional domains. These variants may alter the progestin responsiveness of a tissue and contribute to the abnormal growth associated with breast cancer. Absence of specific functional domains from these spliced variants may also make them undetectable or indistinguishable from full length progesterone receptor by conventional antibodies. A comprehensive investigation into the expression profile and activity of progesterone receptor spliced variants in breast cancer is required to advance our understanding of tumour hormone receptor status. This, in turn, may aid the development of new biomarkers of disease prognosis and improve adjuvant treatment decisions. PMID:18557990

Functional Implications of Novel Human Acid Sphingomyelinase Splice Variants

PubMed Central

Rhein, Cosima; Tripal, Philipp; Seebahn, Angela; Konrad, Alice; Kramer, Marcel; Nagel, Christine; Kemper, Jonas; Bode, Jens; Mühle, Christiane; Gulbins, Erich; Reichel, Martin; Becker, Cord-Michael; Kornhuber, Johannes

2012-01-01

Background Acid sphingomyelinase (ASM) hydrolyses sphingomyelin and generates the lipid messenger ceramide, which mediates a variety of stress-related cellular processes. The pathological effects of dysregulated ASM activity are evident in several human diseases and indicate an important functional role for ASM regulation. We investigated alternative splicing as a possible mechanism for regulating cellular ASM activity. Methodology/Principal Findings We identified three novel ASM splice variants in human cells, termed ASM-5, -6 and -7, which lack portions of the catalytic- and/or carboxy-terminal domains in comparison to full-length ASM-1. Differential expression patterns in primary blood cells indicated that ASM splicing might be subject to regulatory processes. The newly identified ASM splice variants were catalytically inactive in biochemical in vitro assays, but they decreased the relative cellular ceramide content in overexpression studies and exerted a dominant-negative effect on ASM activity in physiological cell models. Conclusions/Significance These findings indicate that alternative splicing of ASM is of functional significance for the cellular stress response, possibly representing a mechanism for maintaining constant levels of cellular ASM enzyme activity. PMID:22558155

Validation of alternative transcript splicing in chicken lines that differ in genetic resistance to Marek’s disease

USDA-ARS?s Scientific Manuscript database

Utilizing RNA-seq data, 1,574 candidate genes with alternative splicing were previously identified between two chicken lines that differ in Marek’s disease (MD) genetic resistance under control and Marek’s disease virus infection conditions. After filtering out 1,530 genes with splice variants in th...

Profiling the array of Ca(v)3.1 variants from the human T-type calcium channel gene CACNA1G: alternative structures, developmental expression, and biophysical variations.

PubMed

Emerick, Mark C; Stein, Rebecca; Kunze, Robin; McNulty, Megan M; Regan, Melissa R; Hanck, Dorothy A; Agnew, William S

2006-08-01

We describe the regulated transcriptome of CACNA1G, a human gene for T-type Ca(v)3.1 calcium channels that is subject to extensive alternative RNA splicing. Fifteen sites of transcript variation include 2 alternative 5'-UTR promoter sites, 2 alternative 3'-UTR polyadenylation sites, and 11 sites of alternative splicing within the open reading frame. A survey of 1580 fetal and adult human brain full-length complementary DNAs reveals a family of 30 distinct transcripts, including multiple functional forms that vary in expression with development. Statistical analyses of fetal and adult transcript populations reveal patterns of linkages among intramolecular splice site configurations that change dramatically with development. A shift from nearly independent, biased splicing in fetal transcripts to strongly concerted splicing in adult transcripts suggests progressive activation of multiple "programs" of splicing regulation that reorganize molecular structures in differentiating cells. Patch-clamp studies of nine selected variants help relate splicing regulation to permutations of the gating parameters most likely to modify T-channel physiology in expressing neurons. Gating behavior reflects combinatorial interactions between variable domains so that molecular phenotype depends on ensembles of coselected domains, consistent with the observed emergence of concerted splicing during development. We conclude that the structural gene and networks of splicing regulatory factors define an integrated system for the phenotypic variation of Ca(v)3.1 biophysics during nervous system development. Copyright 2006 Wiley-Liss, Inc.

Subgroup Specific Alternative Splicing in Medulloblastoma

PubMed Central

Kloosterhof, Nanne K; Northcott, Paul A; Yu, Emily PY; Shih, David; Peacock, John; Grajkowska, Wieslawa; van Meter, Timothy; Eberhart, Charles G; Pfister, Stefan; Marra, Marco A; Weiss, William A; Scherer, Stephen W; Rutka, James T; French, Pim J; Taylor, Michael D

2014-01-01

Medulloblastoma is comprised of four distinct molecular variants: WNT, SHH, Group 3, and Group 4. We analyzed alternative splicing usage in 14 normal cerebellar samples and 103 medulloblastomas of known subgroup. Medulloblastoma samples have a statistically significant increase in alternative splicing as compared to normal fetal cerebella (2.3-times; P<6.47E-8). Splicing patterns are distinct and specific between molecular subgroups. Unsupervised hierarchical clustering of alternative splicing events accurately assigns medulloblastomas to their correct subgroup. Subgroup-specific splicing and alternative promoter usage was most prevalent in Group 3 (19.4%) and SHH (16.2%) medulloblastomas, while observed less frequently in WNT (3.2%), and Group 4 (9.3%) tumors. Functional annotation of alternatively spliced genes reveals over-representation of genes important for neuronal development. Alternative splicing events in medulloblastoma may be regulated in part by the correlative expression of antisense transcripts, suggesting a possible mechanism affecting subgroup specific alternative splicing. Our results identify additional candidate markers for medulloblastoma subgroup affiliation, further support the existence of distinct subgroups of the disease, and demonstrate an additional level of transcriptional heterogeneity between medulloblastoma subgroups. PMID:22358458

Alternative Splicing of NOX4 in the Failing Human Heart

PubMed Central

Varga, Zoltán V.; Pipicz, Márton; Baán, Júlia A.; Baranyai, Tamás; Koncsos, Gábor; Leszek, Przemyslaw; Kuśmierczyk, Mariusz; Sánchez-Cabo, Fátima; García-Pavía, Pablo; Brenner, Gábor J.; Giricz, Zoltán; Csont, Tamás; Mendler, Luca; Lara-Pezzi, Enrique; Pacher, Pál; Ferdinandy, Péter

2017-01-01

Increased oxidative stress is a major contributor to the development and progression of heart failure, however, our knowledge on the role of the distinct NADPH oxidase (NOX) isoenzymes, especially on NOX4 is controversial. Therefore, we aimed to characterize NOX4 expression in human samples from healthy and failing hearts. Explanted human heart samples (left and right ventricular, and septal regions) were obtained from patients suffering from heart failure of ischemic or dilated origin. Control samples were obtained from donor hearts that were not used for transplantation. Deep RNA sequencing of the cardiac transcriptome indicated extensive alternative splicing of the NOX4 gene in heart failure as compared to samples from healthy donor hearts. Long distance PCR analysis with a universal 5′-3′ end primer pair, allowing amplification of different splice variants, confirmed the presence of the splice variants. To assess translation of the alternatively spliced transcripts we determined protein expression of NOX4 by using a specific antibody recognizing a conserved region in all variants. Western blot analysis showed up-regulation of the full-length NOX4 in ischemic cardiomyopathy samples and confirmed presence of shorter isoforms both in control and failing samples with disease-associated expression pattern. We describe here for the first time that NOX4 undergoes extensive alternative splicing in human hearts which gives rise to the expression of different enzyme isoforms. The full length NOX4 is significantly upregulated in ischemic cardiomyopathy suggesting a role for NOX4 in ROS production during heart failure. PMID:29204124

New CD20 alternative splice variants: molecular identification and differential expression within hematological B cell malignancies.

PubMed

Gamonet, Clémentine; Bole-Richard, Elodie; Delherme, Aurélia; Aubin, François; Toussirot, Eric; Garnache-Ottou, Francine; Godet, Yann; Ysebaert, Loïc; Tournilhac, Olivier; Caroline, Dartigeas; Larosa, Fabrice; Deconinck, Eric; Saas, Philippe; Borg, Christophe; Deschamps, Marina; Ferrand, Christophe

2015-01-01

CD20 is a B cell lineage-specific marker expressed by normal and leukemic B cells and targeted by several antibody immunotherapies. We have previously shown that the protein from a CD20 mRNA splice variant (D393-CD20) is expressed at various levels in leukemic B cells or lymphoma B cells but not in resting, sorted B cells from the peripheral blood of healthy donors. Western blot (WB) analysis of B malignancy primary samples showed additional CD20 signals. Deep molecular PCR analysis revealed four new sequences corresponding to in-frame CD20 splice variants (D657-CD20, D618-CD20, D480-CD20, and D177-CD20) matching the length of WB signals. We demonstrated that the cell spliceosome machinery can process ex vivo D480-, D657-, and D618-CD20 transcript variants by involving canonical sites associated with cryptic splice sites. Results of specific and quantitative RT-PCR assays showed that these CD20 splice variants are differentially expressed in B malignancies. Moreover, Epstein-Barr virus (EBV) transformation modified the CD20 splicing profile and mainly increased the D393-CD20 variant transcripts. Finally, investigation of three cohorts of chronic lymphocytic leukemia (CLL) patients showed that the total CD20 splice variant expression was higher in a stage B and C sample collection compared to routinely collected CLL samples or relapsed refractory stage A, B, or C CLL. The involvement of these newly discovered alternative CD20 transcript variants in EBV transformation makes them interesting molecular indicators, as does their association with oncogenesis rather than non-oncogenic B cell diseases, differential expression in B cell malignancies, and correlation with CLL stage and some predictive CLL markers. This potential should be investigated in further studies.

Quantitative imaging of single mRNA splice variants in living cells

NASA Astrophysics Data System (ADS)

Lee, Kyuwan; Cui, Yi; Lee, Luke P.; Irudayaraj, Joseph

2014-06-01

Alternative messenger RNA (mRNA) splicing is a fundamental process of gene regulation, and errors in RNA splicing are known to be associated with a variety of different diseases. However, there is currently a lack of quantitative technologies for monitoring mRNA splice variants in cells. Here, we show that a combination of plasmonic dimer probes and hyperspectral imaging can be used to detect and quantify mRNA splice variants in living cells. The probes are made from gold nanoparticles functionalized with oligonucleotides and can hybridize to specific mRNA sequences, forming nanoparticle dimers that exhibit distinct spectral shifts due to plasmonic coupling. With this approach, we show that the spatial and temporal distribution of three selected splice variants of the breast cancer susceptibility gene, BRCA1, can be monitored at single-copy resolution by measuring the hybridization dynamics of the nanoplasmonic dimers. Our study provides insights into RNA and its transport in living cells, which could improve our understanding of cellular protein complexes, pharmacogenomics, genetic diagnosis and gene therapies.

Characterization of Conserved Tandem Donor Sites and Intronic Motifs Required for Alternative Splicing in Corticosteroid Receptor Genes

PubMed Central

Qian, Xiaoxiao; Matthews, Laura; Lightman, Stafford; Ray, David; Norman, Michael

2015-01-01

Alternative splicing events from tandem donor sites result in mRNA variants coding for additional amino acids in the DNA binding domain of both the glucocorticoid (GR) and mineralocorticoid (MR) receptors. We now show that expression of both splice variants is extensively conserved in mammalian species, providing strong evidence for their functional significance. An exception to the conservation of the MR tandem splice site (an A at position +5 of the MR+12 donor site in the mouse) was predicted to decrease U1 small nuclear RNA binding. In accord with this prediction, we were unable to detect the MR+12 variant in this species. The one exception to the conservation of the GR tandem splice site, an A at position +3 of the platypus GRγ donor site that was predicted to enhance binding of U1 snRNA, was unexpectedly associated with decreased expression of the variant from the endogenous gene as well as a minigene. An intronic pyrimidine motif present in both GR and MR genes was found to be critical for usage of the downstream donor site, and overexpression of TIA1/TIAL1 RNA binding proteins, which are known to bind such motifs, led to a marked increase in the proportion of GRγ and MR+12. These results provide striking evidence for conservation of a complex splicing mechanism that involves processes other than stochastic spliceosome binding and identify a mechanism that would allow regulation of variant expression. PMID:19819975

GETPrime: a gene- or transcript-specific primer database for quantitative real-time PCR.

PubMed

Gubelmann, Carine; Gattiker, Alexandre; Massouras, Andreas; Hens, Korneel; David, Fabrice; Decouttere, Frederik; Rougemont, Jacques; Deplancke, Bart

2011-01-01

The vast majority of genes in humans and other organisms undergo alternative splicing, yet the biological function of splice variants is still very poorly understood in large part because of the lack of simple tools that can map the expression profiles and patterns of these variants with high sensitivity. High-throughput quantitative real-time polymerase chain reaction (qPCR) is an ideal technique to accurately quantify nucleic acid sequences including splice variants. However, currently available primer design programs do not distinguish between splice variants and also differ substantially in overall quality, functionality or throughput mode. Here, we present GETPrime, a primer database supported by a novel platform that uniquely combines and automates several features critical for optimal qPCR primer design. These include the consideration of all gene splice variants to enable either gene-specific (covering the majority of splice variants) or transcript-specific (covering one splice variant) expression profiling, primer specificity validation, automated best primer pair selection according to strict criteria and graphical visualization of the latter primer pairs within their genomic context. GETPrime primers have been extensively validated experimentally, demonstrating high transcript specificity in complex samples. Thus, the free-access, user-friendly GETPrime database allows fast primer retrieval and visualization for genes or groups of genes of most common model organisms, and is available at http://updepla1srv1.epfl.ch/getprime/. Database URL: http://deplanckelab.epfl.ch.

GETPrime: a gene- or transcript-specific primer database for quantitative real-time PCR

PubMed Central

Gubelmann, Carine; Gattiker, Alexandre; Massouras, Andreas; Hens, Korneel; David, Fabrice; Decouttere, Frederik; Rougemont, Jacques; Deplancke, Bart

2011-01-01

The vast majority of genes in humans and other organisms undergo alternative splicing, yet the biological function of splice variants is still very poorly understood in large part because of the lack of simple tools that can map the expression profiles and patterns of these variants with high sensitivity. High-throughput quantitative real-time polymerase chain reaction (qPCR) is an ideal technique to accurately quantify nucleic acid sequences including splice variants. However, currently available primer design programs do not distinguish between splice variants and also differ substantially in overall quality, functionality or throughput mode. Here, we present GETPrime, a primer database supported by a novel platform that uniquely combines and automates several features critical for optimal qPCR primer design. These include the consideration of all gene splice variants to enable either gene-specific (covering the majority of splice variants) or transcript-specific (covering one splice variant) expression profiling, primer specificity validation, automated best primer pair selection according to strict criteria and graphical visualization of the latter primer pairs within their genomic context. GETPrime primers have been extensively validated experimentally, demonstrating high transcript specificity in complex samples. Thus, the free-access, user-friendly GETPrime database allows fast primer retrieval and visualization for genes or groups of genes of most common model organisms, and is available at http://updepla1srv1.epfl.ch/getprime/. Database URL: http://deplanckelab.epfl.ch. PMID:21917859

Expression of Kir7.1 and a Novel Kir7.1 Splice Variant in Native Human Retinal Pigment Epithelium

PubMed Central

Yang, Dongli; Swaminathan, Anuradha; Zhang, Xiaoming; Hughes, Bret A.

2009-01-01

Previous studies on bovine retinal pigment epithelium (RPE) established that Kir7.1 channels compose this epithelium’s large apical membrane K+ conductance. The purpose of this study was to determine whether Kir7.1 and potential Kir7.1 splice variants are expressed in native adult human RPE and, if so, to determine their function and how they are generated. RT-PCR analysis indicated that human RPE expresses full-length Kir7.1 and a novel Kir7.1 splice variant, designated Kir7.1S. Analysis of the human Kir7.1 gene (KCNJ13) organization revealed that it contains 3 exons, 2 introns, and a novel alternative 5′ splice site in exon 2. In human RPE, the alternative usage of two competing 5′ splice sites in exon 2 gives rise to transcripts encoding full-length Kir7.1 and Kir7.1S, which is predicted to encode a truncated protein. Real-time PCR indicated that Kir7.1 transcript is nearly as abundant as GAPDH mRNA in human RPE whereas Kir7.1S transcript expression is 4-fold lower. Western blot analysis showed that the splice variant is translated in Xenopus oocytes injected with Kir7.1S cRNA and revealed the expression of full-length Kir7.1 but not Kir7.1S in adult human RPE. Co-expression of Kir7.1 with Kir7.1S in Xenopus oocytes had no effect on either the kinetics or amplitude of Kir7.1 currents. This study confirms the expression of Kir7.1 in human RPE, identifies a Kir7.1 splice variant resulting in predicted changes in protein sequence, and indicates that there no functional interaction between this splice variant and full-length Kir7.1. PMID:18035352

Alternatively spliced Spalax heparanase inhibits extracellular matrix degradation, tumor growth, and metastasis

PubMed Central

Nasser, Nicola J.; Avivi, Aaron; Shafat, Itay; Edovitsky, Evgeny; Zcharia, Eyal; Ilan, Neta; Vlodavsky, Israel; Nevo, Eviatar

2009-01-01

Heparanase is an endoglycosidase that degrades heparan sulfate (HS) at the cell surface and in the extracellular matrix. Heparanase is expressed mainly by cancer cells, and its expression is correlated with increased tumor aggressiveness, metastasis, and angiogenesis. Here, we report the cloning of a unique splice variant (splice 36) of heparanase from the subterranean blind mole rat (Spalax). This splice variant results from skipping part of exon 3, exons 4 and 5, and part of exon 6 and functions as a dominant negative to the wild-type enzyme. It inhibits HS degradation, suppresses glioma tumor growth, and decreases experimental B16–BL6 lung colonization in a mouse model. Intriguingly, Spalax splice variant 7 of heparanase (which results from skipping of exon 7) is devoid of enzymatic activity, but unlike splice 36 it enhances tumor growth. Our results demonstrate that alternative splicing of heparanase regulates its enzymatic activity and might adapt the heparanase function to the fluctuating normoxic–hypoxic subterranean environment that Spalax experiences. Development of anticancer drugs designed to suppress tumor growth, angiogenesis, and metastasis is a major challenge, of which heparanase inhibition is a promising approach. We anticipate that the heparanase splicing model, evolved during 40 million years of Spalacid adaptation to underground life, would pave the way for the development of heparanase-based therapeutic modalities directed against angiogenesis, tumor growth, and metastasis. PMID:19164514

Tissue-specific alternative splicing of TCF7L2

PubMed Central

Prokunina-Olsson, Ludmila; Welch, Cullan; Hansson, Ola; Adhikari, Neeta; Scott, Laura J.; Usher, Nicolle; Tong, Maurine; Sprau, Andrew; Swift, Amy; Bonnycastle, Lori L.; Erdos, Michael R.; He, Zhi; Saxena, Richa; Harmon, Brennan; Kotova, Olga; Hoffman, Eric P.; Altshuler, David; Groop, Leif; Boehnke, Michael; Collins, Francis S.; Hall, Jennifer L.

2009-01-01

Common variants in the transcription factor 7-like 2 (TCF7L2) gene have been identified as the strongest genetic risk factors for type 2 diabetes (T2D). However, the mechanisms by which these non-coding variants increase risk for T2D are not well-established. We used 13 expression assays to survey mRNA expression of multiple TCF7L2 splicing forms in up to 380 samples from eight types of human tissue (pancreas, pancreatic islets, colon, liver, monocytes, skeletal muscle, subcutaneous adipose tissue and lymphoblastoid cell lines) and observed a tissue-specific pattern of alternative splicing. We tested whether the expression of TCF7L2 splicing forms was associated with single nucleotide polymorphisms (SNPs), rs7903146 and rs12255372, located within introns 3 and 4 of the gene and most strongly associated with T2D. Expression of two splicing forms was lower in pancreatic islets with increasing counts of T2D-associated alleles of the SNPs: a ubiquitous splicing form (P = 0.018 for rs7903146 and P = 0.020 for rs12255372) and a splicing form found in pancreatic islets, pancreas and colon but not in other tissues tested here (P = 0.009 for rs12255372 and P = 0.053 for rs7903146). Expression of this form in glucose-stimulated pancreatic islets correlated with expression of proinsulin (r2 = 0.84–0.90, P < 0.00063). In summary, we identified a tissue-specific pattern of alternative splicing of TCF7L2. After adjustment for multiple tests, no association between expression of TCF7L2 in eight types of human tissue samples and T2D-associated genetic variants remained significant. Alternative splicing of TCF7L2 in pancreatic islets warrants future studies. GenBank Accession Numbers: FJ010164–FJ010174. PMID:19602480

Functions of Ion Transport Peptide and Ion Transport Peptide-Like in the Red Flour Beetle Tribolium castaneum

USDA-ARS?s Scientific Manuscript database

Ion transport peptide (ITP) and ITP-like (ITPL) are highly conserved neuropeptides in insects and crustaceans. We investigated the alternatively spliced variants of ITP/ITPL in Tribolium castaneum to understand their functions. We identified three alternatively spliced transcripts named itp, itpl-...

Identification and characterization of yak (Bos grunniens) b-Boule gene and its alternative splice variants.

PubMed

Li, Bojiang; Ngo, Sherry; Wu, Wangjun; Xu, Hongtao; Xie, Zhuang; Li, Qifa; Pan, Zengxiang

2014-10-25

Boule is responsible for meiotic arrest of sperms and male sterility during mammalian spermatogenesis. In the present study, we first identified yak b-Boule gene and its two alternative splice variants. The full length coding region of yak b-Boule is 888bp and encodes a 295-amino acid protein with a typical RNA-recognition motif (RRM) and a Deleted in Azoospermia (DAZ) repetitive sequence motif. Two alternative splice variants of yak b-Boule were generated following the consensus "GT-AG" rule and named b-Boule1 (36bp deletion in exon 3) and b-Boule2 (deletion of integral exon 7), respectively. In male yak, b-Boule, b-Boule1 and b-Boule2 were found to be exclusively expressed in the testes at a ratio of 81:0.1:1. Intriguingly, the mRNA expression levels of b-Boule and b-Boule1 in yak testis were significantly higher than those in cattle-yak, although no significant difference was observed for b-Boule2 expression between the yak and cattle-yak. These results suggest that b-Boule gene, which is partially regulated by alternative splicing, may be involved in the process of yak spermatogenesis. Copyright © 2014 Elsevier B.V. All rights reserved.

Effects of airborne particulate matter on alternative pre-mRNA splicing in colon cancer cells

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

Buggiano, Valeria; Petrillo, Ezequiel; Alló, Mariano

2015-07-15

Alternative pre-mRNA splicing plays key roles in determining tissue- and species-specific cell differentiation as well as in the onset of hereditary disease and cancer, being controlled by multiple post- and co-transcriptional regulatory mechanisms. We report here that airborne particulate matter, resulting from industrial pollution, inhibits expression and specifically affects alternative splicing at the 5′ untranslated region of the mRNA encoding the bone morphogenetic protein BMP4 in human colon cells in culture. These effects are consistent with a previously reported role for BMP4 in preventing colon cancer development, suggesting that ingestion of particulate matter could contribute to the onset of colonmore » cell proliferation. We also show that the underlying mechanism might involve changes in transcriptional elongation. This is the first study to demonstrate that particulate matter causes non-pleiotropic changes in alternative splicing. - Highlights: • Airborne particulate matter (PM10) affects alternative splicing in colon cells. • PM10 upregulates one of the two mRNA variants of the growth factor BMP-4. • This variant has a longer 5′ unstranslated region and introduces an upstream AUG. • By regulating BMP-4 mRNA splicing PM10 inhibits total expression of BMP-4 protein. • BMP-4 downregulation was previously reported to be associated to colon cancer.« less

The emerging role of alternative splicing in senescence and aging.

PubMed

Deschênes, Mathieu; Chabot, Benoit

2017-10-01

Deregulation of precursor mRNA splicing is associated with many illnesses and has been linked to age-related chronic diseases. Here we review recent progress documenting how defects in the machinery that performs intron removal and controls splice site selection contribute to cellular senescence and organismal aging. We discuss the functional association linking p53, IGF-1, SIRT1, and ING-1 splice variants with senescence and aging, and review a selection of splicing defects occurring in accelerated aging (progeria), vascular aging, and Alzheimer's disease. Overall, it is becoming increasingly clear that changes in the activity of splicing factors and in the production of key splice variants can impact cellular senescence and the aging phenotype. © 2017 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

SpliceSeq: a resource for analysis and visualization of RNA-Seq data on alternative splicing and its functional impacts.

PubMed

Ryan, Michael C; Cleland, James; Kim, RyangGuk; Wong, Wing Chung; Weinstein, John N

2012-09-15

SpliceSeq is a resource for RNA-Seq data that provides a clear view of alternative splicing and identifies potential functional changes that result from splice variation. It displays intuitive visualizations and prioritized lists of results that highlight splicing events and their biological consequences. SpliceSeq unambiguously aligns reads to gene splice graphs, facilitating accurate analysis of large, complex transcript variants that cannot be adequately represented in other formats. SpliceSeq is freely available at http://bioinformatics.mdanderson.org/main/SpliceSeq:Overview. The application is a Java program that can be launched via a browser or installed locally. Local installation requires MySQL and Bowtie. mryan@insilico.us.com Supplementary data are available at Bioinformatics online.

Differential upregulation in DRG neurons of an α2δ-1 splice variant with a lower affinity for gabapentin after peripheral sensory nerve injury

PubMed Central

Lana, Beatrice; Schlick, Bettina; Martin, Stuart; Pratt, Wendy S.; Page, Karen M.; Goncalves, Leonor; Rahman, Wahida; Dickenson, Anthony H.; Bauer, Claudia S.; Dolphin, Annette C.

2014-01-01

The α2δ-1 protein is an auxiliary subunit of voltage-gated calcium channels, critical for neurotransmitter release. It is upregulated in dorsal root ganglion (DRG) neurons following sensory nerve injury, and is also the therapeutic target of the gabapentinoid drugs, which are efficacious in both experimental and human neuropathic pain conditions. α2δ-1 has 3 spliced regions: A, B, and C. A and C are cassette exons, whereas B is introduced via an alternative 3′ splice acceptor site. Here we have examined the presence of α2δ-1 splice variants in DRG neurons, and have found that although the main α2δ-1 splice variant in DRG is the same as that in brain (α2δ-1 ΔA+B+C), there is also another α2δ-1 splice variant (ΔA+BΔC), which is expressed in DRG neurons and is differentially upregulated compared to the main DRG splice variant α2δ-1 ΔA+B+C following spinal nerve ligation. Furthermore, this differential upregulation occurs preferentially in a small nonmyelinated DRG neuron fraction, obtained by density gradient separation. The α2δ-1 ΔA+BΔC splice variant supports CaV2 calcium currents with unaltered properties compared to α2δ-1 ΔA+B+C, but shows a significantly reduced affinity for gabapentin. This variant could therefore play a role in determining the efficacy of gabapentin in neuropathic pain. PMID:24315988

A novel TFF2 splice variant (ΔEX2TFF2) correlates with longer overall survival time in cholangiocarcinoma

PubMed Central

KAMLUA, SURASEE; PATRAKITKOMJORN, SIRIPORN; JEARANAIKOON, PATCHAREE; MENHENIOTT, TREVELYAN R.; GIRAUD, ANDREW S.; LIMPAIBOON, TEMDUANG

2012-01-01

Trefoil factor 2 (TFF2) is a member of trefoil factor family found to be overexpressed in many cancers including cholangiocarcinoma (CCA). The majority of studies have focused on wild-type TFF2 (wtTFF2) expression, but information regarding alternative splicing variants of TFF2 mRNA has not been reported. In this study, we aimed to identify and quantify a novel TFF2 splice variant in cholangiocarcinoma (CCA). Seventy-eight tumors and 15 normal adjacent tissues were quantified for the expression of the TFF2 splice variant relative to wild-type (wt) TFF2 mRNA using quantitative reverse transcriptase polymerase chain reaction (QRT-PCR). The ratio of TFF2 splice variant against wtTFF2 was analyzed for associations with clinical parameters. We found a novel TFF2 splice variant, exon 2 skipping (ΔEX2TFF2), resulting in a stop codon (TAG) at exon 1. The ΔEX2TFF2/wtTFF2 ratio in tumors was significantly higher than in normal tissue (P<0.01). Interestingly, high ΔEX2TFF2/wtTFF2 ratio was significantly associated with good prognosis compared with low ratio (P=0.017). In contrast, the presence of wtTFF2 protein was associated with poor survival of CCA patients (P=0.034). This is the first report of a trefoil factor splice variant and its potential application as a prognostic biomarker in CCA. PMID:22159958

17β-estradiol regulates the RNA-binding protein Nova1, which then regulates the alternative splicing of estrogen receptor β in the aging female rat brain.

PubMed

Shults, Cody L; Dingwall, Caitlin B; Kim, Chun K; Pinceti, Elena; Rao, Yathindar S; Pak, Toni R

2018-01-01

Alternative RNA splicing results in the translation of diverse protein products arising from a common nucleotide sequence. These alternative protein products are often functional and can have widely divergent actions from the canonical protein. Studies in humans and other vertebrate animals have demonstrated that alternative splicing events increase with advanced age, sometimes resulting in pathological consequences. Menopause represents a critical transition for women, where the beneficial effects of estrogens are no longer evident; therefore, factors underlying increased pathological conditions in women are confounded by the dual factors of aging and declining estrogens. Estrogen receptors (ERs) are subject to alternative splicing, the spliced variants increase following menopause, and they fail to efficiently activate estrogen-dependent signaling pathways. However, the factors that regulate the alternative splicing of ERs remain unknown. We demonstrate novel evidence supporting a potential biological feedback loop where 17β-estradiol regulates the RNA-binding protein Nova1, which, in turn, regulates the alternative splicing of ERβ. These data increase our understanding of ER alternative splicing and could have potential implications for women taking hormone replacement therapy after menopause. Copyright © 2017 Elsevier Inc. All rights reserved.

Alternative Splicing in Breast Cancer and the Potential Development of Therapeutic Tools.

PubMed

Martínez-Montiel, Nancy; Anaya-Ruiz, Maricruz; Pérez-Santos, Martín; Martínez-Contreras, Rebeca D

2017-10-05

Alternative splicing is a key molecular mechanism now considered as a hallmark of cancer that has been associated with the expression of distinct isoforms during the onset and progression of the disease. The leading cause of cancer-related deaths in women worldwide is breast cancer, and even when the role of alternative splicing in this type of cancer has been established, the function of this mechanism in breast cancer biology is not completely decoded. In order to gain a comprehensive view of the role of alternative splicing in breast cancer biology and development, we summarize here recent findings regarding alternative splicing events that have been well documented for breast cancer evolution, considering its prognostic and therapeutic value. Moreover, we analyze how the response to endocrine and chemical therapies could be affected due to alternative splicing and differential expression of variant isoforms. With all this knowledge, it becomes clear that targeting alternative splicing represents an innovative approach for breast cancer therapeutics and the information derived from current studies could guide clinical decisions with a direct impact in the clinical advances for breast cancer patients nowadays.

Bidirectional regulation of neurite elaboration by alternatively spliced metabotropic glutamate receptor 5 (mGluR5) isoforms.

PubMed

Mion, S; Corti, C; Neki, A; Shigemoto, R; Corsi, M; Fumagalli, G; Ferraguti, F

2001-06-01

Alternative splicing in the mGluR5 gene generates two different receptor isoforms, of which expression is developmentally regulated. However, little is known about the functional significance of mGluR5 splice variants. We have examined the functional coupling, subcellular targeting, and effect on neuronal differentiation of epitope-tagged mGluR5 isoforms by expression in neuroblastoma NG108-15 cells. We found that both mGluR5 splice variants give rise to comparable [Ca2+]i transients and have similar pharmacological profile. Tagged receptors were shown by immunofluorescence to be inserted in the plasma membrane. In undifferentiated cells the subcellular localization of the two mGluR5 isoforms was partially segregated, whereas in differentiated cells the labeling largely redistributed to the newly formed neurites. Interestingly, we demonstrate that mGluR5 splice variants dramatically influence the formation and maturation of neurites; mGluR5a hinders the acquisition of mature neuronal traits and mGluR5b fosters the elaboration and extension of neurites. These effects are partly inhibited by MPEP. Copyright 2001 Academic Press.

Reconciling newborn screening and a novel splice variant in BTD associated with partial biotinidase deficiency: A BabySeq Project case report.

PubMed

Murry, Jaclyn B; Machini, Kalotina; Ceyhan-Birsoy, Ozge; Kritzer, Amy; Krier, Joel B; Lebo, Matthew S; Fayer, Shawn; Genetti, Casie A; Vannoy, Grace E; Yu, Timothy W; Agrawal, Pankaj B; Parad, Richard B; Holm, Ingrid A; McGuire, Amy L; Green, Robert C; Beggs, Alan H; Rehm, Heidi L; Project, The BabySeq

2018-05-04

Here, we report a newborn female infant from the well-baby cohort of the BabySeq Project who was identified with compound heterozygous BTD gene variants. The two identified variants included a well-established pathogenic variant (c.1612C>T, p.Arg538Cys) that causes profound biotinidase deficiency (BTD) in homozygosity. In addition, a novel splice variant (c.44+1G>A, p.?) was identified in the invariant splice donor region of intron 1, potentially predictive of loss of function. The novel variant was predicted to impact splicing of exon 1; however, given the absence of any reported pathogenic variants in exon 1 and the presence of alternative splicing with exon 1 absent in most tissues in the GTEx database, we assigned an initial classification of uncertain significance. Follow-up medical record review of state mandated newborn screen (NBS) results revealed an initial out-of-range biotinidase activity level. Levels from a repeat NBS sample barely passed cut-off into the normal range. To determine whether the infant was biotinidase deficient, subsequent diagnostic enzyme activity testing was performed, confirming partial BTD, and resulted in a change of management for this patient. This led to reclassification of the novel splice variant based on these results. In conclusion, combining the genetic and NBS results together prompted clinical follow-up that confirmed partial biotinidase deficiency, and informed this novel splice site's reclassification emphasizing the importance of combining iterative genetic and phenotypic evaluations. Cold Spring Harbor Laboratory Press.

Designing oligo libraries taking alternative splicing into account

NASA Astrophysics Data System (ADS)

Shoshan, Avi; Grebinskiy, Vladimir; Magen, Avner; Scolnicov, Ariel; Fink, Eyal; Lehavi, David; Wasserman, Alon

2001-06-01

We have designed sequences for DNA microarrays and oligo libraries, taking alternative splicing into account. Alternative splicing is a common phenomenon, occurring in more than 25% of the human genes. In many cases, different splice variants have different functions, are expressed in different tissues or may indicate different stages of disease. When designing sequences for DNA microarrays or oligo libraries, it is very important to take into account the sequence information of all the mRNA transcripts. Therefore, when a gene has more than one transcript (as a result of alternative splicing, alternative promoter sites or alternative poly-adenylation sites), it is very important to take all of them into account in the design. We have used the LEADS transcriptome prediction system to cluster and assemble the human sequences in GenBank and design optimal oligonucleotides for all the human genes with a known mRNA sequence based on the LEADS predictions.

TSVdb: a web-tool for TCGA splicing variants analysis.

PubMed

Sun, Wenjie; Duan, Ting; Ye, Panmeng; Chen, Kelie; Zhang, Guanling; Lai, Maode; Zhang, Honghe

2018-05-29

Collaborative projects such as The Cancer Genome Atlas (TCGA) have generated various -omics and clinical data on cancer. Many computational tools have been developed to facilitate the study of the molecular characterization of tumors using data from the TCGA. Alternative splicing of a gene produces splicing variants, and accumulating evidence has revealed its essential role in cancer-related processes, implying the urgent need to discover tumor-specific isoforms and uncover their potential functions in tumorigenesis. We developed TSVdb, a web-based tool, to explore alternative splicing based on TCGA samples with 30 clinical variables from 33 tumors. TSVdb has an integrated and well-proportioned interface for visualization of the clinical data, gene expression, usage of exons/junctions and splicing patterns. Researchers can interpret the isoform expression variations between or across clinical subgroups and estimate the relationships between isoforms and patient prognosis. TSVdb is available at http://www.tsvdb.com , and the source code is available at https://github.com/wenjie1991/TSVdb . TSVdb will inspire oncologists and accelerate isoform-level advances in cancer research.

SpliceSeq: a resource for analysis and visualization of RNA-Seq data on alternative splicing and its functional impacts

PubMed Central

Ryan, Michael C.; Cleland, James; Kim, RyangGuk; Wong, Wing Chung; Weinstein, John N.

2012-01-01

Summary: SpliceSeq is a resource for RNA-Seq data that provides a clear view of alternative splicing and identifies potential functional changes that result from splice variation. It displays intuitive visualizations and prioritized lists of results that highlight splicing events and their biological consequences. SpliceSeq unambiguously aligns reads to gene splice graphs, facilitating accurate analysis of large, complex transcript variants that cannot be adequately represented in other formats. Availability and implementation: SpliceSeq is freely available at http://bioinformatics.mdanderson.org/main/SpliceSeq:Overview. The application is a Java program that can be launched via a browser or installed locally. Local installation requires MySQL and Bowtie. Contact: mryan@insilico.us.com Supplementary Information: Supplementary data are available at Bioinformatics online. PMID:22820202

Differential upregulation in DRG neurons of an α2δ-1 splice variant with a lower affinity for gabapentin after peripheral sensory nerve injury.

PubMed

Lana, Beatrice; Schlick, Bettina; Martin, Stuart; Pratt, Wendy S; Page, Karen M; Goncalves, Leonor; Rahman, Wahida; Dickenson, Anthony H; Bauer, Claudia S; Dolphin, Annette C

2014-03-01

The α2δ-1 protein is an auxiliary subunit of voltage-gated calcium channels, critical for neurotransmitter release. It is upregulated in dorsal root ganglion (DRG) neurons following sensory nerve injury, and is also the therapeutic target of the gabapentinoid drugs, which are efficacious in both experimental and human neuropathic pain conditions. α2δ-1 has 3 spliced regions: A, B, and C. A and C are cassette exons, whereas B is introduced via an alternative 3' splice acceptor site. Here we have examined the presence of α2δ-1 splice variants in DRG neurons, and have found that although the main α2δ-1 splice variant in DRG is the same as that in brain (α2δ-1 ΔA+B+C), there is also another α2δ-1 splice variant (ΔA+BΔC), which is expressed in DRG neurons and is differentially upregulated compared to the main DRG splice variant α2δ-1 ΔA+B+C following spinal nerve ligation. Furthermore, this differential upregulation occurs preferentially in a small nonmyelinated DRG neuron fraction, obtained by density gradient separation. The α2δ-1 ΔA+BΔC splice variant supports CaV2 calcium currents with unaltered properties compared to α2δ-1 ΔA+B+C, but shows a significantly reduced affinity for gabapentin. This variant could therefore play a role in determining the efficacy of gabapentin in neuropathic pain. Copyright © 2013 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.

Analysis of RNA-Seq datasets reveals enrichment of tissue-specific splice variants for nuclear envelope proteins.

PubMed

Capitanchik, Charlotte; Dixon, Charles; Swanson, Selene K; Florens, Laurence; Kerr, Alastair R W; Schirmer, Eric C

2018-06-18

Nuclear envelopathies/laminopathies yield tissue-specific pathologies, yet arise from mutation of ubiquitously-expressed genes. One possible explanation of this tissue specificity is that tissue-specific partners become disrupted from larger complexes, but a little investigated alternate hypothesis is that the mutated proteins themselves have tissue-specific splice variants. Here, we analyze RNA-Seq datasets to identify muscle-specific splice variants of nuclear envelope genes that could be relevant to the study of laminopathies, particularly muscular dystrophies, that are not currently annotated in sequence databases. Notably, we found novel isoforms or tissue-specificity of isoforms for: Lap2, linked to cardiomyopathy; Nesprin 2, linked to Emery-Dreifuss muscular dystrophy and Lmo7, a regulator of the emerin gene that is linked to Emery-Dreifuss muscular dystrophy. Interestingly, the muscle-specific exon in Lmo7 is rich in serine phosphorylation motifs, suggesting an important regulatory function. Evidence for muscle-specific splice variants in non-nuclear envelope proteins linked to other muscular dystrophies was also found. Tissue-specific variants were also indicated for several nucleoporins including Nup54, Nup133, Nup153 and Nup358/RanBP2. We confirmed expression of novel Lmo7 and RanBP2 variants with RT-PCR and found that specific knockdown of the Lmo7 variant caused a reduction in myogenic index during mouse C2C12 myogenesis. Global analysis revealed an enrichment of tissue-specific splice variants for nuclear envelope proteins in general compared to the rest of the genome, suggesting that splice variants contribute to regulating its tissue-specific functions.

Isoforms of receptors of fibroblast growth factors.

PubMed

Gong, Siew-Ging

2014-12-01

The breadth and scope of Fibroblast Growth Factor signaling is immense, with documentation of its role in almost every organism and system studied so far. FGF ligands signal through a family of four distinct tyrosine kinase receptors, the FGF receptors (FGFRs). One contribution to the diversity of function and signaling of FGFs and their receptors arises from the numerous alternative splicing variants that have been documented in the FGFR literature. The present review discusses the types and roles of alternatively spliced variants of the FGFR family members and the significant impact of alternative splicing on the physiological functions of five broad classes of FGFR isoforms. Some characterized known regulatory mechanisms of alternative splicing and future directions in studies of FGFR alternative splicing are also discussed. Presence, absence, and/or the combination of specific exons within each FGFR protein impart upon each individual isoform its unique function and expression pattern during normal function and in diseased states (e.g., in cancers and birth defects). A better understanding of the diversity of FGF signaling in different developmental contexts and diseased states can be achieved through increased knowledge of the presence of specific FGFR isoforms and their impact on downstream signaling and functions. Modern high-throughput techniques afford an opportunity to explore the distribution and function of isoforms of FGFR during development and in diseases. © 2014 Wiley Periodicals, Inc.

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.

Diabetes-induced changes in the alternative splicing of the slo gene in corporal tissue.

PubMed

Davies, Kelvin P; Zhao, Weixin; Tar, Moses; Figueroa, Johanna C; Desai, Pratik; Verselis, Vytas K; Kronengold, Jack; Wang, Hong-Zhan; Melman, Arnold; Christ, George J

2007-10-01

Erectile dysfunction is a common diabetic complication. Preclinical studies have documented that the Slo gene (encoding the BK or Maxi-K channel alpha-subunit) plays a critical role in erectile function. Therefore, we determined whether diabetes induces changes in the splicing of the Slo gene relevant to erectile function. Reverse transcriptase-polymerase chain reaction was used to compare Slo splice variant expression in corporal tissue excised from control and streptozotocin (STZ)-induced diabetic Fischer F-344 rats. Splice variants were sequenced, characterized by patch clamping, and fused to green fluorescent protein to determine cellular localization. The impact of altered Slo expression on erectile function was further evaluated in vivo. A novel Slo splice variant (SVcyt, with a cytoplasmic location) was predominantly expressed in corporal tissue from control rats. STZ-diabetes caused upregulation of a channel-forming transcript SV0. Preliminary results suggest that SV0 was also more prevalent in the corporal tissue of human diabetic compared with nondiabetic patients. The change in isoform expression in STZ-treated rats was partially reversed by insulin treatment. Intracorporal injection of a plasmid expressing the SV0 transcript, but not SVcyt, restored erectile function in STZ-diabetic rats. Alternative splicing of the Slo transcript may represent an important compensatory mechanism to increase the ease with which relaxation of corporal tissue may be triggered as a result of a diabetes-related decline in erectile capacity.

Identification of a novel alternative splicing variant of hemocyanin from shrimp Litopenaeus vannamei.

PubMed

Zhao, Shan; Lu, Xin; Zhang, Yueling; Zhao, Xianliang; Zhong, Mingqi; Li, Shengkang; Lun, Jingsheng

2013-01-01

Recent evidences suggest that invertebrates express families of immune molecules with high levels of sequence diversity. Hemocyanin is an important non-specific immune molecule present in the hemolymph of both mollusks and arthropods. In the present study, we characterized a novel alternative splicing variant of hemocyanin (cHE1) from Litopenaeus vannamei that produced mRNA transcript of 2579 bp in length. The isoform contained two additional sequences of 296 and 267 bp in the 5'- and 3'-terminus respectively, in comparison to that of wild type hemocyanin (cHE). Sequence of cHE1 shows 100% identity to that of hemocyanin genomic DNA (HE, which does not form an open reading frame), suggesting that cHE1 might be an alternative splicing variant due to intron retention. Moreover, cHE1 could be detected by RT-PCR from five tissues (heart, gill, stomach, intestine and brain), and from shrimps at stages from nauplius to mysis larva. Further, cHE1 mRNA transcripts were significantly increased in hearts after 12h of infection with Vibrio parahemolyticus or poly I: C, while no significant difference in the transcript levels of hepatopancreas cHE was detected in the pathogen-treated shrimps during the period. In summary, these studies suggested a novel splicing variant of hemocyanin in shrimp, which might be involved in shrimp resistance to pathogenic infection. Copyright © 2013 Elsevier B.V. All rights reserved.

Digital PCR (dPCR) analysis reveals that the homozygous c.315-48T>C variant in the FECH gene might cause erythropoietic protoporphyria (EPP).

PubMed

Brancaleoni, Valentina; Granata, Francesca; Missineo, Pasquale; Fustinoni, Silvia; Graziadei, Giovanna; Di Pierro, Elena

2018-06-13

Alterations in the ferrochelatase gene (FECH) are the basis of the phenotypic expressions in erythropoietic protoporphyria. The phenotype is due to the presence of a mutation in the FECH gene associated in trans to the c.315-48 T > C variant in the intron 3. The latter is able to increase the physiological quota of alternative splicing events in the intron 3. Other two variants in the FECH gene (c.1-252A > G and c.68-23C > T) have been found to be associated to the intron 3 variant in some populations and together, they constitute a haplotype (ACT/GTC), but eventually, their role in the alternative splicing event has never been elucidated. The absolute number of the aberrantly spliced FECH mRNA molecules and the absolute expression of the FECH gene were evaluated by digital PCR technique in a comprehensive cohort. The number of splicing events that rose in the presence of the c.315-48 T > C variant, both in the heterozygous and homozygous condition was reported for the first time. Also, the percentage of the inserted FECH mRNA increased, even doubled in the T/C cases, compared to T/T cases. The constant presence of variants in the promoter and intron 2 did not influence or modulate the aberrant splicing. The results of FECH gene expression suggested that the homozygosity for the c.315-48 T > C variant could be considered pathological. Thus, this study identified the homozygotes for the c.315-48 T > C variant as pathological. By extension, when the samples were categorised according to the haplotypes, the GTC haplotype in homozygosis was pathological. Copyright © 2018 Elsevier Inc. All rights reserved.

Differential HFE Gene Expression Is Regulated by Alternative Splicing in Human Tissues

PubMed Central

Proença, Daniela; Faustino, Paula

2011-01-01

Background The pathophysiology of HFE-derived Hereditary Hemochromatosis and the function of HFE protein in iron homeostasis remain uncertain. Also, the role of alternative splicing in HFE gene expression regulation and the possible function of the corresponding protein isoforms are still unknown. The aim of this study was to gain insights into the physiological significance of these alternative HFE variants. Methodology/Principal Findings Alternatively spliced HFE transcripts in diverse human tissues were identified by RT-PCR, cloning and sequencing. Total HFE transcripts, as well as two alternative splicing transcripts were quantified using a real-time PCR methodology. Intracellular localization, trafficking and protein association of GFP-tagged HFE protein variants were analysed in transiently transfected HepG2 cells by immunoprecipitation and immunofluorescence assays. Alternatively spliced HFE transcripts present both level- and tissue-specificity. Concerning the exon 2 skipping and intron 4 inclusion transcripts, the liver presents the lowest relative level, while duodenum presents one of the highest amounts. The protein resulting from exon 2 skipping transcript is unable to associate with β2M and TfR1 and reveals an ER retention. Conversely, the intron 4 inclusion transcript gives rise to a truncated, soluble protein (sHFE) that is mostly secreted by cells to the medium in association with β2M. Conclusions/Significance HFE gene post-transcriptional regulation is clearly affected by a tissue-dependent alternative splicing mechanism. Among the corresponding proteins, a sHFE isoform stands out, which upon being secreted into the bloodstream, may act in remote tissues. It could be either an agonist or antagonist of the full length HFE, through hepcidin expression regulation in the liver or by controlling dietary iron absorption in the duodenum. PMID:21407826

Differential HFE gene expression is regulated by alternative splicing in human tissues.

PubMed

Martins, Rute; Silva, Bruno; Proença, Daniela; Faustino, Paula

2011-03-03

The pathophysiology of HFE-derived Hereditary Hemochromatosis and the function of HFE protein in iron homeostasis remain uncertain. Also, the role of alternative splicing in HFE gene expression regulation and the possible function of the corresponding protein isoforms are still unknown. The aim of this study was to gain insights into the physiological significance of these alternative HFE variants. Alternatively spliced HFE transcripts in diverse human tissues were identified by RT-PCR, cloning and sequencing. Total HFE transcripts, as well as two alternative splicing transcripts were quantified using a real-time PCR methodology. Intracellular localization, trafficking and protein association of GFP-tagged HFE protein variants were analysed in transiently transfected HepG2 cells by immunoprecipitation and immunofluorescence assays. Alternatively spliced HFE transcripts present both level- and tissue-specificity. Concerning the exon 2 skipping and intron 4 inclusion transcripts, the liver presents the lowest relative level, while duodenum presents one of the highest amounts. The protein resulting from exon 2 skipping transcript is unable to associate with β2M and TfR1 and reveals an ER retention. Conversely, the intron 4 inclusion transcript gives rise to a truncated, soluble protein (sHFE) that is mostly secreted by cells to the medium in association with β2M. HFE gene post-transcriptional regulation is clearly affected by a tissue-dependent alternative splicing mechanism. Among the corresponding proteins, a sHFE isoform stands out, which upon being secreted into the bloodstream, may act in remote tissues. It could be either an agonist or antagonist of the full length HFE, through hepcidin expression regulation in the liver or by controlling dietary iron absorption in the duodenum.

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.

Functional Properties of a Newly Identified C-terminal Splice Variant of Cav1.3 L-type Ca2+ Channels*

PubMed Central

Bock, Gabriella; Gebhart, Mathias; Scharinger, Anja; Jangsangthong, Wanchana; Busquet, Perrine; Poggiani, Chiara; Sartori, Simone; Mangoni, Matteo E.; Sinnegger-Brauns, Martina J.; Herzig, Stefan; Striessnig, Jörg; Koschak, Alexandra

2011-01-01

An intramolecular interaction between a distal (DCRD) and a proximal regulatory domain (PCRD) within the C terminus of long Cav1.3 L-type Ca2+ channels (Cav1.3L) is a major determinant of their voltage- and Ca2+-dependent gating kinetics. Removal of these regulatory domains by alternative splicing generates Cav1.342A channels that activate at a more negative voltage range and exhibit more pronounced Ca2+-dependent inactivation. Here we describe the discovery of a novel short splice variant (Cav1.343S) that is expressed at high levels in the brain but not in the heart. It lacks the DCRD but, in contrast to Cav1.342A, still contains PCRD. When expressed together with α2δ1 and β3 subunits in tsA-201 cells, Cav1.343S also activated at more negative voltages like Cav1.342A but Ca2+-dependent inactivation was less pronounced. Single channel recordings revealed much higher channel open probabilities for both short splice variants as compared with Cav1.3L. The presence of the proximal C terminus in Cav1.343S channels preserved their modulation by distal C terminus-containing Cav1.3- and Cav1.2-derived C-terminal peptides. Removal of the C-terminal modulation by alternative splicing also induced a faster decay of Ca2+ influx during electrical activities mimicking trains of neuronal action potentials. Our findings extend the spectrum of functionally diverse Cav1.3 L-type channels produced by tissue-specific alternative splicing. This diversity may help to fine tune Ca2+ channel signaling and, in the case of short variants lacking a functional C-terminal modulation, prevent excessive Ca2+ accumulation during burst firing in neurons. This may be especially important in neurons that are affected by Ca2+-induced neurodegenerative processes. PMID:21998310

Alternative splicing modulates Kv channel clustering through a molecular ball and chain mechanism

NASA Astrophysics Data System (ADS)

Zandany, Nitzan; Marciano, Shir; Magidovich, Elhanan; Frimerman, Teddy; Yehezkel, Rinat; Shem-Ad, Tzilhav; Lewin, Limor; Abdu, Uri; Orr, Irit; Yifrach, Ofer

2015-03-01

Ion channel clustering at the post-synaptic density serves a fundamental role in action potential generation and transmission. Here, we show that interaction between the Shaker Kv channel and the PSD-95 scaffold protein underlying channel clustering is modulated by the length of the intrinsically disordered C terminal channel tail. We further show that this tail functions as an entropic clock that times PSD-95 binding. We thus propose a ‘ball and chain’ mechanism to explain Kv channel binding to scaffold proteins, analogous to the mechanism describing channel fast inactivation. The physiological relevance of this mechanism is demonstrated in that alternative splicing of the Shaker channel gene to produce variants of distinct tail lengths resulted in differential channel cell surface expression levels and clustering metrics that correlate with differences in affinity of the variants for PSD-95. We suggest that modulating channel clustering by specific spatial-temporal spliced variant targeting serves a fundamental role in nervous system development and tuning.

Identification of Alternative Splicing and Fusion Transcripts in Non-Small Cell Lung Cancer by RNA Sequencing.

PubMed

Hong, Yoonki; Kim, Woo Jin; Bang, Chi Young; Lee, Jae Cheol; Oh, Yeon-Mok

2016-04-01

Lung cancer is the most common cause of cancer related death. Alterations in gene sequence, structure, and expression have an important role in the pathogenesis of lung cancer. Fusion genes and alternative splicing of cancer-related genes have the potential to be oncogenic. In the current study, we performed RNA-sequencing (RNA-seq) to investigate potential fusion genes and alternative splicing in non-small cell lung cancer. RNA was isolated from lung tissues obtained from 86 subjects with lung cancer. The RNA samples from lung cancer and normal tissues were processed with RNA-seq using the HiSeq 2000 system. Fusion genes were evaluated using Defuse and ChimeraScan. Candidate fusion transcripts were validated by Sanger sequencing. Alternative splicing was analyzed using multivariate analysis of transcript sequencing and validated using quantitative real time polymerase chain reaction. RNA-seq data identified oncogenic fusion genes EML4-ALK and SLC34A2-ROS1 in three of 86 normal-cancer paired samples. Nine distinct fusion transcripts were selected using DeFuse and ChimeraScan; of which, four fusion transcripts were validated by Sanger sequencing. In 33 squamous cell carcinoma, 29 tumor specific skipped exon events and six mutually exclusive exon events were identified. ITGB4 and PYCR1 were top genes that showed significant tumor specific splice variants. In conclusion, RNA-seq data identified novel potential fusion transcripts and splice variants. Further evaluation of their functional significance in the pathogenesis of lung cancer is required.

Genome-wide association between DNA methylation and alternative splicing in an invertebrate

PubMed Central

2012-01-01

Background Gene bodies are the most evolutionarily conserved targets of DNA methylation in eukaryotes. However, the regulatory functions of gene body DNA methylation remain largely unknown. DNA methylation in insects appears to be primarily confined to exons. Two recent studies in Apis mellifera (honeybee) and Nasonia vitripennis (jewel wasp) analyzed transcription and DNA methylation data for one gene in each species to demonstrate that exon-specific DNA methylation may be associated with alternative splicing events. In this study we investigated the relationship between DNA methylation, alternative splicing, and cross-species gene conservation on a genome-wide scale using genome-wide transcription and DNA methylation data. Results We generated RNA deep sequencing data (RNA-seq) to measure genome-wide mRNA expression at the exon- and gene-level. We produced a de novo transcriptome from this RNA-seq data and computationally predicted splice variants for the honeybee genome. We found that exons that are included in transcription are higher methylated than exons that are skipped during transcription. We detected enrichment for alternative splicing among methylated genes compared to unmethylated genes using fisher’s exact test. We performed a statistical analysis to reveal that the presence of DNA methylation or alternative splicing are both factors associated with a longer gene length and a greater number of exons in genes. In concordance with this observation, a conservation analysis using BLAST revealed that each of these factors is also associated with higher cross-species gene conservation. Conclusions This study constitutes the first genome-wide analysis exhibiting a positive relationship between exon-level DNA methylation and mRNA expression in the honeybee. Our finding that methylated genes are enriched for alternative splicing suggests that, in invertebrates, exon-level DNA methylation may play a role in the construction of splice variants by positively influencing exon inclusion during transcription. The results from our cross-species homology analysis suggest that DNA methylation and alternative splicing are genetic mechanisms whose utilization could contribute to a longer gene length and a slower rate of gene evolution. PMID:22978521

Integrating alternative splicing detection into gene prediction.

PubMed

Foissac, Sylvain; Schiex, Thomas

2005-02-10

Alternative splicing (AS) is now considered as a major actor in transcriptome/proteome diversity and it cannot be neglected in the annotation process of a new genome. Despite considerable progresses in term of accuracy in computational gene prediction, the ability to reliably predict AS variants when there is local experimental evidence of it remains an open challenge for gene finders. We have used a new integrative approach that allows to incorporate AS detection into ab initio gene prediction. This method relies on the analysis of genomically aligned transcript sequences (ESTs and/or cDNAs), and has been implemented in the dynamic programming algorithm of the graph-based gene finder EuGENE. Given a genomic sequence and a set of aligned transcripts, this new version identifies the set of transcripts carrying evidence of alternative splicing events, and provides, in addition to the classical optimal gene prediction, alternative optimal predictions (among those which are consistent with the AS events detected). This allows for multiple annotations of a single gene in a way such that each predicted variant is supported by a transcript evidence (but not necessarily with a full-length coverage). This automatic combination of experimental data analysis and ab initio gene finding offers an ideal integration of alternatively spliced gene prediction inside a single annotation pipeline.

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

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.

RNA-Seq of Arabidopsis Pollen Uncovers Novel Transcription and Alternative Splicing1[C][W][OA

PubMed Central

Loraine, Ann E.; McCormick, Sheila; Estrada, April; Patel, Ketan; Qin, Peng

2013-01-01

Pollen grains of Arabidopsis (Arabidopsis thaliana) contain two haploid sperm cells enclosed in a haploid vegetative cell. Upon germination, the vegetative cell extrudes a pollen tube that carries the sperm to an ovule for fertilization. Knowing the identity, relative abundance, and splicing patterns of pollen transcripts will improve our understanding of pollen and allow investigation of tissue-specific splicing in plants. Most Arabidopsis pollen transcriptome studies have used the ATH1 microarray, which does not assay splice variants and lacks specific probe sets for many genes. To investigate the pollen transcriptome, we performed high-throughput sequencing (RNA-Seq) of Arabidopsis pollen and seedlings for comparison. Gene expression was more diverse in seedling, and genes involved in cell wall biogenesis were highly expressed in pollen. RNA-Seq detected at least 4,172 protein-coding genes expressed in pollen, including 289 assayed only by nonspecific probe sets. Additional exons and previously unannotated 5′ and 3′ untranslated regions for pollen-expressed genes were revealed. We detected regions in the genome not previously annotated as expressed; 14 were tested and 12 were confirmed by polymerase chain reaction. Gapped read alignments revealed 1,908 high-confidence new splicing events supported by 10 or more spliced read alignments. Alternative splicing patterns in pollen and seedling were highly correlated. For most alternatively spliced genes, the ratio of variants in pollen and seedling was similar, except for some encoding proteins involved in RNA splicing. This study highlights the robustness of splicing patterns in plants and the importance of ongoing annotation and visualization of RNA-Seq data using interactive tools such as Integrated Genome Browser. PMID:23590974

Allele-Specific Alternative mRNA processing (ASARP) | Informatics Technology for Cancer Research (ITCR)

Cancer.gov

A software pipeline for prediction of allele-specific alternative RNA processing events using single RNA-seq data. The current version focuses on prediction of alternative splicing and alternative polyadenylation modulated by genetic variants.

Skipping of Exons by Premature Termination of Transcription and Alternative Splicing within Intron-5 of the Sheep SCF Gene: A Novel Splice Variant

PubMed Central

Saravanaperumal, Siva Arumugam; Pediconi, Dario; Renieri, Carlo; La Terza, Antonietta

2012-01-01

Stem cell factor (SCF) is a growth factor, essential for haemopoiesis, mast cell development and melanogenesis. In the hematopoietic microenvironment (HM), SCF is produced either as a membrane-bound (−) or soluble (+) forms. Skin expression of SCF stimulates melanocyte migration, proliferation, differentiation, and survival. We report for the first time, a novel mRNA splice variant of SCF from the skin of white merino sheep via cloning and sequencing. Reverse transcriptase (RT)-PCR and molecular prediction revealed two different cDNA products of SCF. Full-length cDNA libraries were enriched by the method of rapid amplification of cDNA ends (RACE-PCR). Nucleotide sequencing and molecular prediction revealed that the primary 1519 base pair (bp) cDNA encodes a precursor protein of 274 amino acids (aa), commonly known as ‘soluble’ isoform. In contrast, the shorter (835 and/or 725 bp) cDNA was found to be a ‘novel’ mRNA splice variant. It contains an open reading frame (ORF) corresponding to a truncated protein of 181 aa (vs 245 aa) with an unique C-terminus lacking the primary proteolytic segment (28 aa) right after the D175G site which is necessary to produce ‘soluble’ form of SCF. This alternative splice (AS) variant was explained by the complete nucleotide sequencing of splice junction covering exon 5-intron (5)-exon 6 (948 bp) with a premature termination codon (PTC) whereby exons 6 to 9/10 are skipped (Cassette Exon, CE 6–9/10). We also demonstrated that the Northern blot analysis at transcript level is mediated via an intron-5 splicing event. Our data refine the structure of SCF gene; clarify the presence (+) and/or absence (−) of primary proteolytic-cleavage site specific SCF splice variants. This work provides a basis for understanding the functional role and regulation of SCF in hair follicle melanogenesis in sheep beyond what was known in mice, humans and other mammals. PMID:22719917

Simultaneous quantification of alternatively spliced transcripts in a single droplet digital PCR reaction.

PubMed

Sun, Bing; Tao, Lian; Zheng, Yun-Ling

2014-06-01

Human telomerase reverse transcriptase (hTERT) is an essential component required for telomerase activity and telomere maintenance. Several alternatively spliced forms of hTERT mRNA have been reported in human primary and tumor cells. Currently, however, there is no sensitive and accurate method for the simultaneous quantification of multiple alternatively spliced RNA transcripts, such as in the case of hTERT. Here we show droplet digital PCR (ddPCR) provides sensitive, simultaneous digital quantification in a single reaction of two alternatively spliced single deletion hTERT transcripts (α-/β+ and α+/β-) as well as the opportunity to manually quantify non-deletion (α+/β+) and double deletion (α-/β-) transcripts. Our ddPCR method enables direct comparison among four alternatively spliced mRNAs without the need for internal standards or multiple primer pairs specific for each variant as real-time PCR (qPCR) requires, thus eliminating potential variation due to differences in PCR amplification efficiency.

Identification of a functionally distinct truncated BDNF mRNA splice variant and protein in Trachemys scripta elegans.

PubMed

Ambigapathy, Ganesh; Zheng, Zhaoqing; Li, Wei; Keifer, Joyce

2013-01-01

Brain-derived neurotrophic factor (BDNF) has a diverse functional role and complex pattern of gene expression. Alternative splicing of mRNA transcripts leads to further diversity of mRNAs and protein isoforms. Here, we describe the regulation of BDNF mRNA transcripts in an in vitro model of eyeblink classical conditioning and a unique transcript that forms a functionally distinct truncated BDNF protein isoform. Nine different mRNA transcripts from the BDNF gene of the pond turtle Trachemys scripta elegans (tBDNF) are selectively regulated during classical conditioning: exon I mRNA transcripts show no change, exon II transcripts are downregulated, while exon III transcripts are upregulated. One unique transcript that codes from exon II, tBDNF2a, contains a 40 base pair deletion in the protein coding exon that generates a truncated tBDNF protein. The truncated transcript and protein are expressed in the naïve untrained state and are fully repressed during conditioning when full-length mature tBDNF is expressed, thereby having an alternate pattern of expression in conditioning. Truncated BDNF is not restricted to turtles as a truncated mRNA splice variant has been described for the human BDNF gene. Further studies are required to determine the ubiquity of truncated BDNF alternative splice variants across species and the mechanisms of regulation and function of this newly recognized BDNF protein.

Identification of a Functionally Distinct Truncated BDNF mRNA Splice Variant and Protein in Trachemys scripta elegans

PubMed Central

Ambigapathy, Ganesh; Zheng, Zhaoqing; Li, Wei; Keifer, Joyce

2013-01-01

Brain-derived neurotrophic factor (BDNF) has a diverse functional role and complex pattern of gene expression. Alternative splicing of mRNA transcripts leads to further diversity of mRNAs and protein isoforms. Here, we describe the regulation of BDNF mRNA transcripts in an in vitro model of eyeblink classical conditioning and a unique transcript that forms a functionally distinct truncated BDNF protein isoform. Nine different mRNA transcripts from the BDNF gene of the pond turtle Trachemys scripta elegans (tBDNF) are selectively regulated during classical conditioning: exon I mRNA transcripts show no change, exon II transcripts are downregulated, while exon III transcripts are upregulated. One unique transcript that codes from exon II, tBDNF2a, contains a 40 base pair deletion in the protein coding exon that generates a truncated tBDNF protein. The truncated transcript and protein are expressed in the naïve untrained state and are fully repressed during conditioning when full-length mature tBDNF is expressed, thereby having an alternate pattern of expression in conditioning. Truncated BDNF is not restricted to turtles as a truncated mRNA splice variant has been described for the human BDNF gene. Further studies are required to determine the ubiquity of truncated BDNF alternative splice variants across species and the mechanisms of regulation and function of this newly recognized BDNF protein. PMID:23825634

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.

Diabetes-Induced Changes in the Alternative Splicing of the Slo Gene in Corporal Tissue

PubMed Central

Davies, Kelvin P.; Zhao, Weixin; Tar, Moses; Figueroa, Johanna C.; Desai, Pratik; Verselis, Vytas K.; Kronengold, Jack; Wang, Hong-Zhan; Melman, Arnold; Christ, George J.

2007-01-01

Objectives Erectile dysfunction is a common diabetic complication. Preclinical studies have documented that the Slo gene (encoding the BK or Maxi-K channel α-subunit) plays a critical role in erectile function. Therefore, we determined whether diabetes induces changes in the splicing of the Slo gene relevant to erectile function. Methods Reverse transcriptase-polymerase chain reaction was used to compare Slo splice variant expression in corporal tissue excised from control and streptozotocin (STZ)-induced diabetic Fischer F-344 rats. Splice variants were sequenced, characterized by patch clamping, and fused to green fluorescent protein to determine cellular localization. The impact of altered Slo expression on erectile function was further evaluated in vivo. Results A novel Slo splice variant (SVcyt, with a cytoplasmic location) was predominantly expressed in corporal tissue from control rats. STZ-diabetes caused upregulation of a channel-forming transcript SV0. Preliminary results suggest that SV0 was also more prevalent in the corporal tissue of human diabetic compared with nondiabetic patients. The change in isoform expression in STZ-treated rats was partially reversed by insulin treatment. Intracorporal injection of a plasmid expressing the SV0 transcript, but not SVcyt, restored erectile function in STZ-diabetic rats. Conclusions Alternative splicing of the Slo transcript may represent an important compensatory mechanism to increase the ease with which relaxation of corporal tissue may be triggered as a result of a diabetes-related decline in erectile capacity. PMID:17150299

Human osteopontin splicing isoforms: known roles, potential clinical applications and activated signaling pathways.

PubMed

Gimba, E R; Tilli, T M

2013-04-30

Human osteopontin is subject to alternative splicing, which generates three isoforms, termed OPNa, OPNb and OPNc. These variants show specific expression and roles in different cell contexts. We present an overview of current knowledge of the expression profile of human OPN splicing isoforms (OPN-SIs), their tissue-specific roles, and the pathways mediating their functional properties in different pathophysiological conditions. We also describe their putative application as biomarkers, and their potential use as therapeutic targets by using antibodies, oligonucleotides or siRNA molecules. This synthesis provides new clues for a better understanding of human OPN splice variants, their roles in normal and pathological conditions, and their possible clinical applications. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Mutually Exclusive Splicing of the Insect Dscam Pre-mRNA Directed by Competing Intronic RNA Secondary Structures

PubMed Central

Graveley, Brenton R.

2008-01-01

Summary Drosophila Dscam encodes 38,016 distinct axon guidance receptors through the mutually exclusive alternative splicing of 95 variable exons. Importantly, known mechanisms that ensure the mutually exclusive splicing of pairs of exons cannot explain this phenomenon in Dscam. I have identified two classes of conserved elements in the Dscam exon 6 cluster, which contains 48 alternative exons—the docking site, located in the intron downstream of constitutive exon 5, and the selector sequences, which are located upstream of each exon 6 variant. Strikingly, each selector sequence is complementary to a portion of the docking site, and this pairing juxtaposes one, and only one, alternative exon to the upstream constitutive exon. The mutually exclusive nature of the docking site:selector sequence interactions suggests that the formation of these competing RNA structures is a central component of the mechanism guaranteeing that only one exon 6 variant is included in each Dscam mRNA. PMID:16213213

Sequence variants of KHDRBS1 as high penetrance susceptibility risks for primary ovarian insufficiency by mis-regulating mRNA alternative splicing.

PubMed

Wang, Binbin; Li, Lin; Zhu, Ying; Zhang, Wei; Wang, Xi; Chen, Beili; Li, Tengyan; Pan, Hong; Wang, Jing; Kee, Kehkooi; Cao, Yunxia

2017-10-01

Does a novel heterozygous KHDRBS1 variant, identified using whole-exome sequencing (WES) in two patients with primary ovarian insufficiency (POI) in a pedigree, cause defects in mRNA alternative splicing? The heterozygous variant of KHDRBS1 was confirmed to cause defects in alternative splicing of many genes involved in DNA replication and repair. Studies in mice revealed that Khdrbs1 deficient females are subfertile, which manifests as delayed sexual maturity and significantly reduced numbers of secondary and pre-antral follicles. No mutation of KHDRBS1, however, has been reported in patients with POI. This genetic and functional study used WES to find putative mutations in a POI pedigree. Altogether, 215 idiopathic POI patients and 400 healthy controls were screened for KHDRBS1 mutations. Two POI patients were subjected to WES to identify sequence variants. Mutational analysis of the KHDRBS1 gene in 215 idiopathic POI patients and 400 healthy controls were performed. RNA-sequencing was carried out to find the mis-regulation of gene expression due to KHDRBS1 mutation. Bioinformatics was used to analyze the change in alternative splicing events. We identified a heterozygous mutation (c.460A > G, p.M154V) in KHDRBS1 in two patients. Further mutational analysis of 215 idiopathic POI patients with the KHDRBS1 gene found one heterozygous mutation (c.263C > T, p.P88L). We failed to find these two mutations in 400 healthy control women. Using RNA-sequencing, we found that the KGN cells expressing the M154V KHDRBS1 mutant had different expression of 66 genes compared with wild-type (WT) cells. Furthermore, 145 genes were alternatively spliced in M154V cells, and these genes were enriched for DNA replication and repair function, revealing a potential underlying mechanism of the pathology that leads to POI. Although the in vitro assays demonstrated the effect of the KHDRBS1 variant on alternative splicing, further studies are needed to validate the in vivo effects on germ cell and follicle development. This finding provides researchers and clinicians a better understanding of the etiology and molecular mechanism of POI. This study was supported by the Ministry of Science and Technology of China (2012CB944704; 2012CB966702), National Research Institute for Family Planning (2017GJZ05), the National Natural Science Foundation of China (31171429) and Beijing Advanced Innovation Center for Structural Biology. The authors declare no conflict of interest. © The Author 2017. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com

Simultaneous Quantification of Multiple Alternatively Spliced mRNA Transcripts Using Droplet Digital PCR.

PubMed

Sun, Bing; Zheng, Yun-Ling

2018-01-01

Currently there is no sensitive, precise, and reproducible method to quantitate alternative splicing of mRNA transcripts. Droplet digital™ PCR (ddPCR™) analysis allows for accurate digital counting for quantification of gene expression. Human telomerase reverse transcriptase (hTERT) is one of the essential components required for telomerase activity and for the maintenance of telomeres. Several alternatively spliced forms of hTERT mRNA in human primary and tumor cells have been reported in the literature. Using one pair of primers and two probes for hTERT, four alternatively spliced forms of hTERT (α-/β+, α+/β- single deletions, α-/β- double deletion, and nondeletion α+/β+) were accurately quantified through a novel analysis method via data collected from a single ddPCR reaction. In this chapter, we describe this ddPCR method that enables direct quantitative comparison of four alternatively spliced forms of the hTERT messenger RNA without the need for internal standards or multiple pairs of primers specific for each variant, eliminating the technical variation due to differential PCR amplification efficiency for different amplicons and the challenges of quantification using standard curves. This simple and straightforward method should have general utility for quantifying alternatively spliced gene transcripts.

A few nucleotide polymorphisms are sufficient to recruit nuclear factors differentially to the intron 1 of HPV-16 intratypic variants.

PubMed

López-Urrutia, Eduardo; Valdés, Jesús; Bonilla-Moreno, Raúl; Martínez-Salazar, Martha; Martínez-Garcia, Martha; Berumen, Jaime; Villegas-Sepúlveda, Nicolás

2012-06-01

The HPV-16 E6/E7 genes, which contain intron 1, are processed by alternative splicing and its transcripts are detected with a heterogeneous profile in tumours cells. Frequently, the HPV-16 positive carcinoma cells bear viral variants that contain single nucleotide polymorphisms into its DNA sequence. We were interested in analysing the contribution of this polymorphism to the heterogeneity in the pattern of the E6/E7 spliced transcripts. Using the E6/E7 sequences from three closely related HPV-16 variants, we have shown that a few nucleotide changes are sufficient to produce heterogeneity in the splicing profile. Furthermore, using mutants that contained a single SNP, we also showed that one nucleotide change was sufficient to reproduce the heterogeneous splicing profile. Additionally, a difference of two or three SNPs among these viral sequences was sufficient to recruit differentially several splicing factors to the polymorphic E6/E7 transcripts. Moreover, only one SNP was sufficient to alter the binding site of at least one splicing factor, changing the ability of splicing factors to bind the transcript. Finally, the factors that were differentially bound to the short form of intron 1 of one of these E6/E7 variants were identified as TIA1 and/or TIAR and U1-70k, while U2AF65, U5-52k and PTB were preferentially bound to the transcript of the other variants. Copyright © 2012 Elsevier B.V. All rights reserved.

Enhanced expression of Rubisco activase splicing variants differentially affects Rubisco activity during low temperature treatment in Lolium perenne.

PubMed

Jurczyk, Barbara; Pociecha, Ewa; Grzesiak, Maciej; Kalita, Katarzyna; Rapacz, Marcin

2016-07-01

Alternative splicing of the Rubisco activase gene was shown to be a point for optimization of photosynthetic carbon assimilation. It can be expected to be a stress-regulated event that depends on plant freezing tolerance. The aim of the study was to examine the relationships among Rubisco activity, the expression of two Rubisco activase splicing variants and photoacclimation to low temperature. The experiment was performed on two Lolium perenne genotypes with contrasting levels of freezing tolerance. The study investigated the effect of pre-hardening (15°C) and cold acclimation (4°C) on net photosynthesis, photosystem II photochemical activity, Rubisco activity and the expression of two splicing variants of the Rubisco activase gene. The results showed an induction of Rubisco activity at both 15°C and 4°C only in a highly freezing-tolerant genotype. The enhanced Rubisco activity after pre-hardening corresponded to increased expression of the splicing variant representing the large isoform, while the increase in Rubisco activity during cold acclimation was due to the activation of both transcript variants. These boosts in Rubisco activity also corresponded to an activation of non-photochemical mechanism of photoacclimation induced at low temperature exclusively in the highly freezing-tolerant genotype. In conclusion, enhanced expression of Rubisco activase splicing variants caused an increase in Rubisco activity during pre-hardening and cold acclimation in the more freezing-tolerant Lolium perenne genotype. The induction of the transcript variant representing the large isoform may be an important element of increasing the carbon assimilation rate supporting the photochemical mechanism of photosynthetic acclimation to cold. Copyright © 2016 Elsevier GmbH. All rights reserved.

A study of alternative splicing in the pig

PubMed Central

2010-01-01

Background Since at least half of the genes in mammalian genomes are subjected to alternative splicing, alternative pre-mRNA splicing plays an important contribution to the complexity of the mammalian proteome. Expressed sequence tags (ESTs) provide evidence of a great number of possible alternative isoforms. With the EST resource for the domestic pig now containing more than one million porcine ESTs, it is possible to identify alternative splice forms of the individual transcripts in this species from the EST data with some confidence. Results The pig EST data generated by the Sino-Danish Pig Genome project has been assembled with publicly available ESTs and made available in the PigEST database. Using the Distiller package 2,515 EST clusters with candidate alternative isoforms were identified in the EST data with high confidence. In agreement with general observations in human and mouse, we find putative splice variants in about 30% of the contigs with more than 50 ESTs. Based on the criteria that a minimum of two EST sequences confirmed each splice event, a list of 100 genes with the most distinct tissue-specific alternative splice events was generated from the list of candidates. To confirm the tissue specificity of the splice events, 10 genes with functional annotation were randomly selected from which 16 individual splice events were chosen for experimental verification by quantitative PCR (qPCR). Six genes were shown to have tissue specific alternatively spliced transcripts with expression patterns matching those of the EST data. The remaining four genes had tissue-restricted expression of alternative spliced transcripts. Five out of the 16 splice events that were experimentally verified were found to be putative pig specific. Conclusions In accordance with human and rodent studies we estimate that approximately 30% of the porcine genes undergo alternative splicing. We found a good correlation between EST predicted tissue-specificity and experimentally validated splice events in different porcine tissue. This study indicates that a cluster size of around 50 ESTs is optimal for in silico detection of alternative splicing. Although based on a limited number of splice events, the study supports the notion that alternative splicing could have an important impact on species differentiation since 31% of the splice events studied appears to be species specific. PMID:20444244

Selective expression of a splice variant of decay-accelerating factor in c-erbB-2-positive mammary carcinoma cells showing increased transendothelial invasiveness

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

Brandt, Burkhard; Mikesch, Jan-Hendrik; Simon, Ronald

2005-04-01

By differential-display-PCR a subclone of the SK-BR-3 cell line with high in vitro transendothelial invasiveness was identified to express increased levels of a new alternative splice variant of decay-accelerating factor (DAF). DAF seems to play an important role in some malignant tumours since on the one hand the expression of complement inhibitors on the surface of tumour cells prevents the accumulation of complement factors and in consequence cell lysis. On the other hand, DAF has been identified as a ligand for the CD97 surface receptor which induces cell migration. Immunofluorescence procedures, Western blot analyses, and cDNA clone sequencing were employedmore » to confirm the expression of DAF restricted to invasive tumour cells. Using a radioactive RNA-in situ hybridisation on freshly frozen tissue microarrays and RT-PCR on native tumour tissue, the expression of alternative spliced DAF mRNA was demonstrated in invasive breast cancer. Due to the fact that it could thereby not be detected in normal mammary tissues, it has to be confirmed in larger studies that the DAF splice variant might be a specific tumour marker for invasive breast cancer.« less

Discovery of a new mechanism for regulation of plant triacylglycerol metabolism: The peanut diacylglycerol acyltransferase-1 gene family transcriptome is highly enriched in alternative splicing variants.

PubMed

Zheng, Ling; Shockey, Jay; Guo, Feng; Shi, Lingmin; Li, Xinguo; Shan, Lei; Wan, Shubo; Peng, Zhenying

2017-12-01

Triacylglycerols (TAGs) are the most important energy storage form in oilseed crops. Diacylglycerol acyltransferase (DGAT) catalyzes the rate-limiting step of the Kennedy pathway of TAG biosynthesis. To date, little is known about the regulation of DGAT activity in peanut (Arachis hypogaea), an agronomically important oilseed crop that is cultivated in many parts of the world. In this study, seven distinct forms of type 1 DGAT (AhDGAT1.1-AhDGAT1.7) were identified, cloned, and characterized. Comparisons of the nucleotide sequences and gene structures revealed many different splicing variants of AhDGAT1, some of which displayed different organ-specific expression patterns. A representative gene (AhDGAT1.1) was transformed into wild-type tobacco and was shown to increase seed fatty acid (FA) content by 14.7%-20.9%. All seven AhDGAT1s were expressed in TAG-deficient Saccharomyces cerevisiae strain H1246; the five longest AhDGAT1 variants generated high levels of acyltransferase activity and complemented the free fatty acid lethality phenotype in this strain. The alternative splicing that gives rise to AhDGAT1.2 and AhDGAT1.4 creates predicted protein C-terminal truncations. The proteins encoded by these two variants were not active and did not complement the fatty acid sensitivity in H1246. These results were verified by visualization of intracellular lipid droplets using Nile Red staining. Collectively, the results presented here represent the first comprehensive analysis of the peanut DGAT1 gene family, which, unlike in other published plant DGAT1 sequences, shows widespread alternative splicing that may affect the expression patterns and enzyme activities of some members of the gene family. Copyright © 2017. Published by Elsevier GmbH.

Functional properties of a newly identified C-terminal splice variant of Cav1.3 L-type Ca2+ channels.

PubMed

Bock, Gabriella; Gebhart, Mathias; Scharinger, Anja; Jangsangthong, Wanchana; Busquet, Perrine; Poggiani, Chiara; Sartori, Simone; Mangoni, Matteo E; Sinnegger-Brauns, Martina J; Herzig, Stefan; Striessnig, Jörg; Koschak, Alexandra

2011-12-09

An intramolecular interaction between a distal (DCRD) and a proximal regulatory domain (PCRD) within the C terminus of long Ca(v)1.3 L-type Ca(2+) channels (Ca(v)1.3(L)) is a major determinant of their voltage- and Ca(2+)-dependent gating kinetics. Removal of these regulatory domains by alternative splicing generates Ca(v)1.3(42A) channels that activate at a more negative voltage range and exhibit more pronounced Ca(2+)-dependent inactivation. Here we describe the discovery of a novel short splice variant (Ca(v)1.3(43S)) that is expressed at high levels in the brain but not in the heart. It lacks the DCRD but, in contrast to Ca(v)1.3(42A), still contains PCRD. When expressed together with α2δ1 and β3 subunits in tsA-201 cells, Ca(v)1.3(43S) also activated at more negative voltages like Ca(v)1.3(42A) but Ca(2+)-dependent inactivation was less pronounced. Single channel recordings revealed much higher channel open probabilities for both short splice variants as compared with Ca(v)1.3(L). The presence of the proximal C terminus in Ca(v)1.3(43S) channels preserved their modulation by distal C terminus-containing Ca(v)1.3- and Ca(v)1.2-derived C-terminal peptides. Removal of the C-terminal modulation by alternative splicing also induced a faster decay of Ca(2+) influx during electrical activities mimicking trains of neuronal action potentials. Our findings extend the spectrum of functionally diverse Ca(v)1.3 L-type channels produced by tissue-specific alternative splicing. This diversity may help to fine tune Ca(2+) channel signaling and, in the case of short variants lacking a functional C-terminal modulation, prevent excessive Ca(2+) accumulation during burst firing in neurons. This may be especially important in neurons that are affected by Ca(2+)-induced neurodegenerative processes.

Qualitative research of alternatively splice variants of fibronectin in different development stage of mice heart.

PubMed

Lu, Feng; Ma, Fang-Fang; Zhang, Wei; Li, Ying; Wei, Fei-Yu; Zhou, Lei

2015-12-01

Fibronectin (FN) plays vital roles in cell adhesion, differentiation, proliferation and migration. It is involved in the process of embryonic development and is highly conserved during evolution. The EIIIA and EIIIB of FN show a very high degree of homology among vertebrates. Embryos deleting both EIIIA and EIIIB displayed multiple embryonic cardiovascular defects, implying their crucial role during embryogenesis. The correlation of spliced EIIIB, EIIIA, and IIICS of FN to heart development was studied by observing their chronological expression in mice heart. C57 mice embryos at E11.5, E12.5, E13.5, E14.5, E15.5, E16.5, E17.5, E18.5, E19.5 days, postnatal day 1 (P1d), and adult male mice (3 months) were used. For each alternatively spliced FN1 domain (EIIIB, EIIIA and IIICS), primer pairs were designed for specific amplification. Total RNA was extracted from the heart tissue, reverse transcripted to cDNA, followed by RT-PCR with specific primers. The PCR amplification was verified by agarose gel electrophoresis, showing specific fragments of the expected sizes. In adult mice heart, only alternatively splice variants of EIIIA-, EIIIB-, IIICS+ were expressed. While in embryonic mice, spliced variant of EIIIA+/-, EIIIB+/-, IIICS+ were observed. The expression of EIIIA and EIIIB changed during heart development. FN is crucial for the normal development of the embryonic heart by modulating cardiac neural crest (CNC) proliferation and survival, and maintenance of CNC cells. FN1 gene seems to play a significant role by expression of highly conserved EIIIA and EIIIB in embryonic heart development.

Aberrant and alternative splicing in skeletal system disease.

PubMed

Fan, Xin; Tang, Liling

2013-10-01

The main function of skeletal system is to support the body and help movement. A variety of factors can lead to skeletal system disease, including age, exercise, and of course genetic makeup and expression. Pre-mRNA splicing plays a crucial role in gene expression, by creating multiple protein variants with different biological functions. The recent studies show that several skeletal system diseases are related to pre-mRNA splicing. This review focuses on the relationship between pre-mRNA splicing and skeletal system disease. On the one hand, splice site mutation that leads to aberrant splicing often causes genetic skeletal system disease, like COL1A1, SEDL and LRP5. On the other hand, alternative splicing without genomic mutation may generate some marker protein isoforms, for example, FN, VEGF and CD44. Therefore, understanding the relationship between pre-mRNA splicing and skeletal system disease will aid in uncovering the mechanism of disease and contribute to the future development of gene therapy. © 2013 Elsevier B.V. All rights reserved.

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.

CD44 Splice Variants as Potential Players in Alzheimer's Disease Pathology.

PubMed

Pinner, Elhanan; Gruper, Yaron; Ben Zimra, Micha; Kristt, Don; Laudon, Moshe; Naor, David; Zisapel, Nava

2017-01-01

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by cognitive deficits, deposition of amyloid-β (Aβ) plaques, intracellular neurofibrillary tangles, and neuronal cell death. Neuroinflammation is commonly believed to participate in AD pathogenesis. CD44 is an inflammation-related gene encoding a widely-distributed family of alternatively spliced cell surface glycoproteins that have been implicated in inflammation, metastases, and inflammation-linked neuronal injuries. Here we investigated the expression patterns of CD44S (which does not contain any alternative exon) and CD44 splice variants in postmortem hippocampal samples from AD patients and matched non-AD controls. The expression of CD44S and CD44 splice variants CD44V3, CD44V6, and CD44V10 was significantly higher in AD patients compared to non-AD controls. Immunohistochemistry of human hippocampal sections revealed that CD44S differentially localized to neuritic plaques and astrocytes, whereas CD44V3, CD44V6, and CD44V10 expression was mostly neuronal. Consistent with these findings, we found that the expression of CD44V6 and CD44V10 was induced by Aβ peptide in neuroblastoma cells and primary neurons. Furthermore, in loss of function studies we found that both CD44V10-specific siRNA and CD44V10 antibody protected neuronal cells from Aβ-induced toxicity, suggesting a causal relationship between CD44V10 and neuronal cell death. These data indicate that certain CD44 splice variants contribute to AD pathology and that CD44V10 inhibition may serve as a new neuroprotective treatment strategy for this disease.

RBFOX2 Is an Important Regulator of Mesenchymal Tissue-Specific Splicing in both Normal and Cancer Tissues

PubMed Central

Venables, Julian P.; Brosseau, Jean-Philippe; Gadea, Gilles; Klinck, Roscoe; Prinos, Panagiotis; Beaulieu, Jean-François; Lapointe, Elvy; Durand, Mathieu; Thibault, Philippe; Tremblay, Karine; Rousset, François; Tazi, Jamal; Abou Elela, Sherif

2013-01-01

Alternative splicing provides a critical and flexible layer of regulation intervening in many biological processes to regulate the diversity of proteins and impact cell phenotype. To identify alternative splicing differences that distinguish epithelial from mesenchymal tissues, we have investigated hundreds of cassette exons using a high-throughput reverse transcription-PCR (RT-PCR) platform. Extensive changes in splicing were noted between epithelial and mesenchymal tissues in both human colon and ovarian tissues, with many changes from mostly one splice variant to predominantly the other. Remarkably, many of the splicing differences that distinguish normal mesenchymal from normal epithelial tissues matched those that differentiate normal ovarian tissues from ovarian cancer. Furthermore, because splicing profiling could classify cancer cell lines according to their epithelial/mesenchymal characteristics, we used these cancer cell lines to identify regulators for these specific splicing signatures. By knocking down 78 potential splicing factors in five cell lines, we provide an extensive view of the complex regulatory landscape associated with the epithelial and mesenchymal states, thus revealing that RBFOX2 is an important driver of mesenchymal tissue-specific splicing. PMID:23149937

Alternative role of HuD splicing variants in neuronal differentiation.

PubMed

Hayashi, Satoru; Yano, Masato; Igarashi, Mana; Okano, Hirotaka James; Okano, Hideyuki

2015-03-01

HuD is a neuronal RNA-binding protein that plays an important role in neuronal differentiation of the nervous system. HuD has been reported to have three RNA recognition motifs (RRMs) and three splice variants (SVs) that differ in their amino acid sequences between RRM2 and RRM3. This study investigates whether these SVs have specific roles in neuronal differentiation. In primary neural epithelial cells under differentiating conditions, HuD splice variant 1 (HuD-sv1), which is a general form, and HuD-sv2 were expressed at all tested times, whereas HuD-sv4 was transiently expressed at the beginning of differentiation, indicating that HuD-sv4 might play a role compared different from that of HuD-sv1. Indeed, HuD-sv4 did not promote neuronal differentiation in epithelial cells, whereas HuD-sv1 did promote neuronal differentiation. HuD-sv4 overexpression showed less neurite-inducing activity than HuD-sv1 in mouse neuroblastoma N1E-115 cells; however, HuD-sv4 showed stronger growth-arresting activity. HuD-sv1 was localized only in the cytoplasm, whereas HuD-sv4 was localized in both the cytoplasm and the nuclei. The Hu protein has been reported to be involved in translation and alternative splicing in the cytoplasm and nuclei, respectively. Consistent with this observation, HuD-sv1 showed translational activity on p21, which plays a role in growth arrest and neuronal differentiation, whereas HuD-sv4 did not. By contrast, HuD-sv4 showed stronger pre-mRNA splicing activity than did HuD-sv1 on Clasp2, which participates in cell division. Therefore, HuD SVs might play a role in controlling the timing of proliferation/differentiation switching by controlling the translation and alternative splicing of target genes. © 2014 Wiley Periodicals, Inc.

The combinatorial control of alternative splicing in C. elegans

PubMed Central

2017-01-01

Normal development requires the right splice variants to be made in the right tissues at the right time. The core splicing machinery is engaged in all splicing events, but which precise splice variant is made requires the choice between alternative splice sites—for this to occur, a set of splicing factors (SFs) must recognize and bind to short RNA motifs in the pre-mRNA. In C. elegans, there is known to be extensive variation in splicing patterns across development, but little is known about the targets of each SF or how multiple SFs combine to regulate splicing. Here we combine RNA-seq with in vitro binding assays to study how 4 different C. elegans SFs, ASD-1, FOX-1, MEC-8, and EXC-7, regulate splicing. The 4 SFs chosen all have well-characterised biology and well-studied loss-of-function genetic alleles, and all contain RRM domains. Intriguingly, while the SFs we examined have varied roles in C. elegans development, they show an unexpectedly high overlap in their targets. We also find that binding sites for these SFs occur on the same pre-mRNAs more frequently than expected suggesting extensive combinatorial control of splicing. We confirm that regulation of splicing by multiple SFs is often combinatorial and show that this is functionally significant. We also find that SFs appear to combine to affect splicing in two modes—they either bind in close proximity within the same intron or they appear to bind to separate regions of the intron in a conserved order. Finally, we find that the genes whose splicing are regulated by multiple SFs are highly enriched for genes involved in the cytoskeleton and in ion channels that are key for neurotransmission. Together, this shows that specific classes of genes have complex combinatorial regulation of splicing and that this combinatorial regulation is critical for normal development to occur. PMID:29121637

Epigenetic Machinery Regulates Alternative Splicing of Androgen Receptor (AR) Gene in Castration Resistant Prostate Cancer

DTIC Science & Technology

2017-09-01

AWARD NUMBER: W81XWH-16-1-0531 TITLE: Epigenetic machinery regulates alternative splicing of androgen receptor ( AR ) gene in castration...DISTRIBUTION STATEMENT: Approved for Public Release Distribution Unlimited The views, opinions and/or findings contained in this report are those of...One of the reasons for the resistance to ADT and newer anti-androgen drugs is the emergence of constitutively active AR variants ( AR -Vs) such as AR

Histone demethylase JMJD1A promotes alternative splicing of AR variant 7 (AR-V7) in prostate cancer cells.

PubMed

Fan, Lingling; Zhang, Fengbo; Xu, Songhui; Cui, Xiaolu; Hussain, Arif; Fazli, Ladan; Gleave, Martin; Dong, Xuesen; Qi, Jianfei

2018-05-15

Formation of the androgen receptor splicing variant 7 (AR-V7) is one of the major mechanisms by which resistance of prostate cancer to androgen deprivation therapy occurs. The histone demethylase JMJD1A (Jumonji domain containing 1A) functions as a key coactivator for AR by epigenetic regulation of H3K9 methylation marks. Here, we describe a role for JMJD1A in AR-V7 expression. While JMJD1A knockdown had no effect on full-length AR (AR-FL), it reduced AR-V7 levels in prostate cancer cells. Reexpression of AR-V7 in the JMJD1A-knockdown cells elevated expression of select AR targets and partially rescued prostate cancer cell growth in vitro and in vivo. The AR-V7 protein level correlated positively with JMJD1A in a subset of human prostate cancer specimens. Mechanistically, we found that JMJD1A promoted alternative splicing of AR-V7 through heterogeneous nuclear ribonucleoprotein F (HNRNPF), a splicing factor known to regulate exon inclusion. Knockdown of JMJD1A or HNRNPF inhibited splicing of AR-V7, but not AR-FL, in a minigene reporter assay. JMJD1A was found to interact with and promote the recruitment of HNRNPF to a cryptic exon 3b on AR pre-mRNA for the generation of AR-V7. Taken together, the role of JMJD1A in AR-FL coactivation and AR-V7 alternative splicing highlights JMJD1A as a potentially promising target for prostate cancer therapy.

The transcription factor FBI-1 inhibits SAM68-mediated BCL-X alternative splicing and apoptosis.

PubMed

Bielli, Pamela; Busà, Roberta; Di Stasi, Savino M; Munoz, Manuel J; Botti, Flavia; Kornblihtt, Alberto R; Sette, Claudio

2014-04-01

Alternative splicing (AS) is tightly coupled to transcription for the majority of human genes. However, how these two processes are linked is not well understood. Here, we unveil a direct role for the transcription factor FBI-1 in the regulation of AS. FBI-1 interacts with the splicing factor SAM68 and reduces its binding to BCL-X mRNA. This, in turn, results in the selection of the proximal 5' splice site in BCL-X exon 2, thereby favoring the anti-apoptotic BCL-XL variant and counteracting SAM68-mediated apoptosis. Conversely, depletion of FBI-1, or expression of a SAM68 mutant lacking the FBI-1 binding region, restores the ability of SAM68 to induce BCL-XS splicing and apoptosis. FBI-1's role in splicing requires the activity of histone deacetylases, whose pharmacological inhibition recapitulates the effects of FBI-1 knockdown. Our study reveals an unexpected function for FBI-1 in splicing modulation with a direct impact on cell survival.

The transcription factor FBI-1 inhibits SAM68-mediated BCL-X alternative splicing and apoptosis

PubMed Central

Bielli, Pamela; Busà, Roberta; Di Stasi, Savino M; Munoz, Manuel J; Botti, Flavia; Kornblihtt, Alberto R; Sette, Claudio

2014-01-01

Alternative splicing (AS) is tightly coupled to transcription for the majority of human genes. However, how these two processes are linked is not well understood. Here, we unveil a direct role for the transcription factor FBI-1 in the regulation of AS. FBI-1 interacts with the splicing factor SAM68 and reduces its binding to BCL-X mRNA. This, in turn, results in the selection of the proximal 5′ splice site in BCL-X exon 2, thereby favoring the anti-apoptotic BCL-XL variant and counteracting SAM68-mediated apoptosis. Conversely, depletion of FBI-1, or expression of a SAM68 mutant lacking the FBI-1 binding region, restores the ability of SAM68 to induce BCL-XS splicing and apoptosis. FBI-1's role in splicing requires the activity of histone deacetylases, whose pharmacological inhibition recapitulates the effects of FBI-1 knockdown. Our study reveals an unexpected function for FBI-1 in splicing modulation with a direct impact on cell survival. PMID:24514149

regSNPs-splicing: a tool for prioritizing synonymous single-nucleotide substitution.

PubMed

Zhang, Xinjun; Li, Meng; Lin, Hai; Rao, Xi; Feng, Weixing; Yang, Yuedong; Mort, Matthew; Cooper, David N; Wang, Yue; Wang, Yadong; Wells, Clark; Zhou, Yaoqi; Liu, Yunlong

2017-09-01

While synonymous single-nucleotide variants (sSNVs) have largely been unstudied, since they do not alter protein sequence, mounting evidence suggests that they may affect RNA conformation, splicing, and the stability of nascent-mRNAs to promote various diseases. Accurately prioritizing deleterious sSNVs from a pool of neutral ones can significantly improve our ability of selecting functional genetic variants identified from various genome-sequencing projects, and, therefore, advance our understanding of disease etiology. In this study, we develop a computational algorithm to prioritize sSNVs based on their impact on mRNA splicing and protein function. In addition to genomic features that potentially affect splicing regulation, our proposed algorithm also includes dozens structural features that characterize the functions of alternatively spliced exons on protein function. Our systematical evaluation on thousands of sSNVs suggests that several structural features, including intrinsic disorder protein scores, solvent accessible surface areas, protein secondary structures, and known and predicted protein family domains, show significant differences between disease-causing and neutral sSNVs. Our result suggests that the protein structure features offer an added dimension of information while distinguishing disease-causing and neutral synonymous variants. The inclusion of structural features increases the predictive accuracy for functional sSNV prioritization.

Partial androgen insensitivity syndrome caused by a deep intronic mutation creating an alternative splice acceptor site of the AR gene.

PubMed

Ono, Hiroyuki; Saitsu, Hirotomo; Horikawa, Reiko; Nakashima, Shinichi; Ohkubo, Yumiko; Yanagi, Kumiko; Nakabayashi, Kazuhiko; Fukami, Maki; Fujisawa, Yasuko; Ogata, Tsutomu

2018-02-02

Although partial androgen insensitivity syndrome (PAIS) is caused by attenuated responsiveness to androgens, androgen receptor gene (AR) mutations on the coding regions and their splice sites have been identified only in <25% of patients with a diagnosis of PAIS. We performed extensive molecular studies including whole exome sequencing in a Japanese family with PAIS, identifying a deep intronic variant beyond the branch site at intron 6 of AR (NM_000044.4:c.2450-42 G > A). This variant created the splice acceptor motif that was accompanied by pyrimidine-rich sequence and two candidate branch sites. Consistent with this, reverse transcriptase (RT)-PCR experiments for cycloheximide-treated lymphoblastoid cell lines revealed a relatively large amount of aberrant mRNA produced by the newly created splice acceptor site and a relatively small amount of wildtype mRNA produced by the normal splice acceptor site. Furthermore, most of the aberrant mRNA was shown to undergo nonsense mediated decay (NMD) and, if a small amount of aberrant mRNA may have escaped NMD, such mRNA was predicted to generate a truncated AR protein missing some functional domains. These findings imply that the deep intronic mutation creating an alternative splice acceptor site resulted in the production of a relatively small amount of wildtype AR mRNA, leading to PAIS.

Global Profiling of the Cellular Alternative RNA Splicing Landscape during Virus-Host Interactions

PubMed Central

Boudreault, Simon; Martenon-Brodeur, Camille; Caron, Marie; Garant, Jean-Michel; Tremblay, Marie-Pier; Armero, Victoria E. S.; Durand, Mathieu; Lapointe, Elvy; Thibault, Philippe; Tremblay-Létourneau, Maude; Perreault, Jean-Pierre; Scott, Michelle S.; Lemay, Guy; Bisaillon, Martin

2016-01-01

Alternative splicing (AS) is a central mechanism of genetic regulation which modifies the sequence of RNA transcripts in higher eukaryotes. AS has been shown to increase both the variability and diversity of the cellular proteome by changing the composition of resulting proteins through differential choice of exons to be included in mature mRNAs. In the present study, alterations to the global RNA splicing landscape of cellular genes upon viral infection were investigated using mammalian reovirus as a model. Our study provides the first comprehensive portrait of global changes in the RNA splicing signatures that occur in eukaryotic cells following infection with a human virus. We identify 240 modified alternative splicing events upon infection which belong to transcripts frequently involved in the regulation of gene expression and RNA metabolism. Using mass spectrometry, we also confirm modifications to transcript-specific peptides resulting from AS in virus-infected cells. These findings provide additional insights into the complexity of virus-host interactions as these splice variants expand proteome diversity and function during viral infection. PMID:27598998

Global Profiling of the Cellular Alternative RNA Splicing Landscape during Virus-Host Interactions.

PubMed

Boudreault, Simon; Martenon-Brodeur, Camille; Caron, Marie; Garant, Jean-Michel; Tremblay, Marie-Pier; Armero, Victoria E S; Durand, Mathieu; Lapointe, Elvy; Thibault, Philippe; Tremblay-Létourneau, Maude; Perreault, Jean-Pierre; Scott, Michelle S; Lemay, Guy; Bisaillon, Martin

2016-01-01

Alternative splicing (AS) is a central mechanism of genetic regulation which modifies the sequence of RNA transcripts in higher eukaryotes. AS has been shown to increase both the variability and diversity of the cellular proteome by changing the composition of resulting proteins through differential choice of exons to be included in mature mRNAs. In the present study, alterations to the global RNA splicing landscape of cellular genes upon viral infection were investigated using mammalian reovirus as a model. Our study provides the first comprehensive portrait of global changes in the RNA splicing signatures that occur in eukaryotic cells following infection with a human virus. We identify 240 modified alternative splicing events upon infection which belong to transcripts frequently involved in the regulation of gene expression and RNA metabolism. Using mass spectrometry, we also confirm modifications to transcript-specific peptides resulting from AS in virus-infected cells. These findings provide additional insights into the complexity of virus-host interactions as these splice variants expand proteome diversity and function during viral infection.

Identification of pathogenic gene mutations in LMNA and MYBPC3 that alter RNA splicing.

PubMed

Ito, Kaoru; Patel, Parth N; Gorham, Joshua M; McDonough, Barbara; DePalma, Steven R; Adler, Emily E; Lam, Lien; MacRae, Calum A; Mohiuddin, Syed M; Fatkin, Diane; Seidman, Christine E; Seidman, J G

2017-07-18

Genetic variants that cause haploinsufficiency account for many autosomal dominant (AD) disorders. Gene-based diagnosis classifies variants that alter canonical splice signals as pathogenic, but due to imperfect understanding of RNA splice signals other variants that may create or eliminate splice sites are often clinically classified as variants of unknown significance (VUS). To improve recognition of pathogenic splice-altering variants in AD disorders, we used computational tools to prioritize VUS and developed a cell-based minigene splicing assay to confirm aberrant splicing. Using this two-step procedure we evaluated all rare variants in two AD cardiomyopathy genes, lamin A/C ( LMNA ) and myosin binding protein C ( MYBPC3 ). We demonstrate that 13 LMNA and 35 MYBPC3 variants identified in cardiomyopathy patients alter RNA splicing, representing a 50% increase in the numbers of established damaging splice variants in these genes. Over half of these variants are annotated as VUS by clinical diagnostic laboratories. Familial analyses of one variant, a synonymous LMNA VUS, demonstrated segregation with cardiomyopathy affection status and altered cardiac LMNA splicing. Application of this strategy should improve diagnostic accuracy and variant classification in other haploinsufficient AD disorders.

X-linked CHARGE-like Abruzzo-Erickson syndrome and classic cleft palate with ankyloglossia result from TBX22 splicing mutations.

PubMed

Pauws, E; Peskett, E; Boissin, C; Hoshino, A; Mengrelis, K; Carta, E; Abruzzo, M A; Lees, M; Moore, G E; Erickson, R P; Stanier, P

2013-04-01

X-linked cleft palate (CPX) is caused by mutations in the gene encoding the TBX22 transcription factor and is known to exhibit phenotypic variability, usually involving either a complete, partial or submucous cleft palate, with or without ankyloglossia. This study hypothesized a possible involvement of TBX22 in a family with X-linked, CHARGE-like Abruzzo-Erickson syndrome, of unknown etiology. The phenotype extends to additional features including sensorineural deafness and coloboma, which are suggested by the Tbx22 developmental expression pattern but not previously associated in CPX patients. A novel TBX22 splice acceptor mutation (c.593-5T>A) was identified that tracked with the phenotype in this family. A novel splice donor variant (c.767+5G>A) and a known canonical splice donor mutation (c.767+1G>A) affecting the same exon were identified in patients with classic CPX phenotypes and were comparatively analyzed using both in silico and in vitro splicing studies. All three variants were predicted to abolish normal mRNA splicing and an in vitro assay indicated that use of alternative splice sites was a likely outcome. Collectively, the data showed the functional effect of several novel intronic splice site variants but most importantly confirms that TBX22 is the gene underlying Abruzzo-Erickson syndrome, expanding the phenotypic spectrum of TBX22 mutations. © 2012 John Wiley & Sons A/S. Published by Blackwell Publishing Ltd.

Compound heterozygous NEK1 variants in two siblings with oral-facial-digital syndrome type II (Mohr syndrome)

PubMed Central

Monroe, Glen R; Kappen, Isabelle FPM; Stokman, Marijn F; Terhal, Paulien A; van den Boogaard, Marie-José H; Savelberg, Sanne MC; van der Veken, Lars T; van Es, Robert JJ; Lens, Susanne M; Hengeveld, Rutger C; Creton, Marijn A; Janssen, Nard G; Mink van der Molen, Aebele B; Ebbeling, Michelle B; Giles, Rachel H; Knoers, Nine V; van Haaften, Gijs

2016-01-01

The oral-facial-digital (OFD) syndromes comprise a group of related disorders with a combination of oral, facial and digital anomalies. Variants in several ciliary genes have been associated with subtypes of OFD syndrome, yet in most OFD patients the underlying cause remains unknown. We investigated the molecular basis of disease in two brothers with OFD type II, Mohr syndrome, by performing single-nucleotide polymorphism (SNP)-array analysis on the brothers and their healthy parents to identify homozygous regions and candidate genes. Subsequently, we performed whole-exome sequencing (WES) on the family. Using WES, we identified compound heterozygous variants c.[464G>C][1226G>A] in NIMA (Never in Mitosis Gene A)-Related Kinase 1 (NEK1). The novel variant c.464G>C disturbs normal splicing in an essential region of the kinase domain. The nonsense variant c.1226G>A, p.(Trp409*), results in nonsense-associated alternative splicing, removing the first coiled-coil domain of NEK1. Candidate variants were confirmed with Sanger sequencing and alternative splicing assessed with cDNA analysis. Immunocytochemistry was used to assess cilia number and length. Patient-derived fibroblasts showed severely reduced ciliation compared with control fibroblasts (18.0 vs 48.9%, P<0.0001), but showed no significant difference in cilia length. In conclusion, we identified compound heterozygous deleterious variants in NEK1 in two brothers with Mohr syndrome. Ciliation in patient fibroblasts is drastically reduced, consistent with a ciliary defect pathogenesis. Our results establish NEK1 variants involved in the etiology of a subset of patients with OFD syndrome type II and support the consideration of including (routine) NEK1 analysis in patients suspected of OFD. PMID:27530628

Compound heterozygous NEK1 variants in two siblings with oral-facial-digital syndrome type II (Mohr syndrome).

PubMed

Monroe, Glen R; Kappen, Isabelle Fpm; Stokman, Marijn F; Terhal, Paulien A; van den Boogaard, Marie-José H; Savelberg, Sanne Mc; van der Veken, Lars T; van Es, Robert Jj; Lens, Susanne M; Hengeveld, Rutger C; Creton, Marijn A; Janssen, Nard G; Mink van der Molen, Aebele B; Ebbeling, Michelle B; Giles, Rachel H; Knoers, Nine V; van Haaften, Gijs

2016-12-01

The oral-facial-digital (OFD) syndromes comprise a group of related disorders with a combination of oral, facial and digital anomalies. Variants in several ciliary genes have been associated with subtypes of OFD syndrome, yet in most OFD patients the underlying cause remains unknown. We investigated the molecular basis of disease in two brothers with OFD type II, Mohr syndrome, by performing single-nucleotide polymorphism (SNP)-array analysis on the brothers and their healthy parents to identify homozygous regions and candidate genes. Subsequently, we performed whole-exome sequencing (WES) on the family. Using WES, we identified compound heterozygous variants c.[464G>C];[1226G>A] in NIMA (Never in Mitosis Gene A)-Related Kinase 1 (NEK1). The novel variant c.464G>C disturbs normal splicing in an essential region of the kinase domain. The nonsense variant c.1226G>A, p.(Trp409*), results in nonsense-associated alternative splicing, removing the first coiled-coil domain of NEK1. Candidate variants were confirmed with Sanger sequencing and alternative splicing assessed with cDNA analysis. Immunocytochemistry was used to assess cilia number and length. Patient-derived fibroblasts showed severely reduced ciliation compared with control fibroblasts (18.0 vs 48.9%, P<0.0001), but showed no significant difference in cilia length. In conclusion, we identified compound heterozygous deleterious variants in NEK1 in two brothers with Mohr syndrome. Ciliation in patient fibroblasts is drastically reduced, consistent with a ciliary defect pathogenesis. Our results establish NEK1 variants involved in the etiology of a subset of patients with OFD syndrome type II and support the consideration of including (routine) NEK1 analysis in patients suspected of OFD.

Complex Tissue-Specific Patterns and Distribution of Multiple RAGE Splice Variants in Different Mammals

PubMed Central

López-Díez, Raquel; Rastrojo, Alberto; Villate, Olatz; Aguado, Begoña

2013-01-01

The receptor for advanced glycosylation end products (RAGE) is a multiligand receptor involved in diverse cell signaling pathways. Previous studies show that this gene expresses several splice variants in human, mouse, and dog. Alternative splicing (AS) plays an important role in expanding transcriptomic and proteomic diversity, and it has been related to disease. AS is also one of the main evolutionary mechanisms in mammalian genomes. However, limited information is available regarding the AS of RAGE in a wide context of mammalian tissues. In this study, we examined in detail the different RAGE mRNAs generated by AS from six mammals, including two primates (human and monkey), two artiodactyla (cow and pig), and two rodentia (mouse and rat) in 6–18 different tissues including fetal, adult, and tumor. By nested reverse transcription-polymerase chain reaction (RT-PCR) we identified a high number of splice variants including noncoding transcripts and predicted coding ones with different potential protein modifications affecting mainly the transmembrane and ligand-binding domains that could influence their biological function. However, analysis of RNA-seq data enabled detecting only the most abundant splice variants. More than 80% of the detected RT-PCR variants (87 of 101 transcripts) are novel (different exon/intron structure to the previously described ones), and interestingly, 20–60% of the total transcripts (depending on the species) are noncoding ones that present tissue specificity. Our results suggest that RAGE undergoes extensive AS in mammals, with different expression patterns among adult, fetal, and tumor tissues. Moreover, most splice variants seem to be species specific, especially the noncoding variants, with only two (canonical human Tv1-RAGE, and human N-truncated or Tv10-RAGE) conserved among the six different species. This could indicate a special evolution pattern of this gene at mRNA level. PMID:24273313

Roles of Alternative RNA Splicing of the Bif-1 Gene by SRRM4 During the Development of Treatment-induced Neuroendocrine Prostate Cancer.

PubMed

Gan, Yu; Li, Yinan; Long, Zhi; Lee, Ahn R; Xie, Ning; Lovnicki, Jessica M; Tang, Yuxin; Chen, Xiang; Huang, Jiaoti; Dong, Xuesen

2018-05-01

Treatment-induced neuroendocrine prostate cancer (t-NEPC) is an aggressive subtype of prostate cancer (PCa) that becomes more prevalent when hormonal therapy, chemotherapy, or radiation therapy is applied to patients with metastatic prostate adenocarcinoma (AdPC). How AdPC cells survive these anti-cancer therapies and progress into t-NEPC remains unclear. By comparing the whole transcriptomes between AdPC and t-NEPC, we identified Bif-1, an apoptosis-associated gene, which undergoes alternative RNA splicing in t-NEPC. We found that while Bif-1a is the predominant variant of the Bif-1 gene in AdPC, two neural-specific variants, Bif-1b and Bif-1c, are highly expressed in t-NEPC patients, patient derived xenografts, and cell models. The neural-specific RNA splicing factor, SRRM4, promotes Bif-1b and Bif-1c splicing, and the expression of SRRM4 in tumors is strongly associated with Bif-1b/-1c levels. Furthermore, we showed that Bif-1a is pro-apoptotic, while Bif-1b and Bif-1c are anti-apoptotic in PCa cells under camptothecin and UV light irritation treatments. Taken together, our data indicate that SRRM4 regulates alternative RNA splicing of the Bif-1 gene that enables PCa cells resistant to apoptotic stimuli under anti-cancer therapies, and may contribute to AdPC progression into t-NEPC. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

Corepressors: custom tailoring and alterations while you wait

PubMed Central

Goodson, Michael; Jonas, Brian A.; Privalsky, Martin A.

2005-01-01

A diverse cadre of metazoan transcription factors mediate repression by recruiting protein complexes containing the SMRT (silencing mediator of retinoid and thyroid hormone receptor) or N-CoR (nuclear receptor corepressor) corepressors. SMRT and N-CoR nucleate the assembly of still larger corepressor complexes that perform the specific molecular incantations necessary to confer transcriptional repression. Although SMRT and N-CoR are paralogs and possess similar molecular architectures and mechanistic strategies, they nonetheless exhibit distinct molecular and biological properties. It is now clear that the functions of both SMRT and N-CoR are further diversified through alternative mRNA splicing, yielding a series of corepressor protein variants that participate in distinctive transcription factor partnerships and display distinguishable repression properties. This review will discuss what is known about the structure and actions of SMRT, N-CoR, and their splicing variants, and how alternative splicing may allow the functions of these corepressors to be adapted and tailored to different cells and to different developmental stages. PMID:16604171

Molecular Characterization of the α-Subunit of Na+/K+ ATPase from the Euryhaline Barnacle Balanus improvisus Reveals Multiple Genes and Differential Expression of Alternative Splice Variants

PubMed Central

Lind, Ulrika; Alm Rosenblad, Magnus; Wrange, Anna-Lisa; Sundell, Kristina S.; Jonsson, Per R.; André, Carl; Havenhand, Jonathan; Blomberg, Anders

2013-01-01

The euryhaline bay barnacle Balanus improvisus has one of the broadest salinity tolerances of any barnacle species. It is able to complete its life cycle in salinities close to freshwater (3 PSU) up to fully marine conditions (35 PSU) and is regarded as one of few truly brackish-water species. Na+/K+ ATPase (NAK) has been shown to be important for osmoregulation when marine organisms are challenged by changing salinities, and we therefore cloned and examined the expression of different NAKs from B. improvisus. We found two main gene variants, NAK1 and NAK2, which were approximately 70% identical at the protein level. The NAK1 mRNA existed in a long and short variant with the encoded proteins differing only by 27 N-terminal amino acids. This N-terminal stretch was coded for by a separate exon, and the two variants of NAK1 mRNAs appeared to be created by alternative splicing. We furthermore showed that the two NAK1 isoforms were differentially expressed in different life stages and in various tissues of adult barnacle, i.e the long isoform was predominant in cyprids and in adult cirri. In barnacle cyprid larvae that were exposed to a combination of different salinities and pCO2 levels, the expression of the long NAK1 mRNA increased relative to the short in low salinities. We suggest that the alternatively spliced long variant of the Nak1 protein might be of importance for osmoregulation in B. improvisus in low salinity conditions. PMID:24130836

A liver X receptor (LXR)-{beta} alternative splicing variant (LXRBSV) acts as an RNA co-activator of LXR-{beta}

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

Hashimoto, Koshi, E-mail: khashi@med.gunma-u.ac.jp; Ishida, Emi; Matsumoto, Shunichi

2009-12-25

We report the isolation and functional characterization of a novel transcriptional co-activator, termed LXRBSV. LXRBSV is an alternative splicing variant of liver X receptor (LXR)-{beta} LXRBSV has an intronic sequence between exons 2 and 3 in the mouse LXR-{beta} gene. The LXRBSV gene is expressed in various tissues including the liver and brain. We sub-cloned LXRBSV into pSG5, a mammalian expression vector, and LXRBSV in pSG5 augmented human Sterol Response Element Binding Protein (SREBP)-1c promoter activity in HepG2 cells in a ligand (TO901317) dependent manner. The transactivation mediated by LXRBSV is selective for LXR-{beta}. The LXRBSV protein was deduced tomore » be 64 amino acids in length; however, a GAL4-LXRBSV fusion protein was not able to induce transactivation. Serial deletion constructs of LXRBSV demonstrated that the intronic sequence inserted in LXRBSV is required for its transactivation activity. An ATG mutant of LXRBSV was able to induce transactivation as wild type. Furthermore, LXRBSV functions in the presence of cycloheximide. Taken together, we have concluded that LXRBSV acts as an RNA transcript not as a protein. In the current study, we have demonstrated for the first time that an alternative splicing variant of a nuclear receptor acts as an RNA co-activator.« less

α6-Integrin alternative splicing: distinct cytoplasmic variants in stem cell fate specification and niche interaction.

PubMed

Zhou, Zijing; Qu, Jing; He, Li; Peng, Hong; Chen, Ping; Zhou, Yong

2018-05-02

α6-Integrin subunit (also known as CD49f) is a stemness signature that has been found on the plasma membrane of more than 30 stem cell populations. A growing body of studies have focused on the critical role of α6-containing integrins (α6β1 and α6β4) in the regulation of stem cell properties, lineage-specific differentiation, and niche interaction. α6-Integrin subunit can be alternatively spliced at the post-transcriptional level, giving rise to divergent isoforms which differ in the cytoplasmic and/or extracellular domains. The cytoplasmic domain of integrins is an important functional part of integrin-mediated signals. Structural changes in the cytoplasmic domain of α6 provide an efficient means for the regulation of stem cell responses to biochemical stimuli and/or biophysical cues in the stem cell niche, thus impacting stem cell fate determination. In this review, we summarize the current knowledge on the structural variants of the α6-integrin subunit and spatiotemporal expression of α6 cytoplasmic variants in embryonic and adult stem/progenitor cells. We highlight the roles of α6 cytoplasmic variants in stem cell fate decision and niche interaction, and discuss the potential mechanisms involved. Understanding of the distinct functions of α6 splicing variants in stem cell biology may inform the rational design of novel stem cell-based therapies for a range of human diseases.

Astrocytes express specific variants of CaM KII delta and gamma, but not alpha and beta, that determine their cellular localizations.

PubMed

Vallano, M L; Beaman-Hall, C M; Mathur, A; Chen, Q

2000-04-01

Multiple isoforms of type II Ca(2+)-calmodulin-dependent kinase (CaM KII) are composed of two major neuron-specific subunits, designated alpha and beta, and two less well-characterized subunits that are also expressed in non-neuronal tissues, designated delta and gamma. Regulated expression of these 4 gene products, and several variants produced by alternative splicing, shows temporal and regional specificity and influences intracellular targeting. We used immunoblotting and RT-PCR to analyze subunit and variant expression and distribution in cultured cerebellar astrocytes and neurons, and whole cerebellar cortex from rodent brain. The data indicate that: (i) astrocytes express a single splice variant of delta, namely delta(2); (ii) like neurons, astrocytes express two forms of CaM KII gamma; gamma(B) and gamma(A); (iii) these CaM KII variants are enriched in the supernate fraction in astrocytes, and the particulate fraction in neurons; (iv) unlike neurons, astrocytes do not express detectable levels of alpha or beta subunits or their respective splice variants. The results indicate that neurons and astrocytes express distinct CaM KII subunits and variants that localize to distinct subcellular compartments and, by inference, exert distinct cellular functions. Copyright 2000 Wiley-Liss, Inc.

Global impact of RNA splicing on transcriptome remodeling in the heart.

PubMed

Gao, Chen; Wang, Yibin

2012-08-01

In the eukaryotic transcriptome, both the numbers of genes and different RNA species produced by each gene contribute to the overall complexity. These RNA species are generated by the utilization of different transcriptional initiation or termination sites, or more commonly, from different messenger RNA (mRNA) splicing events. Among the 30,000+ genes in human genome, it is estimated that more than 95% of them can generate more than one gene product via alternative RNA splicing. The protein products generated from different RNA splicing variants can have different intracellular localization, activity, or tissue-distribution. Therefore, alternative RNA splicing is an important molecular process that contributes to the overall complexity of the genome and the functional specificity and diversity among different cell types. In this review, we will discuss current efforts to unravel the full complexity of the cardiac transcriptome using a deep-sequencing approach, and highlight the potential of this technology to uncover the global impact of RNA splicing on the transcriptome during development and diseases of the heart.

Alternative splicing and differential gene expression in colon cancer detected by a whole genome exon array

PubMed Central

Gardina, Paul J; Clark, Tyson A; Shimada, Brian; Staples, Michelle K; Yang, Qing; Veitch, James; Schweitzer, Anthony; Awad, Tarif; Sugnet, Charles; Dee, Suzanne; Davies, Christopher; Williams, Alan; Turpaz, Yaron

2006-01-01

Background Alternative splicing is a mechanism for increasing protein diversity by excluding or including exons during post-transcriptional processing. Alternatively spliced proteins are particularly relevant in oncology since they may contribute to the etiology of cancer, provide selective drug targets, or serve as a marker set for cancer diagnosis. While conventional identification of splice variants generally targets individual genes, we present here a new exon-centric array (GeneChip Human Exon 1.0 ST) that allows genome-wide identification of differential splice variation, and concurrently provides a flexible and inclusive analysis of gene expression. Results We analyzed 20 paired tumor-normal colon cancer samples using a microarray designed to detect over one million putative exons that can be virtually assembled into potential gene-level transcripts according to various levels of prior supporting evidence. Analysis of high confidence (empirically supported) transcripts identified 160 differentially expressed genes, with 42 genes occupying a network impacting cell proliferation and another twenty nine genes with unknown functions. A more speculative analysis, including transcripts based solely on computational prediction, produced another 160 differentially expressed genes, three-fourths of which have no previous annotation. We also present a comparison of gene signal estimations from the Exon 1.0 ST and the U133 Plus 2.0 arrays. Novel splicing events were predicted by experimental algorithms that compare the relative contribution of each exon to the cognate transcript intensity in each tissue. The resulting candidate splice variants were validated with RT-PCR. We found nine genes that were differentially spliced between colon tumors and normal colon tissues, several of which have not been previously implicated in cancer. Top scoring candidates from our analysis were also found to substantially overlap with EST-based bioinformatic predictions of alternative splicing in cancer. Conclusion Differential expression of high confidence transcripts correlated extremely well with known cancer genes and pathways, suggesting that the more speculative transcripts, largely based solely on computational prediction and mostly with no previous annotation, might be novel targets in colon cancer. Five of the identified splicing events affect mediators of cytoskeletal organization (ACTN1, VCL, CALD1, CTTN, TPM1), two affect extracellular matrix proteins (FN1, COL6A3) and another participates in integrin signaling (SLC3A2). Altogether they form a pattern of colon-cancer specific alterations that may particularly impact cell motility. PMID:17192196

Overexpressed long noncoding RNA CRNDE with distinct alternatively spliced isoforms in multiple cancers.

PubMed

Ma, Xuefei; Zhang, Wei; Zhang, Rong; Li, Jingming; Li, Shufen; Ma, Yunlin; Jin, Wen; Wang, Kankan

2018-05-26

Alternative splicing is a tightly regulated process that contributes to cancer development. CRNDE is a long noncoding RNA with alternative splicing and is implicated in the pathogenesis of several cancers. However, whether deregulated expression of CRNDE is common and which isoforms are mainly involved in cancers remain unclear. In this study, we report that CRNDE is aberrantly expressed in the majority of solid and hematopoietic malignancies. The investigation of CRNDE expression in normal samples revealed that CRNDE was expressed in a tissue- and cell-specific manner. Further comparison of CRNDE expression in 2938 patient samples from 15 solid and hematopoietic tumors showed that CRNDE was significantly overexpressed in 11 malignancies, including 3 reported and 8 unreported, and also implicated that the overexpressed isoforms differed in various cancer types. Furthermore, anti-cancer drugs could efficiently repress CRNDE overexpression in cancer cell lines and primary samples, and even had different impacts on the expression of CRNDE isoforms. Finally, experimental profiles of 12 alternatively spliced isoforms demonstrated that the spliced variant CRNDE-g was the most highly expressed isoform in multiple cancer types. Collectively, our results emphasize the cancer-associated feature of CRNDE and its spliced isoforms, and may provide promising targets for cancer diagnosis and therapy.

Splicing analysis for exonic and intronic mismatch repair gene variants associated with Lynch syndrome confirms high concordance between minigene assays and patient RNA analyses

PubMed Central

van der Klift, Heleen M; Jansen, Anne M L; van der Steenstraten, Niki; Bik, Elsa C; Tops, Carli M J; Devilee, Peter; Wijnen, Juul T

2015-01-01

A subset of DNA variants causes genetic disease through aberrant splicing. Experimental splicing assays, either RT-PCR analyses of patient RNA or functional splicing reporter minigene assays, are required to evaluate the molecular nature of the splice defect. Here, we present minigene assays performed for 17 variants in the consensus splice site regions, 14 exonic variants outside these regions, and two deep intronic variants, all in the DNA mismatch-repair (MMR) genes MLH1, MSH2, MSH6, and PMS2, associated with Lynch syndrome. We also included two deep intronic variants in APC and PKD2. For one variant (MLH1 c.122A>G), our minigene assay and patient RNA analysis could not confirm the previously reported aberrant splicing. The aim of our study was to further investigate the concordance between minigene splicing assays and patient RNA analyses. For 30 variants results from patient RNA analyses were available, either performed by our laboratory or presented in literature. Some variants were deliberately included in this study because they resulted in multiple aberrant transcripts in patient RNA analysis, or caused a splice effect other than the prevalent exon skip. While both methods were completely concordant in the assessment of splice effects, four variants exhibited major differences in aberrant splice patterns. Based on the present and earlier studies, together showing an almost 100% concordance of minigene assays with patient RNA analyses, we discuss the weight given to minigene splicing assays in the current criteria proposed by InSiGHT for clinical classification of MMR variants. PMID:26247049

Differential expression and alternative splicing of rice sulphate transporter family members regulate sulphur status during plant growth, development and stress conditions.

PubMed

Kumar, Smita; Asif, Mehar Hasan; Chakrabarty, Debasis; Tripathi, Rudra Deo; Trivedi, Prabodh Kumar

2011-06-01

Sulphur, an essential nutrient required for plant growth and development, is mainly taken up by the plants as inorganic sulphate from the soil and assimilated into the sulphur reductive pathway. The uptake and transport of sulphate in plants is carried out by transporters encoded by the sulphate transporter gene family. Plant sulphate transporters have been classified with respect to their protein sequences, kinetic properties and tissue-specific localization in Arabidopsis. Though sulphate transporter genes from few other plants have also been characterized, no detailed study with respect to the structure and expression of this family from rice has been carried out. Here, we present genome-wide identification, structural and expression analyses of the rice sulphate transporter gene family. Our analysis using microarray data and MPSS database suggests that 14 rice sulphate transporters are differentially expressed during growth and development in various tissues and during biotic and abiotic stresses. Our analysis also suggests differential accumulation of splice variants of OsSultr1;1 and OsSultr4;1 transcripts during these processes. Apart from known spliced variants, we report an unusual alternative splicing of OsSultr1;1 transcript related to sulphur supply in growth medium and during stress response. Taken together, our study suggests that differential expression and alternative splicing of members of the sulphate transporter family plays an important role in regulating cellular sulphur status required for growth and development and during stress conditions. These findings significantly advance our understanding of the posttranscriptional regulatory mechanisms operating to regulate sulphur demand by the plant.

The RNA helicase DDX39B and its paralog DDX39A regulate androgen receptor splice variant AR-V7 generation.

PubMed

Nakata, Daisuke; Nakao, Shoichi; Nakayama, Kazuhide; Araki, Shinsuke; Nakayama, Yusuke; Aparicio, Samuel; Hara, Takahito; Nakanishi, Atsushi

2017-01-29

Mounting evidence suggests that constitutively active androgen receptor (AR) splice variants, typified by AR-V7, are associated with poor prognosis and resistance to androgen deprivation therapy in prostate cancer patients. However, mechanisms governing the generation of AR splice variants are not fully understood. In this study, we aimed to investigate the dynamics of AR splice variant generation using the JDCaP prostate cancer model that expresses AR splice variants under androgen depletion. Microarray analysis of JDCaP xenografts before and after expression of AR splice variants suggested that dysregulation of RNA processing pathways is likely involved in AR splice variant generation. To explore factors contributing to generation of AR-V7 mRNA, we conducted a focused RNA interference screen in AR-V7-positive JDCaP-hr cells using an shRNA library targeting spliceosome-related genes. This screen identified DDX39B as a regulator of AR-V7 mRNA expression. Simultaneous knockdown of DDX39B and its paralog DDX39A drastically and selectively downregulated AR-V7 mRNA expression in multiple AR-V7-positive prostate cancer cell lines. DDX39B was upregulated in relapsed JDCaP xenografts expressing AR splice variants, suggesting its role in expression of AR splice variants. Taken together, our findings offer insight into the mechanisms of AR splice variant generation and identify DDX39 as a potential drug target for the treatment of AR splice variant-positive prostate cancer. Copyright © 2016 Elsevier Inc. All rights reserved.

Complexity of the Alternative Splicing Landscape in Plants[C][W][OPEN

PubMed Central

Reddy, Anireddy S.N.; Marquez, Yamile; Kalyna, Maria; Barta, Andrea

2013-01-01

Alternative splicing (AS) of precursor mRNAs (pre-mRNAs) from multiexon genes allows organisms to increase their coding potential and regulate gene expression through multiple mechanisms. Recent transcriptome-wide analysis of AS using RNA sequencing has revealed that AS is highly pervasive in plants. Pre-mRNAs from over 60% of intron-containing genes undergo AS to produce a vast repertoire of mRNA isoforms. The functions of most splice variants are unknown. However, emerging evidence indicates that splice variants increase the functional diversity of proteins. Furthermore, AS is coupled to transcript stability and translation through nonsense-mediated decay and microRNA-mediated gene regulation. Widespread changes in AS in response to developmental cues and stresses suggest a role for regulated splicing in plant development and stress responses. Here, we review recent progress in uncovering the extent and complexity of the AS landscape in plants, its regulation, and the roles of AS in gene regulation. The prevalence of AS in plants has raised many new questions that require additional studies. New tools based on recent technological advances are allowing genome-wide analysis of RNA elements in transcripts and of chromatin modifications that regulate AS. Application of these tools in plants will provide significant new insights into AS regulation and crosstalk between AS and other layers of gene regulation. PMID:24179125

Alternative splicing of SMPD1 coding for acid sphingomyelinase in major depression.

PubMed

Rhein, Cosima; Reichel, Martin; Kramer, Marcel; Rotter, Andrea; Lenz, Bernd; Mühle, Christiane; Gulbins, Erich; Kornhuber, Johannes

2017-02-01

Major depressive disorder (MDD) is a psychiatric disorder characterized by key symptoms that include depressed mood and a loss of interest and pleasure. A recently developed pathogenic model of MDD involves disturbed neurogenesis in the hippocampus, where the acid sphingomyelinase (ASM)/ceramide system plays an important role and is proposed as a molecular target for antidepressant action. Because alternative splicing of SMPD1 mRNA, coding for ASM, is relevant for the regulation of ASM enzymatic activity, we investigated the frequency of alternatively spliced ASM isoforms in peripheral blood cells of MDD patients versus healthy controls. Because the full-length transcript variant 1 of SMPD1 (termed ASM-1) is the only known form within the splicing pattern that encodes an enzymatically fully active ASM, we determined a fraction of splice isoforms deviating from ASM-1 using PCR amplification and capillary electrophoresis with laser-induced fluorescence analysis. ASM alternative splicing events occurred significantly less frequently in MDD patients compared to healthy subjects. After 5 days of antidepressant treatment, the frequency of alternatively spliced ASM isoforms decreased in those patients who were treated with a functional inhibitor of ASM activity (FIASMA) but remained constant in MDD patients treated with other antidepressant drugs. This effect was more pronounced when healthy male volunteers were treated with the FIASMAs fluoxetine or paroxetine, in contrast to a placebo group. Patients were treated with different antidepressant drugs, depending on individual parameters and disease courses. This study shows that the ASM alternative splicing pattern could be a biological target with diagnostic relevance and could serve as a novel biomarker for MDD. Copyright © 2016 Elsevier B.V. All rights reserved.

Alternative splicing of iodothyronine deiodinases in pituitary adenomas. Regulation by oncoprotein SF2/ASF.

PubMed

Piekielko-Witkowska, Agnieszka; Kedzierska, Hanna; Poplawski, Piotr; Wojcicka, Anna; Rybicka, Beata; Maksymowicz, Maria; Grajkowska, Wieslawa; Matyja, Ewa; Mandat, Tomasz; Bonicki, Wieslaw; Nauman, Pawel

2013-06-01

Pituitary tumors belong to the group of most common neoplasms of the sellar region. Iodothyronine deiodinase types 1 (DIO1) and 2 (DIO2) are enzymes contributing to the levels of locally synthesized T3, a hormone regulating key physiological processes in the pituitary, including its development, cellular proliferation, and hormone secretion. Previous studies revealed that the expression of deiodinases in pituitary tumors is variable and, moreover, there is no correlation between mRNA and protein products of the particular gene, suggesting the potential role of posttranscriptional regulatory mechanisms. In this work we hypothesized that one of such mechanisms could be the alternative splicing. Therefore, we analyzed expression and sequences of DIO1 and DIO2 splicing variants in 30 pituitary adenomas and 9 non-tumorous pituitary samples. DIO2 mRNA was expressed as only two mRNA isoforms. In contrast, nine splice variants of DIO1 were identified. Among them, five were devoid of exon 3. In silico sequence analysis of DIO1 revealed multiple putative binding sites for splicing factor SF2/ASF, of which the top-ranked sites were located in exon 3. Silencing of SF2/ASF in pituitary tumor GH3 cells resulted in change of ratio between DIO1 isoforms with or without exon 3, favoring the expression of variants without exon 3. The expression of SF2/ASF mRNA in pituitary tumors was increased when compared with non-neoplastic control samples. In conclusion, we provide a new mechanism of posttranscriptional regulation of DIO1 and show deregulation of DIO1 expression in pituitary adenoma, possibly resulting from disturbed expression of SF2/ASF. Copyright © 2013 Elsevier B.V. All rights reserved.

Isolation and characterization of novel RECK tumor suppressor gene splice variants

PubMed Central

Trombetta-Lima, Marina; Winnischofer, Sheila Maria Brochado; Demasi, Marcos Angelo Almeida; Filho, Renato Astorino; Carreira, Ana Claudia Oliveira; Wei, Beiyang; de Assis Ribas, Thais; Konig, Michelle Silberspitz; Bowman-Colin, Christian; Oba-Shinjo, Sueli Mieko; Marie, Suely Kazue Nagahashi; Stetler-Stevenson, William; Sogayar, Mari Cleide

2015-01-01

Glioblastoma multiforme is the most common and lethal of the central nervous system glial-derived tumors. RECK suppresses tumor invasion by negatively regulating at least three members of the matrix metalloproteinase family: MMP-9, MMP-2, and MT1-MMP. A positive correlation has been observed between the abundance of RECK expression in tumor samples and a more favorable prognosis for patients with several types of tumors. In the present study, novel alternatively spliced variants of the RECK gene: RECK-B and RECK-I were isolated by RT-PCR and sequenced. The expression levels and profiles of these alternative RECK transcripts, as well as canonical RECK were determined in tissue samples of malignant astrocytomas of different grades and in a normal tissue RNA panel by qRT-PCR. Our results show that higher canonical RECK expression, accompanied by a higher canonical to alternative transcript expression ratio, positively correlates with higher overall survival rate after chemotherapeutic treatment of GBM patients. U87MG and T98G cells over-expressing the RECK-B alternative variant display higher anchorage-independent clonal growth and do not display modulation of, respectively, MMP-2 and MMP-9 expression. Our findings suggest that RECK transcript variants might have opposite roles in GBM biology and the ratio of their expression levels may be informative for the prognostic outcome of GBM patients. PMID:26431549

Comprehensive splicing functional analysis of DNA variants of the BRCA2 gene by hybrid minigenes

PubMed Central

2012-01-01

Introduction The underlying pathogenic mechanism of a large fraction of DNA variants of disease-causing genes is the disruption of the splicing process. We aimed to investigate the effect on splicing of the BRCA2 variants c.8488-1G > A (exon 20) and c.9026_9030del (exon 23), as well as 41 BRCA2 variants reported in the Breast Cancer Information Core (BIC) mutation database. Methods DNA variants were analyzed with the splicing prediction programs NNSPLICE and Human Splicing Finder. Functional analyses of candidate variants were performed by lymphocyte RT-PCR and/or hybrid minigene assays. Forty-one BIC variants of exons 19, 20, 23 and 24 were bioinformatically selected and generated by PCR-mutagenesis of the wild type minigenes. Results Lymphocyte RT-PCR of c.8488-1G > A showed intron 19 retention and a 12-nucleotide deletion in exon 20, whereas c.9026_9030del did not show any splicing anomaly. Minigene analysis of c.8488-1G > A displayed the aforementioned aberrant isoforms but also exon 20 skipping. We further evaluated the splicing outcomes of 41 variants of four BRCA2 exons by minigene analysis. Eighteen variants presented splicing aberrations. Most variants (78.9%) disrupted the natural splice sites, whereas four altered putative enhancers/silencers and had a weak effect. Fluorescent RT-PCR of minigenes accurately detected 14 RNA isoforms generated by cryptic site usage, exon skipping and intron retention events. Fourteen variants showed total splicing disruptions and were predicted to truncate or eliminate essential domains of BRCA2. Conclusions A relevant proportion of BRCA2 variants are correlated with splicing disruptions, indicating that RNA analysis is a valuable tool to assess the pathogenicity of a particular DNA change. The minigene system is a straightforward and robust approach to detect variants with an impact on splicing and contributes to a better knowledge of this gene expression step. PMID:22632462

A genome-wide aberrant RNA splicing in patients with acute myeloid leukemia identifies novel potential disease markers and therapeutic targets.

PubMed

Adamia, Sophia; Haibe-Kains, Benjamin; Pilarski, Patrick M; Bar-Natan, Michal; Pevzner, Samuel; Avet-Loiseau, Herve; Lode, Laurence; Verselis, Sigitas; Fox, Edward A; Burke, John; Galinsky, Ilene; Dagogo-Jack, Ibiayi; Wadleigh, Martha; Steensma, David P; Motyckova, Gabriela; Deangelo, Daniel J; Quackenbush, John; Stone, Richard; Griffin, James D

2014-03-01

Despite new treatments, acute myeloid leukemia (AML) remains an incurable disease. More effective drug design requires an expanded view of the molecular complexity that underlies AML. Alternative splicing of RNA is used by normal cells to generate protein diversity. Growing evidence indicates that aberrant splicing of genes plays a key role in cancer. We investigated genome-wide splicing abnormalities in AML and based on these abnormalities, we aimed to identify novel potential biomarkers and therapeutic targets. We used genome-wide alternative splicing screening to investigate alternative splicing abnormalities in two independent AML patient cohorts [Dana-Farber Cancer Institute (DFCI) (Boston, MA) and University Hospital de Nantes (UHN) (Nantes, France)] and normal donors. Selected splicing events were confirmed through cloning and sequencing analysis, and than validated in 193 patients with AML. Our results show that approximately 29% of expressed genes genome-wide were differentially and recurrently spliced in patients with AML compared with normal donors bone marrow CD34(+) cells. Results were reproducible in two independent AML cohorts. In both cohorts, annotation analyses indicated similar proportions of differentially spliced genes encoding several oncogenes, tumor suppressor proteins, splicing factors, and heterogeneous-nuclear-ribonucleoproteins, proteins involved in apoptosis, cell proliferation, and spliceosome assembly. Our findings are consistent with reports for other malignances and indicate that AML-specific aberrations in splicing mechanisms are a hallmark of AML pathogenesis. Overall, our results suggest that aberrant splicing is a common characteristic for AML. Our findings also suggest that splice variant transcripts that are the result of splicing aberrations create novel disease markers and provide potential targets for small molecules or antibody therapeutics for this disease. ©2013 AACR

Cancer-Associated Perturbations in Alternative Pre-messenger RNA Splicing.

PubMed

Shkreta, Lulzim; Bell, Brendan; Revil, Timothée; Venables, Julian P; Prinos, Panagiotis; Elela, Sherif Abou; Chabot, Benoit

2013-01-01

For most of our 25,000 genes, the removal of introns by pre-messenger RNA (pre-mRNA) splicing represents an essential step toward the production of functional messenger RNAs (mRNAs). Alternative splicing of a single pre-mRNA results in the production of different mRNAs. Although complex organisms use alternative splicing to expand protein function and phenotypic diversity, patterns of alternative splicing are often altered in cancer cells. Alternative splicing contributes to tumorigenesis by producing splice isoforms that can stimulate cell proliferation and cell migration or induce resistance to apoptosis and anticancer agents. Cancer-specific changes in splicing profiles can occur through mutations that are affecting splice sites and splicing control elements, and also by alterations in the expression of proteins that control splicing decisions. Recent progress in global approaches that interrogate splicing diversity should help to obtain specific splicing signatures for cancer types. The development of innovative approaches for annotating and reprogramming splicing events will more fully establish the essential contribution of alternative splicing to the biology of cancer and will hopefully provide novel targets and anticancer strategies. Metazoan genes are usually made up of several exons interrupted by introns. The introns are removed from the pre-mRNA by RNA splicing. In conjunction with other maturation steps, such as capping and polyadenylation, the spliced mRNA is then transported to the cytoplasm to be translated into a functional protein. The basic mechanism of splicing requires accurate recognition of each extremity of each intron by the spliceosome. Introns are identified by the binding of U1 snRNP to the 5' splice site and the U2AF65/U2AF35 complex to the 3' splice site. Following these interactions, other proteins and snRNPs are recruited to generate the complete spliceosomal complex needed to excise the intron. While many introns are constitutively removed by the spliceosome, other splice junctions are not used systematically, generating the phenomenon of alternative splicing. Alternative splicing is therefore the process by which a single species of pre-mRNA can be matured to produce different mRNA molecules (Fig. 1). Depending on the number and types of alternative splicing events, a pre-mRNA can generate from two to several thousands different mRNAs leading to the production of a corresponding number of proteins. It is now believed that the expression of at least 70 % of human genes is subjected to alternative splicing, implying an enormous contribution to proteomic diversity, and by extension, to the development and the evolution of complex animals. Defects in splicing have been associated with human diseases (Caceres and Kornblihtt, Trends Genet 18(4):186-93, 2002, Cartegni et al., Nat Rev Genet 3(4):285-98, 2002, Pagani and Baralle, Nat Rev Genet 5(5):389-96, 2004), including cancer (Brinkman, Clin Biochem 37(7):584-94, 2004, Venables, Bioessays 28(4):378-86, 2006, Srebrow and Kornblihtt, J Cell Sci 119(Pt 13):2635-2641, 2006, Revil et al., Bull Cancer 93(9):909-919, 2006, Venables, Transworld Res Network, 2006, Pajares et al., Lancet Oncol 8(4):349-57, 2007, Skotheim and Nees, Int J Biochem Cell Biol 39:1432-1449, 2007). Numerous studies have now confirmed the existence of specific differences in the alternative splicing profiles between normal and cancer tissues. Although there are a few cases where specific mutations are the primary cause for these changes, global alterations in alternative splicing in cancer cells may be primarily derived from changes in the expression of RNA-binding proteins that control splice site selection. Overall, these cancer-specific differences in alternative splicing offer an immense potential to improve the diagnosis and the prognosis of cancer. This review will focus on the functional impact of cancer-associated alternative splicing variants, the molecular determinants that alter the splicing decisions in cancer cells, and future therapeutic strategies.

Opioid Receptors: Toward Separation of Analgesic from Undesirable Effects

PubMed Central

Law, P.Y.; Reggio, Patricia H.; Loh, H.H.

2013-01-01

The use of opioid analgesics for pain has always been hampered by their many side effects; in particular, the addictive liability associated with chronic use. Recently, attempts to develop analgesic agents with reduced side effects have targeted either the putative opioid receptor splice variants or the receptor heterooligomers. This review discusses the potential for receptor splice variant- and the hetero-oligomer-based discovery of new opioid analgesics. We also examine an alternative approach of using receptor mutants for pain management. Finally, we discuss the role of the biased agonism observed and the recently reported opioid receptor crystal structures in guiding the future development of opioid analgesics PMID:23598157

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

Huang, Ze; Shuldiner, A.R.; Zenilman, M.E.

There are two insulin receptor (IR) isoforms (designated type A and type B), derived from alternative splicing of exon 11 of the IR gene. Recently, we reported that an increase in the exon 11- (i.e. lacking exon 11) (type A) IR messenger RNA (mRNA) variant in muscle is associated with hyperinsulinemia, an early risk factor for noninsulin-dependent diabetes mellitus (NIDDM), in the spontaneously obese, diabetic rhesus monkey. To explore further the role of IR mRNA splicing in insulin resistance of NIDDM, we studied liver, another target organ that is resistant to insulin action in NIDDM. The relative amounts of themore » two IR mRNA-splicing variants in liver were quantitated by RT-PCR in normal, prediabetic, and diabetic (NIDDM) monkeys. The percentage of the exon 11- mRNA variant in liver (n = 24) was significantly correlated with fasting plasma glucose (r = 0.55, P < 0.01) and intravenous glucose disappearance rate (r = -0.45, P < 0.05). The exon 11- mRNA variant was increased significantly from 29.8 {+-} 1.6% in monkeys with normal fasting glucose to 39.2 {+-} 2.9% in monkeys with elevated fasting glucose (P < 0.01). These studies provide the first direct evidence in vivo that the relative expression of the two IR mRNA-splicing variants is altered in liver and suggest that increased expression of the exon 11- IR isoform may contribute to hepatic insulin resistance and NIDDM or may compensate for some yet unidentified defect. 33 refs., 3 figs., 1 tab.« less

Alternative splicing variants of human Fbx4 disturb cyclin D1 proteolysis in human cancer

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

Chu, Xiufeng; Zhang, Ting; Wang, Jie

2014-04-25

Highlights: • The expression of Fbx4 was significantly lower in HCC tissues. • Novel splicing variants of Fbx4 were identified. • These novel variants are much more abundant in human cancer tissues and cells. • The novel Fbx4 isoforms could promote cell proliferation and migration in vitro. • These isoforms showed less capability for cyclin D1 binding and degradation. - Abstract: Fbx4 is a specific substrate recognition component of SCF ubiquitin ligases that catalyzes the ubiquitination and subsequent degradation of cyclin D1 and Trx1. Two isoforms of human Fbx4 protein, the full length Fbx4α and the C-terminal truncated Fbx4β havemore » been identified, but their functions remain elusive. In this study, we demonstrated that the mRNA level of Fbx4 was significantly lower in hepatocellular carcinoma tissues than that in the corresponding non-tumor tissues. More importantly, we identified three novel splicing variants of Fbx4: Fbx4γ (missing 168–245nt of exon1), Fbx4δ (missing exon6) and a N-terminal reading frame shift variant (missing exon2). Using cloning sequencing and RT-PCR, we demonstrated these novel splice variants are much more abundant in human cancer tissues and cell lines than that in normal tissues. When expressed in Sk-Hep1 and NIH3T3 cell lines, Fbx4β, Fbx4γ and Fbx4δ could promote cell proliferation and migration in vitro. Concordantly, these isoforms could disrupt cyclin D1 degradation and therefore increase cyclin D1 expression. Moreover, unlike the full-length isoform Fbx4α that mainly exists in cytoplasm, Fbx4β, Fbx4γ, and Fbx4δ locate in both cytoplasm and nucleus. Since cyclin D1 degradation takes place in cytoplasm, the nuclear distribution of these Fbx4 isoforms may not be involved in the down-regulation of cytoplasmic cyclin D1. These results define the impact of alternative splicing on Fbx4 function, and suggest that the attenuated cyclin D1 degradation by these novel Fbx4 isoforms provides a new insight for aberrant cyclin D1 expression in human cancers.« less

Differentially regulated splice variants and systems biology analysis of Kaposi's sarcoma-associated herpesvirus-infected lymphatic endothelial cells.

PubMed

Chang, Ting-Yu; Wu, Yu-Hsuan; Cheng, Cheng-Chung; Wang, Hsei-Wei

2011-09-01

Alternative RNA splicing greatly increases proteome diversity, and the possibility of studying genome-wide alternative splicing (AS) events becomes available with the advent of high-throughput genomics tools devoted to this issue. Kaposi's sarcoma associated herpesvirus (KSHV) is the etiological agent of KS, a tumor of lymphatic endothelial cell (LEC) lineage, but little is known about the AS variations induced by KSHV. We analyzed KSHV-controlled AS using high-density microarrays capable of detecting all exons in the human genome. Splicing variants and altered exon-intron usage in infected LEC were found, and these correlated with protein domain modification. The different 3'-UTR used in new transcripts also help isoforms to escape microRNA-mediated surveillance. Exome-level analysis further revealed information that cannot be disclosed using classical gene-level profiling: a significant exon usage difference existed between LEC and CD34(+) precursor cells, and KSHV infection resulted in LEC-to-precursor, dedifferentiation-like exon level reprogramming. Our results demonstrate the application of exon arrays in systems biology research, and suggest the regulatory effects of AS in endothelial cells are far more complex than previously observed. This extra layer of molecular diversity helps to account for various aspects of endothelial biology, KSHV life cycle and disease pathogenesis that until now have been unexplored.

Functional analysis of alternative transcripts of the soybean Rj2 gene that restricts nodulation with specific rhizobial strains.

PubMed

Tang, F; Yang, S; Zhu, H

2016-05-01

The Rj2 gene is a TIR-NBS-LRR-type resistance gene in soybean (Glycine max) that restricts root nodule symbiosis with a group of Bradyrhizobium japonicum strains including USDA122. Rj2 generates two distinct transcript variants in its expression profile through alternative splicing. Alternative splicing of Rj2 is caused by the retention of the 86-bp intron 4. Inclusion of intron 4 in mature mRNA introduces an in-frame stop codon; as such, the alternative transcript is predicted to encode a truncated protein consisting of the entire portion of the TIR, NBS and LRR domains but missing the C-terminal domain of the full-length Rj2 protein encoded by the regular transcript. Since alternative splicing has been shown to be essential for full activity of several plant R genes, we attempted to test whether the alternative splicing is required for Rj2-mediated nodulation restriction. Here we demonstrated that the Rj2-mediated nodulation restriction does not require the combined presence of the regular and alternative transcripts, and the expression of the regular transcript alone is sufficient to confer nodulation restriction. © 2016 German Botanical Society and The Royal Botanical Society of the Netherlands.

Experimental Assessment of Splicing Variants Using Expression Minigenes and Comparison with In Silico Predictions

PubMed Central

Sharma, Neeraj; Sosnay, Patrick R.; Ramalho, Anabela S.; Douville, Christopher; Franca, Arianna; Gottschalk, Laura B.; Park, Jeenah; Lee, Melissa; Vecchio-Pagan, Briana; Raraigh, Karen S.; Amaral, Margarida D.; Karchin, Rachel; Cutting, Garry R.

2015-01-01

Assessment of the functional consequences of variants near splice sites is a major challenge in the diagnostic laboratory. To address this issue, we created expression minigenes (EMGs) to determine the RNA and protein products generated by splice site variants (n = 10) implicated in cystic fibrosis (CF). Experimental results were compared with the splicing predictions of eight in silico tools. EMGs containing the full-length Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) coding sequence and flanking intron sequences generated wild-type transcript and fully processed protein in Human Embryonic Kidney (HEK293) and CF bronchial epithelial (CFBE41o-) cells. Quantification of variant induced aberrant mRNA isoforms was concordant using fragment analysis and pyrosequencing. The splicing patterns of c.1585−1G>A and c.2657+5G>A were comparable to those reported in primary cells from individuals bearing these variants. Bioinformatics predictions were consistent with experimental results for 9/10 variants (MES), 8/10 variants (NNSplice), and 7/10 variants (SSAT and Sroogle). Programs that estimate the consequences of mis-splicing predicted 11/16 (HSF and ASSEDA) and 10/16 (Fsplice and SplicePort) experimentally observed mRNA isoforms. EMGs provide a robust experimental approach for clinical interpretation of splice site variants and refinement of in silico tools. PMID:25066652

L-Dopa decarboxylase expression profile in human cancer cells.

PubMed

Chalatsa, Ioanna; Nikolouzou, Eleftheria; Fragoulis, Emmanuel G; Vassilacopoulou, Dido

2011-02-01

L-Dopa decarboxylase (DDC) catalyses the decarboxylation of L-Dopa. It has been shown that the DDC gene undergoes alternative splicing within its 5'-untranslated region (UTR), in a tissue-specific manner, generating identical protein products. The employment of two alternative 5'UTRs is thought to be responsible for tissue-specific expression of the human DDC mRNA. In this study, we focused on the investigation of the nature of the mRNA expression in human cell lines of neural and non-neural origin. Our results show the expression of a neural-type DDC mRNA splice variant, lacking exon 3 in all cell lines studied. Co-expression of the full length non-neural DDC mRNA and the neural-type DDC splice variant lacking exon 3 was detected in all cell lines. The alternative DDC protein isoform, Alt-DDC, was detected in SH-SY5Y and HeLa cells. Our findings suggest that the human DDC gene undergoes complex processing, leading to the formation of multiple mRNA isoforms. The study of the significance of this phenomenon of multiple DDC mRNA isoforms could provide us with new information leading to the elucidation of the complex biological pathways that the human enzyme is involved in.

Intragenic motifs regulate the transcriptional complexity of Pkhd1/PKHD1

PubMed Central

Boddu, Ravindra; Yang, Chaozhe; O’Connor, Amber K.; Hendrickson, Robert Curtis; Boone, Braden; Cui, Xiangqin; Garcia-Gonzalez, Miguel; Igarashi, Peter; Onuchic, Luiz F.; Germino, Gregory G.

2014-01-01

Autosomal recessive polycystic kidney disease (ARPKD) results from mutations in the human PKHD1 gene. Both this gene, and its mouse ortholog, Pkhd1, are primarily expressed in renal and biliary ductal structures. The mouse protein product, fibrocystin/polyductin complex (FPC), is a 445-kDa protein encoded by a 67-exon transcript that spans >500 kb of genomic DNA. In the current study, we observed multiple alternatively spliced Pkhd1 transcripts that varied in size and exon composition in embryonic mouse kidney, liver, and placenta samples, as well as among adult mouse pancreas, brain, heart, lung, testes, liver, and kidney. Using reverse transcription PCR and RNASeq, we identified 22 novel Pkhd1 kidney transcripts with unique exon junctions. Various mechanisms of alternative splicing were observed, including exon skipping, use of alternate acceptor/donor splice sites, and inclusion of novel exons. Bioinformatic analyses identified, and exon-trapping minigene experiments validated, consensus binding sites for serine/arginine-rich proteins that modulate alternative splicing. Using site-directed mutagenesis, we examined the functional importance of selected splice enhancers. In addition, we demonstrated that many of the novel transcripts were polysome bound, thus likely translated. Finally, we determined that the human PKHD1 R760H missense variant alters a splice enhancer motif that disrupts exon splicing in vitro and is predicted to truncate the protein. Taken together, these data provide evidence of the complex transcriptional regulation of Pkhd1/PKHD1 and identified motifs that regulate its splicing. Our studies indicate that Pkhd1/PKHD1 transcription is modulated, in part by intragenic factors, suggesting that aberrant PKHD1 splicing represents an unappreciated pathogenic mechanism in ARPKD. PMID:24984783

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.

Genome wide identification of aberrant alternative splicing events in myotonic dystrophy type 2.

PubMed

Perfetti, Alessandra; Greco, Simona; Fasanaro, Pasquale; Bugiardini, Enrico; Cardani, Rosanna; Garcia-Manteiga, Jose M; Manteiga, Jose M Garcia; Riba, Michela; Cittaro, Davide; Stupka, Elia; Meola, Giovanni; Martelli, Fabio

2014-01-01

Myotonic dystrophy type 2 (DM2) is a genetic, autosomal dominant disease due to expansion of tetraplet (CCTG) repetitions in the first intron of the ZNF9/CNBP gene. DM2 is a multisystemic disorder affecting the skeletal muscle, the heart, the eye and the endocrine system. According to the proposed pathological mechanism, the expanded tetraplets have an RNA toxic effect, disrupting the splicing of many mRNAs. Thus, the identification of aberrantly spliced transcripts is instrumental for our understanding of the molecular mechanisms underpinning the disease. The aim of this study was the identification of new aberrant alternative splicing events in DM2 patients. By genome wide analysis of 10 DM2 patients and 10 controls (CTR), we identified 273 alternative spliced exons in 218 genes. While many aberrant splicing events were already identified in the past, most were new. A subset of these events was validated by qPCR assays in 19 DM2 and 15 CTR subjects. To gain insight into the molecular pathways involving the identified aberrantly spliced genes, we performed a bioinformatics analysis with Ingenuity system. This analysis indicated a deregulation of development, cell survival, metabolism, calcium signaling and contractility. In conclusion, our genome wide analysis provided a database of aberrant splicing events in the skeletal muscle of DM2 patients. The affected genes are involved in numerous pathways and networks important for muscle physio-pathology, suggesting that the identified variants may contribute to DM2 pathogenesis.

Genome Wide Identification of Aberrant Alternative Splicing Events in Myotonic Dystrophy Type 2

PubMed Central

Fasanaro, Pasquale; Bugiardini, Enrico; Cardani, Rosanna; Manteiga, Jose M. Garcia.; Riba, Michela; Cittaro, Davide; Stupka, Elia; Meola, Giovanni; Martelli, Fabio

2014-01-01

Myotonic dystrophy type 2 (DM2) is a genetic, autosomal dominant disease due to expansion of tetraplet (CCTG) repetitions in the first intron of the ZNF9/CNBP gene. DM2 is a multisystemic disorder affecting the skeletal muscle, the heart, the eye and the endocrine system. According to the proposed pathological mechanism, the expanded tetraplets have an RNA toxic effect, disrupting the splicing of many mRNAs. Thus, the identification of aberrantly spliced transcripts is instrumental for our understanding of the molecular mechanisms underpinning the disease. The aim of this study was the identification of new aberrant alternative splicing events in DM2 patients. By genome wide analysis of 10 DM2 patients and 10 controls (CTR), we identified 273 alternative spliced exons in 218 genes. While many aberrant splicing events were already identified in the past, most were new. A subset of these events was validated by qPCR assays in 19 DM2 and 15 CTR subjects. To gain insight into the molecular pathways involving the identified aberrantly spliced genes, we performed a bioinformatics analysis with Ingenuity system. This analysis indicated a deregulation of development, cell survival, metabolism, calcium signaling and contractility. In conclusion, our genome wide analysis provided a database of aberrant splicing events in the skeletal muscle of DM2 patients. The affected genes are involved in numerous pathways and networks important for muscle physio-pathology, suggesting that the identified variants may contribute to DM2 pathogenesis. PMID:24722564

ABCA4 midigenes reveal the full splice spectrum of all reported noncanonical splice site variants in Stargardt disease.

PubMed

Sangermano, Riccardo; Khan, Mubeen; Cornelis, Stéphanie S; Richelle, Valerie; Albert, Silvia; Garanto, Alejandro; Elmelik, Duaa; Qamar, Raheel; Lugtenberg, Dorien; van den Born, L Ingeborgh; Collin, Rob W J; Cremers, Frans P M

2018-01-01

Stargardt disease is caused by variants in the ABCA4 gene, a significant part of which are noncanonical splice site (NCSS) variants. In case a gene of interest is not expressed in available somatic cells, small genomic fragments carrying potential disease-associated variants are tested for splice abnormalities using in vitro splice assays. We recently discovered that when using small minigenes lacking the proper genomic context, in vitro results do not correlate with splice defects observed in patient cells. We therefore devised a novel strategy in which a bacterial artificial chromosome was employed to generate midigenes, splice vectors of varying lengths (up to 11.7 kb) covering almost the entire ABCA4 gene. These midigenes were used to analyze the effect of all 44 reported and three novel NCSS variants on ABCA4 pre-mRNA splicing. Intriguingly, multi-exon skipping events were observed, as well as exon elongation and intron retention. The analysis of all reported NCSS variants in ABCA4 allowed us to reveal the nature of aberrant splicing events and to classify the severity of these mutations based on the residual fraction of wild-type mRNA. Our strategy to generate large overlapping splice vectors carrying multiple exons, creating a toolbox for robust and high-throughput analysis of splice variants, can be applied to all human genes. © 2018 Sangermano et al.; Published by Cold Spring Harbor Laboratory Press.

Splicing factor NSSR1 reduces neuronal injury after mouse transient global cerebral ischemia.

PubMed

Qi, Yao; Li, Ya; Cui, Shi-Chao; Zhao, Jing-Jing; Liu, Xiao-Yan; Ji, Chun-Xia; Sun, Feng-Yan; Xu, Ping; Chen, Xian-Hua

2015-05-01

This study focuses on the function of NSSR1, a splicing factor, in neuronal injury in the ischemic mouse brain using the transient global cerebral ischemic mouse model and the cultured cells treated with oxygen-glucose deprivation (OGD). The results showed that the cerebral ischemia triggers the expression of NSSR1 in hippocampal astrocytes, predominantly the dephosphorylated NSSR1 proteins, and the Exon3 inclusive NCAM-L1 variant and the Exon4 inclusive CREB variant. While in the hippocampus of astrocyte-specific NSSR1 conditional knockdown (cKD) mice, where cerebral ischemia no longer triggers NSSR1 expression in astrocytes, the expression of Exon3 inclusive NCAM-L1 variant and Exon4 inclusive CREB variant were no longer triggered as well. In addition, the injury of hippocampal neurons was more severe in astrocyte-specific NSSR1 cKD mice compared with in wild-type mice after brain ischemia. Of note, the culture media harvested from the astrocytes with overexpression of NSSR1 or the Exon3 inclusive NCAM-L1 variant, or Exon4 inclusive CREB variant were all able to reduce the neuronal injury induced by OGD. The results provide the evidence demonstrating that: (1) Splicing factor NSSR1 is a new factor involved in reducing ischemic injury. (2) Ischemia induces NSSR1 expression in astrocytes, not in neurons. (3) NSSR1-mediated pathway in astrocytes is required for reducing ischemic neuronal injury. (4) NCAM-L1 and CREB are probably mediators in NSSR1-mediated pathway. In conclusion, our results suggest for the first time that NSSR1 may provide a novel mechanism for reducing neuronal injury after ischemia, probably through regulation on alternative splicing of NCAM-L1 and CREB in astrocytes. © 2014 Wiley Periodicals, Inc.

Splicing-related single nucleotide polymorphism of RAB, member of RAS oncogene family like 2B (RABL2B) jeopardises semen quality in Chinese Holstein bulls.

PubMed

Wang, Xiuge; Cui, Xiaohui; Zhang, Yan; Hao, Haisheng; Ju, Zhihua; Liu, Deyu; Jiang, Qiang; Yang, Chunhong; Sun, Yan; Wang, Changfa; Huang, Jinming; Zhu, Huabin

2017-11-01

RAB, member of RAS oncogene family like 2B (RABL2B) is a member of a poorly characterised clade of the RAS GTPase superfamily, which plays an essential role in male fertility, sperm intraflagellar transport and tail assembly. In the present study, we identified a novel RABL2B splice variant in bovine testis and spermatozoa. This splice variant, designated RABL2B-TV, is characterised by exon 2 skipping. Moreover, a single nucleotide polymorphism (SNP), namely c.125G>A, was found within the exonic splicing enhancer (ESE) motif, indicating that the SNP caused the production of the RABL2B-TV aberrant splice variant. This was demonstrated by constructing a pSPL3 exon capturing vector with different genotypes and transfecting these vectors into murine Leydig tumour cell line (MLTC-1) cells. Expression of the RABL2B-TV transcript was lower in semen from high- versus low-performance bulls. Association analysis showed that sperm deformity rate was significantly lower in Chinese Holstein bulls with the GG or GA genotype than in bulls with the AA genotype (P<0.05). In addition, initial sperm motility was significantly higher in individuals with the GG or GA genotype than in individuals with the AA genotype (P<0.05). The findings of the present study suggest that the difference in semen quality in bulls with different RABL2B genotypes is generated via an alternative splicing mechanism caused by a functional SNP within the ESE motif.

Region-specific expression of brain-derived neurotrophic factor splice variants in morphine conditioned place preference in mice.

PubMed

Meng, Min; Zhao, Xinhan; Dang, Yonghui; Ma, Jingyuan; Li, Lixu; Gu, Shanzhi

2013-06-26

It is well established that brain-derived neurotrophic factor (BDNF) plays a pivotal role in brain plasticity-related processes, such as learning, memory and drug addiction. However, changes in expression of BDNF splice variants after acquisition, extinction and reinstatement of cue-elicited morphine seeking behavior have not yet been investigated. Real-time PCR was used to assess BDNF splice variants (I, II, IV and VI) in various brain regions during acquisition, extinction and reinstatement of morphine-conditioned place preference (CPP) in mice. Repeated morphine injections (10mg/kg, i.p.) increased expression of BDNF splice variants II, IV and VI in the hippocampus, caudate putamen (CPu) and nucleus accumbens (NAcc). Levels of BDNF splice variants decreased after extinction training and continued to decrease during reinstatement induced by a morphine priming injection (10mg/kg, i.p.). However, after reinstatement induced by exposure to 6 min of forced swimming (FS), expression of BDNF splice variants II, IV and VI was increased in the hippocampus, CPu, NAcc and prefrontal cortex (PFC). After reinstatement induced by 40 min of restraint, expression of BDNF splice variants was increased in PFC. These results show that exposure to either morphine or acute stress can induce reinstatement of drug-seeking, but expression of BDNF splice variants is differentially affected by chronic morphine and acute stress. Furthermore, BDNF splice variants II, IV and VI may play a role in learning and memory for morphine addiction in the hippocampus, CPu and NAcc. Crown Copyright © 2013. Published by Elsevier B.V. All rights reserved.

A Heroin Addiction Severity-Associated Intronic Single Nucleotide Polymorphism Modulates Alternative Pre-mRNA Splicing of the μ Opioid Receptor Gene OPRM1 via hnRNPH Interactions

PubMed Central

Xu, Jin; Lu, Zhigang; Xu, Mingming; Pan, Ling; Deng, Yi; Xie, Xiaohu; Liu, Huifen; Ding, Shixiong; Hurd, Yasmin L.; Pasternak, Gavril W.; Klein, Robert J.; Cartegni, Luca

2014-01-01

Single nucleotide polymorphisms (SNPs) in the OPRM1 gene have been associated with vulnerability to opioid dependence. The current study identifies an association of an intronic SNP (rs9479757) with the severity of heroin addiction among Han-Chinese male heroin addicts. Individual SNP analysis and haplotype-based analysis with additional SNPs in the OPRM1 locus showed that mild heroin addiction was associated with the AG genotype, whereas severe heroin addiction was associated with the GG genotype. In vitro studies such as electrophoretic mobility shift assay, minigene, siRNA, and antisense morpholino oligonucleotide studies have identified heterogeneous nuclear ribonucleoprotein H (hnRNPH) as the major binding partner for the G-containing SNP site. The G-to-A transition weakens hnRNPH binding and facilitates exon 2 skipping, leading to altered expressions of OPRM1 splice-variant mRNAs and hMOR-1 proteins. Similar changes in splicing and hMOR-1 proteins were observed in human postmortem prefrontal cortex with the AG genotype of this SNP when compared with the GG genotype. Interestingly, the altered splicing led to an increase in hMOR-1 protein levels despite decreased hMOR-1 mRNA levels, which is likely contributed by a concurrent increase in single transmembrane domain variants that have a chaperone-like function on MOR-1 protein stability. Our studies delineate the role of this SNP as a modifier of OPRM1 alternative splicing via hnRNPH interactions, and suggest a functional link between an SNP-containing splicing modifier and the severity of heroin addiction. PMID:25122903

The E2A splice variant E47 regulates the differentiation of projection neurons via p57(KIP2) during cortical development.

PubMed

Pfurr, Sabrina; Chu, Yu-Hsuan; Bohrer, Christian; Greulich, Franziska; Beattie, Robert; Mammadzada, Könül; Hils, Miriam; Arnold, Sebastian J; Taylor, Verdon; Schachtrup, Kristina; Uhlenhaut, N Henriette; Schachtrup, Christian

2017-11-01

During corticogenesis, distinct classes of neurons are born from progenitor cells located in the ventricular and subventricular zones, from where they migrate towards the pial surface to assemble into highly organized layer-specific circuits. However, the precise and coordinated transcriptional network activity defining neuronal identity is still not understood. Here, we show that genetic depletion of the basic helix-loop-helix (bHLH) transcription factor E2A splice variant E47 increased the number of Tbr1-positive deep layer and Satb2-positive upper layer neurons at E14.5, while depletion of the alternatively spliced E12 variant did not affect layer-specific neurogenesis. While ChIP-Seq identified a big overlap for E12- and E47-specific binding sites in embryonic NSCs, including sites at the cyclin-dependent kinase inhibitor (CDKI) Cdkn1c gene locus, RNA-Seq revealed a unique transcriptional regulation by each splice variant. E47 activated the expression of the CDKI Cdkn1c through binding to a distal enhancer. Finally, overexpression of E47 in embryonic NSCs in vitro impaired neurite outgrowth, and overexpression of E47 in vivo by in utero electroporation disturbed proper layer-specific neurogenesis and upregulated p57(KIP2) expression. Overall, this study identifies E2A target genes in embryonic NSCs and demonstrates that E47 regulates neuronal differentiation via p57(KIP2). © 2017. Published by The Company of Biologists Ltd.

The consensus sequence of FAMLF alternative splice variants is overexpressed in undifferentiated hematopoietic cells.

PubMed

Chen, W L; Luo, D F; Gao, C; Ding, Y; Wang, S Y

2015-07-01

The familial acute myeloid leukemia related factor gene (FAMLF) was previously identified from a familial AML subtractive cDNA library and shown to undergo alternative splicing. This study used real-time quantitative PCR to investigate the expression of the FAMLF alternative-splicing transcript consensus sequence (FAMLF-CS) in peripheral blood mononuclear cells (PBMCs) from 119 patients with de novo acute leukemia (AL) and 104 healthy controls, as well as in CD34+ cells from 12 AL patients and 10 healthy donors. A 429-bp fragment from a novel splicing variant of FAMLF was obtained, and a 363-bp consensus sequence was targeted to quantify total FAMLF expression. Kruskal-Wallis, Nemenyi, Spearman's correlation, and Mann-Whitney U-tests were used to analyze the data. FAMLF-CS expression in PBMCs from AL patients and CD34+ cells from AL patients and controls was significantly higher than in control PBMCs (P < 0.0001). Moreover, FAMLF-CS expression in PBMCs from the AML group was positively correlated with red blood cell count (rs =0.317, P=0.006), hemoglobin levels (rs = 0.210, P = 0.049), and percentage of peripheral blood blasts (rs = 0.256, P = 0.027), but inversely correlated with hemoglobin levels in the control group (rs = -0.391, P < 0.0001). AML patients with high CD34+ expression showed significantly higher FAMLF-CS expression than those with low CD34+ expression (P = 0.041). Our results showed that FAMLF is highly expressed in both normal and malignant immature hematopoietic cells, but that expression is lower in normal mature PBMCs.

Cardiac CRFR1 Expression Is Elevated in Human Heart Failure and Modulated by Genetic Variation and Alternative Splicing

PubMed Central

Lewis, Kathy A.; Perrin, Marilyn H.; Sweet, Wendy E.; Moravec, Christine S.; Tang, W. H. Wilson; Huising, Mark O.; Troughton, Richard W.; Cameron, Vicky A.

2016-01-01

Corticotropin-releasing factor (CRF) and the CRF-related peptides, urocortin (Ucn)-1, Ucn2, and Ucn3 signal through receptors CRFR1 and CRFR2 to restore homeostasis in response to stress. The Ucns exert potent cardioprotective effects and may have clinical utility in heart failure. To explore the activity of this system in the heart, we measured the levels of myocardial gene expression of the CRF/Ucn family of ligands/receptors and investigated genetic variation and alternative splicing of CRFR1 in 110 heart failure patients and 108 heart donors. Using quantitative real-time PCR, we detected CRFR1, CRFR2, CRF, Ucn1, Ucn2, and Ucn3 in all samples. CRFR2α was the most abundant receptor and Ucn3 the most abundant ligand, both in patients and donors. Compared with donors, cardiac expression of CRFR1, CRF, and Ucn3 was higher (P < .001) and CRFR2α lower (P = .012) in patients. In patients and donors, genetic variation within CRFR1, represented by the chromosome 17q21.31 inversion polymorphism, was associated with markedly higher CRFR1 expression (P < .001), making CRFR1 and CRFR2α expression almost equivalent in some patients. A novel, truncated splice variant of CRFR1, designated CRFR1j, was identified and shown to exert a dominant-negative effect on CRFR1 signaling in vitro. The novel variant was expressed in a greater proportion of patients (60%) than donors (3%, P < .001). In summary, cardiac expression of CRFR1, CRF, and Ucn3 genes is elevated in heart failure and may contribute to the activation of the CRF/Ucn system in these patients. A common variant within the CRFR1 gene and a novel CRFR1 splice variant may modulate CRFR1 expression and signaling. PMID:27754786

Pig StAR: mRNA expression and alternative splicing in testis and Leydig cells, and association analyses with testicular morphology traits.

PubMed

Zhang, Yanghai; Cui, Yang; Zhang, Xuelian; Wang, Yimin; Gao, Jiayang; Yu, Ting; Lv, Xiaoyan; Pan, Chuanying

2018-05-31

Steroidogenic acute regulatory protein (StAR), primarily expressed in Leydig cells (LCs) in the mammalian testes, is essential for testosterone biosynthesis and male fertility. However, no previous reports have explored the expression profiles, alternative splicing and genetic variations of StAR gene in pig. The aim of current study was to explore the expression profiles in different tissues and different types of testicular cells (LCs; spermatogonial stem cells, SSCs; Sertoli cells, SCs), to identify different splice variants and their expression levels, as well as to detect the indel polymorphism in pig StAR gene. Expression analysis results revealed that StAR was widely expressed in all tested tissues and the expression level in testis was significantly higher than that in other tissues (P < 0.01); among different types of testicular cells, the StAR mRNA expression level was significantly higher in LCs than others (P < 0.05). Furthermore, three splice variants, StAR-a, StAR-b and StAR-c, were first found in pig. Further study showed StAR-a was highly expressed in both testis and LCs when compared with other variants (P < 0.01), suggesting StAR-a was the primary variant at StAR gene post-transcription and may facilitate the combination and transportation of cholesterol with StAR. In addition, a 5-bp duplicated deletion (NC_010457.5:g.5524-5528 delACTTG) was verified in the porcine StAR gene, which was closely related to male testicular morphology traits (P < 0.05), and we speculated that the allele "D" of StAR gene might be a positive allele. Briefly, the current findings suggest that StAR and StAR-a play imperative roles in male fertility and the 5-bp indel can be a potential DNA marker for the marker-assisted selection in boar. Copyright © 2018 Elsevier Inc. All rights reserved.

Intron retention generates ANKRD1 splice variants that are co-regulated with the main transcript in normal and failing myocardium.

PubMed

Torrado, Mario; Iglesias, Raquel; Nespereira, Beatriz; Centeno, Alberto; López, Eduardo; Mikhailov, Alexander T

2009-07-01

The cardiac ankyrin repeat domain 1 protein (ANKRD1, also known as CARP) has been extensively characterized with regard to its proposed functions as a cardio-enriched transcriptional co-factor and stress-inducible myofibrillar protein. The present results show the occurrence of alternative splicing by intron retention events in the pig and human ankrd1 gene. In pig heart, ankrd1 is expressed as four alternatively spliced transcripts, three of which have non-excised introns: ankrd1-contained introns 6, 7 and 8 (i.e., ankrd1-i6,7,8), ankrd1-contained introns 7 and 8 (i.e., ankrd1-i7,8), and ankrd1 retained only intron 8 (i.e., ankrd1-i8). In the human heart, two orthologues of porcine intron-retaining ankrd1 variants (i.e., ankrd1-i8 and ankrd1-i7,8) are detected. We demonstrate that these newly-identified intron-retaining ankrd1 transcripts are functionally intact, efficiently translated into protein in vitro and exported to the cytoplasm in cardiomyocytes in vivo. In the piglet heart, both the intronless and intron-retaining ankrd1 mRNAs are co-expressed in a chamber-dependent manner being more abundant in the left as compared to the right myocardium. Our data further indicate co-upregulation of the ankrd1 spliced variants in myocardium in the porcine model of diastolic heart failure. Most significantly, we demonstrate that in vivo forced expression of recombinant intronless ankrd1 markedly increases the levels of intron-retaining ankrd1 variants (but not of the endogenous main transcript) in piglet myocardium, suggesting that ANKRD1 may positively regulate the expression of its own intron-containing RNAs in response to cardiac stress. Overall, our findings demonstrate that in cardiomyocytes ANKRD1 can exist in multiple isoforms which may contribute to the functional diversity of this factor in heart development and disease.

Functional analysis of a large set of BRCA2 exon 7 variants highlights the predictive value of hexamer scores in detecting alterations of exonic splicing regulatory elements.

PubMed

Di Giacomo, Daniela; Gaildrat, Pascaline; Abuli, Anna; Abdat, Julie; Frébourg, Thierry; Tosi, Mario; Martins, Alexandra

2013-11-01

Exonic variants can alter pre-mRNA splicing either by changing splice sites or by modifying splicing regulatory elements. Often these effects are difficult to predict and are only detected by performing RNA analyses. Here, we analyzed, in a minigene assay, 26 variants identified in the exon 7 of BRCA2, a cancer predisposition gene. Our results revealed eight new exon skipping mutations in this exon: one directly altering the 5' splice site and seven affecting potential regulatory elements. This brings the number of splicing regulatory mutations detected in BRCA2 exon 7 to a total of 11, a remarkably high number considering the total number of variants reported in this exon (n = 36), all tested in our minigene assay. We then exploited this large set of splicing data to test the predictive value of splicing regulator hexamers' scores recently established by Ke et al. (). Comparisons of hexamer-based predictions with our experimental data revealed high sensitivity in detecting variants that increased exon skipping, an important feature for prescreening variants before RNA analysis. In conclusion, hexamer scores represent a promising tool for predicting the biological consequences of exonic variants and may have important applications for the interpretation of variants detected by high-throughput sequencing. © 2013 WILEY PERIODICALS, INC.

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.

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

Alternative splicing within the ligand binding domain of the human constitutive androstane receptor.

PubMed

Savkur, Rajesh S; Wu, Yifei; Bramlett, Kelli S; Wang, Minmin; Yao, Sufang; Perkins, Douglas; Totten, Michelle; Searfoss, George; Ryan, Timothy P; Su, Eric W; Burris, Thomas P

2003-01-01

The human constitutive androstane receptor (hCAR; NR1I3) is a member of the nuclear receptor superfamily. The activity of hCAR is regulated by a variety of xenobiotics including clotrimazole and acetaminophen metabolites. hCAR, in turn, regulates a number of genes responsible for xenobiotic metabolism and transport including several cytochrome P450s (CYP 2B5, 2C9, and 3A4) and the multidrug resistance-associated protein 2 (MRP2, ABCC2). Thus, hCAR is believed to be a mediator of drug-drug interactions. We identified two novel hCAR splice variants: hCAR2 encodes a receptor in which alternative splice acceptor sites are utilized resulting in a 4 amino acid insert between exons 6 and 7, and a 5 amino acid insert between 7 and 8, and hCAR3 encodes a receptor with exon 7 completely deleted resulting in a 39 amino acid deletion. Both hCAR2 and hCAR3 mRNAs are expressed in a pattern similar to the initially described MB67 (hCAR1) with some key distinctions. Although the levels of expression vary depending on the tissue examined, hCAR2 and hCAR3 contribute 6-8% of total hCAR mRNA in liver. Analysis of the activity of these variants indicates that both hCAR2 and hCAR3 lose the ability to heterodimerize with RXR and lack transactivation activity in cotransfection experiments where either full-length receptor or GAL4 DNA-binding domain/CAR ligand binding domain chimeras were utilized. Although the role of hCAR2 and hCAR3 is currently unclear, these additional splice variants may provide for increased diversity in terms of responsiveness to xenobiotics.

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

Heart failure-associated changes in RNA splicing of sarcomere genes.

PubMed

Kong, Sek Won; Hu, Yong Wu; Ho, Joshua W K; Ikeda, Sadakatsu; Polster, Sean; John, Ranjit; Hall, Jennifer L; Bisping, Egbert; Pieske, Burkert; dos Remedios, Cristobal G; Pu, William T

2010-04-01

Alternative mRNA splicing is an important mechanism for regulation of gene expression. Altered mRNA splicing occurs in association with several types of cancer, and a small number of disease-associated changes in splicing have been reported in heart disease. However, genome-wide approaches have not been used to study splicing changes in heart disease. We hypothesized that mRNA splicing is different in diseased hearts compared with control hearts. We used the Affymetrix Exon array to globally evaluate mRNA splicing in left ventricular myocardial RNA from controls (n=15) and patients with ischemic cardiomyopathy (n=15). We observed a broad and significant decrease in mRNA splicing efficiency in heart failure, which affected some introns to a greater extent than others. The profile of mRNA splicing separately clustered ischemic cardiomyopathy and control samples, suggesting distinct changes in mRNA splicing between groups. Reverse transcription-polymerase chain reaction validated 9 previously unreported alternative splicing events. Furthermore, we demonstrated that splicing of 4 key sarcomere genes, cardiac troponin T (TNNT2), cardiac troponin I (TNNI3), myosin heavy chain 7 (MYH7), and filamin C, gamma (FLNC), was significantly altered in ischemic cardiomyopathy and in dilated cardiomyopathy and aortic stenosis. In aortic stenosis samples, these differences preceded the onset of heart failure. Remarkably, the ratio of minor to major splice variants of TNNT2, MYH7, and FLNC classified independent test samples as control or disease with >98% accuracy. Our data indicate that mRNA splicing is broadly altered in human heart disease and that patterns of aberrant RNA splicing accurately assign samples to control or disease classes.

RNA splicing. The human splicing code reveals new insights into the genetic determinants of disease.

PubMed

Xiong, Hui Y; Alipanahi, Babak; Lee, Leo J; Bretschneider, Hannes; Merico, Daniele; Yuen, Ryan K C; Hua, Yimin; Gueroussov, Serge; Najafabadi, Hamed S; Hughes, Timothy R; Morris, Quaid; Barash, Yoseph; Krainer, Adrian R; Jojic, Nebojsa; Scherer, Stephen W; Blencowe, Benjamin J; Frey, Brendan J

2015-01-09

To facilitate precision medicine and whole-genome annotation, we developed a machine-learning technique that scores how strongly genetic variants affect RNA splicing, whose alteration contributes to many diseases. Analysis of more than 650,000 intronic and exonic variants revealed widespread patterns of mutation-driven aberrant splicing. Intronic disease mutations that are more than 30 nucleotides from any splice site alter splicing nine times as often as common variants, and missense exonic disease mutations that have the least impact on protein function are five times as likely as others to alter splicing. We detected tens of thousands of disease-causing mutations, including those involved in cancers and spinal muscular atrophy. Examination of intronic and exonic variants found using whole-genome sequencing of individuals with autism revealed misspliced genes with neurodevelopmental phenotypes. Our approach provides evidence for causal variants and should enable new discoveries in precision medicine. Copyright © 2015, American Association for the Advancement of Science.

Validation and Interrogation of Differentially Expressed and Alternatively Spliced Genes in African American Prostate Cancer

DTIC Science & Technology

2016-10-01

These analyses have led to two submitted manuscripts. The first manuscript, “Variants of stemness -related genes predicted to regulate RNA splicing...and Table 1-3 at the end of this progress report. The second manuscript, “Single nucleotide polymorphisms of stemness pathway genes predicted to...cancer and support a contribution of the stemness pathway to prostate cancer patient outcome. Please see Figure 5-7 and Table 4-6 at the end of this

Adaptive thermal control of stem gravitropism through alternative RNA splicing in Arabidopsis

PubMed Central

Ryu, Jae Yong; Kim, Joo-Young; Park, Chung-Mo

2015-01-01

Gravitropism is an important growth movement in response to gravity in virtually all higher plants: the roots showing positive gravitropism and the shoots showing negative gravitropism. The gravitropic orientation of plant organs is also influenced by environmental factors, such as light and temperature. It is known that a zinc finger (ZF)-containing transcription factor SHOOT GRAVITROPISM 5/INDETERMINATE DOMAIN 15 (SGR5/IDD15) mediates the early events of gravitropic responses occurring in inflorescence stems. We have recently found that SGR5 gene undergoes alternative splicing to produce 2 protein variants, the full-size SGR5α transcription factor and the truncated SGR5β form lacking functional ZF motifs. The SGR5β form inhibits SGR5α function possibly by forming nonfunctional heterodimers that are excluded from DNA binding. Notably, SGR5 alternative splicing is accelerated at high temperatures, resulting in a high-level accumulation of SGR5β proteins. Accordingly, transgenic plants overexpressing SGR5β exhibit a reduction in the negative gravitropism of inflorescence stems, as observed in the SGR5-defective mutant. It is proposed that the thermos-responsive alternative splicing of SGR5 gene provides an adaptation strategy by which plants protect the shoots from aerial heat frequently occurring in natural habitats. PMID:26452406

Adaptive thermal control of stem gravitropism through alternative RNA splicing in Arabidopsis.

PubMed

Ryu, Jae Yong; Kim, Joo-Young; Park, Chung-Mo

2015-01-01

Gravitropism is an important growth movement in response to gravity in virtually all higher plants: the roots showing positive gravitropism and the shoots showing negative gravitropism. The gravitropic orientation of plant organs is also influenced by environmental factors, such as light and temperature. It is known that a zinc finger (ZF)-containing transcription factor SHOOT GRAVITROPISM 5/INDETERMINATE DOMAIN 15 (SGR5/IDD15) mediates the early events of gravitropic responses occurring in inflorescence stems. We have recently found that SGR5 gene undergoes alternative splicing to produce 2 protein variants, the full-size SGR5α transcription factor and the truncated SGR5β form lacking functional ZF motifs. The SGR5β form inhibits SGR5α function possibly by forming nonfunctional heterodimers that are excluded from DNA binding. Notably, SGR5 alternative splicing is accelerated at high temperatures, resulting in a high-level accumulation of SGR5β proteins. Accordingly, transgenic plants overexpressing SGR5β exhibit a reduction in the negative gravitropism of inflorescence stems, as observed in the SGR5-defective mutant. It is proposed that the thermos-responsive alternative splicing of SGR5 gene provides an adaptation strategy by which plants protect the shoots from aerial heat frequently occurring in natural habitats.

An alternatively spliced heat shock transcription factor, OsHSFA2dI, functions in the heat stress-induced unfolded protein response in rice.

PubMed

Cheng, Q; Zhou, Y; Liu, Z; Zhang, L; Song, G; Guo, Z; Wang, W; Qu, X; Zhu, Y; Yang, D

2015-03-01

As sessile organisms, plants have evolved a wide range of defence pathways to cope with environmental stress such as heat shock. However, the molecular mechanism of these defence pathways remains unclear in rice. In this study, we found that OsHSFA2d, a heat shock transcriptional factor, encodes two main splice variant proteins, OsHSFA2dI and OsHSFA2dII in rice. Under normal conditions, OsHSFA2dII is the dominant but transcriptionally inactive spliced form. However, when the plant suffers heat stress, OsHSFA2d is alternatively spliced into a transcriptionally active form, OsHSFA2dI, which participates in the heat stress response (HSR). Further study found that this alternative splicing was induced by heat shock rather than photoperiod. We found that OsHSFA2dI is localised to the nucleus, whereas OsHSFA2dII is localised to the nucleus and cytoplasm. Moreover, expression of the unfolded protein response (UNFOLDED PROTEIN RESPONSE) sensors, OsIRE1, OsbZIP39/OsbZIP60 and the UNFOLDED PROTEIN RESPONSE marker OsBiP1, was up-regulated. Interestingly, OsbZIP50 was also alternatively spliced under heat stress, indicating that UNFOLDED PROTEIN RESPONSE signalling pathways were activated by heat stress to re-establish cellular protein homeostasis. We further demonstrated that OsHSFA2dI participated in the unfolded protein response by regulating expression of OsBiP1. © 2014 German Botanical Society and The Royal Botanical Society of the Netherlands.

Nanoplasmonic probes of RNA folding and assembly during pre-mRNA splicing

NASA Astrophysics Data System (ADS)

Nguyen, Anh H.; Lee, Jong Uk; Sim, Sang Jun

2016-02-01

RNA splicing plays important roles in transcriptome and proteome diversity. Herein, we describe the use of a nanoplasmonic system that unveils RNA folding and assembly during pre-mRNA splicing wherein the quantification of mRNA splice variants is not taken into account. With a couple of SERS-probes and plasmonic probes binding at the boundary sites of exon-2/intron-2 and intron-2/exon-3 of the pre-mature RNA of the β-globin gene, the splicing process brings the probes into the plasmonic bands. For plasmonic probes, a plasmon shift increase of ~29 nm, corresponding to intron removal and exon-2 and exon-3 connection to form the mRNA molecule, is measured by plasmonic coupling. The increased scattering intensity and surface-enhanced Raman scattering (SERS) fingerprinting reveal the clear dynamics of pre-mRNA splicing. Moreover, a time-resolved experiment of individual RNA molecules exhibited a successful splicing and an inhibited splicing event by 33 μM biflavonoid isoginkgetin, a general inhibitor of RNA splicing. The results suggest that the RNA splicing is successfully monitored with the nanoplasmonic system. Thus, this platform can be useful for studying RNA nanotechnology, biomolecular folding, alternative splicing, and maturation of microRNA.

A case of cervical cancer expressed three mRNA variant of Hyaluronan-mediated motility receptor

PubMed Central

Villegas-Ruíz, Vanessa; Salcedo, Mauricio; Zentella-Dehesa, Alejandro; de Oca, Edén V Montes; Román-Basaure, Edgar; Mantilla-Morales, Alejandra; Dávila-Borja, Víctor M; Juárez-Méndez, Sergio

2014-01-01

Cervical cancer is the second malignancy in Mexico, little is known about the prognostic factors associated with this disease. Several cellular components are important in their transformation and progression. Alternative mRNA splice is an important mechanism for generating protein diversity, nevertheless, in cancer unknown mRNA diversity is expressed. Hyaluronan-mediated motility receptor (HMMR, RHAMM, CD168) is a family member of proteins, hyaluronan acid dependent, and has been associated with different malignant processes such as: angiogenesis, cell invasiveness, proliferation, metastasis and poor outcome in some tumors. In the present study we identified expression of HMMR in cervical cancer by means of RT-PCR and sequencing. Our results indicate co-expression of two HMMR variants in all samples, and one case expressed three alternative HMMR splice transcripts. These results showed the heterogeneity of mRNA transcripts of HMMR that could express in cancer and the expression of HMMR could be marker of malignancy in CC. PMID:24966934

An Intron 9 CYP19 Gene Variant (IVS9+5G>A), Present in an Aromatase-Deficient Girl, Affects Normal Splicing and Is Also Present in Normal Human Steroidogenic Tissues.

PubMed

Saraco, Nora; Nesi-Franca, Suzana; Sainz, Romina; Marino, Roxana; Marques-Pereira, Rosana; La Pastina, Julia; Perez Garrido, Natalia; Sandrini, Romolo; Rivarola, Marco Aurelio; de Lacerda, Luiz; Belgorosky, Alicia

2015-01-01

Splicing CYP19 gene variants causing aromatase deficiency in 46,XX disorder of sexual development (DSD) patients have been reported in a few cases. A misbalance between normal and aberrant splicing variants was proposed to explain spontaneous pubertal breast development but an incomplete sex maturation progress. The aim of this study was to functionally characterize a novel CYP19A1 intronic homozygote mutation (IVS9+5G>A) in a 46,XX DSD girl presenting spontaneous breast development and primary amenorrhea, and to evaluate similar splicing variant expression in normal steroidogenic tissues. Genomic DNA analysis, splicing prediction programs, splicing assays, and in vitro protein expression and enzyme activity analyses were carried out. CYP19A1 mRNA expression in human steroidogenic tissues was also studied. A novel IVS9+5G>A homozygote mutation was found. In silico analysis predicts the disappearance of the splicing donor site in intron 9, confirmed by patient peripheral leukocyte cP450arom and in vitro studies. Protein analysis showed a shorter and inactive protein. The intron 9 transcript variant was also found in human steroidogenic tissues. The mutation IVS9+5G>A generates a splicing variant that includes intron 9 which is also present in normal human steroidogenic tissues, suggesting that a misbalance between normal and aberrant splicing variants might occur in target tissues, explaining the clinical phenotype in the affected patient. © 2015 S. Karger AG, Basel.

Alternative splice isoforms of small conductance calcium-activated SK2 channels differ in molecular interactions and surface levels

PubMed Central

Scholl, Elizabeth Storer; Pirone, Antonella; Cox, Daniel H; Duncan, R Keith; Jacob, Michele H

2014-01-01

Small conductance Ca2+-sensitive potassium (SK2) channels are voltage-independent, Ca2+-activated ion channels that conduct potassium cations and thereby modulate the intrinsic excitability and synaptic transmission of neurons and sensory hair cells. In the cochlea, SK2 channels are functionally coupled to the highly Ca2+ permeant α9/10-nicotinic acetylcholine receptors (nAChRs) at olivocochlear postsynaptic sites. SK2 activation leads to outer hair cell hyperpolarization and frequency-selective suppression of afferent sound transmission. These inhibitory responses are essential for normal regulation of sound sensitivity, frequency selectivity, and suppression of background noise. However, little is known about the molecular interactions of these key functional channels. Here we show that SK2 channels co-precipitate with α9/10-nAChRs and with the actin-binding protein α-actinin-1. SK2 alternative splicing, resulting in a 3 amino acid insertion in the intracellular 3′ terminus, modulates these interactions. Further, relative abundance of the SK2 splice variants changes during developmental stages of synapse maturation in both the avian cochlea and the mammalian forebrain. Using heterologous cell expression to separately study the 2 distinct isoforms, we show that the variants differ in protein interactions and surface expression levels, and that Ca2+ and Ca2+-bound calmodulin differentially regulate their protein interactions. Our findings suggest that the SK2 isoforms may be distinctly modulated by activity-induced Ca2+ influx. Alternative splicing of SK2 may serve as a novel mechanism to differentially regulate the maturation and function of olivocochlear and neuronal synapses. PMID:24394769

High diversification of CD94 by alternative splicing in New World primates.

PubMed

Galindo, John A; Cadavid, Luis F

2013-04-01

CD94 forms heterodimers with NKG2A, -C, or -E to constitute lectin-like natural killer cell receptors for MHC-E. Its structure differs from other C-type lectins in that the second α-helix is replaced by a loop that forms the interacting interface with the NKG2 molecules. Although CD94 has remained highly conserved mammals, several alternative splicing variants have been detected in some species. To evaluate the prevalence and significance of this phenomenon, we have cloned and sequenced CD94 cDNAs in six species of New World primates from the Cebidae and Atelidae families. Full-length sequences had a mean similarity of 96 % amongst New World primates and of 90 % to the human orthologue, with little variation in the residues interacting with NKG2 or MHC-E molecules. Despite this high conservation, a total of 14 different splice variants were identified, half of which were shared by two or more primate species. Homology-based modeling of the C-type lectin domain showed that most isoforms folded stably, although they had modifications that prevented its interaction with NKG2 and MHC-E. Two isoforms were predicted to replace the typical CD94 loop by a second α-helix, evidencing a domain fold transition from a CD94 structure to a canonical C-type lectin. These two structures were more similar to members of the CLEC lectin family than to the native CD94. Thus, CD94 has remained conserved in primates to maintain functional interactions with NKG2 and MHC-E, while at the same time has diversified by alternative splicing potentially providing additional functional scenarios.

Splice variants and promoter methylation status of the Bovine Vasa Homology (Bvh) gene may be involved in bull spermatogenesis

PubMed Central

2013-01-01

Background Vasa is a member of the DEAD-box protein family that plays an indispensable role in mammalian spermatogenesis, particularly during meiosis. Bovine vasa homology (Bvh) of Bos taurus has been reported, however, its function in bovine testicular tissue remains obscure. This study aimed to reveal the functions of Bvh and to determine whether Bvh is a candidate gene in the regulation of spermatogenesis in bovine, and to illustrate whether its transcription is regulated by alternative splicing and DNA methylation. Results Here we report the molecular characterization, alternative splicing pattern, expression and promoter methylation status of Bvh. The full-length coding region of Bvh was 2190 bp, which encodes a 729 amino acid (aa) protein containing nine consensus regions of the DEAD box protein family. Bvh is expressed only in the ovary and testis of adult cattle. Two splice variants were identified and termed Bvh-V4 (2112 bp and 703 aa) and Bvh-V45 (2040 bp and 679 aa). In male cattle, full-length Bvh (Bvh-FL), Bvh-V4 and Bvh-V45 are exclusively expressed in the testes in the ratio of 2.2:1.6:1, respectively. Real-time PCR revealed significantly reduced mRNA expression of Bvh-FL, Bvh-V4 and Bvh-V45 in testes of cattle-yak hybrids, with meiotic arrest compared with cattle and yaks with normal spermatogenesis (P < 0.01). The promoter methylation level of Bvh in the testes of cattle-yak hybrids was significantly greater than in cattle and yaks (P < 0.01). Conclusion In the present study, Bvh was isolated and characterized. These data suggest that Bvh functions in bovine spermatogenesis, and that transcription of the gene in testes were regulated by alternative splice and promoter methylation. PMID:23815438

Deciphering the Developmental Dynamics of the Mouse Liver Transcriptome

PubMed Central

Gunewardena, Sumedha S.; Yoo, Byunggil; Peng, Lai; Lu, Hong; Zhong, Xiaobo; Klaassen, Curtis D.; Cui, Julia Yue

2015-01-01

During development, liver undergoes a rapid transition from a hematopoietic organ to a major organ for drug metabolism and nutrient homeostasis. However, little is known on a transcriptome level of the genes and RNA-splicing variants that are differentially regulated with age, and which up-stream regulators orchestrate age-specific biological functions in liver. We used RNA-Seq to interrogate the developmental dynamics of the liver transcriptome in mice at 12 ages from late embryonic stage (2-days before birth) to maturity (60-days after birth). Among 21,889 unique NCBI RefSeq-annotated genes, 9,641 were significantly expressed in at least one age, 7,289 were differently regulated with age, and 859 had multiple (> = 2) RNA splicing-variants. Factor analysis showed that the dynamics of hepatic genes fall into six distinct groups based on their temporal expression. The average expression of cytokines, ion channels, kinases, phosphatases, transcription regulators and translation regulators decreased with age, whereas the average expression of peptidases, enzymes and transmembrane receptors increased with age. The average expression of growth factors peak between Day-3 and Day-10, and decrease thereafter. We identified critical biological functions, upstream regulators, and putative transcription modules that seem to govern age-specific gene expression. We also observed differential ontogenic expression of known splicing variants of certain genes, and 1,455 novel splicing isoform candidates. In conclusion, the hepatic ontogeny of the transcriptome ontogeny has unveiled critical networks and up-stream regulators that orchestrate age-specific biological functions in liver, and suggest that age contributes to the complexity of the alternative splicing landscape of the hepatic transcriptome. PMID:26496202

Deciphering the Developmental Dynamics of the Mouse Liver Transcriptome.

PubMed

Gunewardena, Sumedha S; Yoo, Byunggil; Peng, Lai; Lu, Hong; Zhong, Xiaobo; Klaassen, Curtis D; Cui, Julia Yue

2015-01-01

During development, liver undergoes a rapid transition from a hematopoietic organ to a major organ for drug metabolism and nutrient homeostasis. However, little is known on a transcriptome level of the genes and RNA-splicing variants that are differentially regulated with age, and which up-stream regulators orchestrate age-specific biological functions in liver. We used RNA-Seq to interrogate the developmental dynamics of the liver transcriptome in mice at 12 ages from late embryonic stage (2-days before birth) to maturity (60-days after birth). Among 21,889 unique NCBI RefSeq-annotated genes, 9,641 were significantly expressed in at least one age, 7,289 were differently regulated with age, and 859 had multiple (> = 2) RNA splicing-variants. Factor analysis showed that the dynamics of hepatic genes fall into six distinct groups based on their temporal expression. The average expression of cytokines, ion channels, kinases, phosphatases, transcription regulators and translation regulators decreased with age, whereas the average expression of peptidases, enzymes and transmembrane receptors increased with age. The average expression of growth factors peak between Day-3 and Day-10, and decrease thereafter. We identified critical biological functions, upstream regulators, and putative transcription modules that seem to govern age-specific gene expression. We also observed differential ontogenic expression of known splicing variants of certain genes, and 1,455 novel splicing isoform candidates. In conclusion, the hepatic ontogeny of the transcriptome ontogeny has unveiled critical networks and up-stream regulators that orchestrate age-specific biological functions in liver, and suggest that age contributes to the complexity of the alternative splicing landscape of the hepatic transcriptome.

Genome-Wide Analysis of Alternative Splicing Landscapes Modulated during Plant-Virus Interactions in Brachypodium distachyon

PubMed Central

Scholthof, Karen-Beth G.

2015-01-01

In eukaryotes, alternative splicing (AS) promotes transcriptome and proteome diversity. The extent of genome-wide AS changes occurring during a plant-microbe interaction is largely unknown. Here, using high-throughput, paired-end RNA sequencing, we generated an isoform-level spliceome map of Brachypodium distachyon infected with Panicum mosaic virus and its satellite virus. Overall, we detected ∼44,443 transcripts in B. distachyon, ∼30% more than those annotated in the reference genome. Expression of ∼28,900 transcripts was ≥2 fragments per kilobase of transcript per million mapped fragments, and ∼42% of multi-exonic genes were alternatively spliced. Comparative analysis of AS patterns in B. distachyon, rice (Oryza sativa), maize (Zea mays), sorghum (Sorghum bicolor), Arabidopsis thaliana, potato (Solanum tuberosum), Medicago truncatula, and poplar (Populus trichocarpa) revealed conserved ratios of the AS types between monocots and dicots. Virus infection quantitatively altered AS events in Brachypodium with little effect on the AS ratios. We discovered AS events for >100 immune-related genes encoding receptor-like kinases, NB-LRR resistance proteins, transcription factors, RNA silencing, and splicing-associated proteins. Cloning and molecular characterization of SCL33, a serine/arginine-rich splicing factor, identified multiple novel intron-retaining splice variants that are developmentally regulated and modulated during virus infection. B. distachyon SCL33 splicing patterns are also strikingly conserved compared with a distant Arabidopsis SCL33 ortholog. This analysis provides new insights into AS landscapes conserved among monocots and dicots and uncovered AS events in plant defense-related genes. PMID:25634987

Analysis of prostate-specific antigen transcripts in chimpanzees, cynomolgus monkeys, baboons, and African green monkeys.

PubMed

Mubiru, James N; Yang, Alice S; Olsen, Christian; Nayak, Sudhir; Livi, Carolina B; Dick, Edward J; Owston, Michael; Garcia-Forey, Magdalena; Shade, Robert E; Rogers, Jeffrey

2014-01-01

The function of prostate-specific antigen (PSA) is to liquefy the semen coagulum so that the released sperm can fuse with the ovum. Fifteen spliced variants of the PSA gene have been reported in humans, but little is known about alternative splicing in nonhuman primates. Positive selection has been reported in sex- and reproductive-related genes from sea urchins to Drosophila to humans; however, there are few studies of adaptive evolution of the PSA gene. Here, using polymerase chain reaction (PCR) product cloning and sequencing, we study PSA transcript variant heterogeneity in the prostates of chimpanzees (Pan troglodytes), cynomolgus monkeys (Macaca fascicularis), baboons (Papio hamadryas anubis), and African green monkeys (Chlorocebus aethiops). Six PSA variants were identified in the chimpanzee prostate, but only two variants were found in cynomolgus monkeys, baboons, and African green monkeys. In the chimpanzee the full-length transcript is expressed at the same magnitude as the transcripts that retain intron 3. We have found previously unidentified splice variants of the PSA gene, some of which might be linked to disease conditions. Selection on the PSA gene was studied in 11 primate species by computational methods using the sequences reported here for African green monkey, cynomolgus monkey, baboon, and chimpanzee and other sequences available in public databases. A codon-based analysis (dN/dS) of the PSA gene identified potential adaptive evolution at five residue sites (Arg45, Lys70, Gln144, Pro189, and Thr203).

Genome-wide analysis of alternative splicing in medulloblastoma identifies splicing patterns characteristic of normal cerebellar development

PubMed Central

Menghi, Francesca; Jacques, Thomas S.; Barenco, Martino; Schwalbe, Ed C.; Clifford, Steven C.; Hubank, Mike; Ham, Jonathan

2011-01-01

Alternative splicing is an important mechanism for the generation of protein diversity at a post-transcriptional level. Modifications in the splicing patterns of several genes have been shown to contribute to the malignant transformation of different tissue types. In this study, we used the Affymetrix Exon arrays to investigate patterns of differential splicing between paediatric medulloblastomas and normal cerebellum on a genome-wide scale. Of the 1262 genes identified as potentially generating tumour-associated splice forms, we selected 14 examples of differential splicing of known cassette exons and successfully validated 11 of them by RT-PCR. The pattern of differential splicing of three validated events was characteristic for the molecular subset of Sonic Hedgehog (Shh)-driven medulloblastomas, suggesting that their unique gene signature includes the expression of distinctive transcript variants. Generally, we observed that tumour and normal fetal cerebellar samples shared significantly lower exon inclusion rates compared to normal adult cerebellum. We investigated whether tumour-associated splice forms were expressed in primary cultures of Shh-dependent mouse cerebellar granule cell precursors (GCPs) and found that Shh caused a decrease in the cassette exon inclusion rate of five out of the seven tested genes. Furthermore, we observed a significant increase in exon inclusion between post-natal days 7 and 14 of mouse cerebellar development, at the time when GCPs mature into post-mitotic neurons. We conclude that inappropriate splicing frequently occurs in human medulloblastomas and may be linked to the activation of developmental signalling pathways and a failure of cerebellar precursor cells to differentiate. PMID:21248070

Alternative RNA splicing of the MEAF6 gene facilitates neuroendocrine prostate cancer progression.

PubMed

Lee, Ahn R; Li, Yinan; Xie, Ning; Gleave, Martin E; Cox, Michael E; Collins, Colin C; Dong, Xuesen

2017-04-25

Although potent androgen receptor pathway inhibitors (ARPI) improve overall survival of metastatic prostate cancer patients, treatment-induced neuroendocrine prostate cancer (t-NEPC) as a consequence of the selection pressures of ARPI is becoming a more common clinical issue. Improved understanding of the molecular biology of t-NEPC is essential for the development of new effective management approaches for t-NEPC. In this study, we identify a splice variant of the MYST/Esa1-associated factor 6 (MEAF6) gene, MEAF6-1, that is highly expressed in both t-NEPC tumor biopsies and neuroendocrine cell lines of prostate and lung cancers. We show that MEAF6-1 splicing is stimulated by neuronal RNA splicing factor SRRM4. Rather than inducing neuroendocrine trans-differentiation of cells in prostate adenocarcinoma, MEAF6-1 upregulation stimulates cell proliferation, anchorage-independent cell growth, invasion and xenograft tumor growth. Gene microarray identifies that these MEAF6-1 actions are in part mediated by the ID1 and ID3 genes. These findings suggest that the MEAF6-1 variant does not induce neuroendocrine differentiation of prostate cancer cells, but rather facilitates t-NEPC progression by increasing the proliferation rate of cells that have acquired neuroendocrine phenotypes.

Disturbed alternative splicing of FIR (PUF60) directed cyclin E overexpression in esophageal cancers.

PubMed

Ogura, Yukiko; Hoshino, Tyuji; Tanaka, Nobuko; Ailiken, Guzhanuer; Kobayashi, Sohei; Kitamura, Kouichi; Rahmutulla, Bahityar; Kano, Masayuki; Murakami, Kentarou; Akutsu, Yasunori; Nomura, Fumio; Itoga, Sakae; Matsubara, Hisahiro; Matsushita, Kazuyuki

2018-05-01

Overexpression of alternative splicing of far upstream element binding protein 1 (FUBP1) interacting repressor (FIR; poly(U) binding splicing factor 60 [PUF60]) and cyclin E were detected in esophageal squamous cell carcinomas (ESCC). Accordingly, the expression of FBW7 was examined by which cyclin E is degraded as a substrate via the proteasome system. Expectedly, FBW7 expression was decreased significantly in ESCC. Conversely, c-myc gene transcriptional repressor FIR (alias PUF60; U2AF-related protein) and its alternative splicing variant form (FIRΔexon2) were overexpressed in ESCC. Further, anticancer drugs (cis-diaminedichloroplatinum/cisplatin [CDDP] or 5-fluorouracil [5-FU]) and knockdown of FIR by small interfering RNA (siRNA) increased cyclin E while knockdown of FIRΔexon2 by siRNA decreased cyclin E expression in ESCC cell lines (TE1, TE2, and T.Tn) or cervical SCC cells (HeLa cells). Especially, knockdown of SAP155 (SF3b1), a splicing factor required for proper alternative splicing of FIR pre-mRNA, decreased cyclin E. Therefore, disturbed alternative splicing of FIR generated FIR/FIRΔexon2 with cyclin E overexpression in esophageal cancers, indicating that SAP155 siRNA potentially rescued FBW7 function by reducing expression of FIR and/or FIRΔexon2. Remarkably, Three-dimensional structure analysis revealed the hypothetical inhibitory mechanism of FBW7 function by FIR/FIRΔexon2, a novel mechanism of cyclin E overexpression by FIR/FIRΔexon2-FBW7 interaction was discussed. Clinically, elevated FIR expression potentially is an indicator of the number of lymph metastases and anti-FIR/FIRΔexon2 antibodies in sera as cancer diagnosis, indicating chemical inhibitors of FIR/FIRΔexon2-FBW7 interaction could be potential candidate drugs for cancer therapy. In conclusion, elevated cyclin E expression was, in part, induced owing to potential FIR/FIRΔexon2-FBW7 interaction in ESCC.

Polypyrimidine Tract Binding Protein Homologs from Arabidopsis Are Key Regulators of Alternative Splicing with Implications in Fundamental Developmental Processes[W

PubMed Central

Rühl, Christina; Stauffer, Eva; Kahles, André; Wagner, Gabriele; Drechsel, Gabriele; Rätsch, Gunnar; Wachter, Andreas

2012-01-01

Alternative splicing (AS) generates transcript variants by variable exon/intron definition and massively expands transcriptome diversity. Changes in AS patterns have been found to be linked to manifold biological processes, yet fundamental aspects, such as the regulation of AS and its functional implications, largely remain to be addressed. In this work, widespread AS regulation by Arabidopsis thaliana Polypyrimidine tract binding protein homologs (PTBs) was revealed. In total, 452 AS events derived from 307 distinct genes were found to be responsive to the levels of the splicing factors PTB1 and PTB2, which predominantly triggered splicing of regulated introns, inclusion of cassette exons, and usage of upstream 5′ splice sites. By contrast, no major AS regulatory function of the distantly related PTB3 was found. Dependent on their position within the mRNA, PTB-regulated events can both modify the untranslated regions and give rise to alternative protein products. We find that PTB-mediated AS events are connected to diverse biological processes, and the functional implications of selected instances were further elucidated. Specifically, PTB misexpression changes AS of PHYTOCHROME INTERACTING FACTOR6, coinciding with altered rates of abscisic acid–dependent seed germination. Furthermore, AS patterns as well as the expression of key flowering regulators were massively changed in a PTB1/2 level-dependent manner. PMID:23192226

Conservation of CD44 exon v3 functional elements in mammals

PubMed Central

Vela, Elena; Hilari, Josep M; Delclaux, María; Fernández-Bellon, Hugo; Isamat, Marcos

2008-01-01

Background The human CD44 gene contains 10 variable exons (v1 to v10) that can be alternatively spliced to generate hundreds of different CD44 protein isoforms. Human CD44 variable exon v3 inclusion in the final mRNA depends on a multisite bipartite splicing enhancer located within the exon itself, which we have recently described, and provides the protein domain responsible for growth factor binding to CD44. Findings We have analyzed the sequence of CD44v3 in 95 mammalian species to report high conservation levels for both its splicing regulatory elements (the 3' splice site and the exonic splicing enhancer), and the functional glycosaminglycan binding site coded by v3. We also report the functional expression of CD44v3 isoforms in peripheral blood cells of different mammalian taxa with both consensus and variant v3 sequences. Conclusion CD44v3 mammalian sequences maintain all functional splicing regulatory elements as well as the GAG binding site with the same relative positions and sequence identity previously described during alternative splicing of human CD44. The sequence within the GAG attachment site, which in turn contains the Y motif of the exonic splicing enhancer, is more conserved relative to the rest of exon. Amplification of CD44v3 sequence from mammalian species but not from birds, fish or reptiles, may lead to classify CD44v3 as an exclusive mammalian gene trait. PMID:18710510

Alternative Splicing Studies of the Reactive Oxygen Species Gene Network in Populus Reveal Two Isoforms of High-Isoelectric-Point Superoxide Dismutase1[C][W

PubMed Central

Srivastava, Vaibhav; Srivastava, Manoj Kumar; Chibani, Kamel; Nilsson, Robert; Rouhier, Nicolas; Melzer, Michael; Wingsle, Gunnar

2009-01-01

Recent evidence has shown that alternative splicing (AS) is widely involved in the regulation of gene expression, substantially extending the diversity of numerous proteins. In this study, a subset of expressed sequence tags representing members of the reactive oxygen species gene network was selected from the PopulusDB database to investigate AS mechanisms in Populus. Examples of all known types of AS were detected, but intron retention was the most common. Interestingly, the closest Arabidopsis (Arabidopsis thaliana) homologs of half of the AS genes identified in Populus are not reportedly alternatively spliced. Two genes encoding the protein of most interest in our study (high-isoelectric-point superoxide dismutase [hipI-SOD]) have been found in black cottonwood (Populus trichocarpa), designated PthipI-SODC1 and PthipI-SODC2. Analysis of the expressed sequence tag libraries has indicated the presence of two transcripts of PthipI-SODC1 (hipI-SODC1b and hipI-SODC1s). Alignment of these sequences with the PthipI-SODC1 gene showed that hipI-SODC1b was 69 bp longer than hipI-SODC1s due to an AS event involving the use of an alternative donor splice site in the sixth intron. Transcript analysis showed that the splice variant hipI-SODC1b was differentially expressed, being clearly expressed in cambial and xylem, but not phloem, regions. In addition, immunolocalization and mass spectrometric data confirmed the presence of hipI-SOD proteins in vascular tissue. The functionalities of the spliced gene products were assessed by expressing recombinant hipI-SOD proteins and in vitro SOD activity assays. PMID:19176719

Alternative splicing studies of the reactive oxygen species gene network in Populus reveal two isoforms of high-isoelectric-point superoxide dismutase.

PubMed

Srivastava, Vaibhav; Srivastava, Manoj Kumar; Chibani, Kamel; Nilsson, Robert; Rouhier, Nicolas; Melzer, Michael; Wingsle, Gunnar

2009-04-01

Recent evidence has shown that alternative splicing (AS) is widely involved in the regulation of gene expression, substantially extending the diversity of numerous proteins. In this study, a subset of expressed sequence tags representing members of the reactive oxygen species gene network was selected from the PopulusDB database to investigate AS mechanisms in Populus. Examples of all known types of AS were detected, but intron retention was the most common. Interestingly, the closest Arabidopsis (Arabidopsis thaliana) homologs of half of the AS genes identified in Populus are not reportedly alternatively spliced. Two genes encoding the protein of most interest in our study (high-isoelectric-point superoxide dismutase [hipI-SOD]) have been found in black cottonwood (Populus trichocarpa), designated PthipI-SODC1 and PthipI-SODC2. Analysis of the expressed sequence tag libraries has indicated the presence of two transcripts of PthipI-SODC1 (hipI-SODC1b and hipI-SODC1s). Alignment of these sequences with the PthipI-SODC1 gene showed that hipI-SODC1b was 69 bp longer than hipI-SODC1s due to an AS event involving the use of an alternative donor splice site in the sixth intron. Transcript analysis showed that the splice variant hipI-SODC1b was differentially expressed, being clearly expressed in cambial and xylem, but not phloem, regions. In addition, immunolocalization and mass spectrometric data confirmed the presence of hipI-SOD proteins in vascular tissue. The functionalities of the spliced gene products were assessed by expressing recombinant hipI-SOD proteins and in vitro SOD activity assays.

Splice Site Variants in the KCNQ1 and SCN5A Genes: Transcript Analysis as a Tool in Supporting Pathogenicity

PubMed Central

Leong, Ivone U.S.; Dryland, Philippa A.; Prosser, Debra O.; Lai, Stella W.-S.; Graham, Mandy; Stiles, Martin; Crawford, Jackie; Skinner, Jonathan R.; Love, Donald R.

2017-01-01

Background Approximately 75% of clinically definite long QT syndrome (LQTS) cases are caused by mutations in the KCNQ1, KCNH2 and SCN5A genes. Of these mutations, a small proportion (3.2-9.2%) are predicted to affect splicing. These mutations present a particular challenge in ascribing pathogenicity. Methods Here we report an analysis of the transcriptional consequences of two mutations, one in the KCNQ1 gene (c.781_782delinsTC) and one in the SCN5A gene (c.2437-5C>A), which are predicted to affect splicing. We isolated RNA from lymphocytes and used a directed PCR amplification strategy of cDNA to show mis-spliced transcripts in mutation-positive patients. Results The loss of an exon in each mis-spliced transcript had no deduced effect on the translational reading frame. The clinical phenotype corresponded closely with genotypic status in family members carrying the KCNQ1 splice variant, but not in family members with the SCN5A splice variant. These results are put in the context of a literature review, where only 20% of all splice variants reported in the KCNQ1, KCNH2 and SCN5A gene entries in the HGMDPro 2015.4 database have been evaluated using transcriptional assays. Conclusions Prediction programmes play a strong role in most diagnostic laboratories in classifying variants located at splice sites; however, transcriptional analysis should be considered critical to confirm mis-splicing. Critically, this study shows that genuine mis- splicing may not always imply clinical significance, and genotype/phenotype cosegregation remains important even when mis-splicing is confirmed. PMID:28725320

Lin28 induces resistance to anti-androgens via promotion of AR splice variant generation.

PubMed

Tummala, Ramakumar; Nadiminty, Nagalakshmi; Lou, Wei; Evans, Christopher P; Gao, Allen C

2016-04-01

Prostate cancer (PCa) is androgen-dependent initially and progresses to a castration-resistant state after androgen deprivation therapy. Treatment options for castration-resistant PCa include the potent second-generation anti-androgen enzalutamide or CYP17A1 inhibitor abiraterone. Recent clinical observations point to the development of resistance to these therapies which may be mediated by constitutively active alternative splice variants of the androgen receptor (AR). Sensitivity of LNCaP cells overexpressing Lin28 (LN-Lin28) to enzalutamide, abiraterone, or bicalutamide was compared to that of control LN-neo cells using cell growth assays, proliferation assays using MTT, anchorage-dependent clonogenic ability assays and soft agar assays. Ability of LN-Lin28 cells to maintain AR activation after treatment with enzalutamide, abiraterone, or bicalutamide was tested using immunofluorescence, Western blotting, ChIP assays, and qRT-PCR. Importance of Lin28 in enzalutamide resistance was assessed by the downregulation of Lin28 expression in C4-2B and 22Rv1 cells chronically treated with enzalutamide. Requirement for sustained AR signaling in LN-Lin28 cells was examined by the downregulation of either full length AR or AR-V7 using siRNA. We show that Lin28 promotes the development of resistance to currently used targeted therapeutics by enhancing the expression of AR splice variants such as AR-V7. PCa cells overexpressing Lin28 exhibit resistance to treatment with enzalutamide, abiraterone, or bicalutamide. Downregulation of Lin28 resensitizes enzalutamide-resistant PCa cells to enzalutamide treatment. We also show that the upregulation of splicing factors such as hnRNPA1 by Lin28 may mediate the enhanced generation of AR splice variants in Lin28-expressing cells. Our findings suggest that Lin28 plays a key role in the acquisition of resistance to AR-targeted therapies by PCa cells and establish the importance of Lin28 in PCa progression. © 2015 Wiley Periodicals, Inc.

The genomic basis of circadian and circalunar timing adaptations in a midge.

PubMed

Kaiser, Tobias S; Poehn, Birgit; Szkiba, David; Preussner, Marco; Sedlazeck, Fritz J; Zrim, Alexander; Neumann, Tobias; Nguyen, Lam-Tung; Betancourt, Andrea J; Hummel, Thomas; Vogel, Heiko; Dorner, Silke; Heyd, Florian; von Haeseler, Arndt; Tessmar-Raible, Kristin

2016-12-01

Organisms use endogenous clocks to anticipate regular environmental cycles, such as days and tides. Natural variants resulting in differently timed behaviour or physiology, known as chronotypes in humans, have not been well characterized at the molecular level. We sequenced the genome of Clunio marinus, a marine midge whose reproduction is timed by circadian and circalunar clocks. Midges from different locations show strain-specific genetic timing adaptations. We examined genetic variation in five C. marinus strains from different locations and mapped quantitative trait loci for circalunar and circadian chronotypes. The region most strongly associated with circadian chronotypes generates strain-specific differences in the abundance of calcium/calmodulin-dependent kinase II.1 (CaMKII.1) splice variants. As equivalent variants were shown to alter CaMKII activity in Drosophila melanogaster, and C. marinus (Cma)-CaMKII.1 increases the transcriptional activity of the dimer of the circadian proteins Cma-CLOCK and Cma-CYCLE, we suggest that modulation of alternative splicing is a mechanism for natural adaptation in circadian timing.

Developmentally regulated switch in alternatively spliced SNAP-25 isoforms alters facilitation of synaptic transmission.

PubMed

Bark, Christina; Bellinger, Frederick P; Kaushal, Ashutosh; Mathews, James R; Partridge, L Donald; Wilson, Michael C

2004-10-06

Although the basic molecular components that promote regulated neurotransmitter release are well established, the contribution of these proteins as regulators of the plasticity of neurotransmission and refinement of synaptic connectivity during development is elaborated less fully. For example, during the period of synaptic growth and maturation in brain, the expression of synaptosomal protein 25 kDa (SNAP-25), a neuronal t-SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) essential for action potential-dependent neuroexocytosis, is altered through alternative splicing of pre-mRNA transcripts. We addressed the role of the two splice-variant isoforms of SNAP-25 with a targeted mouse mutation that impairs the shift from SNAP-25a to SNAP-25b. Most of these mutant mice die between 3 and 5 weeks of age, which coincides with the time when SNAP-25b expression normally reaches mature levels in brain and synapse formation is essentially completed. The altered expression of these SNAP-25 isoforms influences short-term synaptic function by affecting facilitation but not the initial probability of release. This suggests that mechanisms controlling alternative splicing between SNAP-25 isoforms contribute to a molecular switch important for survival that helps to guide the transition from immature to mature synaptic connections, as well as synapse regrowth and remodeling after neural injury.

In silico study of breast cancer associated gene 3 using LION Target Engine and other tools.

PubMed

León, Darryl A; Cànaves, Jaume M

2003-12-01

Sequence analysis of individual targets is an important step in annotation and validation. As a test case, we investigated human breast cancer associated gene 3 (BCA3) with LION Target Engine and with other bioinformatics tools. LION Target Engine confirmed that the BCA3 gene is located on 11p15.4 and that the two most likely splice variants (lacking exon 3 and exons 3 and 5, respectively) exist. Based on our manual curation of sequence data, it is proposed that an additional variant (missing only exon 5) published in a public sequence repository, is a prediction artifact. A significant number of new orthologs were also identified, and these were the basis for a high-quality protein secondary structure prediction. Moreover, our research confirmed several distinct functional domains as described in earlier reports. Sequence conservation from multiple sequence alignments, splice variant identification, secondary structure predictions, and predicted phosphorylation sites suggest that the removal of interaction sites through alternative splicing might play a modulatory role in BCA3. This in silico approach shows the depth and relevance of an analysis that can be accomplished by including a variety of publicly available tools with an integrated and customizable life science informatics platform.

Amelogenin exons 8 and 9 encoded peptide enhances leucine rich amelogenin peptide mediated dental pulp repair.

PubMed

Huang, Yulei; Goldberg, Michel; Le, Thuan; Qiang, Ran; Warner, Douglas; Witkowska, Halina Ewa; Liu, Haichuan; Zhu, Li; Denbesten, Pamela; Li, Wu

2012-01-01

Amelogenins containing exons 8 and 9 are alternatively spliced variants of amelogenin. Some amelogenin spliced variants have been found to promote pulp regeneration following pulp exposure. The function of the amelogenin spliced variants with the exons 8 and 9 remains unknown. In this study, we synthesized recombinant leucine rich amelogenin peptide (LRAP, A-4), LRAP plus exons 8 and 9 peptide (LRAP 8, 9) or exons 8 and 9 peptide (P89), to determine their effects on odontoblasts. In vivo analyses were completed following the insertion of agarose beads containing LRAP or LRAP 8, 9 into exposed cavity preparations of rat molars. After 8, 15 or 30 days' exposure, the pulp tissues were analyzed for changes in histomorphometry and cell proliferation by PCNA stainings. In vitro analyses included the effects of the addition of the recombinant proteins or peptide on cell proliferation, differentiation and adhesion of postnatal human dental pulp cells (DPCs). These studies showed that in vivo LRAP 8, 9 enhanced the reparative dentin formation as compared to LRAP. In vitro LRAP 8, 9 promoted DPC proliferation and differentiation to a greater extent than LRAP. These data suggest that amelogenin exons 8 and 9 may be useful in amelogenin-mediated pulp repair. Copyright © 2012 S. Karger AG, Basel.

Novel FGFR1 mutations in Kallmann syndrome and normosmic idiopathic hypogonadotropic hypogonadism: evidence for the involvement of an alternatively spliced isoform.

PubMed

Gonçalves, Catarina; Bastos, Margarida; Pignatelli, Duarte; Borges, Teresa; Aragüés, José M; Fonseca, Fernando; Pereira, Bernardo D; Socorro, Sílvia; Lemos, Manuel C

2015-11-01

To determine the prevalence of fibroblast growth factor receptor 1 (FGFR1) mutations and their predicted functional consequences in patients with idiopathic hypogonadotropic hypogonadism (IHH). Cross-sectional study. Multicentric. Fifty unrelated patients with IHH (21 with Kallmann syndrome and 29 with normosmic IHH). None. Patients were screened for mutations in FGFR1. The functional consequences of mutations were predicted by in silico structural and conservation analysis. Heterozygous FGFR1 mutations were identified in six (12%) kindreds. These consisted of frameshift mutations (p.Pro33-Alafs*17 and p.Tyr654*) and missense mutations in the signal peptide (p.Trp4Cys), in the D1 extracellular domain (p.Ser96Cys) and in the cytoplasmic tyrosine kinase domain (p.Met719Val). A missense mutation was identified in the alternatively spliced exon 8A (p.Ala353Thr) that exclusively affects the D3 extracellular domain of FGFR1 isoform IIIb. Structure-based and sequence-based prediction methods and the absence of these variants in 200 normal controls were all consistent with a critical role for the mutations in the activity of the receptor. Oligogenic inheritance (FGFR1/CHD7/PROKR2) was found in one patient. Two FGFR1 isoforms, IIIb and IIIc, result from alternative splicing of exons 8A and 8B, respectively. Loss-of-function of isoform IIIc is a cause of IHH, whereas isoform IIIb is thought to be redundant. Ours is the first report of normosmic IHH associated with a mutation in the alternatively spliced exon 8A and suggests that this disorder can be caused by defects in either of the two alternatively spliced FGFR1 isoforms. Copyright © 2015 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Increased frequency of FBN1 truncating and splicing variants in Marfan syndrome patients with aortic events.

PubMed

Baudhuin, Linnea M; Kotzer, Katrina E; Lagerstedt, Susan A

2015-03-01

Marfan syndrome is a systemic disorder that typically involves FBN1 mutations and cardiovascular manifestations. We investigated FBN1 genotype-phenotype correlations with aortic events (aortic dissection and prophylactic aortic surgery) in patients with Marfan syndrome. Genotype and phenotype information from probands (n = 179) with an FBN1 pathogenic or likely pathogenic variant were assessed. A higher frequency of truncating or splicing FBN1 variants was observed in Ghent criteria-positive patients with an aortic event (n = 34) as compared with all other probands (n = 145) without a reported aortic event (79 vs. 39%; P < 0.0001), as well as Ghent criteria-positive probands (n = 54) without an aortic event (79 vs. 48%; P = 0.0039). Most probands with an early aortic event had a truncating or splicing variant (100% (n = 12) and 95% (n = 21) of patients younger than 30 and 40 years old, respectively). Aortic events occurred at a younger median age in patients with truncating/splicing variants (29 years) as compared with those with missense variants (51 years). A trend toward a higher frequency of truncating/splicing variants in patients with aortic dissection (n = 21) versus prophylactic surgery (n = 13) (85.7 vs. 69.3%; not significant) was observed. These aortic event- and age-associated findings may have important implications for the management of Marfan syndrome patients with FBN1 truncating and splicing variants.Genet Med 17 3, 177-187.

Mutations in SNRPB, encoding components of the core splicing machinery, cause cerebro-costo-mandibular syndrome.

PubMed

Bacrot, Séverine; Doyard, Mathilde; Huber, Céline; Alibeu, Olivier; Feldhahn, Niklas; Lehalle, Daphné; Lacombe, Didier; Marlin, Sandrine; Nitschke, Patrick; Petit, Florence; Vazquez, Marie-Paule; Munnich, Arnold; Cormier-Daire, Valérie

2015-02-01

Cerebro-costo-mandibular syndrome (CCMS) is a developmental disorder characterized by the association of Pierre Robin sequence and posterior rib defects. Exome sequencing and Sanger sequencing in five unrelated CCMS patients revealed five heterozygous variants in the small nuclear ribonucleoprotein polypeptides B and B1 (SNRPB) gene. This gene includes three transcripts, namely transcripts 1 and 2, encoding components of the core spliceosomal machinery (SmB' and SmB) and transcript 3 undergoing nonsense-mediated mRNA decay. All variants were located in the premature termination codon (PTC)-introducing alternative exon of transcript 3. Quantitative RT-PCR analysis revealed a significant increase in transcript 3 levels in leukocytes of CCMS individuals compared to controls. We conclude that CCMS is due to heterozygous mutations in SNRPB, enhancing inclusion of a SNRPB PTC-introducing alternative exon, and show that this developmental disease is caused by defects in the splicing machinery. Our finding confirms the report of SNRPB mutations in CCMS patients by Lynch et al. (2014) and further extends the clinical and molecular observations. © 2014 WILEY PERIODICALS, INC.

Do Androgen Receptor Splice Variants Facilitate Growth of Bone Metastases

DTIC Science & Technology

2016-11-01

therapy is expression of constitutively active AR splice variants, which lack the carboxyl terminal hormone binding domain. The best characterized...resistance is expression of constitutively active AR splice variants, which lack the carboxyl terminal hormone binding domain. Of these, the most...removes hormone , but also many other factors. We plan to retest the effects of AR-V7 in the complete medium to determine whether effects would be

Alternative Splicing Substantially Diversifies the Transcriptome during Early Photomorphogenesis and Correlates with the Energy Availability in Arabidopsis.

PubMed

Hartmann, Lisa; Drewe-Boß, Philipp; Wießner, Theresa; Wagner, Gabriele; Geue, Sascha; Lee, Hsin-Chieh; Obermüller, Dominik M; Kahles, André; Behr, Jonas; Sinz, Fabian H; Rätsch, Gunnar; Wachter, Andreas

2016-11-01

Plants use light as source of energy and information to detect diurnal rhythms and seasonal changes. Sensing changing light conditions is critical to adjust plant metabolism and to initiate developmental transitions. Here, we analyzed transcriptome-wide alterations in gene expression and alternative splicing (AS) of etiolated seedlings undergoing photomorphogenesis upon exposure to blue, red, or white light. Our analysis revealed massive transcriptome reprogramming as reflected by differential expression of ∼20% of all genes and changes in several hundred AS events. For more than 60% of all regulated AS events, light promoted the production of a presumably protein-coding variant at the expense of an mRNA with nonsense-mediated decay-triggering features. Accordingly, AS of the putative splicing factor REDUCED RED-LIGHT RESPONSES IN CRY1CRY2 BACKGROUND1, previously identified as a red light signaling component, was shifted to the functional variant under light. Downstream analyses of candidate AS events pointed at a role of photoreceptor signaling only in monochromatic but not in white light. Furthermore, we demonstrated similar AS changes upon light exposure and exogenous sugar supply, with a critical involvement of kinase signaling. We propose that AS is an integration point of signaling pathways that sense and transmit information regarding the energy availability in plants. © 2016 American Society of Plant Biologists. All rights reserved.

Antitumorigenic Effects of ZAKβ, an Alternative Splicing Isoform of ZAK.

PubMed

Lee, Jin-Sun; Lin, Yuh-Yih; Wang, Tsu-Shing; Liu, Jer-Yuh; Lin, Wei-Wen; Yang, Jaw-Ji

2018-02-28

Sterile alpha motif (SAM)- and leucine-zipper-containing kinase (ZAK) plays a role in the regulation of cell cycle progression and oncogenic transformation. The ZAK gene generates two transcript variants, ZAKα and ZAKβ, through alternative splicing. In this study, we identified that ZAKα proteins were upregulated in tumor tissues, whereas ZAKβ proteins were mostly expressed in corresponding normal tissues. The ectopically expressed ZAKβ proteins in cancer cells inhibited cancer cell proliferation as well as anchorage-independent growth. The ZAKβ:ZAKα protein ratio played a role in the regulation of the cyclic adenosine monophosphate (cAMP) signaling pathway, whereas high ZAKβ protein levels led to the activation of cAMP response element binding protein 1 (CREB1) and exerted antitumor properties. Overexpression of ZAKβ or CREB1 cDNAs in cancer cells inhibited anchorage-independent growth and also reduced the levels of cyclooxygenase 2 (Cox2) and β-catenin proteins. Cancer cells treated with doxorubicin (Doxo) resulted in the switching from the expression of ZAKα to ZAKβ and also inhibited cancer cell growth in soft agar, demonstrating that pharmacological drugs could be used to manipulate endogenous reprogramming splicing events and resulting in the activation of endogenous antitumorigenic properties. We showed that the two ZAK transcript variants, ZAKα and ZAKβ, had opposite biological functions in the regulation of tumor cell proliferation in that ZAKβ had powerful antitumor properties and that ZAKα could promote tumor growth.

Restricting calcium currents is required for correct fiber type specification in skeletal muscle

PubMed Central

Sultana, Nasreen; Dienes, Beatrix; Benedetti, Ariane; Tuluc, Petronel; Szentesi, Peter; Sztretye, Monika; Rainer, Johannes; Hess, Michael W.; Schwarzer, Christoph; Obermair, Gerald J.; Csernoch, Laszlo

2016-01-01

ABSTRACT Skeletal muscle excitation-contraction (EC) coupling is independent of calcium influx. In fact, alternative splicing of the voltage-gated calcium channel CaV1.1 actively suppresses calcium currents in mature muscle. Whether this is necessary for normal development and function of muscle is not known. However, splicing defects that cause aberrant expression of the calcium-conducting developmental CaV1.1e splice variant correlate with muscle weakness in myotonic dystrophy. Here, we deleted CaV1.1 (Cacna1s) exon 29 in mice. These mice displayed normal overall motor performance, although grip force and voluntary running were reduced. Continued expression of the developmental CaV1.1e splice variant in adult mice caused increased calcium influx during EC coupling, altered calcium homeostasis, and spontaneous calcium sparklets in isolated muscle fibers. Contractile force was reduced and endurance enhanced. Key regulators of fiber type specification were dysregulated and the fiber type composition was shifted toward slower fibers. However, oxidative enzyme activity and mitochondrial content declined. These findings indicate that limiting calcium influx during skeletal muscle EC coupling is important for the secondary function of the calcium signal in the activity-dependent regulation of fiber type composition and to prevent muscle disease. PMID:26965373

Proliferation marker pKi-67 occurs in different isoforms with various cellular effects.

PubMed

Schmidt, Mirko H H; Broll, Rainer; Bruch, Hans-Peter; Finniss, Susan; Bögler, Oliver; Duchrow, Michael

2004-04-15

The Ki-67 antigen, pKi-67, is a commonly used proliferation marker in research and pathology. It has been recognized that the protein exists in two different splice variants that differ in one exon. In the current work, we present three new splice variants of human pKi-67 consisting of two naturally occurring isoforms and one atypical version. Additionally, data is presented indicating that alternative splicing of the pKi-67 N-terminus is common in tumor cell lines. Analyzing 93 tissues mainly consisting of brain tumor specimens, we found evidence that long and short isoform can be expressed independently of each other. Induction of mitosis in human peripheral blood mononuclear cells revealed that short pKi-67 appears earlier in the cell cycle than the long isoform and reaches its expression maximum when transcription of the latter sets in. Finally, transfection of mammalian culture cells with exon 7 (specific for the long pKi-67 isoform and not present in the short isoform) in a tetracycline regulated expression system decreased the rate of cell proliferation without affecting the cell cycle. In summary, we present evidence that the pKi-67 N-terminus is differentially spliced resulting in at least five different isoforms with different functions. Copyright 2004 Wiley-Liss, Inc.

Evaluation of Bioinformatic Programmes for the Analysis of Variants within Splice Site Consensus Regions

PubMed Central

Tang, Rongying; Prosser, Debra O.; Love, Donald R.

2016-01-01

The increasing diagnostic use of gene sequencing has led to an expanding dataset of novel variants that lie within consensus splice junctions. The challenge for diagnostic laboratories is the evaluation of these variants in order to determine if they affect splicing or are merely benign. A common evaluation strategy is to use in silico analysis, and it is here that a number of programmes are available online; however, currently, there are no consensus guidelines on the selection of programmes or protocols to interpret the prediction results. Using a collection of 222 pathogenic mutations and 50 benign polymorphisms, we evaluated the sensitivity and specificity of four in silico programmes in predicting the effect of each variant on splicing. The programmes comprised Human Splice Finder (HSF), Max Entropy Scan (MES), NNSplice, and ASSP. The MES and ASSP programmes gave the highest performance based on Receiver Operator Curve analysis, with an optimal cut-off of score reduction of 10%. The study also showed that the sensitivity of prediction is affected by the level of conservation of individual positions, with in silico predictions for variants at positions −4 and +7 within consensus splice sites being largely uninformative. PMID:27313609

Unexpected dependence of RyR1 splice variant expression in human lower limb muscles on fiber-type composition.

PubMed

Willemse, Hermia; Theodoratos, Angelo; Smith, Paul N; Dulhunty, Angela F

2016-02-01

The skeletal muscle ryanodine receptor Ca(2+) release channel (RyR1), essential for excitation-contraction (EC) coupling, demonstrates a known developmentally regulated alternative splicing in the ASI region. We now find unexpectedly that the expression of the splice variants is closely related to fiber type in adult human lower limb muscles. We examined the distribution of myosin heavy chain isoforms and ASI splice variants in gluteus minimus, gluteus medius and vastus medialis from patients aged 45 to 85 years. There was a strong positive correlation between ASI(+)RyR1 and the percentage of type 2 fibers in the muscles (r = 0.725), and a correspondingly strong negative correlation between the percentages of ASI(+)RyR1 and percentage of type 1 fibers. When the type 2 fiber data were separated into type 2X and type 2A, the correlation with ASI(+)RyR1 was stronger in type 2X fibers (r = 0.781) than in type 2A fibers (r = 0.461). There was no significant correlation between age and either fiber-type composition or ASI(+)RyR1/ASI(-)RyR1 ratio. The results suggest that the reduced expression of ASI(-)RyR1 during development may reflect a reduction in type 1 fibers during development. Preferential expression of ASI(-) RyR1, having a higher gain of in Ca(2+) release during EC coupling than ASI(+)RyR1, may compensate for the reduced terminal cisternae volume, fewer junctional contacts and reduced charge movement in type 1 fibers.

Functional analysis of U1-70K interacting SR proteins in pre-mRNA splicing in Arabidopsis

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

A.S.N. Reddy

Proteins of a serine/arginine-rich (SR) family are part of the spliceosome and are implicated in both constitutive and alternative splicing of pre-mRNAs. With the funding from DOE we have been studying alternative of splicing of genes encoding serine/arginine-rich (SR) proteins and the roles of SR proteins that interact with U1-70K in regulating basic and alternative splicing. Alternative splicing of pre-mRNAs of Arabidopsis serine/arginine-rich proteins and its regulation by hormones and stresses: We analyzed the splicing of all 19 Arabidopsis genes in different tissues, during different seedling stages and in response to various hormonal and stress treatments. Remarkably, about 90 differentmore » transcripts are produced from 15 SR genes, thereby increasing the transcriptome complexity of SR genes by about five fold. Using the RNA isolated from polysomes we have shown that most of the splice variants are recruited for translation. Alternative splicing of some SR genes is controlled in a developmental and tissue-specific manner (Palusa et al., 2007). Interestingly, among the various hormones and abiotic stresses tested, temperature stress (cold and heat) and ultraviolet light dramatically altered alternative splicing of pre-mRNAs of several SR genes whereas hormones altered the splicing of only two SR genes (Palusa et al., 2007). Localization and dynamics of a novel serine/arginine-rich protein that interacts with U1-70K: We analyzed the intranuclear movement of SR45 fused to GFP by fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP). We demonstrate that the movement of GFP-SR45 is ATP-dependent. Interestingly, inhibition of transcription or phosphorylation slowed the mobility of GFP-SR45 (Ali et al., 2006). Our studies have revealed that the nuclear localization signals are located in arg/ser-rich domains (RS) 1 and 2, whereas the speckle targeting signals are exclusively present in RS2 (Ali et al., 2006). The regulation of SR45 mobility by ATP and a transcriptional inhibitor is in contrast to the mobility of SR family splicing factors in animals and suggests fundamental differences in the movement of plant and animals splicing factors. In vivo interaction of U170K with SR45: To analyze the interaction of U170K with SR45, we expressed these proteins fused to RFP and GFP respectively, in protoplasts. Both the reporters co-localized to the same subnuclear domains. To determine direct interaction of these proteins, we fused full-length U170K to one part of split YFP and full-length or truncated version of SR45 to the second half of split YFP. Coexpession of these split YFP constructs resulted in reconstitution of YFP in speckles, suggesting direction interaction of these proteins in vivo (Ali et al., 2008). SR45 is a Novel Plant-Specific Splicing Factor and is Involved in Regulating Multiple Developmental Processes: Using an in vitro splicing complementation assay, we showed that SR45 is an essential splicing factor. The sr45-1 mutant exhibited a number of developmental abnormalities. Further analysis of flowering time has shown that the autonomous pathway of flowering is affected in the mutant. Expression analysis of several flowering genes has revealed that FLC, a key flowering repressor, is up-regulated in the SR45 mutant. Further, alternative splicing pattern of several other SR genes was altered in the sr45-1 mutant in a tissue-specific manner. Hence, the observed pleiotropic effects on various aspects of development are likely due to altered level of SR protein isoforms, which in turn regulate the splicing of other pre-mRNAs. Expression of wild-type SR45 in the mutant complemented the phenotypic defects and changes in alternative splicing of SR genes. SR45 thus is a novel plant-specific splicing factor and plays a crucial role in multiple developmental processes.« less

Combined genetic and splicing analysis of BRCA1 c.[594-2A>C; 641A>G] highlights the relevance of naturally occurring in-frame transcripts for developing disease gene variant classification algorithms

PubMed Central

de la Hoya, Miguel; Soukarieh, Omar; López-Perolio, Irene; Vega, Ana; Walker, Logan C.; van Ierland, Yvette; Baralle, Diana; Santamariña, Marta; Lattimore, Vanessa; Wijnen, Juul; Whiley, Philip; Blanco, Ana; Raponi, Michela; Hauke, Jan; Wappenschmidt, Barbara; Becker, Alexandra; Hansen, Thomas V. O.; Behar, Raquel; Investigators, KConFaB; Niederacher, Diether; Arnold, Norbert; Dworniczak, Bernd; Steinemann, Doris; Faust, Ulrike; Rubinstein, Wendy; Hulick, Peter J.; Houdayer, Claude; Caputo, Sandrine M.; Castera, Laurent; Pesaran, Tina; Chao, Elizabeth; Brewer, Carole; Southey, Melissa C.; van Asperen, Christi J.; Singer, Christian F.; Sullivan, Jan; Poplawski, Nicola; Mai, Phuong; Peto, Julian; Johnson, Nichola; Burwinkel, Barbara; Surowy, Harald; Bojesen, Stig E.; Flyger, Henrik; Lindblom, Annika; Margolin, Sara; Chang-Claude, Jenny; Rudolph, Anja; Radice, Paolo; Galastri, Laura; Olson, Janet E.; Hallberg, Emily; Giles, Graham G.; Milne, Roger L.; Andrulis, Irene L.; Glendon, Gord; Hall, Per; Czene, Kamila; Blows, Fiona; Shah, Mitul; Wang, Qin; Dennis, Joe; Michailidou, Kyriaki; McGuffog, Lesley; Bolla, Manjeet K.; Antoniou, Antonis C.; Easton, Douglas F.; Couch, Fergus J.; Tavtigian, Sean; Vreeswijk, Maaike P.; Parsons, Michael; Meeks, Huong D.; Martins, Alexandra; Goldgar, David E.; Spurdle, Amanda B.

2016-01-01

A recent analysis using family history weighting and co-observation classification modeling indicated that BRCA1 c.594-2A > C (IVS9-2A > C), previously described to cause exon 10 skipping (a truncating alteration), displays characteristics inconsistent with those of a high risk pathogenic BRCA1 variant. We used large-scale genetic and clinical resources from the ENIGMA, CIMBA and BCAC consortia to assess pathogenicity of c.594-2A > C. The combined odds for causality considering case-control, segregation and breast tumor pathology information was 3.23 × 10−8. Our data indicate that c.594-2A > C is always in cis with c.641A > G. The spliceogenic effect of c.[594-2A > C;641A > G] was characterized using RNA analysis of human samples and splicing minigenes. As expected, c.[594-2A > C; 641A > G] caused exon 10 skipping, albeit not due to c.594-2A > C impairing the acceptor site but rather by c.641A > G modifying exon 10 splicing regulatory element(s). Multiple blood-based RNA assays indicated that the variant allele did not produce detectable levels of full-length transcripts, with a per allele BRCA1 expression profile composed of ≈70–80% truncating transcripts, and ≈20–30% of in-frame Δ9,10 transcripts predicted to encode a BRCA1 protein with tumor suppression function. We confirm that BRCA1c.[594-2A > C;641A > G] should not be considered a high-risk pathogenic variant. Importantly, results from our detailed mRNA analysis suggest that BRCA-associated cancer risk is likely not markedly increased for individuals who carry a truncating variant in BRCA1 exons 9 or 10, or any other BRCA1 allele that permits 20–30% of tumor suppressor function. More generally, our findings highlight the importance of assessing naturally occurring alternative splicing for clinical evaluation of variants in disease-causing genes. PMID:27008870

Prognostic impact of mRNA levels of osteopontin splice variants in soft tissue sarcoma patients.

PubMed

Hahnel, Antje; Wichmann, Henri; Greither, Thomas; Kappler, Matthias; Würl, Peter; Kotzsch, Matthias; Taubert, Helge; Vordermark, Dirk; Bache, Matthias

2012-04-02

It is well known that osteopontin (OPN) plays an important role in tumor progression and that a high OPN expression level in several tumor entities correlates with poor prognosis in cancer patients. However, little is known about the prognostic relevance of the OPN mRNA splice variants. We analyzed the mRNA expression levels of different OPN splice variants in tumor tissue of 124 soft tissue sarcoma (STS) patients. Quantitative real-time PCR (qRT-PCR) was used to analyze the mRNA expression level of three OPN splice variants (OPN-a, -b and -c). The multivariate Cox's proportional hazard regression model revealed that high mRNA expression levels of OPN splice variants are significantly associated with poor prognosis in STS patients (n = 124). Women (n = 68) with high mRNA expression levels of OPN-a and OPN-b have an especially elevated risk of tumor-related death (OPN-a: RR = 3.0, P = 0.01, CI = 1.3-6.8; OPN-b: RR = 3.4, P = 0.01, CI = 1.4-8.2). In particular, we found that high mRNA expression levels of OPN-b and OPN-c correlated with a high risk of tumor-related death in STS patients that received radiotherapy (n = 52; OPN-b: RR = 10.3, P < 0.01, CI = 2.0-53.7; OPN-c: RR = 11.4, P < 0.01, CI = 2.2-59.3). Our study shows that elevated mRNA expression levels of OPN splice variants are negative prognostic and predictive markers for STS patients. Further studies are needed to clarify the impact of the OPN splice variants on prognosis.

Hypoxia-Induced Expression of VEGF Splice Variants and Protein in Four Retinal Cell Types

PubMed Central

Watkins, William M.; McCollum, Gary W.; Savage, Sara R.; Capozzi, Megan E.; Penn, John S.; Morrison, David G.

2014-01-01

The purpose of this study was to investigate the hypoxia-induced Vegf120, Vegf164 and Vegf188 mRNA expression profiles in rat Müller cells (MC), astrocytes, retinal pigmented epithelial cells (RPE) and retinal microvascular endothelial cells (RMEC) and correlate these findings to VEGF secreted protein. Cultured cells were exposed to normoxia or hypoxia. Total RNA was isolated from cell lysates and Vegf splice variant mRNA copy numbers were assayed by a validated qRT-PCR external calibration curve method. mRNA copy numbers were normalized to input total RNA. Conditioned medium was collected from cells and assayed for total VEGF protein by ELISA. Hypoxia increased total Vegf mRNA and secreted protein in all the retinal cell types, with the highest levels observed in MC and astrocytes ranking second. Total Vegf mRNA levels in hypoxic RPE and RMEC were comparable; however, the greatest hypoxic induction of each Vegf splice variant mRNA was observed in RMEC. RPE and RMEC ranked 3rd and 4th respectively, in terms of secreted total VEGF protein in hypoxia. The Vegf120, Vegf164 and Vegf188 mRNA splice variants were all increased in hypoxic cells compared to normoxic controls. In normoxia, the relative Vegf splice variant mRNA levels ranked from highest to lowest for each cell type were Vegf164>Vegf120>Vegf188. Hypoxic induction did not alter this ranking, although it did favor an increased stoichiometry of Vegf164 mRNA over the other two splice variants. MC and astrocytes are likely to be the major sources of total Vegf, and Vegf164 splice variant mRNAs, and VEGF protein in retinal hypoxia. PMID:24076411

High temperature attenuates the gravitropism of inflorescence stems by inducing SHOOT GRAVITROPISM 5 alternative splicing in Arabidopsis.

PubMed

Kim, Joo-Young; Ryu, Jae Yong; Baek, Kon; Park, Chung-Mo

2016-01-01

In higher plants, gravitropism proceeds through three sequential steps in the responding organs: perception of gravity signals, signal transduction and asymmetric cell elongation. Light and temperature also influence the gravitropic orientation of plant organs. A series of Arabidopsis shoot gravitropism (sgr) mutants has been shown to exhibit disturbed shoot gravitropism. SGR5 is functionally distinct from other SGR members in that it mediates the early events of gravitropic responses in inflorescence stems. Here, we demonstrated that SGR5 alternative splicing produces two protein variants (SGR5α and SGR5β) in modulating the gravitropic response of inflorescence stems at high temperatures. SGR5β inhibits SGR5α function by forming non-DNA-binding heterodimers. Transgenic plants overexpressing SGR5β (35S:SGR5β) exhibit reduced gravitropic growth of inflorescence stems, as observed in the SGR5-deficient sgr5-5 mutant. Interestingly, SGR5 alternative splicing is accelerated at high temperatures, resulting in the high-level accumulation of SGR5β transcripts. When plants were exposed to high temperatures, whereas gravitropic curvature was reduced in Col-0 inflorescence stems, it was uninfluenced in the inflorescence stems of 35S:SGR5β transgenic plants and sgr5-5 mutant. We propose that the thermoresponsive alternative splicing of SGR5 provides an adaptation strategy by which plants protect the shoots from hot air under high temperature stress in natural habitats. © 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.

Osteopontin and splice variant expression level in human malignant glioma: radiobiologic effects and prognosis after radiotherapy.

PubMed

Güttler, Antje; Giebler, Maria; Cuno, Peter; Wichmann, Henri; Keßler, Jacqueline; Ostheimer, Christian; Söling, Ariane; Strauss, Christian; Illert, Jörg; Kappler, Matthias; Vordermark, Dirk; Bache, Matthias

2013-09-01

We investigated the role of the hypoxia-associated secreted glycoprotein osteopontin (OPN) in the response of malignant glioma to radiotherapy by characterizing OPN and its splice variants in vitro and in patient material. The effect of siRNA knockdown of OPN splice variants on cellular and radiobiologic behavior was analyzed in U251MG cells using OpnS siRNA (inhibition of all OPN splice variants) and OpnAC siRNA (knockdown only of OPNa and OPNc). OPN and splice variant mRNA levels were quantified in archival material of 41 glioblastoma tumor samples. Plasma OPN was prospectively measured in 33 malignant glioma patients. Inhibition of OPNa and OPNc (OpnAC) reduced clonogenic survival in U251MG cells but did not affect proliferation, migration or apoptosis. Knockdown of all OPN splice variants (OpnS) resulted in an even stronger inhibition of clonogenic survival, while cell proliferation and migration were reduced and rate of apoptosis was increased. Additional irradiation had additive effects with both siRNAs. Plasma OPN increased continuously in malignant glioma patients and was associated with poor survival. OPNb is partially able to compensate the effects of OPNa and OPNc knockdown in U251MG cells. High OPN plasma levels at the end of radiotherapy are associated with poor survival. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Diverse alternative back-splicing and alternative splicing landscape of circular RNAs

PubMed Central

Zhang, Xiao-Ou; Dong, Rui; Zhang, Yang; Zhang, Jia-Lin; Luo, Zheng; Zhang, Jun; Chen, Ling-Ling; Yang, Li

2016-01-01

Circular RNAs (circRNAs) derived from back-spliced exons have been widely identified as being co-expressed with their linear counterparts. A single gene locus can produce multiple circRNAs through alternative back-splice site selection and/or alternative splice site selection; however, a detailed map of alternative back-splicing/splicing in circRNAs is lacking. Here, with the upgraded CIRCexplorer2 pipeline, we systematically annotated different types of alternative back-splicing and alternative splicing events in circRNAs from various cell lines. Compared with their linear cognate RNAs, circRNAs exhibited distinct patterns of alternative back-splicing and alternative splicing. Alternative back-splice site selection was correlated with the competition of putative RNA pairs across introns that bracket alternative back-splice sites. In addition, all four basic types of alternative splicing that have been identified in the (linear) mRNA process were found within circRNAs, and many exons were predominantly spliced in circRNAs. Unexpectedly, thousands of previously unannotated exons were detected in circRNAs from the examined cell lines. Although these novel exons had similar splice site strength, they were much less conserved than known exons in sequences. Finally, both alternative back-splicing and circRNA-predominant alternative splicing were highly diverse among the examined cell lines. All of the identified alternative back-splicing and alternative splicing in circRNAs are available in the CIRCpedia database (http://www.picb.ac.cn/rnomics/circpedia). Collectively, the annotation of alternative back-splicing and alternative splicing in circRNAs provides a valuable resource for depicting the complexity of circRNA biogenesis and for studying the potential functions of circRNAs in different cells. PMID:27365365

Glucose-dependent insulinotropic peptide stimulates thymidine incorporation in endothelial cells: role of endothelin-1

NASA Technical Reports Server (NTRS)

Ding, Ke-Hong; Zhong, Qing; Isales, Carlos M.; Iscules, C. M. (Principal Investigator)

2003-01-01

We have previously characterized the receptor for glucose-dependent insulinotropic polypeptide (GIPR) in vascular endothelial cells (EC). Different EC types were found to contain distinct GIPR splice variants. To determine whether activation of the GIPR splice variants resulted in different cellular responses, we examined GIP effects on human umbilical vein endothelial cells (HUVEC), which contain two GIPR splice variants, and compared them with a spontaneously transformed human umbilical vein EC line, ECV 304, which contains four GIPR splice variants. GIP dose-dependently stimulated HUVEC and ECV 304 proliferation as measured by [3H]thymidine incorporation. GIP increased endothelin-1 (ET-1) secretion from HUVEC but not from ECV 304. Use of the endothelin B receptor blocker BQ-788 resulted in an inhibition of [3H]thymidine incorporation in HUVEC but not in ECV 304. These findings suggest that, although GIP increases [3H]thymidine incorporation in both HUVEC and ECV 304, this proliferative response is mediated by ET-1 only in HUVEC. These differences in cellular response to GIP may be related to differences in activation of GIPR splice variants.

Comprehensive Characterization of Swine Cardiac Troponin T Proteoforms by Top-Down Mass Spectrometry

NASA Astrophysics Data System (ADS)

Lin, Ziqing; Guo, Fang; Gregorich, Zachery R.; Sun, Ruixiang; Zhang, Han; Hu, Yang; Shanmuganayagam, Dhanansayan; Ge, Ying

2018-04-01

Cardiac troponin T (cTnT) regulates the Ca2+-mediated interaction between myosin thick filaments and actin thin filaments during cardiac contraction and relaxation. cTnT is released into the blood following injury, and increased serum levels of the protein are used clinically as a biomarker for myocardial infarction. Moreover, mutations in cTnT are causative in a number of familial cardiomyopathies. With the increasing use of large animal (swine) model to recapitulate human diseases, it is essential to characterize species-dependent protein sequence variants, alternative RNA splicing, and post-translational modifications (PTMs), but challenges remain due to the incomplete database and lack of validation of the predicted splicing isoforms. Herein, we integrated top-down mass spectrometry (MS) with online liquid chromatography (LC) and immunoaffinity purification to comprehensively characterize miniature swine cTnT proteoforms, including those arising from alternative RNA splicing and PTMs. A total of seven alternative splicing isoforms of cTnT were identified by LC/MS from swine left ventricular tissue, with each isoform containing un-phosphorylated and mono-phosphorylated proteoforms. The phosphorylation site was localized to Ser1 for the mono-phosphorylated proteoforms of cTnT1, 3, 4, and 6 by online MS/MS combining collisionally activated dissociation (CAD) and electron transfer dissociation (ETD). Offline MS/MS on Fourier-transform ion cyclotron resonance (FT-ICR) mass spectrometer with CAD and electron capture dissociation (ECD) was then utilized to achieve deep sequencing of mono-phosphorylated cTnT1 (35.2 kDa) with a high sequence coverage of 87%. Taken together, this study demonstrated the unique advantage of top-down MS in the comprehensive characterization of protein alternative splicing isoforms together with PTMs. [Figure not available: see fulltext.

Exonic Splicing Mutations Are More Prevalent than Currently Estimated and Can Be Predicted by Using In Silico Tools

PubMed Central

Soukarieh, Omar; Gaildrat, Pascaline; Hamieh, Mohamad; Drouet, Aurélie; Baert-Desurmont, Stéphanie; Frébourg, Thierry; Tosi, Mario; Martins, Alexandra

2016-01-01

The identification of a causal mutation is essential for molecular diagnosis and clinical management of many genetic disorders. However, even if next-generation exome sequencing has greatly improved the detection of nucleotide changes, the biological interpretation of most exonic variants remains challenging. Moreover, particular attention is typically given to protein-coding changes often neglecting the potential impact of exonic variants on RNA splicing. Here, we used the exon 10 of MLH1, a gene implicated in hereditary cancer, as a model system to assess the prevalence of RNA splicing mutations among all single-nucleotide variants identified in a given exon. We performed comprehensive minigene assays and analyzed patient’s RNA when available. Our study revealed a staggering number of splicing mutations in MLH1 exon 10 (77% of the 22 analyzed variants), including mutations directly affecting splice sites and, particularly, mutations altering potential splicing regulatory elements (ESRs). We then used this thoroughly characterized dataset, together with experimental data derived from previous studies on BRCA1, BRCA2, CFTR and NF1, to evaluate the predictive power of 3 in silico approaches recently described as promising tools for pinpointing ESR-mutations. Our results indicate that ΔtESRseq and ΔHZEI-based approaches not only discriminate which variants affect splicing, but also predict the direction and severity of the induced splicing defects. In contrast, the ΔΨ-based approach did not show a compelling predictive power. Our data indicates that exonic splicing mutations are more prevalent than currently appreciated and that they can now be predicted by using bioinformatics methods. These findings have implications for all genetically-caused diseases. PMID:26761715

Enhanced risk profiling of implanted defibrillator shocks with circulating SCN5A mRNA splicing variants: a pilot trial

PubMed Central

Gao, Ge; Brahmanandam, Vikram; Raicu, Mihai; Gu, Lianzhi; Zhou, Li; Kasturirangan, Srinivasan; Shah, Anish; Negi, Smita I.; Wood, Melissa R.; Desai, Ankit A.; Tatooles, Antone; Schwartz, Alan; Dudley, Samuel C.

2014-01-01

Objectives The aim of this study was to determine the association of SCN5A cardiac sodium (Na+) channel mRNA splice variants in white blood cells (WBCs) with risk of arrhythmias in heart failure (HF). Background HF is associated with upregulation of two cardiac SCN5A mRNA splice variants. that encode prematurely truncated, nonfunctional Na+ channels. Since circulating WBCs demonstrate similar SCN5A splicing patterns, we hypothesized that these WBC-derived splice variants might further stratify HF patients at risk for arrhythmias. Methods Simultaneously obtained myocardial core samples and WBCs were compared for SCN5A variants C (VC) and D (VD). Circulating variant levels were compared between HF patients divided into three groups: HF without an implantable cardioverter-defibrillator (ICD), HF with an ICD without appropriate intervention, and HF with an ICD with appropriate intervention. Results Myocardial tissue-derived SCN5A variant expression levels strongly correlated with circulating WBC samples for both VC and VD variants (r = 0.78 and 0.75, respectively). After controlling for covariates, HF patients who had received an appropriate ICD intervention had higher expression levels of both WBC-derived SCN5A variants compared to HF patients with ICDs who had not (OR= 3.25 (95% CI 1.64–6.45; p=0.001)). Receiver operating characteristics analysis revealed that circulating SCN5A variants levels were highly associated with the risk for appropriate ICD intervention (area under the curve ≥ 0.97). Conclusions Circulating expression levels of SCN5A variants were strongly associated with myocardial tissue levels. Furthermore, circulating variant levels were correlative with arrhythmic risk as measured by ICD events in a HF population within one year. PMID:24703920

Renoprotective impact of estrogen receptor α and its splice variants in female mice with type 1 diabetes.

PubMed

Irsik, Debra L; Romero-Aleshire, Melissa Jill; Chavez, Erin M; Fallet, Rachel W; Brooks, Heddwen L; Carmines, Pamela K; Lane, Pascale H

2018-04-18

Estrogen has been implicated in the regulation of growth and immune function in the kidney, which expresses the full-length estrogen receptor α (ERα66), its ERα splice variants, and estrogen receptor β (ERβ). Thus, we hypothesized that these splice variants may inhibit glomerular enlargement that occurs early in type 1 diabetes (T1D). T1D was induced by streptozotocin (STZ) injection in 8-12 wk-old female mice lacking ERα66 (ERα66KO) or all ERα variants (αERKO), and their wild-type (WT) littermates. Basal renal ERα36 protein expression was reduced in the ERα66KO model and was downregulated by T1D in WT mice. T1D did not alter ERα46 or ERβ in WT-STZ; however, ERα46 was decreased modestly in ERα66KO. Renal hypertrophy was evident in all diabetic mice. F4/80-positive immunostaining was reduced in ERα66KO, compared with WT and αERKO mice, but was higher in STZ than in WT mice across all genotypes. Glomerular area was greater in WT and αERKO than in ERα66KO mice, with T1D-induced glomerular enlargement apparent in WT-STZ and αERKO-STZ, but not in ERα66KO-STZ. Proteinuria and hyperfiltration were evident in ERα66KO-STZ and αERKO-STZ, but not in WT-STZ mice. These data indicate that ERα splice variants may exert an inhibitory influence on glomerular enlargement and macrophage infiltration during T1D; however, effects of splice variants are masked in the presence of the full-length ERα66, suggesting that ERα66 acts in opposition to its splice variants to influence these parameters. In contrast, hyperfiltration and proteinuria in T1D are attenuated via an ERα66-dependent mechanism that is unaffected by splice variant status.

1-alpha,25-Dihydroxyvitamin D3 up-regulates the expression of 2 types of human intestinal alkaline phosphatase alternative splicing variants in Caco-2 cells and may be an important regulator of their expression in gut homeostasis.

PubMed

Noda, Seiko; Yamada, Asako; Nakaoka, Kanae; Goseki-Sone, Masae

2017-10-01

Vitamin D insufficiency is associated with a greater risk of osteoporosis and also influences skeletal muscle functions, differentiation, and development. The principal function of vitamin D in calcium homeostasis is to increase the absorption of calcium from the intestine, and the level of alkaline phosphatase (ALP) activity, a differentiation marker for intestinal epithelial cells, is regulated by vitamin D. Intestinal-type ALP is expressed at a high concentration in the brush border membrane of intestinal epithelial cells, and is known to be affected by several kinds of nutrients. Recent reviews have highlighted the importance of intestinal-type ALP in gut homeostasis. Intestinal-type ALP controls bacterial endotoxin-induced inflammation by dephosphorylating lipopolysaccharide and is a gut mucosal defense factor. In this study, we investigated the influence of vitamin D on the expression of 2 types of alternative mRNA variants encoding the human alkaline phosphatase, intestinal (ALPI) gene in human Caco-2 cells as an in vitro model of the small intestinal epithelium. After treatment with 1-alpha,25-dihydroxyvitamin D 3 , the biologically active form of vitamin D 3 , there were significant increases in the ALP activities of Caco-2 cells. Inhibitor and thermal inactivation experiments showed that the increased ALP had properties of intestinal-type ALP. Reverse transcription-polymerase chain reaction analysis revealed that expression of the 2 types of alternative mRNA variants from the ALPI gene was markedly enhanced by vitamin D in Caco-2 cells. In conclusion, these findings agree with the hypothesis: vitamin D up-regulated the expression of 2 types of human intestinal alkaline phosphatase alternative splicing variants in Caco-2 cells; vitamin D may be an important regulator of ALPI gene expression in gut homeostasis. Copyright © 2017 Elsevier Inc. All rights reserved.

Investigations into the binding affinities of different human 5-HT4 receptor splice variants.

PubMed

Irving, Helen R; Tochon-Danguy, Nathalie; Chinkwo, Kenneth A; Li, Jian G; Grabbe, Carmen; Shapiro, Marina; Pouton, Colin W; Coupar, Ian M

2010-01-01

This study examined whether the drug-receptor-binding sites of 5 selected human 5-HT(4) receptor splice variants [h5-HT4(a), h5-HT4(b), h5-HT4(c), h5-HT4(d) and h5-HT4(g)] display preferential affinities towards agonists. The agonists selected on the basis of chemical diversity and clinical relevance were: 5-HT4 benzamides, renzapride, zacopride and prucalopride; the benzimidazolones, DAU 6236 and BIMU 1; the aromatic ketone, RS67333, and the indole carbazimidamide tegaserod. The rank order of affinities ranging across the splice variants was: tegaserod (pKi: 7.38-7.91) > or = Y-36912 (pKi: 7.03-7.85) = BIMU 1 (pKi: 6.92-7.78) > or = DAU 6236 (pKi: 6.79-7.99) > or = 5-HT (pKi: 5.82-7.29) > or = 5-MeOT (pKi: 5.64-6.83) > or = renzapride (pKi: 4.85-5.56). We obtained affinity values for the 5-HT4(b), (d) and (g) variants for RS67333 (pKi: 7:48-8.29), prucalopride (pKi: 6.86-7.37) and zacopride (pKi: 5.88-7.0). These results indicate that the ligands interact with the same conserved site in each splice variant. Some splice variants have a higher affinity for certain agonists and the direction of selectivity followed a common trend of lowest affinity at the (d) variant. However, this trend was not evident in functional experiments. Our findings suggest that it may be possible to design splice variant selective ligands, which may be of relevance for experimental drugs but may be difficult to develop clinically. 2010 S. Karger AG, Basel.

CaMKIIβ Association with the Actin Cytoskeleton Is Regulated by Alternative Splicing

PubMed Central

O'Leary, Heather; Lasda, Erika

2006-01-01

The Ca2+/calmodulin (CaM)-dependent protein kinase II (CaMKII)β has morphogenic functions in neurons not shared by the α isoform. CaMKIIβ contains three exons (v1, v3, and v4) not present in the CaMKIIα gene, and two of these exons (v1 and v4) are subject to differential alternative splicing. We show here that CaMKIIβ, but not α, mediated bundling of F-actin filaments in vitro. Most importantly, inclusion of exon v1 was required for CaMKIIβ association with the F-actin cytoskeleton within cells. CaMKIIβe, which is the dominant variant around birth and lacks exon v1 sequences, failed to associate with F-actin. By contrast, CaMKIIβ′, which instead lacks exon v4, associated with F-actin as full-length CaMKIIβ. Previous studies with CaMKIIβ mutants have indicated a role of nonstimulated kinase activity in enhancing dendritic arborization. Here, we show that F-actin–targeted CaMKIIβ, but not α, was able to phosphorylate actin in vitro even by nonstimulated basal activity in absence of Ca2+/CaM. In rat pancreatic islets and in skeletal muscle, the actin-associated CaMKIIβ′ and βM were the predominant variants, respectively. Thus, cytoskeletal targeting may mediate functions of CaMKIIβ variants also outside the nervous system. PMID:16928958

Membrane expression of MRP-1, but not MRP-1 splicing or Pgp expression, predicts survival in patients with ESFT.

PubMed

Roundhill, E; Burchill, S

2013-07-09

Primary Ewing's sarcoma family of tumours (ESFTs) may respond to chemotherapy, although many patients experience subsequent disease recurrence and relapse. The survival of ESFT cells following chemotherapy has been attributed to the development of resistant disease, possibly through the expression of ABC transporter proteins. MRP-1 and Pgp mRNA and protein expression in primary ESFTs was determined by quantitative reverse-transcriptase PCR (RT-qPCR) and immunohistochemistry, respectively, and alternative splicing of MRP-1 by RT-PCR. We observed MRP-1 protein expression in 92% (43 out of 47) of primary ESFTs, and cell membrane MRP-1 was highly predictive of both overall survival (P<0.0001) and event-free survival (P<0.0001). Alternative splicing of MRP-1 was detected in primary ESFTs, although the pattern of splicing variants was not predictive of patient outcome, with the exception of loss of exon 9 in six patients, which predicted relapse (P=0.041). Pgp protein was detected in 6% (38 out of 44) of primary ESFTs and was not associated with patient survival. For the first time we have established that cell membrane expression of MRP-1 or loss of exon 9 is predictive of outcome but not the number of splicing events or expression of Pgp, and both may be valuable factors for the stratification of patients for more intensive therapy.

In silico prediction of splice-altering single nucleotide variants in the human genome.

PubMed

Jian, Xueqiu; Boerwinkle, Eric; Liu, Xiaoming

2014-12-16

In silico tools have been developed to predict variants that may have an impact on pre-mRNA splicing. The major limitation of the application of these tools to basic research and clinical practice is the difficulty in interpreting the output. Most tools only predict potential splice sites given a DNA sequence without measuring splicing signal changes caused by a variant. Another limitation is the lack of large-scale evaluation studies of these tools. We compared eight in silico tools on 2959 single nucleotide variants within splicing consensus regions (scSNVs) using receiver operating characteristic analysis. The Position Weight Matrix model and MaxEntScan outperformed other methods. Two ensemble learning methods, adaptive boosting and random forests, were used to construct models that take advantage of individual methods. Both models further improved prediction, with outputs of directly interpretable prediction scores. We applied our ensemble scores to scSNVs from the Catalogue of Somatic Mutations in Cancer database. Analysis showed that predicted splice-altering scSNVs are enriched in recurrent scSNVs and known cancer genes. We pre-computed our ensemble scores for all potential scSNVs across the human genome, providing a whole genome level resource for identifying splice-altering scSNVs discovered from large-scale sequencing studies.

Updated genetic testing of Italian patients referred with a clinical diagnosis of primary hyperoxaluria.

PubMed

Pelle, Alessandra; Cuccurullo, Alessandra; Mancini, Cecilia; Sebastiano, Regina; Stallone, Giovanni; Negrisolo, Susanna; Benetti, Elisa; Peruzzi, Licia; Petrarulo, Michele; De Marchi, Mario; Marangella, Martino; Amoroso, Antonio; Giachino, Daniela; Mandrile, Giorgia

2017-04-01

Primary hyperoxaluria (PH) is a rare autosomal recessive disease commonly arising in childhood and presenting with nephrolithiasis, nephrocalcinosis and/or chronic renal failure. Three genes are currently known as responsible: alanine-glyoxylate aminotransferase (AGXT, PH type 1), glyoxylate reductase/hydroxypyruvate reductase (GRHPR, PH type 2), and 4-hydroxy-2-oxoglutarate aldolase (HOGA1, PH type 3). In our Centre, at the end of 2014 molecular diagnosis of PH1 had been performed in 80 patients, while one patient received a PH2 diagnosis. Fifteen patients referred to our Centre and suspected to have PH on clinical grounds were negative for pathogenic variants in the entire coding sequence and exon-intron boundaries of the AGXT gene. Therefore, we extended the analysis to the AGXT promoter region and the GRHPR and HOGA1 genes. Two patients were heterozygous for two novel AGXT-promoter variants (c.-647C > T, c.-424C > T) that were probably non pathogenic. One patient was homozygous for a novel HOGA1 variant of intron 2 (c.341-81delT), whose pathogenicity predicted by in silico splicing tools was not confirmed by a minigene splicing assay in COS-7 and HEK293T cells. New genetic subtypes of PH can be hypothesized in our patients, that may be caused by mutations in other gene encoding proteins of glyoxylate metabolism. Alternatively, some kind of mutations (e.g., deletions/duplications, deep intronic splicing regulatory variants) could be missed in a few cases, similarly to other genetic diseases.

Post-transcriptional regulation of inducible nitric oxide synthase in chronic lymphocytic leukemia B cells in pro- and antiapoptotic culture conditions.

PubMed

Tiscornia, A C; Cayota, A; Landoni, A I; Brito, C; Oppezzo, P; Vuillier, F; Robello, C; Dighiero, G; Gabús, R; Pritsch, O

2004-01-01

Functional inducible NOS (iNOS) may be involved in the prolonged lifespan of chronic lymphocytic leukemia cells (B-CLL), although the exact mechanisms implicated remain elusive as yet. In this work, we have examined iNOS expression in normal B lymphocytes and B-CLL cells in pro- and antiapoptotic conditions. Our results demonstrate: (1) The existence of a new splice variant characterized by a complete deletion of exon 14 (iNOS 13-16(14del)), which was preferentially detected in normal B lymphocytes and may represent an isoform that could play a role in the regulation of enzyme activity. (2) The existence of another alternatively spliced iNOS mRNA transcript involving a partial deletion of the flavodoxin region (iNOS 13-16(neg)) was correlated to a decreased B-CLL cell viability. The 9-beta-D-arabinofuranosyl-2-fluoradenine or fludarabine (F-ara) treatment induced iNOS 13-16(neg) transcript variants, whereas IL-4 enhanced both the transcription of variants, including these exons (iNOS 13-16(pos)), and the expression of a 122 kDa iNOS protein. These results suggest that in B-CLL, a regulation process involving nitric oxide (.- NO) levels could occur by a post-transcriptional mechanism mediated by soluble factors. Our results also provide an insight into a new complementary proapoptotic action of F-ara in B-CLL by the induction of particular iNOS splice variants, leading to the activation of a caspase-3-dependent apoptotic pathway.

CRAWview: for viewing splicing variation, gene families, and polymorphism in clusters of ESTs and full-length sequences.

PubMed

Chou, A; Burke, J

1999-05-01

DNA sequence clustering has become a valuable method in support of gene discovery and gene expression analysis. Our interest lies in leveraging the sequence diversity within clusters of expressed sequence tags (ESTs) to model gene structure for the study of gene variants that arise from, among other things, alternative mRNA splicing, polymorphism, and divergence after gene duplication, fusion, and translocation events. In previous work, CRAW was developed to discover gene variants from assembled clusters of ESTs. Most importantly, novel gene features (the differing units between gene variants, for example alternative exons, polymorphisms, transposable elements, etc.) that are specialized to tissue, disease, population, or developmental states can be identified when these tools collate DNA source information with gene variant discrimination. While the goal is complete automation of novel feature and gene variant detection, current methods are far from perfect and hence the development of effective tools for visualization and exploratory data analysis are of paramount importance in the process of sifting through candidate genes and validating targets. We present CRAWview, a Java based visualization extension to CRAW. Features that vary between gene forms are displayed using an automatically generated color coded index. The reporting format of CRAWview gives a brief, high level summary report to display overlap and divergence within clusters of sequences as well as the ability to 'drill down' and see detailed information concerning regions of interest. Additionally, the alignment viewing and editing capabilities of CRAWview make it possible to interactively correct frame-shifts and otherwise edit cluster assemblies. We have implemented CRAWview as a Java application across windows NT/95 and UNIX platforms. A beta version of CRAWview will be freely available to academic users from Pangea Systems (http://www.pangeasystems.com). Contact :

Molecular Cloning and Characterization of the Human ErbB4 Gene: Identification of Novel Splice Isoforms in the Developing and Adult Brain

PubMed Central

Tan, Wei; Dean, Michael; Law, Amanda J.

2010-01-01

ErbB4 is a growth factor receptor tyrosine kinase essential for neurodevelopment. Genetic variation in ErbB4 is associated with schizophrenia and risk-associated polymorphisms predict overexpression of ErbB4 CYT-1 isoforms in the brain in the disorder. The molecular mechanism of association is unclear because the polymorphisms flank exon 3 of the gene and reside 700 kb distal to the CYT-1 defining exon. We hypothesized that the polymorphisms are indirectly associated with ErbB4 CYT-1 via splicing of exon 3 on the CYT-1 background. We report via cloning and sequencing of adult and fetal human brain cDNA libraries the identification of novel splice isoforms of ErbB4, whereby exon 3 is skipped (del.3). ErbB4 del.3 transcripts exist as CYT-2 isoforms and are predicted to produce truncated proteins. Furthermore, our data refine the structure of the human ErbB4 gene, clarify that juxtamembrane (JM) splice variants of ErbB4, JM-a and JM-b respectively, are characterized by the replacement of a 75 nucleotide (nt) sequence with a 45-nt insertion, and demonstrate that there are four alternative exons in the gene. Our analyses reveal that novel splice variants of ErbB4 exist in the developing and adult human brain and, given the failure to identify ErbB4 del.3 CYT-1 transcripts, suggest that the association of risk polymorphisms in the ErbB4 gene with CYT-1 transcript levels is not mediated via an exon 3 splicing event. PMID:20886074

Osteopontin splice variants expression is involved on docetaxel resistance in PC3 prostate cancer cells.

PubMed

Nakamura, K D M; Tilli, T M; Wanderley, J L; Palumbo, A; Mattos, R M; Ferreira, A C; Klumb, C E; Nasciutti, L E; Gimba, E R

2016-02-01

Osteopontin (OPN) is a phosphoprotein that activates several aspects of tumor progression. Alternative splicing of the OPN primary transcript generates three splicing isoforms, OPNa, OPNb and OPNc. In this report, we investigated some cellular mechanisms by which OPN splice variants could mediate PC3 prostate cancer (PCa) cell survival and growth in response to docetaxel (DXT)-induced cell death. Cell survival before and after DXT treatment was analyzed by phase-contrast microscopy and crystal-violet staining assays. Quantitative real-time PCR and immunocytochemical staining assays were used to evaluate the putative involvement of epithelial-mesenchymal transition (EMT) and OPN isoforms on mediating PC3 cell survival. Upon DXT treatment, PC3 cells overexpressing OPNb or OPNc isoforms showed higher cell densities, compared to cells overexpressing OPNa and controls. Notably, cells overexpressing OPNb or OPNc isoforms showed a downregulated pattern of EMT epithelial cell markers, while mesenchymal markers were mostly upregulated in these experimental conditions. We concluded that OPNc or OPNb overexpression in PC3 cells can mediate resistance and cell survival features in response to DXT-induced cell death. Our data also provide evidence the EMT program could be one of the molecular mechanisms mediating survival in OPNb- or OPNc-overexpressing cells in response to DXT treatment. These data could further contribute to a better understanding of the mechanisms by which PCa cells acquire resistance to DXT treatment.

Evolution of the Antisense Overlap between Genes for Thyroid Hormone Receptor and Rev-erbα and Characterization of an Exonic G-Rich Element That Regulates Splicing of TRα2 mRNA

PubMed Central

Munroe, Stephen H.; Morales, Christopher H.; Duyck, Tessa H.; Waters, Paul D.

2015-01-01

The α-thyroid hormone receptor gene (TRα) codes for two functionally distinct proteins: TRα1, the α-thyroid hormone receptor; and TRα2, a non-hormone-binding variant. The final exon of TRα2 mRNA overlaps the 3’ end of Rev-erbα mRNA, which encodes another nuclear receptor on the opposite strand of DNA. To understand the evolution of this antisense overlap, we sequenced these genes and mRNAs in the platypus Orthorhynchus anatinus. Despite its strong homology with other mammals, the platypus TRα/Rev-erbα locus lacks elements essential for expression of TRα2. Comparative analysis suggests that alternative splicing of TRα2 mRNA expression evolved in a stepwise fashion before the divergence of eutherian and marsupial mammals. A short G-rich element (G30) located downstream of the alternative 3’splice site of TRα2 mRNA and antisense to the 3’UTR of Rev-erbα plays an important role in regulating TRα2 splicing. G30 is tightly conserved in eutherian mammals, but is absent in marsupials and monotremes. Systematic deletions and substitutions within G30 have dramatically different effects on TRα2 splicing, leading to either its inhibition or its enhancement. Mutations that disrupt one or more clusters of G residues enhance splicing two- to three-fold. These results suggest the G30 sequence can adopt a highly structured conformation, possibly a G-quadruplex, and that it is part of a complex splicing regulatory element which exerts both positive and negative effects on TRα2 expression. Since mutations that strongly enhance splicing in vivo have no effect on splicing in vitro, it is likely that the regulatory role of G30 is mediated through linkage of transcription and splicing. PMID:26368571

Discovery of a new mechanism for regulation of plant triacylglycerol metabolism: The peanut diacylglycerol acyltransferase-1 gene family transcriptome is highly enriched in alternative splicing variants

USDA-ARS?s Scientific Manuscript database

Triacylglycerols (TAGs) are the most important energy storage form in oilseed crops. Diacylglycerol acyltransferase (DGAT) catalyzes the rate-limiting step of the Kennedy pathway of TAG biosynthesis. To date, little is known about the regulation of DGAT activity in peanut (Arachis hypogaea), an agr...

Coupling between alternative polyadenylation and alternative splicing is limited to terminal introns.

PubMed

Movassat, Maliheh; Crabb, Tara L; Busch, Anke; Yao, Chengguo; Reynolds, Derrick J; Shi, Yongsheng; Hertel, Klemens J

2016-07-02

Alternative polyadenylation has been implicated as an important regulator of gene expression. In some cases, alternative polyadenylation is known to couple with alternative splicing to influence last intron removal. However, it is unknown whether alternative polyadenylation events influence alternative splicing decisions at upstream exons. Knockdown of the polyadenylation factors CFIm25 or CstF64 in HeLa cells was used as an approach in identifying alternative polyadenylation and alternative splicing events on a genome-wide scale. Although hundreds of alternative splicing events were found to be differentially spliced in the knockdown of CstF64, genes associated with alternative polyadenylation did not exhibit an increased incidence of alternative splicing. These results demonstrate that the coupling between alternative polyadenylation and alternative splicing is usually limited to defining the last exon. The striking influence of CstF64 knockdown on alternative splicing can be explained through its effects on UTR selection of known splicing regulators such as hnRNP A2/B1, thereby indirectly influencing splice site selection. We conclude that changes in the expression of the polyadenylation factor CstF64 influences alternative splicing through indirect effects.

Structural insights into alternative splicing-mediated desensitization of jasmonate signaling.

PubMed

Zhang, Feng; Ke, Jiyuan; Zhang, Li; Chen, Rongzhi; Sugimoto, Koichi; Howe, Gregg A; Xu, H Eric; Zhou, Mingguo; He, Sheng Yang; Melcher, Karsten

2017-02-14

Jasmonate ZIM-domain (JAZ) transcriptional repressors play a key role in regulating jasmonate (JA) signaling in plants. Below a threshold concentration of jasmonoyl isoleucine (JA-Ile), the active form of JA, the C-terminal Jas motif of JAZ proteins binds MYC transcription factors to repress JA signaling. With increasing JA-Ile concentration, the Jas motif binds to JA-Ile and the COI1 subunit of the SCF COI1 E3 ligase, which mediates ubiquitination and proteasomal degradation of JAZ repressors, resulting in derepression of MYC transcription factors. JA signaling subsequently becomes desensitized, in part by feedback induction of JAZ splice variants that lack the C-terminal Jas motif but include an N-terminal cryptic MYC-interaction domain (CMID). The CMID sequence is dissimilar to the Jas motif and is incapable of recruiting SCF COI1 , allowing CMID-containing JAZ splice variants to accumulate in the presence of JA and to re-repress MYC transcription factors as an integral part of reestablishing signal homeostasis. The mechanism by which the CMID represses MYC transcription factors remains elusive. Here we describe the crystal structure of the MYC3-CMID JAZ10 complex. In contrast to the Jas motif, which forms a single continuous helix when bound to MYC3, the CMID adopts a loop-helix-loop-helix architecture with modular interactions with both the Jas-binding groove and the backside of the Jas-interaction domain of MYC3. This clamp-like interaction allows the CMID to bind MYC3 tightly and block access of MED25 (a subunit of the Mediator coactivator complex) to the MYC3 transcriptional activation domain, shedding light on the enigmatic mechanism by which JAZ splice variants desensitize JA signaling.

Structural basis for regulation of GPR56/ADGRG1 by its alternatively spliced extracellular domains

PubMed Central

Salzman, Gabriel S.; Ackerman, Sarah D.; Ding, Chen; Koide, Akiko; Leon, Katherine; Luo, Rong; Stoveken, Hannah M.; Fernandez, Celia G.; Tall, Gregory G.; Piao, Xianhua; Monk, Kelly R.; Koide, Shohei; Araç, Demet

2016-01-01

Summary Adhesion G-protein-coupled receptors (aGPCRs) play critical roles in diverse neurobiological processes including brain development, synaptogenesis, and myelination. aGPCRs have large alternatively spliced extracellular regions (ECRs) that likely mediate intercellular signaling; however, the precise roles of ECRs remain unclear. The aGPCR GPR56/ADGRG1 regulates both oligodendrocyte and cortical development. Accordingly, human GPR56 mutations cause myelination defects and brain malformations. Here, we determined the crystal structure of the GPR56 ECR, the first structure of any complete aGPCR ECR, in complex with an inverse-agonist monobody, revealing a GPCR-Autoproteolysis-Inducing domain and a previously unidentified domain that we term Pentraxin/Laminin/neurexin/sex-hormone-binding-globulin-Like (PLL). Strikingly, PLL domain deletion caused increased signaling and characterizes a GPR56 splice variant. Finally, we show that an evolutionarily conserved residue in the PLL domain is critical for oligodendrocyte development in vivo. Thus, our results suggest that the GPR56 ECR has unique and multifaceted regulatory functions, providing novel insights into aGPCR roles in neurobiology. PMID:27657451

Differential Expression Profile of ZFX Variants Discriminates Breast Cancer Subtypes

PubMed

Pourkeramati, Fatemeh; Asadi, Malek Hossein; Shakeri, Shahryar; Farsinejad, Alireza

2018-05-13

ZFX is a transcriptional regulator in embryonic stem cells that plays an important role in pluripotency and self-renewal. ZFX is widely expressed in pluripotent stem cells and is down-regulated during differentiation of embryonic stem cells. ZFX has five different variants that encode three different protein isoforms. While several reports have determined the overexpression of ZFX in a variety of somatic cancers, the expression of ZFX-spliced variants in cancer cells is not well-understood. We investigated the expression of ZFX variants in a series of breast cancer tissues and cell lines using quantitative PCR. The expression of ZFX variant 1/3 was higher in tumor tissue compared to marginal tissue. In contrast, the ZFX variant 5 was down-regulated in tumor tissues. While the ZFX variant 1/3 and ZFX variant 5 expression significantly increased in low-grade tumors, ZFX variant 4 was strongly expressed in high-grade tumors and demonstrating lymphatic invasion. In addition, our result revealed a significant association between the HER2 status and the expression of ZFX-spliced variants. Our data suggest that the expression of ZFX-spliced transcripts varies between different types of breast cancer and may contribute to their tumorigenesis process. Hence, ZFX-spliced transcripts could be considered as novel tumor markers with a probable value in diagnosis, prognosis, and therapy of breast cancer.

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.

Schizophyllum commune has an extensive and functional alternative splicing repertoire

PubMed Central

Gehrmann, Thies; Pelkmans, Jordi F.; Lugones, Luis G.; Wösten, Han A. B.; Abeel, Thomas; Reinders, Marcel J. T.

2016-01-01

Recent genome-wide studies have demonstrated that fungi possess the machinery to alternatively splice pre-mRNA. However, there has not been a systematic categorization of the functional impact of alternative splicing in a fungus. We investigate alternative splicing and its functional consequences in the model mushroom forming fungus Schizophyllum commune. Alternative splicing was demonstrated for 2,285 out of 12,988 expressed genes, resulting in 20% additional transcripts. Intron retentions were the most common alternative splicing events, accounting for 33% of all splicing events, and 43% of the events in coding regions. On the other hand, exon skipping events were rare in coding regions (1%) but enriched in UTRs where they accounted for 57% of the events. Specific functional groups, including transcription factors, contained alternatively spliced genes. Alternatively spliced transcripts were regulated differently throughout development in 19% of the 2,285 alternatively spliced genes. Notably, 69% of alternatively spliced genes have predicted alternative functionality by loss or gain of functional domains, or by acquiring alternative subcellular locations. S. commune exhibits more alternative splicing than any other studied fungus. Taken together, alternative splicing increases the complexity of the S. commune proteome considerably and provides it with a rich repertoire of alternative functionality that is exploited dynamically. PMID:27659065

Schizophyllum commune has an extensive and functional alternative splicing repertoire

DOE PAGES

Gehrmann, Thies; Pelkmans, Jordi F.; Lugones, Luis G.; ...

2016-09-23

Recent genome-wide studies have demonstrated that fungi possess the machinery to alternatively splice pre-mRNA. However, there has not been a systematic categorization of the functional impact of alternative splicing in a fungus. We investigate alternative splicing and its functional consequences in the model mushroom forming fungus Schizophyllum commune. Alternative splicing was demonstrated for 2,285 out of 12,988 expressed genes, resulting in 20% additional transcripts. Intron retentions were the most common alternative splicing events, accounting for 33% of all splicing events, and 43% of the events in coding regions. On the other hand, exon skipping events were rare in coding regionsmore » (1%) but enriched in UTRs where they accounted for 57% of the events. Specific functional groups, including transcription factors, contained alternatively spliced genes. Alternatively spliced transcripts were regulated differently throughout development in 19% of the 2,285 alternatively spliced genes. Notably, 69% of alternatively spliced genes have predicted alternative functionality by loss or gain of functional domains, or by acquiring alternative subcellular locations. S. commune exhibits more alternative splicing than any other studied fungus. Finally, taken together, alternative splicing increases the complexity of the S. commune proteome considerably and provides it with a rich repertoire of alternative functionality that is exploited dynamically.« less

Transcriptional expression analysis of survivin splice variants reveals differential expression of survivin-3α in breast cancer.

PubMed

Moniri Javadhesari, Solmaz; Gharechahi, Javad; Hosseinpour Feizi, Mohammad Ali; Montazeri, Vahid; Halimi, Monireh

2013-04-01

Survivin, which is a novel member of the inhibitor of apoptosis family proteins, is known to play an important role in the regulation of cell cycle and apoptosis. Differential expression of survivin in tumor tissues introduces it as a new candidate molecular marker for cancer. Here we investigated the expression of survivin and its splice variants in breast tumors, as well as normal adjacent tissues obtained from the same patients. Thirty five tumors and 17 normal adjacent tissues from women diagnosed with breast cancer were explored in this study. Differential expression of different survivin splice variants was detected and semiquantitatively analyzed using reverse transcription-polymerase chain reaction. Results showed that survivin and its splice variants were differentially expressed in tumor specimens compared with normal adjacent tissues. The expression of survivin-3B and survivin-3α was specifically detected in tumor tissues compared with normal adjacent ones (53% in tumor tissues compared to 5% in normal adjacent for survivin-3B and 65% in tumor tissues and 0.0% in normal adjacent tissues for survivin-3α). Statistical analysis showed that survivin and survivin-ΔEx3 were upregulated in benign (90%, p<0.034) and malignant (76%, p<0.042) tumors, respectively. On the other hand, our results showed that survivin-2α (100% of the cases) was the dominant expressed variant of survivin in breast cancer. The data presented here showed that survivin splice variants were differentially expressed in benign and malignant breast cancer tissues, suggesting their potential role in breast cancer development. Differential expression of survivin-2α and survivin-3α splice variants highlights their usefulness as new candidate markers for breast cancer diagnosis and prognosis.

SplicingTypesAnno: annotating and quantifying alternative splicing events for RNA-Seq data.

PubMed

Sun, Xiaoyong; Zuo, Fenghua; Ru, Yuanbin; Guo, Jiqiang; Yan, Xiaoyan; Sablok, Gaurav

2015-04-01

Alternative splicing plays a key role in the regulation of the central dogma. Four major types of alternative splicing have been classified as intron retention, exon skipping, alternative 5 splice sites or alternative donor sites, and alternative 3 splice sites or alternative acceptor sites. A few algorithms have been developed to detect splice junctions from RNA-Seq reads. However, there are few tools targeting at the major alternative splicing types at the exon/intron level. This type of analysis may reveal subtle, yet important events of alternative splicing, and thus help gain deeper understanding of the mechanism of alternative splicing. This paper describes a user-friendly R package, extracting, annotating and analyzing alternative splicing types for sequence alignment files from RNA-Seq. SplicingTypesAnno can: (1) provide annotation for major alternative splicing at exon/intron level. By comparing the annotation from GTF/GFF file, it identifies the novel alternative splicing sites; (2) offer a convenient two-level analysis: genome-scale annotation for users with high performance computing environment, and gene-scale annotation for users with personal computers; (3) generate a user-friendly web report and additional BED files for IGV visualization. SplicingTypesAnno is a user-friendly R package for extracting, annotating and analyzing alternative splicing types at exon/intron level for sequence alignment files from RNA-Seq. It is publically available at https://sourceforge.net/projects/splicingtypes/files/ or http://genome.sdau.edu.cn/research/software/SplicingTypesAnno.html. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Regulated capture by exosomes of mRNAs for cytoplasmic tRNA synthetases.

PubMed

Wang, Feng; Xu, Zhiwen; Zhou, Jie; Lo, Wing-Sze; Lau, Ching-Fun; Nangle, Leslie A; Yang, Xiang-Lei; Zhang, Mingjie; Schimmel, Paul

2013-10-11

Although tRNA synthetases are enzymes that catalyze the first step of translation in the cytoplasm, surprising functions unrelated to translation have been reported. These studies, and the demonstration of novel activities of splice variants, suggest a far broader reach of tRNA synthetases into cell biology than previously recognized. Here we show that mRNAs for most tRNA synthetases can be detected in exosomes. Also detected in exosomes was an mRNA encoding a unique splice variant that others had associated with prostate cancer. The exosomal mRNAs encoding the native synthetase and its cancer-associated splice variant could be translated in vitro and in mammalian cells into stable proteins. Other results showed that selection by exosomes of the splice variant mRNA could be regulated by an external stimulus. Thus, a broad and diverse regulated pool of tRNA synthetase-derived mRNAs is packaged for genetic exchange.

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

Gehrmann, Thies; Pelkmans, Jordi F.; Lugones, Luis G.

Recent genome-wide studies have demonstrated that fungi possess the machinery to alternatively splice pre-mRNA. However, there has not been a systematic categorization of the functional impact of alternative splicing in a fungus. We investigate alternative splicing and its functional consequences in the model mushroom forming fungus Schizophyllum commune. Alternative splicing was demonstrated for 2,285 out of 12,988 expressed genes, resulting in 20% additional transcripts. Intron retentions were the most common alternative splicing events, accounting for 33% of all splicing events, and 43% of the events in coding regions. On the other hand, exon skipping events were rare in coding regionsmore » (1%) but enriched in UTRs where they accounted for 57% of the events. Specific functional groups, including transcription factors, contained alternatively spliced genes. Alternatively spliced transcripts were regulated differently throughout development in 19% of the 2,285 alternatively spliced genes. Notably, 69% of alternatively spliced genes have predicted alternative functionality by loss or gain of functional domains, or by acquiring alternative subcellular locations. S. commune exhibits more alternative splicing than any other studied fungus. Finally, taken together, alternative splicing increases the complexity of the S. commune proteome considerably and provides it with a rich repertoire of alternative functionality that is exploited dynamically.« less

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.

Actions of Agonists, Fipronil and Ivermectin on the Predominant In Vivo Splice and Edit Variant (RDLbd, I/V) of the Drosophila GABA Receptor Expressed in Xenopus laevis Oocytes

PubMed Central

Suwanmanee, Siros; Buckingham, Steven David; Biggin, Philip; Sattelle, David

2014-01-01

Ionotropic GABA receptors are the targets for several classes of insecticides. One of the most widely-studied insect GABA receptors is RDL (resistance to dieldrin), originally isolated from Drosophila melanogaster. RDL undergoes alternative splicing and RNA editing, which influence the potency of GABA. Most work has focussed on minority isoforms. Here, we report the first characterisation of the predominant native splice variant and RNA edit, combining functional characterisation with molecular modelling of the agonist-binding region. The relative order of agonist potency is GABA> muscimol> TACA> β-alanine. The I/V edit does not alter the potency of GABA compared to RDLbd. Docking calculations suggest that these agonists bind and activate RDLbdI/V through a similar binding mode. TACA and β-alanine are predicted to bind with lower affinity than GABA, potentially explaining their lower potency, whereas the lower potency of muscimol and isoguvacine cannot be explained structurally from the docking calculations. The A301S (resistance to dieldrin) mutation reduced the potency of antagonists picrotoxin, fipronil and pyrafluprole but the I/V edit had no measurable effect. Ivermectin suppressed responses to GABA of RDLbdI/V, RDLbd and RDLbdI/VA301S. The dieldrin resistant variant also showed reduced sensitivity to Ivermectin. This study of a highly abundant insect GABA receptor isoform will help the design of new insecticides. PMID:24823815

Characterization of a spliced variant of human IRF-3 promoter and its regulation by the transcription factor Sp1.

PubMed

Ren, Wei; Zhu, Liang-Hua; Xu, Hua-Guo; Jin, Rui; Zhou, Guo-Ping

2012-06-01

Interferon regulatory factor 3 (IRF-3), an essential transcriptional regulator of the interferon genes, plays an important role in host defense against viral and microbial infection as well as in cell growth regulation. Promoter plays a crucial role in gene transcription. We have reported the characterization of the wide type of human IRF-3 promoter, but the characterization of the spliced variant of human IRF-3 Int2V1 promoter has not been systematically analyzed. To observe the spliced variant of human IRF-3 promoter, we have cloned the human IRF-3 gene promoter region containing 300 nucleotides upstream the transcription start site (TSS). Transient transfection of 5' deleted promoter-reporter constructs and luciferase assay illustrated the region -159/-100 relative to the TSS is sufficient for full promoter activity. This region contains GATA1 and specific protein-1 (Sp1) transcription factor binding sites. Interestingly, mutation of this Sp1 site reduced the promoter activity by 50%. However, overexpression of Sp1 increased the transcription activity by 2.4-fold. These results indicated that the spliced variant of human IRF-3 gene core promoter was located within the region -159/-100 relative to the TSS. Sp1 transcription factor upregulates the spliced variant of human IRF-3 gene promoter.

Alternative splicing at exon 2 results in the loss of the catalytic activity of mouse DNA polymerase iota in vitro.

PubMed

Kazachenko, Konstantin Y; Miropolskaya, Nataliya A; Gening, Leonid V; Tarantul, Vyacheslav Z; Makarova, Alena V

2017-02-01

Y-family DNA polymerase iota (Pol ι) possesses both DNA polymerase and dRP lyase activities and was suggested to be involved in DNA translesion synthesis and base excision repair in mammals. The 129 strain of mice and its derivatives have a natural nonsense codon mutation in the second exon of the Pol ι gene resulting in truncation of the Pol ι protein. These mice were widely used as a Pol ι-null model for in vivo studies of the Pol ι function. However whether 129-derived strains of mice are fully deficient in the Pol ι functions was a subject of discussion since Pol ι mRNA undergoes alternative splicing at exon 2. Here we report purification of mouse Pol ι lacking the region encoded by exon 2, which includes several conserved residues involved in catalysis. We show that the deletion abrogates both the DNA polymerase and dRP lyase activities of Pol ι in the presence of either Mg 2+ or Mn 2+ ions. Thus, 129-derived strains of mice express catalytically inactive alternatively spliced Pol ι variant, whose cellular functions, if any exist, remain to be established. Copyright © 2017 Elsevier B.V. All rights reserved.

JAK2 Exon 14 Deletion in Patients with Chronic Myeloproliferative Neoplasms

PubMed Central

Ma, Wanlong; Kantarjian, Hagop; Zhang, Xi; Wang, Xiuqiang; Zhang, Zhong; Yeh, Chen-Hsiung; O'Brien, Susan; Giles, Francis; Bruey, Jean Marie; Albitar, Maher

2010-01-01

Background The JAK2 V617F mutation in exon 14 is the most common mutation in chronic myeloproliferative neoplasms (MPNs); deletion of the entire exon 14 is rarely detected. In our previous study of >10,000 samples from patients with suspected MPNs tested for JAK2 mutations by reverse transcription-PCR (RT-PCR) with direct sequencing, complete deletion of exon 14 (Δexon14) constituted <1% of JAK2 mutations. This appears to be an alternative splicing mutation, not detectable with DNA-based testing. Methodology/Principal Findings We investigated the possibility that MPN patients may express the JAK2 Δexon14 at low levels (<15% of total transcript) not routinely detectable by RT-PCR with direct sequencing. Using a sensitive RT-PCR–based fluorescent fragment analysis method to quantify JAK2 Δexon14 mRNA expression relative to wild-type, we tested 61 patients with confirmed MPNs, 183 with suspected MPNs (93 V617F-positive, 90 V617F-negative), and 46 healthy control subjects. The Δexon14 variant was detected in 9 of the 61 (15%) confirmed MPN patients, accounting for 3.96% to 33.85% (mean  = 12.04%) of total JAK2 transcript. This variant was also detected in 51 of the 183 patients with suspected MPNs (27%), including 20 of the 93 (22%) with V617F (mean [range] expression  = 5.41% [2.13%–26.22%]) and 31 of the 90 (34%) without V617F (mean [range] expression  = 3.88% [2.08%–12.22%]). Immunoprecipitation studies demonstrated that patients expressing Δexon14 mRNA expressed a corresponding truncated JAK2 protein. The Δexon14 variant was not detected in the 46 control subjects. Conclusions/Significance These data suggest that expression of the JAK2 Δexon14 splice variant, leading to a truncated JAK2 protein, is common in patients with MPNs. This alternatively spliced transcript appears to be more frequent in MPN patients without V617F mutation, in whom it might contribute to leukemogenesis. This mutation is missed if DNA rather than RNA is used for testing. PMID:20730051

An intronic ncRNA-dependent regulation of SORL1 expression affecting Aβ formation is upregulated in post-mortem Alzheimer's disease brain samples.

PubMed

Ciarlo, Eleonora; Massone, Sara; Penna, Ilaria; Nizzari, Mario; Gigoni, Arianna; Dieci, Giorgio; Russo, Claudio; Florio, Tullio; Cancedda, Ranieri; Pagano, Aldo

2013-03-01

Recent studies indicated that sortilin-related receptor 1 (SORL1) is a risk gene for late-onset Alzheimer's disease (AD), although its role in the aetiology and/or progression of this disorder is not fully understood. Here, we report the finding of a non-coding (nc) RNA (hereafter referred to as 51A) that maps in antisense configuration to intron 1 of the SORL1 gene. 51A expression drives a splicing shift of SORL1 from the synthesis of the canonical long protein variant A to an alternatively spliced protein form. This process, resulting in a decreased synthesis of SORL1 variant A, is associated with impaired processing of amyloid precursor protein (APP), leading to increased Aβ formation. Interestingly, we found that 51A is expressed in human brains, being frequently upregulated in cerebral cortices from individuals with Alzheimer's disease. Altogether, these findings document a novel ncRNA-dependent regulatory pathway that might have relevant implications in neurodegeneration.

Membrane expression of MRP-1, but not MRP-1 splicing or Pgp expression, predicts survival in patients with ESFT

PubMed Central

Roundhill, E; Burchill, S

2013-01-01

Background: Primary Ewing's sarcoma family of tumours (ESFTs) may respond to chemotherapy, although many patients experience subsequent disease recurrence and relapse. The survival of ESFT cells following chemotherapy has been attributed to the development of resistant disease, possibly through the expression of ABC transporter proteins. Methods: MRP-1 and Pgp mRNA and protein expression in primary ESFTs was determined by quantitative reverse-transcriptase PCR (RT-qPCR) and immunohistochemistry, respectively, and alternative splicing of MRP-1 by RT-PCR. Results: We observed MRP-1 protein expression in 92% (43 out of 47) of primary ESFTs, and cell membrane MRP-1 was highly predictive of both overall survival (P<0.0001) and event-free survival (P<0.0001). Alternative splicing of MRP-1 was detected in primary ESFTs, although the pattern of splicing variants was not predictive of patient outcome, with the exception of loss of exon 9 in six patients, which predicted relapse (P=0.041). Pgp protein was detected in 6% (38 out of 44) of primary ESFTs and was not associated with patient survival. Conclusion: For the first time we have established that cell membrane expression of MRP-1 or loss of exon 9 is predictive of outcome but not the number of splicing events or expression of Pgp, and both may be valuable factors for the stratification of patients for more intensive therapy. PMID:23799853

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

Smirnova, Anna S.; Morgun, Andrey; Shulzhenko, Natalia

Two transcript variants (TV) of the T cell immune regulator gene 1 (TCIRG1) have already been characterized. TV1 encodes a subunit of the osteoclast vacuolar proton pump and TV2 encodes a T cell inhibitory receptor. Based on the search in dbEST, we validated by RT-PCR six new alternative splice events in TCIRG1 in most of the 28 human tissues studied. In addition, we observed that transcripts using the TV1 transcription start site and two splice forms previously described in a patient with infantile malignant osteopetrosis are also expressed in various tissues of healthy individuals. Studies of these nine splice formsmore » in cytoplasmic RNA of peripheral blood mononuclear cells showed that at least six of them could be efficiently exported from the nucleus. Since various products with nearly ubiquitous tissue distribution are generated from TCIRG1, this gene may be involved in other processes besides immune response and bone resorption.« less

Differential expression of Oct4 variants and pseudogenes in normal urothelium and urothelial cancer.

PubMed

Wezel, Felix; Pearson, Joanna; Kirkwood, Lisa A; Southgate, Jennifer

2013-10-01

The transcription factor octamer-binding protein 4 (Oct4; encoded by POU5F1) has a key role in maintaining embryonic stem cell pluripotency during early embryonic development and it is required for generation of induced pluripotent stem cells. Controversy exists concerning Oct4 expression in somatic tissues, with reports that Oct4 is expressed in normal and in neoplastic urothelium carrying implications for a bladder cancer stem cell phenotype. Here, we show that the pluripotency-associated Oct4A transcript was absent from cultures of highly regenerative normal human urothelial cells and from low-grade to high-grade urothelial carcinoma cell lines, whereas alternatively spliced variants and transcribed pseudogenes were expressed in abundance. Immunolabeling and immunoblotting studies confirmed the absence of Oct4A in normal and neoplastic urothelial cells and tissues, but indicated the presence of alternative isoforms or potentially translated pseudogenes. The stable forced expression of Oct4A in normal human urothelial cells in vitro profoundly inhibited growth and affected morphology, but protein expression was rapidly down-regulated. Our findings demonstrate that pluripotency-associated isoform Oct4A is not expressed by normal or malignant human urothelium and therefore is unlikely to play a role in a cancer stem cell phenotype. However, our findings also indicate that urothelium expresses a variety of other Oct4 splice-variant isoforms and transcribed pseudogenes that warrant further study. Copyright © 2013 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

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

CD33: increased inclusion of exon 2 implicates the Ig V-set domain in Alzheimer's disease susceptibility

PubMed Central

Raj, Towfique; Ryan, Katie J.; Replogle, Joseph M.; Chibnik, Lori B.; Rosenkrantz, Laura; Tang, Anna; Rothamel, Katie; Stranger, Barbara E.; Bennett, David A.; Evans, Denis A.; De Jager, Philip L.; Bradshaw, Elizabeth M.

2014-01-01

We previously demonstrated that the Alzheimer's disease (AD) associated risk allele, rs3865444C, results in a higher surface density of CD33 on monocytes. Here, we find alternative splicing of exon 2 to be the primary mechanism of the genetically driven differential expression of CD33 protein. We report that the risk allele, rs3865444C, is associated with greater cell surface expression of CD33 in both subjects of European and African–American ancestry and that there is a single haplotype influencing CD33 surface expression. A meta-analysis of the two populations narrowed the number of significant SNPs in high linkage disequilibrium (LD) (r2 > 0.8) with rs3865444 to just five putative causal variants associated with increased protein expression. Using gene expression data from flow-sorted CD14+CD16− monocytes from 398 healthy subjects of three populations, we show that the rs3865444C risk allele is strongly associated with greater expression of CD33 exon 2 (pMETA = 2.36 × 10−60). Western blotting confirms increased protein expression of the full-length CD33 isoform containing exon 2 relative to the rs3865444C allele (P < 0.0001). Of the variants in strong LD with rs3865444, rs12459419, which is located in a putative SRSF2 splice site of exon 2, is the most likely candidate to mediate the altered alternative splicing of CD33's Immunoglobulin V-set domain 2 and ultimately influence AD susceptibility. PMID:24381305

Transcriptome Sequencing Revealed Significant Alteration of Cortical Promoter Usage and Splicing in Schizophrenia

PubMed Central

Wu, Jing Qin; Wang, Xi; Beveridge, Natalie J.; Tooney, Paul A.; Scott, Rodney J.; Carr, Vaughan J.; Cairns, Murray J.

2012-01-01

Background While hybridization based analysis of the cortical transcriptome has provided important insight into the neuropathology of schizophrenia, it represents a restricted view of disease-associated gene activity based on predetermined probes. By contrast, sequencing technology can provide un-biased analysis of transcription at nucleotide resolution. Here we use this approach to investigate schizophrenia-associated cortical gene expression. Methodology/Principal Findings The data was generated from 76 bp reads of RNA-Seq, aligned to the reference genome and assembled into transcripts for quantification of exons, splice variants and alternative promoters in postmortem superior temporal gyrus (STG/BA22) from 9 male subjects with schizophrenia and 9 matched non-psychiatric controls. Differentially expressed genes were then subjected to further sequence and functional group analysis. The output, amounting to more than 38 Gb of sequence, revealed significant alteration of gene expression including many previously shown to be associated with schizophrenia. Gene ontology enrichment analysis followed by functional map construction identified three functional clusters highly relevant to schizophrenia including neurotransmission related functions, synaptic vesicle trafficking, and neural development. Significantly, more than 2000 genes displayed schizophrenia-associated alternative promoter usage and more than 1000 genes showed differential splicing (FDR<0.05). Both types of transcriptional isoforms were exemplified by reads aligned to the neurodevelopmentally significant doublecortin-like kinase 1 (DCLK1) gene. Conclusions This study provided the first deep and un-biased analysis of schizophrenia-associated transcriptional diversity within the STG, and revealed variants with important implications for the complex pathophysiology of schizophrenia. PMID:22558445

Central nervous system involvement in severe congenital neutropenia: neurological and neuropsychological abnormalities associated with specific HAX1 mutations.

PubMed

Carlsson, G; van't Hooft, I; Melin, M; Entesarian, M; Laurencikas, E; Nennesmo, I; Trebińska, A; Grzybowska, E; Palmblad, J; Dahl, N; Nordenskjöld, M; Fadeel, B; Henter, J-I

2008-10-01

Homozygous mutations in the HAX1 gene were recently identified in severe congenital neutropenia patients belonging to the original Kostmann family in northern Sweden. Our observations suggested that these patients also develop neurological and neuropsychological symptoms. Detailed clinical studies and mutation analyses were performed in the surviving patients belonging to the Kostmann kindred and in two patients not related to this family, along with studies of HAX1 splice variant expression in normal human tissues. Five of six Kostmann family patients and one other patient from northern Sweden harboured homozygous HAX1 mutations (568C-->T, Q190X) and one carried a heterozygous ELA2 gene mutation. One Swedish patient of Kurdish extraction carried alternative homozygous HAX1 mutations (131G-->A, W44X). All the three patients with Q190X mutations who were alive and available for evaluation developed neurological disease with decreased cognitive function, and three of four patients who reached 10 years developed epilepsy. In contrast, the patients with the ELA2 and W44X HAX1 mutations, respectively, showed no obvious neurological abnormalities. Moreover, two alternative HAX1 splice variants were identified in normal human tissues, including the brain. Both transcripts contained exon 5, harbouring the Q190X mutation, whereas the 5' end of exon 2 containing the W44X mutation was spliced out from the second transcript. We describe neurological and neuropsychological abnormalities for the first time in Kostmann disease patients. These central nervous system symptoms appear to be associated with specific HAX1 mutations.

Expression analysis of a heat-inducible, Myo-inositol-1-phosphate synthase (MIPS) gene from wheat and the alternatively spliced variants of rice and Arabidopsis.

PubMed

Khurana, Neetika; Chauhan, Harsh; Khurana, Paramjit

2012-01-01

Molecular dissection and a deeper analysis of the heat stress response mechanism in wheat have been poorly understood so far. This study delves into the molecular basis of action of TaMIPS, a heat stress-inducible enzyme that was identified through PCR-select subtraction technology, which is named here as TaMIPS2. MIPS (L-Myo-inositol-phosphate synthase) is important for the normal growth and development in plants. Expression profiling showed that TaMIPS2 is expressed during different developing seed stages upon heat stress. Also, the transcript levels increase in unfertilized ovaries and significant amounts are present during the recovery period providing evidence that MIPS is crucial for its role in heat stress recovery and flower development. Alternatively spliced forms from rice and Arabidopsis were also identified and their expression analysis revealed that apart from heat stress, some of the spliced variants were also inducible by drought, NaCl, Cold, ABA, BR, SA and mannitol. In silico promoter analysis revealed various cis-elements that could contribute for the differential regulation of MIPS in different plant systems. Phylogenetic analysis indicated that MIPS are highly conserved among monocots and dicots and TaMIPS2 grouped specifically with monocots. Comparative analyses was undertaken by different experimental approaches, i.e., semi-quantitative RT-PCR, quantitative RT-PCR, Genevestigator as a reference expression tool and motif analysis to predict the possible function of TaMIPS2 in regulating the different aspects of plant development under abiotic stress in wheat.

Differential splicing and glycosylation of Apoer2 alters synaptic plasticity and fear learning.

PubMed

Wasser, Catherine R; Masiulis, Irene; Durakoglugil, Murat S; Lane-Donovan, Courtney; Xian, Xunde; Beffert, Uwe; Agarwala, Anandita; Hammer, Robert E; Herz, Joachim

2014-11-25

Apoer2 is an essential receptor in the central nervous system that binds to the apolipoprotein ApoE. Various splice variants of Apoer2 are produced. We showed that Apoer2 lacking exon 16, which encodes the O-linked sugar (OLS) domain, altered the proteolytic processing and abundance of Apoer2 in cells and synapse number and function in mice. In cultured cells expressing this splice variant, extracellular cleavage of OLS-deficient Apoer2 was reduced, consequently preventing γ-secretase-dependent release of the intracellular domain of Apoer2. Mice expressing Apoer2 lacking the OLS domain had increased Apoer2 abundance in the brain, hippocampal spine density, and glutamate receptor abundance, but decreased synaptic efficacy. Mice expressing a form of Apoer2 lacking the OLS domain and containing an alternatively spliced cytoplasmic tail region that promotes glutamate receptor signaling showed enhanced hippocampal long-term potentiation (LTP), a phenomenon associated with learning and memory. However, these mice did not display enhanced spatial learning in the Morris water maze, and cued fear conditioning was reduced. Reducing the expression of the mutant Apoer2 allele so that the abundance of the protein was similar to that of Apoer2 in wild-type mice normalized spine density, hippocampal LTP, and cued fear learning. These findings demonstrated a role for ApoE receptors as regulators of synaptic glutamate receptor activity and established differential receptor glycosylation as a potential regulator of synaptic function and memory. Copyright © 2014, American Association for the Advancement of Science.

Differential splicing and glycosylation of Apoer2 alters synaptic plasticity and fear learning

PubMed Central

Wasser, Catherine R.; Masiulis, Irene; Durakoglugil, Murat S.; Lane-Donovan, Courtney; Xian, Xunde; Beffert, Uwe; Agarwala, Anandita; Hammer, Robert E.; Herz, Joachim

2015-01-01

Apoer2 is an essential receptor in the central nervous system that binds to the apolipoprotein ApoE. Various splice variants of Apoer2 are produced. We showed that Apoer2 lacking exon 16, which encodes the O-linked sugar (OLS) domain, altered the proteolytic processing and abundance of Apoer2 in cells and synapse number and function in mice. In cultured cells expressing this splice variant, extracellular cleavage of OLS-deficient Apoer2 was reduced, consequently preventing γ-secretase-dependent release of the intracellular domain of Apoer2. Mice expressing Apoer2 lacking the OLS domain had increased Apoer2 abundance in the brain, hippocampal spine density, and glutamate receptor abundance, but decreased synaptic efficacy. Mice expressing a form of Apoer2 lacking the OLS domain and containing an alternatively spliced cytoplasmic tail region that promotes glutamate receptor signaling showed enhanced hippocampal long-term potentiation (LTP), a phenomenon associated with learning and memory. However, these mice did not display enhanced spatial learning in the Morris water maze, and cued fear conditioning was reduced. Reducing the expression of the mutant Apoer2 allele so that the abundance of the protein was similar to that of Apoer2 in wild-type mice normalized spine density, hippocampal LTP, and cued fear learning. These findings demonstrated a role for ApoE receptors as regulators of synaptic glutamate receptor activity and established differential receptor glycosylation as a potential regulator of synaptic function and memory. PMID:25429077

Expression of REST4 in human gliomas in vivo and influence of pioglitazone on REST in vitro

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

Ren, Huan; Institute of Clinical Pharmacology, Central South University, Hunan Key Laboratory of Pharmacogenetics, Changsha 410078; Gao, Zhangfeng

The repressor element-1 (RE1) silencing transcription factor/neuron-restrictive silencer factor (REST/NRSF) has an irreplaceable role during the differentiation of neurons. REST has multiple splice variants which link to various types of cancer. Previous work had highlighted the role of REST in glioma, where the expression of REST is enhanced. But whether alternative splicing of REST is expressed in glioma has not been described. Here, we show that a specific isoform REST4 is expressed in glioma specimens, and will influence the mRNA level of REST in vivo. Peroxisome proliferator-activated receptor-γ (PPARγ) agonists have a role of antineoplastic in various tumor cells, which includingmore » glioma cells. Moreover, study indicated that PPARγ agonist pioglitazone can promote alternative splicing of REST pre-mRNA. In this study, we selected pioglitazone as a tool drug to explore whether the role of pioglitazone in anti-glioma is mediated by regulating REST expression or promoting alternative splicing of REST in glioma cells. Results show that pioglitazone can inhibit proliferation and induce apoptosis of glioma cell in vitro, which may be mediated by down-regulating REST mRNA level but not by inducing alternative splicing of REST pre-mRNA. Our study firstly reports the expression of REST4 in glioma tissue samples. And we recommend that pioglitazone, which can reduce the expression level of REST, represents a promising drug for therapy of glioma. - Highlights: • A specific isoform REST4 is expressed in glioma specimens in vivo. • REST4 will influence the mRNA level of REST in vivo. • Pioglitazone can inhibit proliferation and induce apoptosis of glioma cells. • The role of pioglitazone in anti-glioma may be mediated by down-regulating REST.« less

Intron-mediated alternative splicing of Arabidopsis P5CS1 and its association with natural variation in proline and climate adaptation

PubMed Central

Kesari, Ravi; Lasky, Jesse R.; Villamor, Joji Grace; Des Marais, David L.; Chen, Ying-Jiun C.; Liu, Tzu-Wen; Juenger, Thomas E.; Verslues, Paul E.

2012-01-01

Drought-induced proline accumulation is widely observed in plants but its regulation and adaptive value are not as well understood. Proline accumulation of the Arabidopsis accession Shakdara (Sha) was threefold less than that of Landsberg erecta (Ler) and quantitative trait loci mapping identified a reduced function allele of the proline synthesis enzyme Δ1-pyrroline-5-carboxylate synthetase1 (P5CS1) as a basis for the lower proline of Sha. Sha P5CS1 had additional TA repeats in intron 2 and a G-to-T transversion in intron 3 that were sufficient to promote alternative splicing and production of a nonfunctional transcript lacking exon 3 (exon 3-skip P5CS1). In Sha, and additional accessions with the same intron polymorphisms, the nonfunctional exon 3-skip P5CS1 splice variant constituted as much as half of the total P5CS1 transcript. In a larger panel of Arabidopsis accessions, low water potential-induced proline accumulation varied by 10-fold and variable production of exon 3-skip P5CS1 among accessions was an important, but not the sole, factor underlying variation in proline accumulation. Population genetic analyses suggest that P5CS1 may have evolved under positive selection, and more extensive correlation of exon 3-skip P5CS1 production than proline abundance with climate conditions of natural accessions also suggest a role of P5CS1 in local adaptation to the environment. These data identify a unique source of alternative splicing in plants, demonstrate a role of exon 3-skip P5CS1 in natural variation of proline metabolism, and suggest an association of P5CS1 and its alternative splicing with environmental adaptation. PMID:22615385

PGC1α -1 Nucleosome Position and Splice Variant Expression and Cardiovascular Disease Risk in Overweight and Obese Individuals.

PubMed

Henagan, Tara M; Stewart, Laura K; Forney, Laura A; Sparks, Lauren M; Johannsen, Neil; Church, Timothy S

2014-01-01

PGC1α, a transcriptional coactivator, interacts with PPARs and others to regulate skeletal muscle metabolism. PGC1α undergoes splicing to produce several mRNA variants, with the NTPGC1α variant having a similar biological function to the full length PGC1α (FLPGC1α). CVD is associated with obesity and T2D and a lower percentage of type 1 oxidative fibers and impaired mitochondrial function in skeletal muscle, characteristics determined by PGC1α expression. PGC1α expression is epigenetically regulated in skeletal muscle to determine mitochondrial adaptations, and epigenetic modifications may regulate mRNA splicing. We report in this paper that skeletal muscle PGC1α  -1 nucleosome (-1N) position is associated with splice variant NTPGC1α but not FLPGC1α expression. Division of participants based on the -1N position revealed that those individuals with a -1N phased further upstream from the transcriptional start site (UP) expressed lower levels of NTPGC1α than those with the -1N more proximal to TSS (DN). UP showed an increase in body fat percentage and serum total and LDL cholesterol. These findings suggest that the -1N may be a potential epigenetic regulator of NTPGC1α splice variant expression, and -1N position and NTPGC1α variant expression in skeletal muscle are linked to CVD risk. This trial is registered with clinicaltrials.gov, identifier NCT00458133.

Functional identification of a novel 14-3-3 epsilon splicing variant suggests dimerization is not necessary for 14-3-3 epsilon to inhibit UV-induced apoptosis

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

Han, Dingding; Ye, Guangming; Liu, Tingting

2010-05-28

14-3-3 proteins function as a dimer and have been identified to involve in diverse signaling pathways. Here we reported the identification of a novel splicing variant of human 14-3-3 epsilon (14-3-3 epsilon sv), which is derived from a novel exon 1' insertion. The insertion contains a stop codon and leads to a truncated splicing variant of 14-3-3 epsilon. The splicing variant is translated from the exon 2 and results in the deletion of an N-terminal {alpha}-helix which is crucial for the dimerization. Therefore, the 14-3-3 epsilon sv could not form a dimer with 14-3-3 zeta. However, after UV irradiation 14-3-3more » epsilon sv could also support cell survival, suggesting monomer of 14-3-3 epsilon is sufficient to protect cell from apoptosis.« less

Functional SNPs of INCENP Affect Semen Quality by Alternative Splicing Mode and Binding Affinity with the Target Bta-miR-378 in Chinese Holstein Bulls

PubMed Central

Zhang, Yan; Jiang, Qiang; Huang, Jinming; Ju, Zhihua; Wang, Xiuge; Zhong, Jifeng; Wang, Changfa

2016-01-01

Inner centromere protein (INCENP) plays an important role in mitosis and meiosis as the main member of chromosomal passenger protein complex (CPC). To investigate the functional markers of the INCENP gene associated with semen quality, the single nucleotide polymorphisms (SNPs) g.19970 A>G and g.34078 T>G were identified and analyzed. The new splice variant INCENP-TV is characterized by the deletion of exon 12. The g.19970 A>G in the exonic splicing enhancer (ESE) motif region results in an aberrant splice variant by constructing two minigene expression vectors using the pSPL3 exon capturing vector and transfecting vectors into MLTC-1 cells. INCENP-TV was more highly expressed than INCENP-reference in adult bull testes. The g.34078 T>G located in the binding region of bta-miR-378 could affect the expression of INCENP, which was verified by luciferase assay. To analyze comprehensively the correlation of SNPs with sperm quality, haplotype combinations constructed by g.19970 A>G and g.34078 T>G, as well as g.-692 C>T and g.-556 G>T reported in our previous studies, were analyzed. The bulls with H1H12 and H2H2 exhibited a higher ejaculate volume than those with H2H10 and H9H12, respectively (P < 0.05). Bulls with H11H11 and H2H10 exhibited higher initial sperm motility than those with H2H2 (P < 0.05). The expression levels of INCENP in bulls with H1H12 and H11H11 were significantly higher than those in bulls with H9H12 (P < 0.05), as determined by qRT-PCR. Findings suggest that g.19970 A>G and g.34078 T>G in INCENP both of which appear to change the molecular and biological characteristics of the mRNA transcribed from the locus may serve as a biomarkers of male bovine fertility by affecting alternative splicing mode and binding affinity with the target bta-miR-378. PMID:27669152

Structural Features of Ion Transport and Allosteric Regulation in Sodium-Calcium Exchanger (NCX) Proteins

PubMed Central

Giladi, Moshe; Tal, Inbal; Khananshvili, Daniel

2016-01-01

Na+/Ca2+ exchanger (NCX) proteins extrude Ca2+ from the cell to maintain cellular homeostasis. Since NCX proteins contribute to numerous physiological and pathophysiological events, their pharmacological targeting has been desired for a long time. This intervention remains challenging owing to our poor understanding of the underlying structure-dynamic mechanisms. Recent structural studies have shed light on the structure-function relationships underlying the ion-transport and allosteric regulation of NCX. The crystal structure of an archaeal NCX (NCX_Mj) along with molecular dynamics simulations and ion flux analyses, have assigned the ion binding sites for 3Na+ and 1Ca2+, which are being transported in separate steps. In contrast with NCX_Mj, eukaryotic NCXs contain the regulatory Ca2+-binding domains, CBD1 and CBD2, which affect the membrane embedded ion-transport domains over a distance of ~80 Å. The Ca2+-dependent regulation is ortholog, isoform, and splice-variant dependent to meet physiological requirements, exhibiting either a positive, negative, or no response to regulatory Ca2+. The crystal structures of the two-domain (CBD12) tandem have revealed a common mechanism involving a Ca2+-driven tethering of CBDs in diverse NCX variants. However, dissociation kinetics of occluded Ca2+ (entrapped at the two-domain interface) depends on the alternative-splicing segment (at CBD2), thereby representing splicing-dependent dynamic coupling of CBDs. The HDX-MS, SAXS, NMR, FRET, equilibrium 45Ca2+ binding and stopped-flow techniques provided insights into the dynamic mechanisms of CBDs. Ca2+ binding to CBD1 results in a population shift, where more constraint conformational states become highly populated without global conformational changes in the alignment of CBDs. This mechanism is common among NCXs. Recent HDX-MS studies have demonstrated that the apo CBD1 and CBD2 are stabilized by interacting with each other, while Ca2+ binding to CBD1 rigidifies local backbone segments of CBD2, but not of CBD1. The extent and strength of Ca2+-dependent rigidification at CBD2 is splice-variant dependent, showing clear correlations with phenotypes of matching NCX variants. Therefore, diverse NCX variants share a common mechanism for the initial decoding of the regulatory signal upon Ca2+ binding at the interface of CBDs, whereas the allosteric message is shaped by CBD2, the dynamic features of which are dictated by the splicing segment. PMID:26903880

Splicing-related genes are alternatively spliced upon changes in ambient temperatures in plants

PubMed Central

Bucher, Johan; Lammers, Michiel; Busscher-Lange, Jacqueline; Bonnema, Guusje; Rodenburg, Nicole; Proveniers, Marcel C. G.; Angenent, Gerco C.

2017-01-01

Plants adjust their development and architecture to small variations in ambient temperature. In a time in which temperatures are rising world-wide, the mechanism by which plants are able to sense temperature fluctuations and adapt to it, is becoming of special interest. By performing RNA-sequencing on two Arabidopsis accession and one Brassica species exposed to temperature alterations, we showed that alternative splicing is an important mechanism in ambient temperature sensing and adaptation. We found that amongst the differentially alternatively spliced genes, splicing related genes are enriched, suggesting that the splicing machinery itself is targeted for alternative splicing when temperature changes. Moreover, we showed that many different components of the splicing machinery are targeted for ambient temperature regulated alternative splicing. Mutant analysis of a splicing related gene that was differentially spliced in two of the genotypes showed an altered flowering time response to different temperatures. We propose a two-step mechanism where temperature directly influences alternative splicing of the splicing machinery genes, followed by a second step where the altered splicing machinery affects splicing of downstream genes involved in the adaptation to altered temperatures. PMID:28257507

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

CD82 suppresses CD44 alternative splicing-dependent melanoma metastasis by mediating U2AF2 ubiquitination and degradation

PubMed Central

Fu, Ailing; Zhu, Huifeng; Ren, Qiao; Wang, Bochu; Xu, Xingran; Bai, Huiyuan; Dong, Cheng

2016-01-01

Melanoma is one of the most lethal forms of skin cancer due to its early metastatic spread. The variant form of CD44 (CD44v), a cell surface glycoprotein, is highly expressed on metastatic melanoma. The mechanisms of regulation of CD44 alternative splicing in melanoma and its pathogenic contributions are so far poorly understood. Here, we investigated the expression level of CD44 in a large set of melanocytic lesions at different stages. We found that the expression of CD44v8-10 and a splicing factor, U2AF2, is significantly increased during melanoma progression, while CD82/KAI1, a tetraspanin family of tumor suppressor, is reduced in metastatic melanoma. CD44v8-10 and U2AF2 expressions which are negatively correlated with CD82 levels are dramatically elevated in primary melanoma compared with dysplastic nevi and further increased in metastatic melanoma. We also showed that patients with higher CD44v8-10 and U2AF2 expression levels tended to have shorter survival. By using both in vivo and in vitro assays, we demonstrated that CD82 inhibits the production of CD44v8-10 on melanoma. Mechanistically, U2AF2 is a downstream target of CD82 and in malignant melanoma facilitates CD44v8-10 alternative splicing. U2AF2-mediated CD44 isoform switch is required for melanoma migration in vitro and lung and liver metastasis in vivo. Notably, overexpression of CD82 suppresses U2AF2 activity by inducing U2AF2 ubiquitination. In addition, our data suggested that enhancement of melanoma migration by U2AF2-dependent CD44v8-10 splicing is mediated by Src/FAK/RhoA activation and formation of stress fibers as well as CD44-E-selectin binding reinforcement. These findings uncovered a hitherto unappreciated function of CD82 in severing the linkage between U2AF2-mediated CD44 alternative splicing and cancer aggressiveness, with potential prognostic and therapeutic implications in melanoma. PMID:27041584

ATP-binding cassette subfamily A, member 4 intronic variants c.4773+3A>G and c.5461-10T>C cause Stargardt disease due to defective splicing.

PubMed

Jonsson, Frida; Westin, Ida Maria; Österman, Lennart; Sandgren, Ola; Burstedt, Marie; Holmberg, Monica; Golovleva, Irina

2018-02-20

Inherited retinal dystrophies (IRDs) represent a group of progressive conditions affecting the retina. There is a great genetic heterogeneity causing IRDs, and to date, more than 260 genes are associated with IRDs. Stargardt disease, type 1 (STGD1) or macular degeneration with flecks, STGD1 represents a disease with early onset, central visual impairment, frequent appearance of yellowish flecks and mutations in the ATP-binding cassette subfamily A, member 4 (ABCA4) gene. A large number of intronic sequence variants in ABCA4 have been considered pathogenic although their functional effect was seldom demonstrated. In this study, we aimed to reveal how intronic variants present in patients with Stargardt from the same Swedish family affect splicing. The splicing of the ABCA4 gene was studied in human embryonic kidney cells, HEK293T, and in human retinal pigment epithelium cells, ARPE-19, using a minigene system containing variants c.4773+3A>G and c.5461-10T>C. We showed that both ABCA4 variants, c.4773+3A>G and c.5461-10T>C, cause aberrant splicing of the ABCA4 minigene resulting in exon skipping. We also demonstrated that splicing of ABCA4 has different outcomes depending on transfected cell type. Two intronic variants c.4773+3A>G and c.5461-10T>C, both predicted to affect splicing, are indeed disease-causing mutations due to skipping of exons 33, 34, 39 and 40 of ABCA4 gene. The experimental proof that ABCA4 mutations in STGD patients affect protein function is crucial for their inclusion to future clinical trials; therefore, functional testing of all ABCA4 intronic variants associated with Stargardt disease by minigene technology is desirable. © 2018 Acta Ophthalmologica Scandinavica Foundation. Published by John Wiley & Sons Ltd.

Expressed sequence tag analysis of adult human lens for the NEIBank Project: over 2000 non-redundant transcripts, novel genes and splice variants.

PubMed

Wistow, Graeme; Bernstein, Steven L; Wyatt, M Keith; Behal, Amita; Touchman, Jeffrey W; Bouffard, Gerald; Smith, Don; Peterson, Katherine

2002-06-15

To explore the expression profile of the human lens and to provide a resource for microarray studies, expressed sequence tag (EST) analysis has been performed on cDNA libraries from adult lenses. A cDNA library was constructed from two adult (40 year old) human lenses. Over two thousand clones were sequenced from the unamplified, un-normalized library. The library was then normalized and a further 2200 sequences were obtained. All the data were analyzed using GRIST (GRouping and Identification of Sequence Tags), a procedure for gene identification and clustering. The lens library (by) contains a low percentage of non-mRNA contaminants and a high fraction (over 75%) of apparently full length cDNA clones. Approximately 2000 reads from the unamplified library yields 810 clusters, potentially representing individual genes expressed in the lens. After normalization, the content of crystallins and other abundant cDNAs is markedly reduced and a similar number of reads from this library (fs) yields 1455 unique groups of which only two thirds correspond to named genes in GenBank. Among the most abundant cDNAs is one for a novel gene related to glutamine synthetase, which was designated "lengsin" (LGS). Analyses of ESTs also reveal examples of alternative transcripts, including a major alternative splice form for the lens specific membrane protein MP19. Variant forms for other transcripts, including those encoding the apoptosis inhibitor Livin and the armadillo repeat protein ARVCF, are also described. The lens cDNA libraries are a resource for gene discovery, full length cDNAs for functional studies and microarrays. The discovery of an abundant, novel transcript, lengsin, and a major novel splice form of MP19 reflect the utility of unamplified libraries constructed from dissected tissue. Many novel transcripts and splice forms are represented, some of which may be candidates for genetic diseases.

The Interplay of Temperature and Genotype on Patterns of Alternative Splicing in Drosophila melanogaster.

PubMed

Jakšić, Ana Marija; Schlötterer, Christian

2016-09-01

Alternative splicing is the highly regulated process of variation in the removal of introns from premessenger-RNA transcripts. The consequences of alternative splicing on the phenotype are well documented, but the impact of the environment on alternative splicing is not yet clear. We studied variation in alternative splicing among four different temperatures, 13, 18, 23, and 29°, in two Drosophila melanogaster genotypes. We show plasticity of alternative splicing with up to 10% of the expressed genes being differentially spliced between the most extreme temperatures for a given genotype. Comparing the two genotypes at different temperatures, we found <1% of the genes being differentially spliced at 18°. At extreme temperatures, however, we detected substantial differences in alternative splicing-with almost 10% of the genes having differential splicing between the genotypes: a magnitude similar to between species differences. Genes with differential alternative splicing between genotypes frequently exhibit dominant inheritance. Remarkably, the pattern of surplus of differences in alternative splicing at extreme temperatures resembled the pattern seen for gene expression intensity. Since different sets of genes were involved for the two phenotypes, we propose that purifying selection results in the reduction of differences at benign temperatures. Relaxed purifying selection at temperature extremes, on the other hand, may cause the divergence in gene expression and alternative splicing between the two strains in rarely encountered environments. Copyright © 2016 by the Genetics Society of America.

Two Novel Variants Affecting CDKL5 Transcript Associated with Epileptic Encephalopathy.

PubMed

Neupauerová, Jana; Štěrbová, Katalin; Vlčková, Markéta; Sebroňová, Věra; Maříková, Tat'ána; Krůtová, Marcela; David, Staněk; Kršek, Pavel; Žaliová, Markéta; Seeman, Pavel; Laššuthová, Petra

2017-10-01

Variants in the human X-linked cyclin-dependent kinase-like 5 (CDKL5) gene have been reported as being etiologically associated with early infantile epileptic encephalopathy type 2 (EIEE2). We report on two patients, a boy and a girl, with EIEE2 that present with early onset epilepsy, hypotonia, severe intellectual disability, and poor eye contact. Massively parallel sequencing (MPS) of a custom-designed gene panel for epilepsy and epileptic encephalopathy containing 112 epilepsy-related genes was performed. Sanger sequencing was used to confirm the novel variants. For confirmation of the functional consequence of an intronic CDKL5 variant in patient 2, an RNA study was done. DNA sequencing revealed de novo variants in CDKL5, a c.2578C>T (p. Gln860*) present in a hemizygous state in a 3-year-old boy, and a potential splice site variant c.463+5G>A in heterozygous state in a 5-year-old girl. Multiple in silico splicing algorithms predicted a highly reduced splice site score for c.463+5G>A. A subsequent mRNA study confirmed an aberrant shorter transcript lacking exon 7. Our data confirmed that variants in the CDKL5 are associated with EIEE2. There is credible evidence that the novel identified variants are pathogenic and, therefore, are likely the cause of the disease in the presented patients. In one of the patients a stop codon variant is predicted to produce a truncated protein, and in the other patient an intronic variant results in aberrant splicing.

Transcriptome Bioinformatical Analysis of Vertebrate Stages of Schistosoma japonicum Reveals Alternative Splicing Events

PubMed Central

Wang, Xinye; Xu, Xindong; Lu, Xingyu; Zhang, Yuanbin; Pan, Weiqing

2015-01-01

Alternative splicing is a molecular process that contributes greatly to the diversification of proteome and to gene functions. Understanding the mechanisms of stage-specific alternative splicing can provide a better understanding of the development of eukaryotes and the functions of different genes. Schistosoma japonicum is an infectious blood-dwelling trematode with a complex lifecycle that causes the tropical disease schistosomiasis. In this study, we analyzed the transcriptome of Schistosoma japonicum to discover alternative splicing events in this parasite, by applying RNA-seq to cDNA library of adults and schistosomula. Results were validated by RT-PCR and sequencing. We found 11,623 alternative splicing events among 7,099 protein encoding genes and average proportion of alternative splicing events per gene was 42.14%. We showed that exon skip is the most common type of alternative splicing events as found in high eukaryotes, whereas intron retention is the least common alternative splicing type. According to intron boundary analysis, the parasite possesses same intron boundaries as other organisms, namely the classic “GT-AG” rule. And in alternative spliced introns or exons, this rule is less strict. And we have attempted to detect alternative splicing events in genes encoding proteins with signal peptides and transmembrane helices, suggesting that alternative splicing could change subcellular locations of specific gene products. Our results indicate that alternative splicing is prevalent in this parasitic worm, and that the worm is close to its hosts. The revealed secretome involved in alternative splicing implies new perspective into understanding interaction between the parasite and its host. PMID:26407301

Alternative splicing and the evolution of phenotypic novelty.

PubMed

Bush, Stephen J; Chen, Lu; Tovar-Corona, Jaime M; Urrutia, Araxi O

2017-02-05

Alternative splicing, a mechanism of post-transcriptional RNA processing whereby a single gene can encode multiple distinct transcripts, has been proposed to underlie morphological innovations in multicellular organisms. Genes with developmental functions are enriched for alternative splicing events, suggestive of a contribution of alternative splicing to developmental programmes. The role of alternative splicing as a source of transcript diversification has previously been compared to that of gene duplication, with the relationship between the two extensively explored. Alternative splicing is reduced following gene duplication with the retention of duplicate copies higher for genes which were alternatively spliced prior to duplication. Furthermore, and unlike the case for overall gene number, the proportion of alternatively spliced genes has also increased in line with the evolutionary diversification of cell types, suggesting alternative splicing may contribute to the complexity of developmental programmes. Together these observations suggest a prominent role for alternative splicing as a source of functional innovation. However, it is unknown whether the proliferation of alternative splicing events indeed reflects a functional expansion of the transcriptome or instead results from weaker selection acting on larger species, which tend to have a higher number of cell types and lower population sizes.This article is part of the themed issue 'Evo-devo in the genomics era, and the origins of morphological diversity'. © 2016 The Author(s).

Alternative splicing and the evolution of phenotypic novelty

PubMed Central

Bush, Stephen J.; Chen, Lu; Tovar-Corona, Jaime M.

2017-01-01

Alternative splicing, a mechanism of post-transcriptional RNA processing whereby a single gene can encode multiple distinct transcripts, has been proposed to underlie morphological innovations in multicellular organisms. Genes with developmental functions are enriched for alternative splicing events, suggestive of a contribution of alternative splicing to developmental programmes. The role of alternative splicing as a source of transcript diversification has previously been compared to that of gene duplication, with the relationship between the two extensively explored. Alternative splicing is reduced following gene duplication with the retention of duplicate copies higher for genes which were alternatively spliced prior to duplication. Furthermore, and unlike the case for overall gene number, the proportion of alternatively spliced genes has also increased in line with the evolutionary diversification of cell types, suggesting alternative splicing may contribute to the complexity of developmental programmes. Together these observations suggest a prominent role for alternative splicing as a source of functional innovation. However, it is unknown whether the proliferation of alternative splicing events indeed reflects a functional expansion of the transcriptome or instead results from weaker selection acting on larger species, which tend to have a higher number of cell types and lower population sizes. This article is part of the themed issue ‘Evo-devo in the genomics era, and the origins of morphological diversity’. PMID:27994117

Regulation of alternative splicing of Slo K+ channels in adrenal and pituitary during the stress-hyporesponsive period of rat development.

PubMed

Lai, Guey-Jen; McCobb, David P

2006-08-01

Stress triggers release of ACTH from the pituitary, glucocorticoids from the adrenal cortex, and epinephrine from the adrenal medulla. Although functions differ, these hormone systems interact in many ways. Previous evidence indicates that pituitary and steroid hormones regulate alternative splicing of the Slo gene at the stress axis-regulated exon (STREX), with functional implications for the calcium-activated K+ channels prominent in adrenal medullary and pituitary cells. Here we examine the role of corticosterone in Slo splicing regulation in pituitary and adrenal tissues during the stress-hyporesponsive period of early rat postnatal life. The sharp drop in plasma corticosterone (CORT) that defines this period offers a unique opportunity to test CORT's role in Slo splicing. We report that in both adrenal and pituitary tissues, the percentage of Slo transcripts having STREX declines and recovers in parallel with CORT. Moreover, addition of 500 nm CORT to cultures of anterior pituitary cells from 13-, 21-, and 30-d postnatal animals increased the percentage of Slo transcripts with STREX, whereas 20 microm CORT reduced STREX representation. Applied to adrenal chromaffin cells, 20 microm CORT decreased STREX inclusion, whereas neither 500 nm nor 2 microm had any effect. The mineralocorticoid receptor antagonist RU28318 abolished the effect of 500 nm CORT on splicing in pituitary cells, whereas the glucocorticoid receptor antagonist RU38486 blocked the effect of 20 microm CORT on adrenal chromaffin cells. These results support the hypothesis that the abrupt, transient drop in CORT during the stress-hyporesponsive period drives the transient decline in STREX splice variant representation in pituitary, but not adrenal.

Predicting Gene Structure Changes Resulting from Genetic Variants via Exon Definition Features.

PubMed

Majoros, William H; Holt, Carson; Campbell, Michael S; Ware, Doreen; Yandell, Mark; Reddy, Timothy E

2018-04-25

Genetic variation that disrupts gene function by altering gene splicing between individuals can substantially influence traits and disease. In those cases, accurately predicting the effects of genetic variation on splicing can be highly valuable for investigating the mechanisms underlying those traits and diseases. While methods have been developed to generate high quality computational predictions of gene structures in reference genomes, the same methods perform poorly when used to predict the potentially deleterious effects of genetic changes that alter gene splicing between individuals. Underlying that discrepancy in predictive ability are the common assumptions by reference gene finding algorithms that genes are conserved, well-formed, and produce functional proteins. We describe a probabilistic approach for predicting recent changes to gene structure that may or may not conserve function. The model is applicable to both coding and noncoding genes, and can be trained on existing gene annotations without requiring curated examples of aberrant splicing. We apply this model to the problem of predicting altered splicing patterns in the genomes of individual humans, and we demonstrate that performing gene-structure prediction without relying on conserved coding features is feasible. The model predicts an unexpected abundance of variants that create de novo splice sites, an observation supported by both simulations and empirical data from RNA-seq experiments. While these de novo splice variants are commonly misinterpreted by other tools as coding or noncoding variants of little or no effect, we find that in some cases they can have large effects on splicing activity and protein products, and we propose that they may commonly act as cryptic factors in disease. The software is available from geneprediction.org/SGRF. bmajoros@duke.edu. Supplementary information is available at Bioinformatics online.

Genomic structure and expression of STM2, the chromosome 1 familial Alzheimer disease gene.

PubMed

Levy-Lahad, E; Poorkaj, P; Wang, K; Fu, Y H; Oshima, J; Mulligan, J; Schellenberg, G D

1996-06-01

Mutations in the gene STM2 result in autosomal dominant familial Alzheimer disease. To screen for mutations and to identify regulatory elements for this gene, the genomic DNA sequence and intron-exon structure were determined. Twelve exons including 10 coding exons were identified in a genomic region spanning 23,737 bp. The first 2 exons encode the 5'-untranslated region. Expression analysis of STM2 indicates that two transcripts of 2.4 and 2.8 kb are found in skeletal muscle, pancreas, and heart. In addition, a splice variant of the 2.4-kb transcript was identified that is the result of the use of an alternative splice acceptor site located in exon 10. The use of this site results in a transcript lacking a single glutamate. The promotor for this gene and the alternatively spliced exons leading to the 2.8-kb form of the gene remain to be identified. Expression of STM2 was high in skeletal muscle and pancreas, with comparatively low levels observed in brain. This expression pattern is intriguing since in Alzheimer disease, pathology and degeneration are observed only in the central nervous system.

Expression of VEGF 111 and other VEGF-A variants in the rat uterus is correlated with stage of pregnancy.

PubMed

Whittington, Camilla M; Danastas, Kevin; Grau, Georges E; Murphy, Christopher R; Thompson, Michael B

2017-02-01

Vascular endothelial growth factor A is a major mediator of angiogenesis, a critically important process in vertebrate growth and development as well as pregnancy. Here we report for the first time the expression of a rare and unusually potent splice variant, VEGF 111 , in vivo in mammals. This variant has previously only been found in mammals in cultured human cells exposed to genotoxic agents. Our discovery of VEGF 111 in the uterus of both a eutherian (rat) and a marsupial (fat-tailed dunnart) suggests that the splice variant may be common to all mammals. As VEGF 111 is also expressed in the uterus of at least one lineage of lizards, the expression of this splice variant may be a widespread amniote phenomenon. We measured expression of VEGF 111 and two major VEGF-A splice variants in the uterus of pregnant rats, showing that the three variants show different expression patterns across pregnancy. Our results suggest that viviparous mammals possess a precisely regulated milieu of VEGF isoforms producing the angiogenesis required for successful pregnancy. The discovery of VEGF 111 in rat uterus paves the way for the development of in vivo models of VEGF 111 activity in a highly tractable laboratory animal, and is particularly significant in the context of early pregnancy loss and cancer research.

A saga of cancer epigenetics: linking epigenetics to alternative splicing.

PubMed

Narayanan, Sathiya Pandi; Singh, Smriti; Shukla, Sanjeev

2017-03-07

The discovery of an increasing number of alternative splicing events in the human genome highlighted that ∼94% of genes generate alternatively spliced transcripts that may produce different protein isoforms with diverse functions. It is now well known that several diseases are a direct and indirect consequence of aberrant splicing events in humans. In addition to the conventional mode of alternative splicing regulation by ' cis ' RNA-binding sites and ' trans' RNA-binding proteins, recent literature provides enormous evidence for epigenetic regulation of alternative splicing. The epigenetic modifications may regulate alternative splicing by either influencing the transcription elongation rate of RNA polymerase II or by recruiting a specific splicing regulator via different chromatin adaptors. The epigenetic alterations and aberrant alternative splicing are known to be associated with various diseases individually, but this review discusses/highlights the latest literature on the role of epigenetic alterations in the regulation of alternative splicing and thereby cancer progression. This review also points out the need for further studies to understand the interplay between epigenetic modifications and aberrant alternative splicing in cancer progression. © 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

Alternative splicing in cancers: From aberrant regulation to new therapeutics.

PubMed

Song, Xiaowei; Zeng, Zhenyu; Wei, Huanhuan; Wang, Zefeng

2018-03-01

Alternative splicing is one of the most common mechanisms for gene regulation in humans, and plays a vital role to increase the complexity of functional proteins. In this article, we seek to provide a general review on the relationships between alternative splicing and tumorigenesis. We briefly introduce the basic rules for regulation of alternative splicing, and discuss recent advances on dynamic regulation of alternative splicing in cancers by highlighting the roles of a variety of RNA splicing factors in tumorigenesis. We further discuss several important questions regarding the splicing of long noncoding RNAs and back-splicing of circular RNAs in cancers. Finally, we discuss the current technologies that can be used to manipulate alternative splicing and serve as potential cancer treatment. Copyright © 2017 Elsevier Ltd. All rights reserved.

Interplay between estrogen receptor and AKT in Estradiol-induced alternative splicing

PubMed Central

2013-01-01

Background Alternative splicing is critical for generating complex proteomes in response to extracellular signals. Nuclear receptors including estrogen receptor alpha (ERα) and their ligands promote alternative splicing. The endogenous targets of ERα:estradiol (E2)-mediated alternative splicing and the influence of extracellular kinases that phosphorylate ERα on E2-induced splicing are unknown. Methods MCF-7 and its anti-estrogen derivatives were used for the majority of the assays. CD44 mini gene was used to measure the effect of E2 and AKT on alternative splicing. ExonHit array analysis was performed to identify E2 and AKT-regulated endogenous alternatively spliced apoptosis-related genes. Quantitative reverse transcription polymerase chain reaction was performed to verify alternative splicing. ERα binding to alternatively spliced genes was verified by chromatin immunoprecipitation assay. Bromodeoxyuridine incorporation-ELISA and Annexin V labeling assays were done to measure cell proliferation and apoptosis, respectively. Results We identified the targets of E2-induced alternative splicing and deconstructed some of the mechanisms surrounding E2-induced splicing by combining splice array with ERα cistrome and gene expression array. E2-induced alternatively spliced genes fall into at least two subgroups: coupled to E2-regulated transcription and ERα binding to the gene without an effect on rate of transcription. Further, AKT, which phosphorylates both ERα and splicing factors, influenced ERα:E2 dependent splicing in a gene-specific manner. Genes that are alternatively spliced include FAS/CD95, FGFR2, and AXIN-1. E2 increased the expression of FGFR2 C1 isoform but reduced C3 isoform at mRNA level. E2-induced alternative splicing of FAS and FGFR2 in MCF-7 cells correlated with resistance to FAS activation-induced apoptosis and response to keratinocyte growth factor (KGF), respectively. Resistance of MCF-7 breast cancer cells to the anti-estrogen tamoxifen was associated with ERα-dependent overexpression of FGFR2, whereas resistance to fulvestrant was associated with ERα-dependent isoform switching, which correlated with altered response to KGF. Conclusion E2 may partly alter cellular proteome through alternative splicing uncoupled to its effects on transcription initiation and aberration in E2-induced alternative splicing events may influence response to anti-estrogens. PMID:23758675

Decoding the Effect of Isobaric Substitutions on Identifying Missing Proteins and Variant Peptides in Human Proteome.

PubMed

Choong, Wai-Kok; Lih, Tung-Shing Mamie; Chen, Yu-Ju; Sung, Ting-Yi

2017-12-01

To confirm the existence of missing proteins, we need to identify at least two unique peptides with length of 9-40 amino acids of a missing protein in bottom-up mass-spectrometry-based proteomic experiments. However, an identified unique peptide of the missing protein, even identified with high level of confidence, could possibly coincide with a peptide of a commonly observed protein due to isobaric substitutions, mass modifications, alternative splice isoforms, or single amino acid variants (SAAVs). Besides unique peptides of missing proteins, identified variant peptides (SAAV-containing peptides) could also alternatively map to peptides of other proteins due to the aforementioned issues. Therefore, we conducted a thorough comparative analysis on data sets in PeptideAtlas Tiered Human Integrated Search Proteome (THISP, 2017-03 release), including neXtProt (2017-01 release), to systematically investigate the possibility of unique peptides in missing proteins (PE2-4), unique peptides in dubious proteins, and variant peptides affected by isobaric substitutions, causing doubtful identification results. In this study, we considered 11 isobaric substitutions. From our analysis, we found <5% of the unique peptides of missing proteins and >6% of variant peptides became shared with peptides of PE1 proteins after isobaric substitutions.

Systematic profiling of alternative splicing signature reveals prognostic predictor for ovarian cancer.

PubMed

Zhu, Junyong; Chen, Zuhua; Yong, Lei

2018-02-01

The majority of genes are alternatively spliced and growing evidence suggests that alternative splicing is modified in cancer and is associated with cancer progression. Systematic analysis of alternative splicing signature in ovarian cancer is lacking and greatly needed. We profiled genome-wide alternative splicing events in 408 ovarian serous cystadenocarcinoma (OV) patients in TCGA. Seven types of alternative splicing events were curated and prognostic analyses were performed with predictive models and splicing network built for OV patients. Among 48,049 mRNA splicing events in 10,582 genes, we detected 2,611 alternative splicing events in 2,036 genes which were significant associated with overall survival of OV patients. Exon skip events were the most powerful prognostic factors among the seven types. The area under the curve of the receiver-operator characteristic curve for prognostic predictor, which was built with top significant alternative splicing events, was 0.937 at 2,000 days of overall survival, indicating powerful efficiency in distinguishing patient outcome. Interestingly, splicing correlation network suggested obvious trends in the role of splicing factors in OV. In summary, we built powerful prognostic predictors for OV patients and uncovered interesting splicing networks which could be underlying mechanisms. Copyright © 2017 Elsevier Inc. All rights reserved.

Non-exomic and synonymous variants in ABCA4 are an important cause of Stargardt disease

PubMed Central

Braun, Terry A.; Mullins, Robert F.; Wagner, Alex H.; Andorf, Jeaneen L.; Johnston, Rebecca M.; Bakall, Benjamin B.; Deluca, Adam P.; Fishman, Gerald A.; Lam, Byron L.; Weleber, Richard G.; Cideciyan, Artur V.; Jacobson, Samuel G.; Sheffield, Val C.; Tucker, Budd A.; Stone, Edwin M.

2013-01-01

Mutations in ABCA4 cause Stargardt disease and other blinding autosomal recessive retinal disorders. However, sequencing of the complete coding sequence in patients with clinical features of Stargardt disease sometimes fails to detect one or both mutations. For example, among 208 individuals with clear clinical evidence of ABCA4 disease ascertained at a single institution, 28 had only one disease-causing allele identified in the exons and splice junctions of the primary retinal transcript of the gene. Haplotype analysis of these 28 probands revealed 3 haplotypes shared among ten families, suggesting that 18 of the 28 missing alleles were rare enough to be present only once in the cohort. We hypothesized that mutations near rare alternate splice junctions in ABCA4 might cause disease by increasing the probability of mis-splicing at these sites. Next-generation sequencing of RNA extracted from human donor eyes revealed more than a dozen alternate exons that are occasionally incorporated into the ABCA4 transcript in normal human retina. We sequenced the genomic DNA containing 15 of these minor exons in the 28 one-allele subjects and observed five instances of two different variations in the splice signals of exon 36.1 that were not present in normal individuals (P < 10−6). Analysis of RNA obtained from the keratinocytes of patients with these mutations revealed the predicted alternate transcript. This study illustrates the utility of RNA sequence analysis of human donor tissue and patient-derived cell lines to identify mutations that would be undetectable by exome sequencing. PMID:23918662

Pit-1/growth hormone factor 1 splice variant expression in the rhesus monkey pituitary gland and the rhesus and human placenta.

PubMed

Schanke, J T; Conwell, C M; Durning, M; Fisher, J M; Golos, T G

1997-03-01

We have examined the expression of Pit-1 messenger RNA (mRNA) splice variants in the nonhuman primate pituitary and in rhesus and human placenta. Full-length complementary DNAs (cDNAs) representing Pit-1 and the Pit-1 beta splice variants were cloned from a rhesus monkey pituitary cDNA library and were readily detectable by RT-PCR with rhesus pituitary gland RNA. The Pit-1T variant previously reported in mouse pituitary tumor cell lines was not detectable in normal rhesus pituitary tissue, although two novel splice variants were detected. A cDNA approximating the rat Pit-1 delta 4 variant was cloned but coded for a truncated and presumably nonfunctional protein. Only by using a nested RT-PCR approach were Pit-1 and Pit-1 beta variants consistently detectable in both human and rhesus placental tissue. The Pit-1 beta variant mRNA was not detectable in JEG-3 choriocarcinoma cells unless the cells were stimulated with 8-Br-cAMP. Immunoblot studies with nuclear extracts from primary rhesus syncytiotrophoblast cultures or JEG-3 choriocarcinoma cells indicated that although mRNA levels were very low, Pit-1 protein was detectable in differentiated cytotrophoblasts, and levels increased after treatment with 8-Br-cAMP. Two major species of Pit-1 protein were detected that corresponded to the two major bands in rat pituitary GH3 cell nuclear extracts. Low levels of slightly larger bands also were seen, which may represent Pit-1 beta protein or phosphorylated species. We conclude that Pit-1 splice variants expressed in the primate pituitary gland differ from those in the rodent gland and that the Pit-1 and Pit-1 beta mRNAs expressed in the placenta give rise to a pattern of protein expression similar to that seen in pituitary cells, which is inducible by treatment with 8-Br-cAMP.

A serine–arginine-rich (SR) splicing factor modulates alternative splicing of over a thousand genes in Toxoplasma gondii

PubMed Central

Yeoh, Lee M.; Goodman, Christopher D.; Hall, Nathan E.; van Dooren, Giel G.; McFadden, Geoffrey I.; Ralph, Stuart A.

2015-01-01

Single genes are often subject to alternative splicing, which generates alternative mature mRNAs. This phenomenon is widespread in animals, and observed in over 90% of human genes. Recent data suggest it may also be common in Apicomplexa. These parasites have small genomes, and economy of DNA is evolutionarily favoured in this phylum. We investigated the mechanism of alternative splicing in Toxoplasma gondii, and have identified and localized TgSR3, a homologue of ASF/SF2 (alternative-splicing factor/splicing factor 2, a serine-arginine–rich, or SR protein) to a subnuclear compartment. In addition, we conditionally overexpressed this protein, which was deleterious to growth. qRT-PCR was used to confirm perturbation of splicing in a known alternatively-spliced gene. We performed high-throughput RNA-seq to determine the extent of splicing modulated by this protein. Current RNA-seq algorithms are poorly suited to compact parasite genomes, and hence we complemented existing tools by writing a new program, GeneGuillotine, that addresses this deficiency by segregating overlapping reads into distinct genes. In order to identify the extent of alternative splicing, we released another program, JunctionJuror, that detects changes in intron junctions. Using this program, we identified about 2000 genes that were constitutively alternatively spliced in T. gondii. Overexpressing the splice regulator TgSR3 perturbed alternative splicing in over 1000 genes. PMID:25870410

Divergent biophysical properties, gating mechanisms, and possible functions of the two skeletal muscle Ca(V)1.1 calcium channel splice variants.

PubMed

Tuluc, Petronel; Flucher, Bernhard E

2011-12-01

Voltage-gated calcium channels are multi-subunit protein complexes that specifically allow calcium ions to enter the cell in response to membrane depolarization. But, for many years it seemed that the skeletal muscle calcium channel Ca(V)1.1 is the exception. The classical splice variant Ca(V)1.1a activates slowly, has a very small current amplitude and poor voltage sensitivity. In fact adult muscle fibers work perfectly well even in the absence of calcium influx. Recently a new splice variant of the skeletal muscle calcium channel Ca(V)1.1e has been characterized. The lack of the 19 amino acid exon 29 in this splice variant results in a rapidly activating calcium channel with high current amplitude and good voltage sensitivity. Ca(V)1.1e is the dominant channel in embryonic muscle, where the expression of this high calcium-conducting Ca(V)1.1 isoform readily explains developmental processes depending on L-type calcium currents. Moreover, the availability of these two structurally similar but functionally distinct channel variants facilitates the analysis of the molecular mechanisms underlying the unique current properties of the classical Ca(V)1.1a channel.

Methods for Characterization of Alternative RNA Splicing.

PubMed

Harvey, Samuel E; Cheng, Chonghui

2016-01-01

Quantification of alternative splicing to detect the abundance of differentially spliced isoforms of a gene in total RNA can be accomplished via RT-PCR using both quantitative real-time and semi-quantitative PCR methods. These methods require careful PCR primer design to ensure specific detection of particular splice isoforms. We also describe analysis of alternative splicing using a splicing "minigene" in mammalian cell tissue culture to facilitate investigation of the regulation of alternative splicing of a particular exon of interest.

The long non-coding RNA GAS5 differentially regulates cell cycle arrest and apoptosis through activation of BRCA1 and p53 in human neuroblastoma

PubMed Central

Mazar, Joseph; Rosado, Amy; Shelley, John; Marchica, John; Westmoreland, Tamarah J

2017-01-01

The long non-coding RNA GAS5 has been shown to modulate cancer proliferation in numerous human cancer systems and has been correlated with successful patient outcome. Our examination of GAS5 in neuroblastoma has revealed robust expression in both MYCN-amplified and non-amplified cell lines. Knockdown of GAS5 In vitro resulted in defects in cell proliferation, apoptosis, and induced cell cycle arrest. Further analysis of GAS5 clones revealed multiple novel splice variants, two of which inversely modulated with MYCN status. Complementation studies of the variants post-knockdown of GAS5 indicated alternate phenotypes, with one variant (FL) considerably enhancing cell proliferation by rescuing cell cycle arrest and the other (C2) driving apoptosis, suggesting a unique role for each in neuroblastoma cancer physiology. Global sequencing and ELISA arrays revealed that the loss of GAS5 induced p53, BRCA1, and GADD45A, which appeared to modulate cell cycle arrest in concert. Complementation with only the FL GAS5 clone could rescue cell cycle arrest, stabilizing HDM2, and leading to the loss of p53. Together, these data offer novel therapeutic targets in the form of lncRNA splice variants for separate challenges against cancer growth and cell death. PMID:28035057

The Human Splice Variant Δ16HER2 Induces Rapid Tumor Onset in a Reporter Transgenic Mouse

PubMed Central

Iezzi, Manuela; Zenobi, Santa; Montani, Maura; Pietrella, Lucia; Kalogris, Cristina; Rossini, Anna; Ciravolo, Valentina; Castagnoli, Lorenzo; Tagliabue, Elda; Pupa, Serenella M.; Musiani, Piero; Monaci, Paolo; Menard, Sylvie; Amici, Augusto

2011-01-01

Several transgenic mice models solidly support the hypothesis that HER2 (ERBB2) overexpression or mutation promotes tumorigenesis. Recently, a HER2 splice variant lacking exon-16 (Δ16HER2) has been detected in human breast carcinomas. This alternative protein, a normal byproduct of HER2, has an increased transforming potency compared to wild-type (wt) HER2 receptors. To examine the ability of Δ16HER2 to transform mammary epithelium in vivo and to monitor Δ16HER2-driven tumorigenesis in live mice, we generated and characterized a mouse line that transgenically expresses both human Δ16HER2 and firefly luciferase under the transcriptional control of the MMTV promoter. All the transgenic females developed multifocal mammary tumors with a rapid onset and an average latency of 15.11 weeks. Immunohistochemical analysis revealed the concurrent expression of luciferase and the human Δ16HER2 oncogene only in the mammary gland and in strict correlation with tumor development. Transgenic Δ16HER2 expressed on the tumor cell plasma membrane from spontaneous mammary adenocarcinomas formed constitutively active homodimers able to activate the oncogenic signal transduction pathway mediated through Src kinase. These new transgenic animals demonstrate the ability of the human Δ16HER2 isoform to transform “per se” mammary epithelium in vivo. The high tumor incidence as well as the short latency strongly suggests that the Δ16HER2 splice variant represents the transforming form of the HER2 oncoprotein. PMID:21559085

Involvement of dominant-negative spliced variants of the intermediate conductance Ca2+-activated K+ channel, K(Ca)3.1, in immune function of lymphoid cells.

PubMed

Ohya, Susumu; Niwa, Satomi; Yanagi, Ayano; Fukuyo, Yuka; Yamamura, Hisao; Imaizumi, Yuji

2011-05-13

The intermediate conductance Ca(2+)-activated K(+) channel (IK(Ca) channel) encoded by K(Ca)3.1 is responsible for the control of proliferation and differentiation in various types of cells. We identified novel spliced variants of K(Ca)3.1 (human (h) K(Ca)3.1b) from the human thymus, which were lacking the N-terminal domains of the original hK(Ca)3.1a as a result of alternative splicing events. hK(Ca)3.1b was significantly expressed in human lymphoid tissues. Western blot analysis showed that hK(Ca)3.1a proteins were mainly expressed in the plasma membrane fraction, whereas hK(Ca)3.1b was in the cytoplasmic fraction. We also identified a similar N terminus lacking K(Ca)3.1 variants from mice and rat lymphoid tissues (mK(Ca)3.1b and rK(Ca)3.1b). In the HEK293 heterologous expression system, the cellular distribution of cyan fluorescent protein-tagged hK(Ca)3.1a and/or YFP-tagged hK(Ca)3.1b isoforms showed that hK(Ca)3.1b suppressed the localization of hK(Ca)3.1a to the plasma membrane. In the Xenopus oocyte translation system, co-expression of hK(Ca)3.1b with hK(Ca)3.1a suppressed IK(Ca) channel activity of hK(Ca)3.1a in a dominant-negative manner. In addition, this study indicated that up-regulation of mK(Ca)3.1b in mouse thymocytes differentiated CD4(+)CD8(+) phenotype thymocytes into CD4(-)CD8(-) ones and suppressed concanavalin-A-stimulated thymocyte growth by down-regulation of mIL-2 transcripts. Anti-proliferative effects and down-regulation of mIL-2 transcripts were also observed in mK(Ca)3.1b-overexpressing mouse thymocytes. These suggest that the N-terminal domain of K(Ca)3.1 is critical for channel trafficking to the plasma membrane and that the fine-tuning of IK(Ca) channel activity modulated through alternative splicing events may be related to the control in physiological and pathophysiological conditions in T-lymphocytes.

Alternative Splicing in Neurogenesis and Brain Development.

PubMed

Su, Chun-Hao; D, Dhananjaya; Tarn, Woan-Yuh

2018-01-01

Alternative splicing of precursor mRNA is an important mechanism that increases transcriptomic and proteomic diversity and also post-transcriptionally regulates mRNA levels. Alternative splicing occurs at high frequency in brain tissues and contributes to every step of nervous system development, including cell-fate decisions, neuronal migration, axon guidance, and synaptogenesis. Genetic manipulation and RNA sequencing have provided insights into the molecular mechanisms underlying the effects of alternative splicing in stem cell self-renewal and neuronal fate specification. Timely expression and perhaps post-translational modification of neuron-specific splicing regulators play important roles in neuronal development. Alternative splicing of many key transcription regulators or epigenetic factors reprograms the transcriptome and hence contributes to stem cell fate determination. During neuronal differentiation, alternative splicing also modulates signaling activity, centriolar dynamics, and metabolic pathways. Moreover, alternative splicing impacts cortical lamination and neuronal development and function. In this review, we focus on recent progress toward understanding the contributions of alternative splicing to neurogenesis and brain development, which has shed light on how splicing defects may cause brain disorders and diseases.

A novel variant of aquaporin 3 is expressed in killifish (Fundulus heteroclitus) intestine

PubMed Central

Jung, Dawoon; Adamo, Meredith A.; Lehman, Rebecca M.; Barnaby, Roxanna; Jackson, Craig E.; Jackson, Brian P.; Shaw, Joseph R.; Stanton, Bruce A.

2015-01-01

Killifish (Fundulus heteroclitus) are euryhaline teleosts that are widely used in environmental and toxicological studies, and they are tolerant to arsenic, in part due to very low assimilation of arsenic from the environment. The mechanism of arsenic uptake by the intestine, a major route of arsenic uptake in humans is unknown. Thus, the goal of this study was to determine if aquaglyceroporins (AQP), which transport water and other small molecules including arsenite across cell membranes, are expressed in the killifish intestine, and whether AQP expression is affected by osmotic stress. Through RT-PCR and sequence analysis of PCR amplicons, we demonstrated that the intestine expresses kfAQP3a and kfAQP3b, two previously identified variants, and also identified a novel variant of killifish AQP3 (kfAQP3c) in the intestine. The variants likely represent alternate splice forms. A BLAST search of the F. heteroclitus reference genome revealed that the AQP3 gene resides on a single locus, while an alignment of the AQP3 sequence among 384 individuals from eight population ranging from Rhode Island to North Carolina revealed that its coding sequence was remarkably conserved with no fixed polymorphism residing in the region that distinguishes these variants. We further demonstrate that the novel variant transports arsenite into HEK293T cells. Whereas kfAQP3a, which does not transport arsenite, was expressed in both freshwater (FW) and saltwater (SW) acclimated fish, kfAQP3b, an arsenic transporter, was expressed only in FW acclimated fish, and kfAQP3c was expressed only in SW acclimated fish. Thus, we have identified a novel, putative splice variant of kfAQP3, kfAQP3c, which transports arsenic and is expressed only in SW acclimated fish. PMID:25766383

A Presumptive Developmental Role for a Sea Urchin Cyclin B Splice Variant

PubMed Central

Lozano, Jean-Claude; Schatt, Philippe; Marquès, François; Peaucellier, Gérard; Fort, Philippe; Féral, Jean-Pierre; Genevière, Anne-Marie; Picard, André

1998-01-01

We show that a splice variant–derived cyclin B is produced in sea urchin oocytes and embryos. This splice variant protein lacks highly conserved sequences in the COOH terminus of the protein. It is found strikingly abundant in growing oocytes and cells committed to differentiation during embryogenesis. Cyclin B splice variant (CBsv) protein associates weakly in the cell with Xenopus cdc2 and with budding yeast CDC28p. In contrast to classical cyclin B, CBsv very poorly complements a triple CLN deletion in budding yeast, and its microinjection prevents an initial step in MPF activation, leading to an important delay in oocyte meiosis reinitiation. CBsv microinjection in fertilized eggs induces cell cycle delay and abnormal development. We assume that CBsv is produced in growing oocytes to keep them in prophase, and during embryogenesis to slow down cell cycle in cells that will be committed to differentiation. PMID:9442104

Widespread alternative and aberrant splicing revealed by lariat sequencing

PubMed Central

Stepankiw, Nicholas; Raghavan, Madhura; Fogarty, Elizabeth A.; Grimson, Andrew; Pleiss, Jeffrey A.

2015-01-01

Alternative splicing is an important and ancient feature of eukaryotic gene structure, the existence of which has likely facilitated eukaryotic proteome expansions. Here, we have used intron lariat sequencing to generate a comprehensive profile of splicing events in Schizosaccharomyces pombe, amongst the simplest organisms that possess mammalian-like splice site degeneracy. We reveal an unprecedented level of alternative splicing, including alternative splice site selection for over half of all annotated introns, hundreds of novel exon-skipping events, and thousands of novel introns. Moreover, the frequency of these events is far higher than previous estimates, with alternative splice sites on average activated at ∼3% the rate of canonical sites. Although a subset of alternative sites are conserved in related species, implying functional potential, the majority are not detectably conserved. Interestingly, the rate of aberrant splicing is inversely related to expression level, with lowly expressed genes more prone to erroneous splicing. Although we validate many events with RNAseq, the proportion of alternative splicing discovered with lariat sequencing is far greater, a difference we attribute to preferential decay of aberrantly spliced transcripts. Together, these data suggest the spliceosome possesses far lower fidelity than previously appreciated, highlighting the potential contributions of alternative splicing in generating novel gene structures. PMID:26261211

Clinico-Pathologic Relevance of Survivin Splice Variant Expression in Cancer

PubMed Central

de Necochea-Campion, Rosalia; Chen, Chien-Shing; Mirshahidi, Saied; Howard, Frank D.; Wall, Nathan R.

2013-01-01

Survivin is a member of the inhibitor of apoptosis (IAP) family and has multifunctional properties that include aspects of proliferation, invasion and cell survival control. Survivin is a promising candidate for targeted cancer therapy as its expression is associated with poor clinical outcome, more aggressive clinico-pathologic features, and resistance to radiation and chemotherapy. In the present review the different properties of the Survivin splice variants are discussed and their activities correlated with different aspects of cancer cell biology, to include subcellular location. Special emphasis is placed on our current understanding of these Survivin splice variants influence on each other and on the phenotypic responses to therapy that they may control. PMID:23791888

Sequencing of mRNA identifies re-expression of fetal splice variants in cardiac hypertrophy

PubMed Central

Ames, EG; Lawson, MJ; Mackey, AJ; Holmes, JW

2013-01-01

Cardiac hypertrophy has been well-characterized at the level of transcription. During cardiac hypertrophy, genes normally expressed primarily during fetal heart development are reexpressed, and this fetal gene program is believed to be a critical component of the hypertrophic process. Recently, alternative splicing of mRNA transcripts has been shown to be temporally regulated during heart development, leading us to consider whether fetal patterns of splicing also reappear during hypertrophy. We hypothesized that patterns of alternative splicing occurring during heart development are recapitulated during cardiac hypertrophy. Here we present a study of isoform expression during pressure-overload cardiac hypertrophy induced by 10 days of transverse aortic constriction (TAC) in rats and in developing fetal rat hearts compared to sham-operated adult rat hearts, using high-throughput sequencing of poly(A) tail mRNA. We find a striking degree of overlap between the isoforms expressed differentially in fetal and pressure-overloaded hearts compared to control: forty-four percent of the isoforms with significantly altered expression in TAC hearts are also expressed at significantly different levels in fetal hearts compared to control (P < 0.001). The isoforms that are shared between hypertrophy and fetal heart development are significantly enriched for genes involved in cytoskeletal organization, RNA processing, developmental processes, and metabolic enzymes. Our data strongly support the concept that mRNA splicing patterns normally associated with heart development recur as part of the hypertrophic response to pressure overload. These findings suggest that cardiac hypertrophy shares post-transcriptional as well as transcriptional regulatory mechanisms with fetal heart development. PMID:23688780

Evolutionary conservation and regulation of particular alternative splicing events in plant SR proteins

PubMed Central

Kalyna, Maria; Lopato, Sergiy; Voronin, Viktor; Barta, Andrea

2006-01-01

Alternative splicing is an important mechanism for fine tuning of gene expression at the post-transcriptional level. SR proteins govern splice site selection and spliceosome assembly. The Arabidopsis genome encodes 19 SR proteins, several of which have no orthologues in metazoan. Three of the plant specific subfamilies are characterized by the presence of a relatively long alternatively spliced intron located in their first RNA recognition motif, which potentially results in an extremely truncated protein. In atRSZ33, a member of the RS2Z subfamily, this alternative splicing event was shown to be autoregulated. Here we show that atRSp31, a member of the RS subfamily, does not autoregulate alternative splicing of its similarily positioned intron. Interestingly, this alternative splicing event is regulated by atRSZ33. We demonstrate that the positions of these long introns and their capability for alternative splicing are conserved from green algae to flowering plants. Moreover, in particular alternative splicing events the splicing signals are embedded into highly conserved sequences. In different taxa, these conserved sequences occur in at least one gene within a subfamily. The evolutionary preservation of alternative splice forms together with highly conserved intron features argues for additional functions hidden in the genes of these plant-specific SR proteins. PMID:16936312

Widespread Use of Non-productive Alternative Splice Sites in Saccharomyces cerevisiae

PubMed Central

Kawashima, Tadashi; Douglass, Stephen; Gabunilas, Jason; Pellegrini, Matteo; Chanfreau, Guillaume F.

2014-01-01

Saccharomyces cerevisiae has been used as a model system to investigate the mechanisms of pre-mRNA splicing but only a few examples of alternative splice site usage have been described in this organism. Using RNA-Seq analysis of nonsense-mediated mRNA decay (NMD) mutant strains, we show that many S. cerevisiae intron-containing genes exhibit usage of alternative splice sites, but many transcripts generated by splicing at these sites are non-functional because they introduce premature termination codons, leading to degradation by NMD. Analysis of splicing mutants combined with NMD inactivation revealed the role of specific splicing factors in governing the use of these alternative splice sites and identified novel functions for Prp17p in enhancing the use of branchpoint-proximal upstream 3′ splice sites and for Prp18p in suppressing the usage of a non-canonical AUG 3′-splice site in GCR1. The use of non-productive alternative splice sites can be increased in stress conditions in a promoter-dependent manner, contributing to the down-regulation of genes during stress. These results show that alternative splicing is frequent in S. cerevisiae but masked by RNA degradation and that the use of alternative splice sites in this organism is mostly aimed at controlling transcript levels rather than increasing proteome diversity. PMID:24722551

A novel mechanism of myostatin regulation by its alternative splicing variant during myogenesis in avian species.

PubMed

Shin, Sangsu; Song, Yan; Ahn, Jinsoo; Kim, Eunsoo; Chen, Paula; Yang, Shujin; Suh, Yeunsu; Lee, Kichoon

2015-11-15

Myostatin (MSTN) is a key negative regulator of muscle growth and development, and an increase of muscle mass is achieved by inhibiting MSTN signaling. In the current study, five alternative splicing isoforms of MSTN mRNAs in avian species were identified in various tissues. Among these five, three truncated forms of myostatin, MSTN-B, -C, and -E created premature stop codons and produced partial MSTN prodomains encoded from exon 1. MSTN-B is the second dominant isoform following full-length MSTN-A, and their expression was dynamically regulated during muscle development of chicken, turkey, and quail in vivo and in vitro. To clarify the function of MSTN-B, two stable cell lines of quail myoblasts (QM7) were generated to overexpress MSTN-A or MSTN-B. Interestingly, MSTN-B promoted both cell proliferation and differentiation similar to the function of the MSTN prodomain to counteract the negative role of MSTN on myogenesis. The coimmunoprecipitation assay revealed that MSTN-B binds to MSTN-A and reduces the generation of mature MSTN. Furthermore, the current study demonstrated that the partial prodomain encoded from exon 1 is critical for binding of MSTN-B to MSTN-A. Altogether, these data imply that alternative splicing isoforms of MSTN could negatively regulate pro-myostatin processing in muscle cells and prevent MSTN-mediated inhibition of myogenesis in avian species. Copyright © 2015 the American Physiological Society.

Reversing Anoikis Resistance in Triple-Negative Breast Cancer

DTIC Science & Technology

2015-10-01

impaired function and therefore is not as efficient at inducing apoptosis. Supervillin is a protein involved in focal adhesions, and the shorter...splice variant archvillin is normally only expressed in muscle cells. GOLGA4 encodes a golgi protein involved in vesicle transport. It shuttles GPI...when miR-200c is induced and another more 3’ exon is retained, so we are still investigating the function of these regions. Figure 4. Alternative

Splice Variant Biomarkers for Parkinson’s Disease

DTIC Science & Technology

2014-05-01

reactions where they are a major source of energy and are critical for carbohydrate, fat and protein metabolism (Massey, 2000). It has been suggested... fat in the diet, saturated or unsat- urated, which is not always specified. Nor is the amount of animal protein consumed to supply the fat intake...neurotoxins, such as those possibly present in milk . Alternatively, saturated fat could modify the risk of PD by affecting PUFA metabolism and inducing adverse

Nemaline myopathy and distal arthrogryposis associated with an autosomal recessive TNNT3 splice variant.

PubMed

Sandaradura, Sarah A; Bournazos, Adam; Mallawaarachchi, Amali; Cummings, Beryl B; Waddell, Leigh B; Jones, Kristi J; Troedson, Christopher; Sudarsanam, Annapurna; Nash, Benjamin M; Peters, Gregory B; Algar, Elizabeth M; MacArthur, Daniel G; North, Kathryn N; Brammah, Susan; Charlton, Amanda; Laing, Nigel G; Wilson, Meredith J; Davis, Mark R; Cooper, Sandra T

2018-03-01

A male neonate presented with severe weakness, hypotonia, contractures and congenital scoliosis. Skeletal muscle specimens showed marked atrophy and degeneration of fast fibers with striking nemaline rods and hypertrophy of slow fibers that were ultrastructurally normal. A neuromuscular gene panel identified a homozygous essential splice variant in TNNT3 (chr11:1956150G > A, NM_006757.3:c.681+1G > A). TNNT3 encodes skeletal troponin-T fast and is associated with autosomal dominant distal arthrogryposis. TNNT3 has not previously been associated with nemaline myopathy (NM), a rare congenital myopathy linked to defects in proteins associated with thin filament structure and regulation. cDNA studies confirmed pathogenic consequences of the splice variant, eliciting exon-skipping and intron retention events leading to a frameshift. Western blot showed deficiency of troponin-T fast protein with secondary loss of troponin-I fast . We establish a homozygous splice variant in TNNT3 as the likely cause of severe congenital NM with distal arthrogryposis, characterized by specific involvement of Type-2 fibers and deficiency of troponin-T fast . © 2017 Wiley Periodicals, Inc.

Functional Studies and In Silico Analyses to Evaluate Non-Coding Variants in Inherited Cardiomyopathies.

PubMed

Frisso, Giulia; Detta, Nicola; Coppola, Pamela; Mazzaccara, Cristina; Pricolo, Maria Rosaria; D'Onofrio, Antonio; Limongelli, Giuseppe; Calabrò, Raffaele; Salvatore, Francesco

2016-11-10

Point mutations are the most common cause of inherited diseases. Bioinformatics tools can help to predict the pathogenicity of mutations found during genetic screening, but they may work less well in determining the effect of point mutations in non-coding regions. In silico analysis of intronic variants can reveal their impact on the splicing process, but the consequence of a given substitution is generally not predictable. The aim of this study was to functionally test five intronic variants ( MYBPC3 -c.506-2A>C, MYBPC3 -c.906-7G>T, MYBPC3 -c.2308+3G>C, SCN5A -c.393-5C>A, and ACTC1 -c.617-7T>C) found in five patients affected by inherited cardiomyopathies in the attempt to verify their pathogenic role. Analysis of the MYBPC3 -c.506-2A>C mutation in mRNA from the peripheral blood of one of the patients affected by hypertrophic cardiac myopathy revealed the loss of the canonical splice site and the use of an alternative splicing site, which caused the loss of the first seven nucleotides of exon 5 ( MYBPC3 -G169AfsX14). In the other four patients, we generated minigene constructs and transfected them in HEK-293 cells. This minigene approach showed that MYBPC3 -c.2308+3G>C and SCN5A -c.393-5C>A altered pre-mRNA processing, thus resulting in the skipping of one exon. No alterations were found in either MYBPC3 -c.906-7G>T or ACTC1 -c.617-7T>C. In conclusion, functional in vitro analysis of the effects of potential splicing mutations can confirm or otherwise the putative pathogenicity of non-coding mutations, and thus help to guide the patient's clinical management and improve genetic counseling in affected families.

Long genes and genes with multiple splice variants are enriched in pathways linked to cancer and other multigenic diseases.

PubMed

Sahakyan, Aleksandr B; Balasubramanian, Shankar

2016-03-12

The role of random mutations and genetic errors in defining the etiology of cancer and other multigenic diseases has recently received much attention. With the view that complex genes should be particularly vulnerable to such events, here we explore the link between the simple properties of the human genes, such as transcript length, number of splice variants, exon/intron composition, and their involvement in the pathways linked to cancer and other multigenic diseases. We reveal a substantial enrichment of cancer pathways with long genes and genes that have multiple splice variants. Although the latter two factors are interdependent, we show that the overall gene length and splicing complexity increase in cancer pathways in a partially decoupled manner. Our systematic survey for the pathways enriched with top lengthy genes and with genes that have multiple splice variants reveal, along with cancer pathways, the pathways involved in various neuronal processes, cardiomyopathies and type II diabetes. We outline a correlation between the gene length and the number of somatic mutations. Our work is a step forward in the assessment of the role of simple gene characteristics in cancer and a wider range of multigenic diseases. We demonstrate a significant accumulation of long genes and genes with multiple splice variants in pathways of multigenic diseases that have already been associated with de novo mutations. Unlike the cancer pathways, we note that the pathways of neuronal processes, cardiomyopathies and type II diabetes contain genes long enough for topoisomerase-dependent gene expression to also be a potential contributing factor in the emergence of pathologies, should topoisomerases become impaired.

Identification of a splicing variant that regulates type 2 diabetes risk factor CDKAL1 level by a coding-independent mechanism in human.

PubMed

Zhou, Bo; Wei, Fan-Yan; Kanai, Narumi; Fujimura, Atsushi; Kaitsuka, Taku; Tomizawa, Kazuhito

2014-09-01

Single-nucleotide polymorphisms (SNPs) in CDKAL1 have been associated with the development of type 2 diabetes (T2D). CDKAL1 catalyzes 2-methylthio modification of adenosine at position 37 of tRNA(Lys)(UUU). A deficit of this modification causes aberrant protein synthesis, and is associated with impairment of insulin secretion in both mouse model and human. However, it is unknown whether the T2D-associated SNPs in CDKAL1 are associated with downregulation of CDKAL1 by regulating the gene expression. Here, we report a specific splicing variant of CDKAL1 termed CDKAL1-v1 that is markedly lower in individuals carrying risk SNPs of CDKAL1. Interestingly, CDKAL1-v1 is a non-coding transcript, which regulates the CDKAL1 level by competitive binding to a CDKAL1-targeting miRNA. By direct editing of the genome, we further show that the nucleotides around the SNP regions are critical for the alternative splicing of CDKAL1-v1. These findings reveal that the T2D-associated SNPs in CDKAL1 reduce CDKAL1-v1 levels by impairing splicing, which in turn increases miRNA-mediated suppression of CDKAL1. Our results suggest that CDKAL1-v1-mediated suppression of CDKAL1 might underlie the pathogenesis of T2D in individuals carrying the risk SNPs. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Alternative Splicing as a Target for Cancer Treatment.

PubMed

Martinez-Montiel, Nancy; Rosas-Murrieta, Nora Hilda; Anaya Ruiz, Maricruz; Monjaraz-Guzman, Eduardo; Martinez-Contreras, Rebeca

2018-02-11

Alternative splicing is a key mechanism determinant for gene expression in metazoan. During alternative splicing, non-coding sequences are removed to generate different mature messenger RNAs due to a combination of sequence elements and cellular factors that contribute to splicing regulation. A different combination of splicing sites, exonic or intronic sequences, mutually exclusive exons or retained introns could be selected during alternative splicing to generate different mature mRNAs that could in turn produce distinct protein products. Alternative splicing is the main source of protein diversity responsible for 90% of human gene expression, and it has recently become a hallmark for cancer with a full potential as a prognostic and therapeutic tool. Currently, more than 15,000 alternative splicing events have been associated to different aspects of cancer biology, including cell proliferation and invasion, apoptosis resistance and susceptibility to different chemotherapeutic drugs. Here, we present well established and newly discovered splicing events that occur in different cancer-related genes, their modification by several approaches and the current status of key tools developed to target alternative splicing with diagnostic and therapeutic purposes.

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.

Analysis of 31-year-old patient with SYNGAP1 gene defect points to importance of variants in broader splice regions and reveals developmental trajectory of SYNGAP1-associated phenotype: case report.

PubMed

Prchalova, Darina; Havlovicova, Marketa; Sterbova, Katalin; Stranecky, Viktor; Hancarova, Miroslava; Sedlacek, Zdenek

2017-06-02

Whole exome sequencing is a powerful tool for the analysis of genetically heterogeneous conditions. The prioritization of variants identified often focuses on nonsense, frameshift and canonical splice site mutations, and highly deleterious missense variants, although other defects can also play a role. The definition of the phenotype range and course of rare genetic conditions requires long-term clinical follow-up of patients. We report an adult female patient with severe intellectual disability, severe speech delay, epilepsy, autistic features, aggressiveness, sleep problems, broad-based clumsy gait and constipation. Whole exome sequencing identified a de novo mutation in the SYNGAP1 gene. The variant was located in the broader splice donor region of intron 10 and replaced G by A at position +5 of the splice site. The variant was predicted in silico and shown experimentally to abolish the regular splice site and to activate a cryptic donor site within exon 10, causing frameshift and premature termination. The overall clinical picture of the patient corresponded well with the characteristic SYNGAP1-associated phenotype observed in previously reported patients. However, our patient was 31 years old which contrasted with most other published SYNGAP1 cases who were much younger. Our patient had a significant growth delay and microcephaly. Both features normalised later, although the head circumference stayed only slightly above the lower limit of the norm. The patient had a delayed puberty. Her cognitive and language performance remained at the level of a one-year-old child even in adulthood and showed a slow decline. Myopathic facial features and facial dysmorphism became more pronounced with age. Although the gait of the patient was unsteady in childhood, more severe gait problems developed in her teens. While the seizures remained well-controlled, her aggressive behaviour worsened with age and required extensive medication. The finding in our patient underscores the notion that the interpretation of variants identified using whole exome sequencing should focus not only on variants in the canonical splice dinucleotides GT and AG, but also on broader splice regions. The long-term clinical follow-up of our patient contributes to the knowledge of the developmental trajectory in individuals with SYNGAP1 gene defects.

Histone and RNA-binding protein interaction creates crosstalk network for regulation of alternative splicing.

PubMed

Kim, Yong-Eun; Park, Chungoo; Kim, Kyoon Eon; Kim, Kee K

2018-04-30

Alternative splicing is an essential process in eukaryotes, as it increases the complexity of gene expression by generating multiple proteins from a single pre-mRNA. However, information on the regulatory mechanisms for alternative splicing is lacking, because splicing occurs over a short period via the transient interactions of proteins within functional complexes of the spliceosome. Here, we investigated in detail the molecular mechanisms connecting alternative splicing with epigenetic mechanisms. We identified interactions between histone proteins and splicing factors such as Rbfox2, Rbfox3, and splicing factor proline and glutamine rich protein (SFPQ) by in vivo crosslinking and immunoprecipitation. Furthermore, we confirmed that splicing factors were bound to specific modified residues of histone proteins. Additionally, changes in histone methylation due to histone methyltransferase inhibitor treatment notably affected alternative splicing in selected genes. Therefore, we suggested that there may be crosstalk mechanisms connecting histone modifications and RNA-binding proteins that increase the local concentration of RNA-binding proteins in alternative exon loci of nucleosomes by binding specific modified histone proteins, leading to alternative splicing. This crosstalk mechanism may play a major role in epigenetic processes such as histone modification and the regulation of alternative splicing. Copyright © 2018 Elsevier Inc. All rights reserved.

Genome-wide survey of human alternative pre-mRNA splicing with exon junction microarrays.

PubMed

Johnson, Jason M; Castle, John; Garrett-Engele, Philip; Kan, Zhengyan; Loerch, Patrick M; Armour, Christopher D; Santos, Ralph; Schadt, Eric E; Stoughton, Roland; Shoemaker, Daniel D

2003-12-19

Alternative pre-messenger RNA (pre-mRNA) splicing plays important roles in development, physiology, and disease, and more than half of human genes are alternatively spliced. To understand the biological roles and regulation of alternative splicing across different tissues and stages of development, systematic methods are needed. Here, we demonstrate the use of microarrays to monitor splicing at every exon-exon junction in more than 10,000 multi-exon human genes in 52 tissues and cell lines. These genome-wide data provide experimental evidence and tissue distributions for thousands of known and novel alternative splicing events. Adding to previous studies, the results indicate that at least 74% of human multi-exon genes are alternatively spliced.

Splicing predictions reliably classify different types of alternative splicing

PubMed Central

Busch, Anke; Hertel, Klemens J.

2015-01-01

Alternative splicing is a key player in the creation of complex mammalian transcriptomes and its misregulation is associated with many human diseases. Multiple mRNA isoforms are generated from most human genes, a process mediated by the interplay of various RNA signature elements and trans-acting factors that guide spliceosomal assembly and intron removal. Here, we introduce a splicing predictor that evaluates hundreds of RNA features simultaneously to successfully differentiate between exons that are constitutively spliced, exons that undergo alternative 5′ or 3′ splice-site selection, and alternative cassette-type exons. Surprisingly, the splicing predictor did not feature strong discriminatory contributions from binding sites for known splicing regulators. Rather, the ability of an exon to be involved in one or multiple types of alternative splicing is dictated by its immediate sequence context, mainly driven by the identity of the exon's splice sites, the conservation around them, and its exon/intron architecture. Thus, the splicing behavior of human exons can be reliably predicted based on basic RNA sequence elements. PMID:25805853

Multiple roles of phosphoinositide-specific phospholipase C isozymes.

PubMed

Suh, Pann-Ghill; Park, Jae-Il; Manzoli, Lucia; Cocco, Lucio; Peak, Joanna C; Katan, Matilda; Fukami, Kiyoko; Kataoka, Tohru; Yun, Sanguk; Ryu, Sung Ho

2008-06-30

Phosphoinositide-specific phospholipase C is an effector molecule in the signal transduction process. It generates two second messengers, inositol-1,4,5-trisphosphate and diacylglycerol from phosphatidylinositol 4,5-bisphosphate. Currently, thirteen mammal PLC isozymes have been identified, and they are divided into six groups: PLC-beta, -gamma, -delta, -epsilon, -zeta and -eta. Sequence analysis studies demonstrated that each isozyme has more than one alternative splicing variant. PLC isozymes contain the X and Y domains that are responsible for catalytic activity. Several other domains including the PH domain, the C2 domain and EF hand motifs are involved in various biological functions of PLC isozymes as signaling proteins. The distribution of PLC isozymes is tissue and organ specific. Recent studies on isolated cells and knockout mice depleted of PLC isozymes have revealed their distinct phenotypes. Given the specificity in distribution and cellular localization, it is clear that each PLC isozyme bears a unique function in the modulation of physiological responses. In this review, we discuss the structural organization, enzymatic properties and molecular diversity of PLC splicing variants and study functional and physiological roles of each isozyme.

COMMUNICATION: Alternative splicing and genomic stability

NASA Astrophysics Data System (ADS)

Cahill, Kevin

2004-06-01

Alternative splicing allows an organism to make different proteins in different cells at different times, all from the same gene. In a cell that uses alternative splicing, the total length of all the exons is much shorter than in a cell that encodes the same set of proteins without alternative splicing. This economical use of exons makes genes more stable during reproduction and development because a genome with a shorter exon length is more resistant to harmful mutations. Genomic stability may be the reason why higher vertebrates splice alternatively. For a broad class of alternatively spliced genes, a formula is given for the increase in their stability.

Mechanisms of alternative splicing regulation: insights from molecular and genomics approaches

PubMed Central

Chen, Mo; Manley, James L.

2010-01-01

Alternative splicing of mRNA precursors provides an important means of genetic control and is a crucial step in the expression of most genes. Alternative splicing markedly affects human development, and its misregulation underlies many human diseases. Although the mechanisms of alternative splicing have been studied extensively, until the past few years we had not begun to realize fully the diversity and complexity of alternative splicing regulation by an intricate protein–RNA network. Great progress has been made by studying individual transcripts and through genome-wide approaches, which together provide a better picture of the mechanistic regulation of alternative pre-mRNA splicing. PMID:19773805

Characterization of a rare variant (c.2635-2A>G) of the MSH2 gene in a family with Lynch syndrome.

PubMed

Cariola, Filomena; Disciglio, Vittoria; Valentini, Anna M; Lotesoriere, Claudio; Fasano, Candida; Forte, Giovanna; Russo, Luciana; Di Carlo, Antonio; Guglielmi, Floranna; Manghisi, Andrea; Lolli, Ivan; Caruso, Maria L; Simone, Cristiano

2018-04-01

Lynch syndrome is caused by germline mutations in one of the mismatch repair genes ( MLH1, MSH2, MSH6, and PMS2) or in the EPCAM gene. Lynch syndrome is defined on the basis of clinical, pathological, and genetic findings. Accordingly, the identification of predisposing genes allows for accurate risk assessment and tailored screening protocols. Here, we report a family case with three family members manifesting the Lynch syndrome phenotype, all of which harbor the rare variant c.2635-2A>G affecting the splice site consensus sequence of intron 15 of the MSH2 gene. This mutation was previously described only in one family with Lynch syndrome, in which mismatch repair protein expression in tumor tissues was not assessed. In this study, we report for the first time the molecular characterization of the MSH2 c.2635-2A>G variant through in silico prediction analysis, microsatellite instability, and mismatch repair protein expression experiments on tumor tissues of Lynch syndrome patients. The potential effect of the splice site variant was revealed by three splicing prediction bioinformatics tools, which suggested the generation of a new cryptic splicing site. The potential pathogenic role of this variant was also revealed by the presence of microsatellite instability and the absence of MSH2/MSH6 heterodimer protein expression in the tumor cells of cancer tissues of the affected family members. We provide compelling evidence in favor of the pathogenic role of the MSH2 variant c.2635-2A>G, which could induce an alteration of the canonical splice site and consequently an aberrant form of the protein product (MSH2).

Exome Sequencing Identified a Splice Site Mutation in FHL1 that Causes Uruguay Syndrome, an X-Linked Disorder With Skeletal Muscle Hypertrophy and Premature Cardiac Death.

PubMed

Xue, Yuan; Schoser, Benedikt; Rao, Aliz R; Quadrelli, Roberto; Vaglio, Alicia; Rupp, Verena; Beichler, Christine; Nelson, Stanley F; Schapacher-Tilp, Gudrun; Windpassinger, Christian; Wilcox, William R

2016-04-01

Previously, we reported a rare X-linked disorder, Uruguay syndrome in a single family. The main features are pugilistic facies, skeletal deformities, and muscular hypertrophy despite a lack of exercise and cardiac ventricular hypertrophy leading to premature death. An ≈19 Mb critical region on X chromosome was identified through identity-by-descent analysis of 3 affected males. Exome sequencing was conducted on one affected male to identify the disease-causing gene and variant. A splice site variant (c.502-2A>G) in the FHL1 gene was highly suspicious among other candidate genes and variants. FHL1A is the predominant isoform of FHL1 in cardiac and skeletal muscle. Sequencing cDNA showed the splice site variant led to skipping of exons 6 of the FHL1A isoform, equivalent to the FHL1C isoform. Targeted analysis showed that this splice site variant cosegregated with disease in the family. Western blot and immunohistochemical analysis of muscle from the proband showed a significant decrease in protein expression of FHL1A. Real-time polymerase chain reaction analysis of different isoforms of FHL1 demonstrated that the FHL1C is markedly increased. Mutations in the FHL1 gene have been reported in disorders with skeletal and cardiac myopathy but none has the skeletal or facial phenotype seen in patients with Uruguay syndrome. Our data suggest that a novel FHL1 splice site variant results in the absence of FHL1A and the abundance of FHL1C, which may contribute to the complex and severe phenotype. Mutation screening of the FHL1 gene should be considered for patients with uncharacterized myopathies and cardiomyopathies. © 2016 American Heart Association, Inc.

FZD4S, a splicing variant of frizzled-4, encodes a soluble-type positive regulator of the WNT signaling pathway.

PubMed

Sagara, N; Kirikoshi, H; Terasaki, H; Yasuhiko, Y; Toda, G; Shiokawa, K; Katoh, M

2001-04-06

Frizzled-1 (FZD1)-FZD10 are seven-transmembrane-type WNT receptors, and SFRP1-SFRP5 are soluble-type WNT antagonists. These molecules are encoded by mutually distinct genes. We have previously isolated and characterized the 7.7-kb FZD4 mRNA, encoding a seven-transmembrane receptor with the extracellular cysteine-rich domain (CRD). Here, we have cloned and characterized FZD4S, a splicing variant of the FZD4 gene. FZD4S, corresponding to the 10.0-kb FZD4 mRNA, consisted of exon 1, intron 1, and exon 2 of the FZD4 gene. FZD4S encoded a soluble-type polypeptide with the N-terminal part of CRD, and was expressed in human fetal kidney. Injection of synthetic FZD4S mRNA into the ventral marginal zone of Xenopus embryos at the 4-cell stage did not induce axis duplication by itself, but augmented the axis duplication potential of coinjected Xwnt-8 mRNA. These results indicate that the FZD4 gene gives rise to soluble-type FZD4S as well as seven-transmembrane-type FZD4 due to alternative splicing, and strongly suggest that FZD4S plays a role as a positive regulator of the WNT signaling pathway. Copyright 2001 Academic Press.

Alternative splicing of inner-ear-expressed genes.

PubMed

Wang, Yanfei; Liu, Yueyue; Nie, Hongyun; Ma, Xin; Xu, Zhigang

2016-09-01

Alternative splicing plays a fundamental role in the development and physiological function of the inner ear. Inner-ear-specific gene splicing is necessary to establish the identity and maintain the function of the inner ear. For example, exon 68 of Cadherin 23 (Cdh23) gene is subject to inner-ear-specific alternative splicing, and as a result, Cdh23(+ 68) is only expressed in inner ear hair cells. Alternative splicing along the tonotopic axis of the cochlea contributes to frequency tuning, particularly in lower vertebrates, such as chickens and turtles. Differential splicing of Kcnma1, which encodes for the α subunit of the Ca(2+)-activated K(+) channel (BK channel), has been suggested to affect the channel gating properties and is important for frequency tuning. Consequently, deficits in alternative splicing have been shown to cause hearing loss, as we can observe in Bronx Waltzer (bv) mice and Sfswap mutant mice. Despite the advances in this field, the regulation of alternative splicing in the inner ear remains elusive. Further investigation is also needed to clarify the mechanism of hearing loss caused by alternative splicing deficits.

High-throughput alternative splicing detection using dually constrained correspondence analysis (DCCA).

PubMed

Baty, Florent; Klingbiel, Dirk; Zappa, Francesco; Brutsche, Martin

2015-12-01

Alternative splicing is an important component of tumorigenesis. Recent advent of exon array technology enables the detection of alternative splicing at a genome-wide scale. The analysis of high-throughput alternative splicing is not yet standard and methodological developments are still needed. We propose a novel statistical approach-Dually Constrained Correspondence Analysis-for the detection of splicing changes in exon array data. Using this methodology, we investigated the genome-wide alteration of alternative splicing in patients with non-small cell lung cancer treated by bevacizumab/erlotinib. Splicing candidates reveal a series of genes related to carcinogenesis (SFTPB), cell adhesion (STAB2, PCDH15, HABP2), tumor aggressiveness (ARNTL2), apoptosis, proliferation and differentiation (PDE4D, FLT3, IL1R2), cell invasion (ETV1), as well as tumor growth (OLFM4, FGF14), tumor necrosis (AFF3) or tumor suppression (TUSC3, CSMD1, RHOBTB2, SERPINB5), with indication of known alternative splicing in a majority of genes. DCCA facilitates the identification of putative biologically relevant alternative splicing events in high-throughput exon array data. Copyright © 2015 Elsevier Inc. All rights reserved.

The influence of Argonaute proteins on alternative RNA splicing.

PubMed

Batsché, Eric; Ameyar-Zazoua, Maya

2015-01-01

Alternative splicing of precursor RNAs is an important process in multicellular species because it impacts several aspects of gene expression: from the increase of protein repertoire to the level of expression. A large body of evidences demonstrates that factors regulating chromatin and transcription impact the outcomes of alternative splicing. Argonaute (AGO) proteins were known to play key roles in the regulation of gene expression at the post-transcriptional level. More recently, their role in the nucleus of human somatic cells has emerged. Here, we will discuss some of the nuclear functions of AGO, with special emphasis on alternative splicing. The AGO-mediated modulation of alternative splicing is based on several properties of these proteins: their binding to transcripts on chromatin and their interactions with many proteins, especially histone tail-modifying enzymes, HP1γ and splicing factors. AGO proteins may favor a decrease in the RNA-polymerase II kinetics at actively transcribed genes leading to the modulation of alternative splicing decisions. They could also influence alternative splicing through their interaction with core components of the splicing machinery and several splicing factors. We will discuss the modes of AGO recruitment on chromatin at active genes. We suggest that long intragenic antisense transcripts (lincRNA) might be an important feature of genes containing splicing events regulated by AGO. © 2014 John Wiley & Sons, Ltd.

Unprecedented multiplicity of Ig transmembrane and secretory mRNA forms in the cartilaginous fish.

PubMed

Rumfelt, Lynn L; Diaz, Marilyn; Lohr, Rebecca L; Mochon, Evonne; Flajnik, Martin F

2004-07-15

In most jawed vertebrates including cartilaginous fish, membrane-bound IgM is expressed as a five Ig superfamily (Igsf)-domain H chain attached to a transmembrane (Tm) region. Heretofore, bony fish IgM was the one exception with IgM mRNA spliced to produce a four-domain Tm H chain. We now demonstrate that the Tm and secretory (Sec) mRNAs of the novel cartilaginous fish Ig isotypes, IgW and IgNAR, are present in multiple forms, most likely generated by alternative splicing. In the nurse shark, Ginglymostoma cirratum, and horn shark, Heterodontus francisci, alternative splicing of Tm exons to the second or the fourth constant (C(H)) exons produces two distinct IgW Tm cDNAs. Although the seven-domain IgW Sec cDNA form contains a canonical secretory tail shared with IgM, IgNAR, and IgA, we report a three-domain cDNA form of shark IgW (IgW(short)) having an unusual Sec tail, which is orthologous to skate IgX(short) cDNA. The IgW and IgW(short) Sec transcripts are restricted in their tissue distribution and expression levels vary among individual sharks, with all forms expressed early in ontogeny. IgNAR mRNA is alternatively spliced to produce a truncated four-domain Tm cDNA and a second Tm cDNA is expressed identical in Igsf domains as the Sec form. PBL is enriched in the Tm cDNA of these Igs. These molecular data suggest that cartilaginous fish have augmented their humoral immune repertoire by diversifying the sizes of their Ig isotypes. Furthermore, these Tm cDNAs are prototypical and the truncated variants may translate as more stable protein at the cell surface.

Analysis of cellulose synthase genes from domesticated apple identifies collinear genes WDR53 and CesA8A: partial co-expression, bicistronic mRNA, and alternative splicing of CESA8A

PubMed Central

Guerriero, Gea; Spadiut, Oliver; Kerschbamer, Christine; Giorno, Filomena; Baric, Sanja; Ezcurra, Inés

2016-01-01

Cellulose synthase (CesA) genes constitute a complex multigene family with six major phylogenetic clades in angiosperms. The recently sequenced genome of domestic apple, Malus×domestica, was mined for CesA genes, by blasting full-length cellulose synthase protein (CESA) sequences annotated in the apple genome against protein databases from the plant models Arabidopsis thaliana and Populus trichocarpa. Thirteen genes belonging to the six angiosperm CesA clades and coding for proteins with conserved residues typical of processive glycosyltransferases from family 2 were detected. Based on their phylogenetic relationship to Arabidopsis CESAs, as well as expression patterns, a nomenclature is proposed to facilitate further studies. Examination of their genomic organization revealed that MdCesA8-A is closely linked and co-oriented with WDR53, a gene coding for a WD40 repeat protein. The WDR53 and CesA8 genes display conserved collinearity in dicots and are partially co-expressed in the apple xylem. Interestingly, the presence of a bicistronic WDR53–CesA8A transcript was detected in phytoplasma-infected phloem tissues of apple. The bicistronic transcript contains a spliced intergenic sequence that is predicted to fold into hairpin structures typical of internal ribosome entry sites, suggesting its potential cap-independent translation. Surprisingly, the CesA8A cistron is alternatively spliced and lacks the zinc-binding domain. The possible roles of WDR53 and the alternatively spliced CESA8 variant during cellulose biosynthesis in M.×domestica are discussed. PMID:23048131

Prognostic alternative mRNA splicing signature in non-small cell lung cancer.

PubMed

Li, Yuan; Sun, Nan; Lu, Zhiliang; Sun, Shouguo; Huang, Jianbing; Chen, Zhaoli; He, Jie

2017-05-01

Alternative splicing provides a major mechanism to generate protein diversity. Increasing evidence suggests a link of dysregulation of splicing associated with cancer. Genome-wide alternative splicing profiling in lung cancer remains largely unstudied. We generated alternative splicing profiles in 491 lung adenocarcinoma (LUAD) and 471 lung squamous cell carcinoma (LUSC) patients in TCGA using RNA-seq data, prognostic models and splicing networks were built by integrated bioinformatics analysis. A total of 3691 and 2403 alternative splicing events were significantly associated with patient survival in LUAD and LUSC, respectively, including EGFR, CD44, PIK3C3, RRAS2, MAPKAP1 and FGFR2. The area under the curve of the receiver-operator characteristic curve for prognostic predictor in NSCLC was 0.817 at 2000 days of overall survival which were also over 0.8 in LUAD and LUSC, separately. Interestingly, splicing correlation networks uncovered opposite roles of splicing factors in LUAD and LUSC. We created prognostic predictors based on alternative splicing events with high performances for risk stratification in NSCLC patients and uncovered interesting splicing networks in LUAD and LUSC which could be underlying mechanisms. Copyright © 2017 Elsevier B.V. All rights reserved.

Global regulation of alternative RNA splicing by the SR-rich protein RBM39.

PubMed

Mai, Sanyue; Qu, Xiuhua; Li, Ping; Ma, Qingjun; Cao, Cheng; Liu, Xuan

2016-08-01

RBM39 is a serine/arginine-rich RNA-binding protein that is highly homologous to the splicing factor U2AF65. However, the role of RBM39 in alternative splicing is poorly understood. In this study, RBM39-mediated global alternative splicing was investigated using RNA-Seq and genome-wide RBM39-RNA interactions were mapped via cross-linking and immunoprecipitation coupled with deep sequencing (CLIP-Seq) in wild-type and RBM39-knockdown MCF-7 cells. RBM39 was involved in the up- or down-regulation of the transcript levels of various genes. Hundreds of alternative splicing events regulated by endogenous RBM39 were identified. The majority of these events were cassette exons. Genes containing RBM39-regulated alternative exons were found to be linked to G2/M transition, cellular response to DNA damage, adherens junctions and endocytosis. CLIP-Seq analysis showed that the binding site of RBM39 was mainly in proximity to 5' and 3' splicing sites. Considerable RBM39 binding to mRNAs encoding proteins involved in translation was observed. Of particular importance, ~20% of the alternative splicing events that were significantly regulated by RBM39 were similarly regulated by U2AF65. RBM39 is extensively involved in alternative splicing of RNA and helps regulate transcript levels. RBM39 may modulate alternative splicing similarly to U2AF65 by either directly binding to RNA or recruiting other splicing factors, such as U2AF65. The current study offers a genome-wide view of RBM39's regulatory function in alternative splicing. RBM39 may play important roles in multiple cellular processes by regulating both alternative splicing of RNA molecules and transcript levels. Copyright © 2016 Elsevier B.V. All rights reserved.

MutPred Splice: machine learning-based prediction of exonic variants that disrupt splicing

PubMed Central

2014-01-01

We have developed a novel machine-learning approach, MutPred Splice, for the identification of coding region substitutions that disrupt pre-mRNA splicing. Applying MutPred Splice to human disease-causing exonic mutations suggests that 16% of mutations causing inherited disease and 10 to 14% of somatic mutations in cancer may disrupt pre-mRNA splicing. For inherited disease, the main mechanism responsible for the splicing defect is splice site loss, whereas for cancer the predominant mechanism of splicing disruption is predicted to be exon skipping via loss of exonic splicing enhancers or gain of exonic splicing silencer elements. MutPred Splice is available at http://mutdb.org/mutpredsplice. PMID:24451234

JMJD6 and U2AF65 co-regulate alternative splicing in both JMJD6 enzymatic activity dependent and independent manner

PubMed Central

Yi, Jia; Shen, Hai-Feng; Qiu, Jin-Song; Huang, Ming-Feng; Zhang, Wen-Juan; Ding, Jian-Cheng; Zhu, Xiao-Yan; Zhou, Yu

2017-01-01

Abstract JMJD6, a jumonji C (Jmj C) domain-containing protein demethylase and hydroxylase, has been implicated in an array of biological processes. It has been shown that JMJD6 interacts with and hydroxylates multiple serine/arginine-rich (SR) proteins and SR related proteins, including U2AF65, all of which are known to function in alternative splicing regulation. However, whether JMJD6 is widely involved in alternative splicing and the molecular mechanism underlying JMJD6-regulated alternative splicing have remained incompletely understood. Here, by using RASL-Seq, we investigated the functional impact of RNA-dependent interaction between JMJD6 and U2AF65, revealing that JMJD6 and U2AF65 co-regulated a large number of alternative splicing events. We further demonstrated the JMJD6 function in alternative splicing in jmjd6 knockout mice. Mechanistically, we showed that the enzymatic activity of JMJD6 was required for a subset of JMJD6-regulated splicing, and JMJD6-mediated lysine hydroxylation of U2AF65 could account for, at least partially, their co-regulated alternative splicing events, suggesting both JMJD6 enzymatic activity-dependent and independent control of alternative splicing. These findings reveal an intimate link between JMJD6 and U2AF65 in alternative splicing regulation, which has important implications in development and disease processes. PMID:27899633

Pre-mRNA mis-splicing of sarcomeric genes in heart failure.

PubMed

Zhu, Chaoqun; Chen, Zhilong; Guo, Wei

2017-08-01

Pre-mRNA splicing is an important biological process that allows production of multiple proteins from a single gene in the genome, and mainly contributes to protein diversity in eukaryotic organisms. Alternative splicing is commonly governed by RNA binding proteins to meet the ever-changing demands of the cell. However, the mis-splicing may lead to human diseases. In the heart of human, mis-regulation of alternative splicing has been associated with heart failure. In this short review, we focus on alternative splicing of sarcomeric genes and review mis-splicing related heart failure with relatively well studied Sarcomeric genes and splicing mechanisms with identified regulatory factors. The perspective of alternative splicing based therapeutic strategies in heart failure has also been discussed. Copyright © 2016 Elsevier B.V. All rights reserved.

Effects of breed, parity, and folic Acid supplement on the expression of folate metabolism genes in endometrial and embryonic tissues from sows in early pregnancy.

PubMed

Vallée, Maud; Guay, Frédéric; Beaudry, Danièle; Matte, Jacques; Blouin, Richard; Laforest, Jean-Paul; Lessard, Martin; Palin, Marie-France

2002-10-01

Folic acid and glycine are factors of great importance in early gestation. In sows, folic acid supplement can increase litter size through a decrease in embryonic mortality, while glycine, the most abundant amino acid in the sow oviduct, uterine, and allantoic fluids, is reported to act as an organic osmoregulator. In this study, we report the characterization of cytoplasmic serine hydroxymethyltransferase (cSHMT), T-protein, and vT-protein (variant T-protein) mRNA expression levels in endometrial and embryonic tissues in gestating sows on Day 25 of gestation according to the breed, parity, and folic acid + glycine supplementation. Expression levels of cSHMT, T-protein, and vT-protein mRNA in endometrial and embryonic tissues were performed using semiquantitative reverse transcription-polymerase chain reaction. We also report, for the first time, an alternative splicing event in the porcine T-protein gene. Results showed that a T-protein splice variant, vT-protein, is present in all the tested sow populations. Further characterizations revealed that this T-protein splice variant contains a coding intron that can adopt a secondary structure. Results demonstrated that cSHMT mRNA expression levels were significantly higher in sows receiving the folic acid + glycine supplementation, independently of the breed or parity and in both endometrial and embryonic tissues. Upon receiving the same treatment, the vT-protein and T-protein mRNA expression levels were significantly reduced in the endometrial tissue of Yorkshire-Landrace sows only. These results indicate that modulation of specific gene expression levels in endometrial and embryonic tissues of sows in early gestation could be one of the mechanism involved with the role of folic acid on improving swine reproduction traits.

Identification and characterization of a human smad3 splicing variant lacking part of the linker region.

PubMed

Kjellman, Christian; Honeth, Gabriella; Järnum, Sofia; Lindvall, Magnus; Darabi, Anna; Nilsson, Ingar; Edvardsen, Klaus; Salford, Leif G; Widegren, Bengt

2004-03-03

Smad3 is one of the signal transducers that are activated in response to transforming growth factor-beta (TGF-beta). We have identified and characterized a splicing variant of smad3. The splicing variant (smad3-Delta3) lacks exon 3 resulting in a truncated linker region. We could detect mRNA expression of smad3-Delta3 in all investigated human tissues. Real-time PCR analyses demonstrated that the fraction of smad3-Delta3 mRNA compared to normal smad3 varies between tissues. The amount of spliced mRNA was estimated to represent 0.5-5% of the normal smad3 mRNA. When smad3-Delta3 is overexpressed in a fibrosarcoma cell line, the Smad3-Delta3 is translocated to the nucleus upon TGF-beta stimulation and binds the Smad responsive element. Using a CAGA luciferase reporter system, we demonstrate that Smad3-Delta3 has transcriptional activity and we conclude that Smad3-Delta3 possesses functional transactivating properties.

Alternative splicing and trans-splicing events revealed by analysis of the Bombyx mori transcriptome

PubMed Central

Shao, Wei; Zhao, Qiong-Yi; Wang, Xiu-Ye; Xu, Xin-Yan; Tang, Qing; Li, Muwang; Li, Xuan; Xu, Yong-Zhen

2012-01-01

Alternative splicing and trans-splicing events have not been systematically studied in the silkworm Bombyx mori. Here, the silkworm transcriptome was analyzed by RNA-seq. We identified 320 novel genes, modified 1140 gene models, and found thousands of alternative splicing and 58 trans-splicing events. Studies of three SR proteins show that both their alternative splicing patterns and mRNA products are conserved from insect to human, and one isoform of Srsf6 with a retained intron is expressed sex-specifically in silkworm gonads. Trans-splicing of mod(mdg4) in silkworm was experimentally confirmed. We identified integrations from a common 5′-gene with 46 newly identified alternative 3′-exons that are located on both DNA strands over a 500-kb region. Other trans-splicing events in B. mori were predicted by bioinformatic analysis, in which 12 events were confirmed by RT-PCR, six events were further validated by chimeric SNPs, and two events were confirmed by allele-specific RT-PCR in F1 hybrids from distinct silkworm lines of JS and L10, indicating that trans-splicing is more widespread in insects than previously thought. Analysis of the B. mori transcriptome by RNA-seq provides valuable information of regulatory alternative splicing events. The conservation of splicing events across species and newly identified trans-splicing events suggest that B. mori is a good model for future studies. PMID:22627775

Pharmacological profile of brain-derived neurotrophic factor (BDNF) splice variant translation using a novel drug screening assay: a "quantitative code".

PubMed

Vaghi, Valentina; Polacchini, Alessio; Baj, Gabriele; Pinheiro, Vera L M; Vicario, Annalisa; Tongiorgi, Enrico

2014-10-03

The neurotrophin brain-derived neurotrophic factor (BDNF) is a key regulator of neuronal development and plasticity. BDNF is a major pharmaceutical target in neurodevelopmental and psychiatric disorders. However, pharmacological modulation of this neurotrophin is challenging because BDNF is generated by multiple, alternatively spliced transcripts with different 5'- and 3'UTRs. Each BDNF mRNA variant is transcribed independently, but translation regulation is unknown. To evaluate the translatability of BDNF transcripts, we developed an in vitro luciferase assay in human neuroblastoma cells. In unstimulated cells, each BDNF 5'- and 3'UTR determined a different basal translation level of the luciferase reporter gene. However, constructs with either a 5'UTR or a 3'UTR alone showed poor translation modulation by BDNF, KCl, dihydroxyphenylglycine, AMPA, NMDA, dopamine, acetylcholine, norepinephrine, or serotonin. Constructs consisting of the luciferase reporter gene flanked by the 5'UTR of one of the most abundant BDNF transcripts in the brain (exons 1, 2c, 4, and 6) and the long 3'UTR responded selectively to stimulation with the different receptor agonists, and only transcripts 2c and 6 were increased by the antidepressants desipramine and mirtazapine. We propose that BDNF mRNA variants represent "a quantitative code" for regulated expression of the protein. Thus, to discriminate the efficacy of drugs in stimulating BDNF synthesis, it is appropriate to use variant-specific in vitro screening tests. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Alternative Splicing in CaV2.2 Regulates Neuronal Trafficking via Adaptor Protein Complex-1 Adaptor Protein Motifs

PubMed Central

Macabuag, Natsuko

2015-01-01

N-type voltage-gated calcium (CaV2.2) channels are expressed in neurons and targeted to the plasma membrane of presynaptic terminals, facilitating neurotransmitter release. Here, we find that the adaptor protein complex-1 (AP-1) mediates trafficking of CaV2.2 from the trans-Golgi network to the cell surface. Examination of splice variants of CaV2.2, containing either exon 37a (selectively expressed in nociceptors) or 37b in the proximal C terminus, reveal that canonical AP-1 binding motifs, YxxΦ and [DE]xxxL[LI], present only in exon 37a, enhance intracellular trafficking of exon 37a-containing CaV2.2 to the axons and plasma membrane of rat DRG neurons. Finally, we identify differential effects of dopamine-2 receptor (D2R) and its agonist-induced activation on trafficking of CaV2.2 isoforms. D2R slowed the endocytosis of CaV2.2 containing exon 37b, but not exon 37a, and activation by the agonist quinpirole reversed the effect of the D2R. Our work thus reveals key mechanisms involved in the trafficking of N-type calcium channels. SIGNIFICANCE STATEMENT CaV2.2 channels are important for neurotransmitter release, but how they are trafficked is still poorly understood. Here, we describe a novel mechanism for trafficking of CaV2.2 from the trans-Golgi network to the cell surface which is mediated by the adaptor protein AP-1. Alternative splicing of exon 37 produces CaV2.2-exon 37a, selectively expressed in nociceptors, or CaV2.2-exon 37b, which is the major splice isoform. Our study reveals that canonical AP-1 binding motifs (YxxΦ and [DE]xxxL[LI]), present in exon 37a, but not 37b, enhance intracellular trafficking of exon 37a-containing CaV2.2 to axons and plasma membrane of DRG neurons. Interaction of APs with CaV2.2 channels may also be key underlying mechanisms for differential effects of the dopamine D2 receptor on trafficking of CaV2.2 splice variants. PMID:26511252

Regulation of Alternative Splicing in Vivo by Overexpression of Antagonistic Splicing Factors

NASA Astrophysics Data System (ADS)

Caceres, Javier F.; Stamm, Stefan; Helfman, David M.; Krainer, Adrian R.

1994-09-01

The opposing effects of SF2/ASF and heterogeneous nuclear ribonucleoprotein (hnRNP) A1 influence alternative splicing in vitro. SF2/ASF or hnRNP A1 complementary DNAs were transiently overexpressed in HeLa cells, and the effect on alternative splicing of several cotransfected reporter genes was measured. Increased expression of SF2/ASF activated proximal 5' splice sites, promoted inclusion of a neuron-specific exon, and prevented abnormal exon skipping. Increased expression of hnRNP A1 activated distal 5' splice sites. Therefore, variations in the intracellular levels of antagonistic splicing factors influence different modes of alternative splicing in vivo and may be a natural mechanism for tissue-specific or developmental regulation of gene expression.

An EMT–Driven Alternative Splicing Program Occurs in Human Breast Cancer and Modulates Cellular Phenotype

PubMed Central

Flytzanis, Nicholas C.; Balsamo, Michele; Condeelis, John S.; Oktay, Maja H.; Burge, Christopher B.; Gertler, Frank B.

2011-01-01

Epithelial-mesenchymal transition (EMT), a mechanism important for embryonic development, plays a critical role during malignant transformation. While much is known about transcriptional regulation of EMT, alternative splicing of several genes has also been correlated with EMT progression, but the extent of splicing changes and their contributions to the morphological conversion accompanying EMT have not been investigated comprehensively. Using an established cell culture model and RNA–Seq analyses, we determined an alternative splicing signature for EMT. Genes encoding key drivers of EMT–dependent changes in cell phenotype, such as actin cytoskeleton remodeling, regulation of cell–cell junction formation, and regulation of cell migration, were enriched among EMT–associated alternatively splicing events. Our analysis suggested that most EMT–associated alternative splicing events are regulated by one or more members of the RBFOX, MBNL, CELF, hnRNP, or ESRP classes of splicing factors. The EMT alternative splicing signature was confirmed in human breast cancer cell lines, which could be classified into basal and luminal subtypes based exclusively on their EMT–associated splicing pattern. Expression of EMT–associated alternative mRNA transcripts was also observed in primary breast cancer samples, indicating that EMT–dependent splicing changes occur commonly in human tumors. The functional significance of EMT–associated alternative splicing was tested by expression of the epithelial-specific splicing factor ESRP1 or by depletion of RBFOX2 in mesenchymal cells, both of which elicited significant changes in cell morphology and motility towards an epithelial phenotype, suggesting that splicing regulation alone can drive critical aspects of EMT–associated phenotypic changes. The molecular description obtained here may aid in the development of new diagnostic and prognostic markers for analysis of breast cancer progression. PMID:21876675

Alternative RNA splicing and cancer

PubMed Central

Liu, Sali; Cheng, Chonghui

2015-01-01

Alternative splicing of pre-messenger RNA (mRNA) is a fundamental mechanism by which a gene can give rise to multiple distinct mRNA transcripts, yielding protein isoforms with different, even opposing, functions. With the recognition that alternative splicing occurs in nearly all human genes, its relationship with cancer-associated pathways has emerged as a rapidly growing field. In this review, we summarize recent findings that have implicated the critical role of alternative splicing in cancer and discuss current understandings of the mechanisms underlying dysregulated alternative splicing in cancer cells. PMID:23765697

Connecting the dots: chromatin and alternative splicing in EMT.

PubMed

Warns, Jessica A; Davie, James R; Dhasarathy, Archana

2016-02-01

Nature has devised sophisticated cellular machinery to process mRNA transcripts produced by RNA Polymerase II, removing intronic regions and connecting exons together, to produce mature RNAs. This process, known as splicing, is very closely linked to transcription. Alternative splicing, or the ability to produce different combinations of exons that are spliced together from the same genomic template, is a fundamental means of regulating protein complexity. Similar to transcription, both constitutive and alternative splicing can be regulated by chromatin and its associated factors in response to various signal transduction pathways activated by external stimuli. This regulation can vary between different cell types, and interference with these pathways can lead to changes in splicing, often resulting in aberrant cellular states and disease. The epithelial to mesenchymal transition (EMT), which leads to cancer metastasis, is influenced by alternative splicing events of chromatin remodelers and epigenetic factors such as DNA methylation and non-coding RNAs. In this review, we will discuss the role of epigenetic factors including chromatin, chromatin remodelers, DNA methyltransferases, and microRNAs in the context of alternative splicing, and discuss their potential involvement in alternative splicing during the EMT process.

Lariat sequencing in a unicellular yeast identifies regulated alternative splicing of exons that are evolutionarily conserved with humans.

PubMed

Awan, Ali R; Manfredo, Amanda; Pleiss, Jeffrey A

2013-07-30

Alternative splicing is a potent regulator of gene expression that vastly increases proteomic diversity in multicellular eukaryotes and is associated with organismal complexity. Although alternative splicing is widespread in vertebrates, little is known about the evolutionary origins of this process, in part because of the absence of phylogenetically conserved events that cross major eukaryotic clades. Here we describe a lariat-sequencing approach, which offers high sensitivity for detecting splicing events, and its application to the unicellular fungus, Schizosaccharomyces pombe, an organism that shares many of the hallmarks of alternative splicing in mammalian systems but for which no previous examples of exon-skipping had been demonstrated. Over 200 previously unannotated splicing events were identified, including examples of regulated alternative splicing. Remarkably, an evolutionary analysis of four of the exons identified here as subject to skipping in S. pombe reveals high sequence conservation and perfect length conservation with their homologs in scores of plants, animals, and fungi. Moreover, alternative splicing of two of these exons have been documented in multiple vertebrate organisms, making these the first demonstrations of identical alternative-splicing patterns in species that are separated by over 1 billion y of evolution.

An alternative splicing program promotes adipose tissue thermogenesis

PubMed Central

Vernia, Santiago; Edwards, Yvonne JK; Han, Myoung Sook; Cavanagh-Kyros, Julie; Barrett, Tamera; Kim, Jason K; Davis, Roger J

2016-01-01

Alternative pre-mRNA splicing expands the complexity of the transcriptome and controls isoform-specific gene expression. Whether alternative splicing contributes to metabolic regulation is largely unknown. Here we investigated the contribution of alternative splicing to the development of diet-induced obesity. We found that obesity-induced changes in adipocyte gene expression include alternative pre-mRNA splicing. Bioinformatics analysis associated part of this alternative splicing program with sequence specific NOVA splicing factors. This conclusion was confirmed by studies of mice with NOVA deficiency in adipocytes. Phenotypic analysis of the NOVA-deficient mice demonstrated increased adipose tissue thermogenesis and improved glycemia. We show that NOVA proteins mediate a splicing program that suppresses adipose tissue thermogenesis. Together, these data provide quantitative analysis of gene expression at exon-level resolution in obesity and identify a novel mechanism that contributes to the regulation of adipose tissue function and the maintenance of normal glycemia. DOI: http://dx.doi.org/10.7554/eLife.17672.001 PMID:27635635

Integrative Analysis of Many RNA-Seq Datasets to Study Alternative Splicing

PubMed Central

Li, Wenyuan; Dai, Chao; Kang, Shuli; Zhou, Xianghong Jasmine

2014-01-01

Alternative splicing is an important gene regulatory mechanism that dramatically increases the complexity of the proteome. However, how alternative splicing is regulated and how transcription and splicing are coordinated are still poorly understood, and functions of transcript isoforms have been studied only in a few limited cases. Nowadays, RNA-seq technology provides an exceptional opportunity to study alternative splicing on genome-wide scales and in an unbiased manner. With the rapid accumulation of data in public repositories, new challenges arise from the urgent need to effectively integrate many different RNA-seq datasets for study alterative splicing. This paper discusses a set of advanced computational methods that can integrate and analyze many RNA-seq datasets to systematically identify splicing modules, unravel the coupling of transcription and splicing, and predict the functions of splicing isoforms on a genome-wide scale. PMID:24583115

Transcriptome analysis using next generation sequencing reveals molecular signatures of diabetic retinopathy and efficacy of candidate drugs.

PubMed

Kandpal, Raj P; Rajasimha, Harsha K; Brooks, Matthew J; Nellissery, Jacob; Wan, Jun; Qian, Jiang; Kern, Timothy S; Swaroop, Anand

2012-01-01

To define gene expression changes associated with diabetic retinopathy in a mouse model using next generation sequencing, and to utilize transcriptome signatures to assess molecular pathways by which pharmacological agents inhibit diabetic retinopathy. We applied a high throughput RNA sequencing (RNA-seq) strategy using Illumina GAIIx to characterize the entire retinal transcriptome from nondiabetic and from streptozotocin-treated mice 32 weeks after induction of diabetes. Some of the diabetic mice were treated with inhibitors of receptor for advanced glycation endproducts (RAGE) and p38 mitogen activated protein (MAP) kinase, which have previously been shown to inhibit diabetic retinopathy in rodent models. The transcripts and alternatively spliced variants were determined in all experimental groups. Next generation sequencing-based RNA-seq profiles provided comprehensive signatures of transcripts that are altered in early stages of diabetic retinopathy. These transcripts encoded proteins involved in distinct yet physiologically relevant disease-associated pathways such as inflammation, microvasculature formation, apoptosis, glucose metabolism, Wnt signaling, xenobiotic metabolism, and photoreceptor biology. Significant upregulation of crystallin transcripts was observed in diabetic animals, and the diabetes-induced upregulation of these transcripts was inhibited in diabetic animals treated with inhibitors of either RAGE or p38 MAP kinase. These two therapies also showed dissimilar regulation of some subsets of transcripts that included alternatively spliced versions of arrestin, neutral sphingomyelinase activation associated factor (Nsmaf), SH3-domain GRB2-like interacting protein 1 (Sgip1), and axin. Diabetes alters many transcripts in the retina, and two therapies that inhibit the vascular pathology similarly inhibit a portion of these changes, pointing to possible molecular mechanisms for their beneficial effects. These therapies also changed the abundance of various alternatively spliced versions of signaling transcripts, suggesting a possible role of alternative splicing in disease etiology. Our studies clearly demonstrate RNA-seq as a comprehensive strategy for identifying disease-specific transcripts, and for determining comparative profiles of molecular changes mediated by candidate drugs.

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.

JMJD6 and U2AF65 co-regulate alternative splicing in both JMJD6 enzymatic activity dependent and independent manner.

PubMed

Yi, Jia; Shen, Hai-Feng; Qiu, Jin-Song; Huang, Ming-Feng; Zhang, Wen-Juan; Ding, Jian-Cheng; Zhu, Xiao-Yan; Zhou, Yu; Fu, Xiang-Dong; Liu, Wen

2017-04-07

JMJD6, a jumonji C (Jmj C) domain-containing protein demethylase and hydroxylase, has been implicated in an array of biological processes. It has been shown that JMJD6 interacts with and hydroxylates multiple serine/arginine-rich (SR) proteins and SR related proteins, including U2AF65, all of which are known to function in alternative splicing regulation. However, whether JMJD6 is widely involved in alternative splicing and the molecular mechanism underlying JMJD6-regulated alternative splicing have remained incompletely understood. Here, by using RASL-Seq, we investigated the functional impact of RNA-dependent interaction between JMJD6 and U2AF65, revealing that JMJD6 and U2AF65 co-regulated a large number of alternative splicing events. We further demonstrated the JMJD6 function in alternative splicing in jmjd6 knockout mice. Mechanistically, we showed that the enzymatic activity of JMJD6 was required for a subset of JMJD6-regulated splicing, and JMJD6-mediated lysine hydroxylation of U2AF65 could account for, at least partially, their co-regulated alternative splicing events, suggesting both JMJD6 enzymatic activity-dependent and independent control of alternative splicing. These findings reveal an intimate link between JMJD6 and U2AF65 in alternative splicing regulation, which has important implications in development and disease processes. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Novel alternative splicings of BPAG1 (bullous pemphigoid antigen 1) including the domain structure closely related to MACF (microtubule actin cross-linking factor).

PubMed

Okumura, Masayo; Yamakawa, Hisashi; Ohara, Osamu; Owaribe, Katsushi

2002-02-22

BPAG1 (bullous pemphigoid antigen 1) was originally identified as a 230-kDa hemidesmosomal protein and belongs to the plakin family, because it consists of a plakin domain, a coiled-coil rod domain and a COOH-terminal intermediate filament binding domain. To date, alternatively spliced products of BPAG1, BPAG1e, and BPAG1n are known. BPAG1e is expressed in epithelial tissues and localized to hemidesmosomes, on the other hand, BPAG1n is expressed in neural tissues and muscles and has an actin binding domain at the NH(2)-terminal of BPAG1e. BPAG1 is also known as a gene responsible for Dystonia musculorum (dt) neurodegeneration syndrome of the mouse. Another plakin family protein MACF (microtubule actin cross-linking factor) has also an actin binding domain and the plakin domain at the NH(2)-terminal. However, in contrast to its high homology with BPAG1 at the NH(2)-terminal, the COOH-terminal structure of MACF, including a microtubule binding domain, resembles dystrophin rather than plakins. Here, we investigated RNAs and proteins expressed from the BPAG1 locus and suggest novel alternative splicing variants, which include one consisting of the COOH-terminal domain structure homologous to MACF. The results indicate that BPAG1 has three kinds of cytoskeletal binding domains and seems to play an important role in linking the different types of cytoskeletons.

Alternative splicing of c-fos pre-mRNA: contribution of the rates of synthesis and degradation to the copy number of each transcript isoform and detection of a truncated c-Fos immunoreactive species.

PubMed

Jurado, Juan; Fuentes-Almagro, Carlos A; Prieto-Alamo, María J; Pueyo, Carmen

2007-09-21

Alternative splicing is a widespread mechanism of gene expression regulation. Previous analyses based on conventional RT-PCR reported the presence of an unspliced c-fos transcript in several mammalian systems. Compared to the well-defined knowledge on the alternative splicing of fosB, the physiological relevance of the unspliced c-fos transcript in regulating c-fos expression remains largely unknown. This work aimed to investigate the functional significance of the alternative splicing c-fos pre-mRNA. A set of primers was designed to demonstrate that, whereas introns 1 and 2 are regularly spliced from primary c-fos transcript, intron 3 remains unspliced in part of total transcript molecules. Here, the two species are referred to as c-fos-2 (+ intron 3) and spliced c-fos (- intron 3) transcripts. Then, we used a quantitatively rigorous approach based on real-time PCR to provide, for the first time, the actual steady-state copy numbers of the two c-fos transcripts. We tested how the mouse-organ context and mouse-gestational age, the synthesis and turnover rates of the investigated transcripts, and the serum stimulation of quiescent cells modulate their absolute-expression profiles. Intron 3 generates an in-frame premature termination codon that predicts the synthesis of a truncated c-Fos protein. This prediction was evaluated by immunoaffinity chromatography purification of c-Fos proteins. We demonstrate that: (i) The c-fos-2 transcript is ubiquitously synthesized either in vivo or in vitro, in amounts that are higher or similar to those of mRNAs coding for other Fos family members, like FosB, DeltaFosB, Fra-1 or Fra-2. (ii) Intron 3 confers to c-fos-2 an outstanding destabilizing effect of about 6-fold. (iii) Major determinant of c-fos-2 steady-state levels in cultured cells is its remarkably high rate of synthesis. (iv) Rapid changes in the synthesis and/or degradation rates of both c-fos transcripts in serum-stimulated cells give rise to rapid and transient changes in their relative proportions. Taken as a whole, these findings suggest a co-ordinated fine-tune of the two c-fos transcript species, supporting the notion that the alternative processing of the precursor mRNA might be physiologically relevant. Moreover, we detected a c-Fos immunoreactive species corresponding in mobility to the predicted truncated variant.

iCLIP Predicts the Dual Splicing Effects of TIA-RNA Interactions

PubMed Central

Briese, Michael; Zarnack, Kathi; Luscombe, Nicholas M.; Rot, Gregor; Zupan, Blaž; Curk, Tomaž; Ule, Jernej

2010-01-01

The regulation of alternative splicing involves interactions between RNA-binding proteins and pre-mRNA positions close to the splice sites. T-cell intracellular antigen 1 (TIA1) and TIA1-like 1 (TIAL1) locally enhance exon inclusion by recruiting U1 snRNP to 5′ splice sites. However, effects of TIA proteins on splicing of distal exons have not yet been explored. We used UV-crosslinking and immunoprecipitation (iCLIP) to find that TIA1 and TIAL1 bind at the same positions on human RNAs. Binding downstream of 5′ splice sites was used to predict the effects of TIA proteins in enhancing inclusion of proximal exons and silencing inclusion of distal exons. The predictions were validated in an unbiased manner using splice-junction microarrays, RT-PCR, and minigene constructs, which showed that TIA proteins maintain splicing fidelity and regulate alternative splicing by binding exclusively downstream of 5′ splice sites. Surprisingly, TIA binding at 5′ splice sites silenced distal cassette and variable-length exons without binding in proximity to the regulated alternative 3′ splice sites. Using transcriptome-wide high-resolution mapping of TIA-RNA interactions we evaluated the distal splicing effects of TIA proteins. These data are consistent with a model where TIA proteins shorten the time available for definition of an alternative exon by enhancing recognition of the preceding 5′ splice site. Thus, our findings indicate that changes in splicing kinetics could mediate the distal regulation of alternative splicing. PMID:21048981

Changes in the transcriptional profile in response to overexpression of the osteopontin-c splice isoform in ovarian (OvCar-3) and prostate (PC-3) cancer cell lines

PubMed Central

2014-01-01

Background Especially in human tumor cells, the osteopontin (OPN) primary transcript is subject to alternative splicing, generating three isoforms termed OPNa, OPNb and OPNc. We previously demonstrated that the OPNc splice variant activates several aspects of the progression of ovarian and prostate cancers. The goal of the present study was to develop cell line models to determine the impact of OPNc overexpression on main cancer signaling pathways and thus obtain insights into the mechanisms of OPNc pro-tumorigenic roles. Methods Human ovarian and prostate cancer cell lines, OvCar-3 and PC-3 cells, respectively, were stably transfected to overexpress OPNc. Transcriptomic profiling was performed on these cells and compared to controls, to identify OPNc overexpression-dependent changes in gene expression levels and pathways by qRT-PCR analyses. Results Among 84 genes tested by using a multiplex real-time PCR Cancer Pathway Array approach, 34 and 16, respectively, were differentially expressed between OvCar-3 and PC-3 OPNc-overexpressing cells in relation to control clones. Differentially expressed genes are included in all main hallmarks of cancer, and several interacting proteins have been identified using an interactome network analysis. Based on marked up-regulation of Vegfa transcript in response to OPNc overexpression, we partially validated the array data by demonstrating that conditioned medium (CM) secreted from OvCar-3 and PC-3 OPNc-overexpressing cells significantly induced endothelial cell adhesion, proliferation and migration, compared to CM secreted from control cells. Conclusions Overall, the present study elucidated transcriptional changes of OvCar-3 and PC-3 cancer cell lines in response to OPNc overexpression, which provides an assessment for predicting the molecular mechanisms by which this splice variant promotes tumor progression features. PMID:24928374

Molecular identification and functional expression of mu 3, a novel alternatively spliced variant of the human mu opiate receptor gene.

PubMed

Cadet, Patrick; Mantione, Kirk J; Stefano, George B

2003-05-15

Studies from our laboratory have revealed a novel mu opiate receptor, mu 3, which is expressed in both vascular tissues and leukocytes. The mu 3 receptor is selective for opiate alkaloids and is insensitive to opioid peptides. We now identify the mu 3 receptor at the molecular level using a 441-bp conserved region of the mu 1 receptor. Sequence analysis of the isolated cDNA suggests that it is a novel, alternatively spliced variant of the mu opiate receptor gene. To determine whether protein expressed from this cDNA exhibits the biochemical characteristics expected of the mu 3 receptor, the cDNA clone was expressed in a heterologous system. At the functional level, COS-1 cells transfected with the mu 3 receptor cDNA exhibited dose-dependent release of NO following treatment with morphine, but not opioid peptides (i.e., Met-enkephalin). Naloxone was able to block the effect of morphine on COS-1 transfected cells. Nontransfected COS-1 cells did not produce NO in the presence of morphine or the opioid peptides at similar concentrations. Receptor binding analysis with [(3)H]dihydromorphine further supports the opiate alkaloid selectivity and opioid peptide insensitivity of this receptor. These data suggest that this new mu opiate receptor cDNA encodes the mu 3 opiate receptor, since it exhibits biochemical characteristics known to be unique to this receptor (opiate alkaloid selective and opioid peptide insensitive). Furthermore, using Northern blot, RT-PCR, and sequence analysis, we have demonstrated the expression of this new mu variant in human vascular tissue, mononuclear cells, polymorphonuclear cells, and human neuroblastoma cells.

Differential agonist sensitivity of glycine receptor α2 subunit splice variants

PubMed Central

Miller, Paul S; Harvey, Robert J; Smart, Trevor G

2004-01-01

The glycine receptor (GlyR) α2A and α2B splice variants differ by a dual, adjacent amino acid substitution from α2AV58,T59 to α2BI58,A59 in the N-terminal extracellular domain. Comparing the effects of the GlyR agonists, glycine, β-alanine and taurine, on the GlyR α2 isoforms, revealed a significant increase in potency for all three agonists at the α2B variant. The sensitivities of the splice variants to the competitive antagonist, strychnine, and to the biphasic modulator Zn2+, were comparable. In contrast, the allosteric inhibitor picrotoxin was more potent on GlyR α2A compared to GlyR α2B receptors. Coexpression of α2A or α2B subunits with the GlyR β subunit revealed that the higher agonist potencies observed with the α2B homomer were retained for the α2Bβ heteromer. The identical sensitivity to strychnine combined with a reduction in the maximum current induced by the partial agonist taurine at the GlyR α2A homomer, suggested that the changed sensitivity to agonists is in accordance with a modulation of agonist efficacy rather than agonist affinity. An effect on agonist efficacy was also supported by using a structural model of the GlyR, localising the region of splice variation to the proposed docking region between GlyR loop 2 and the TM2-3 loop, an area associated with channel activation. The existence of a spasmodic mouse phenotype linked to a GlyR α1A52S mutation, the equivalent position to the source of the α2 splice variation, raises the possibility that the GlyR α2 splice variants may be responsible for distinct roles in neuronal function. PMID:15302677

Regulation of alternative splicing by the circadian clock and food related cues

PubMed Central

2012-01-01

Background The circadian clock orchestrates daily rhythms in metabolism, physiology and behaviour that allow organisms to anticipate regular changes in their environment, increasing their adaptation. Such circadian phenotypes are underpinned by daily rhythms in gene expression. Little is known, however, about the contribution of post-transcriptional processes, particularly alternative splicing. Results Using Affymetrix mouse exon-arrays, we identified exons with circadian alternative splicing in the liver. Validated circadian exons were regulated in a tissue-dependent manner and were present in genes with circadian transcript abundance. Furthermore, an analysis of circadian mutant Vipr2-/- mice revealed the existence of distinct physiological pathways controlling circadian alternative splicing and RNA binding protein expression, with contrasting dependence on Vipr2-mediated physiological signals. This view was corroborated by the analysis of the effect of fasting on circadian alternative splicing. Feeding is an important circadian stimulus, and we found that fasting both modulates hepatic circadian alternative splicing in an exon-dependent manner and changes the temporal relationship with transcript-level expression. Conclusions The circadian clock regulates alternative splicing in a manner that is both tissue-dependent and concurrent with circadian transcript abundance. This adds a novel temporal dimension to the regulation of mammalian alternative splicing. Moreover, our results demonstrate that circadian alternative splicing is regulated by the interaction between distinct physiological cues, and illustrates the capability of single genes to integrate circadian signals at different levels of regulation. PMID:22721557

Systematic Identification of Genes Required for Expression of Androgen Receptor Splice Variants

DTIC Science & Technology

2016-08-01

engineering tool has been developed from bacterial Clustered Regularly Interspaced Short Palindromic Repeats ( CRISPR )/ CRISPR ‐Associated System (Cas...regulation of AR splice variant through CRISPR /Cas screening system. 15. SUBJECT TERMS CRISPR /Cas, Androgen receptor, castration resistance, biomarker 16...control (non-targeting) gRNAs available from Addgene (http://www.addgene.org/ CRISPR /libraries/). Generation of AR3 reporter: We used molecular cloning

CYP3A5 mRNA degradation by nonsense-mediated mRNA decay.

PubMed

Busi, Florent; Cresteil, Thierry

2005-09-01

The total CYP3A5 mRNA level is significantly greater in carriers of the CYP3A5*1 allele than in CYP3A5*3 homozygotes. Most of the CYP3A5*3 mRNA includes an intronic sequence (exon 3B) containing premature termination codons (PTCs) between exons 3 and 4. Two models were used to investigate the degradation of CYP3A5 mRNA: a CYP3A5 minigene consisting of CYP3A5 exons and introns 3 to 6 transfected into MCF7 cells, and the endogenous CYP3A5 gene expressed in HepG2 cells. The 3'-untranslated region g.31611C>T mutation has no effect on CYP3A5 mRNA decay. Splice variants containing exon 3B were more unstable than wild-type (wt) CYP3A5 mRNA. Cycloheximide prevents the recognition of PTCs by ribosomes: in transfected MCF7 and HepG2 cells, cycloheximide slowed down the degradation of exon 3B-containing splice variants, suggesting the participation of nonsense-mediated decay (NMD). When PTCs were removed from pseudoexon 3B or when UPF1 small interfering RNA was used to impair the NMD mechanism, the decay of the splice variant was reduced, confirming the involvement of NMD in the degradation of CYP3A5 splice variants. Induction could represent a source of variability for CYP3A5 expression and could modify the proportion of splice variants. The extent of CYP3A5 induction was investigated after exposure to barbiturates or steroids: CYP3A4 was markedly induced in a pediatric population compared with untreated neonates. However, no effect could be detected in either the total CYP3A5 RNA, the proportion of splice variant RNA, or the protein level. Therefore, in these carriers, induction is unlikely to switch on the phenotypic CYP3A5 expression in carriers of CYP3A5*3/*3.

The Functional Impact of Alternative Splicing in Cancer.

PubMed

Climente-González, Héctor; Porta-Pardo, Eduard; Godzik, Adam; Eyras, Eduardo

2017-08-29

Alternative splicing changes are frequently observed in cancer and are starting to be recognized as important signatures for tumor progression and therapy. However, their functional impact and relevance to tumorigenesis remain mostly unknown. We carried out a systematic analysis to characterize the potential functional consequences of alternative splicing changes in thousands of tumor samples. This analysis revealed that a subset of alternative splicing changes affect protein domain families that are frequently mutated in tumors and potentially disrupt protein-protein interactions in cancer-related pathways. Moreover, there was a negative correlation between the number of these alternative splicing changes in a sample and the number of somatic mutations in drivers. We propose that a subset of the alternative splicing changes observed in tumors may represent independent oncogenic processes that could be relevant to explain the functional transformations in cancer, and some of them could potentially be considered alternative splicing drivers (AS drivers). Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Modulation of p53β and p53γ expression by regulating the alternative splicing of TP53 gene modifies cellular response

PubMed Central

Marcel, V; Fernandes, K; Terrier, O; Lane, D P; Bourdon, J-C

2014-01-01

In addition to the tumor suppressor p53 protein, also termed p53α, the TP53 gene produces p53β and p53γ through alternative splicing of exons 9β and 9γ located within TP53 intron 9. Here we report that both TG003, a specific inhibitor of Cdc2-like kinases (Clk) that regulates the alternative splicing pre-mRNA pathway, and knockdown of SFRS1 increase expression of endogenous p53β and p53γ at mRNA and protein levels. Development of a TP53 intron 9 minigene shows that TG003 treatment and knockdown of SFRS1 promote inclusion of TP53 exons 9β/9γ. In a series of 85 primary breast tumors, a significant association was observed between expression of SFRS1 and α variant, supporting our experimental data. Using siRNA specifically targeting exons 9β/9γ, we demonstrate that cell growth can be driven by modulating p53β and p53γ expression in an opposite manner, depending on the cellular context. In MCF7 cells, p53β and p53γ promote apoptosis, thus inhibiting cell growth. By transient transfection, we show that p53β enhanced p53α transcriptional activity on the p21 and Bax promoters, while p53γ increased p53α transcriptional activity on the Bax promoter only. Moreover, p53β and p53γ co-immunoprecipitate with p53α only in the presence of p53-responsive promoter. Interestingly, although p53β and p53γ promote apoptosis in MCF7 cells, p53β and p53γ maintain cell growth in response to TG003 in a p53α-dependent manner. The dual activities of p53β and p53γ isoforms observed in non-treated and TG003-treated cells may result from the impact of TG003 on both expression and activities of p53 isoforms. Overall, our data suggest that p53β and p53γ regulate cellular response to modulation of alternative splicing pre-mRNA pathway by a small drug inhibitor. The development of novel drugs targeting alternative splicing process could be used as a novel therapeutic approach in human cancers. PMID:24926616

Cotransin induces accumulation of a cytotoxic clusterin variant that cotranslationally rerouted to the cytosol

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

Choi, Ilho; Kim, Jiyeon; Park, Joong-Yeol

2013-05-01

Although clusterin (CLU) was originally identified as a secreted glycoprotein that plays cytoprotective role, several intracellular CLU variants have been recently identified in the diverse pathological conditions. The mechanistic basis of these variants is now believed to be alternative splicing and retrotranslocation. Here, we uncovered, an unglycosylated and signal sequence-unprocessed, CLU variant in the cytosol. This variant proved to be a product that cotranslationally rerouted to the cytosol during translocation. Cytosolic CLU was prone to aggregation at peri-nuclear region of cells and induced cell death. Signal sequence is shown to be an important determinant for cytosolic CLU generation and aggregation.more » These results provide not only a new mechanistic insight into the cytosolic CLU generation but also an idea for therapeutic mislocalization of CLU as a strategy for cancer treatment. - Highlights: ► Intracellular CLU variants have been recently identified in the diverse pathological conditions. ► Translocation of clusterin is less efficient than that of Prl. ► We identified a new cytotoxic clusterin variant whose signal sequence was unprocessed. ► This variant proved to be a product that cotranslationally rerouted to cytosol.« less

C-terminal modulatory domain controls coupling of voltage-sensing to pore opening in Cav1.3 L-type Ca(2+) channels.

PubMed

Lieb, Andreas; Ortner, Nadine; Striessnig, Jörg

2014-04-01

Activity of voltage-gated Cav1.3 L-type Ca(2+) channels is required for proper hearing as well as sinoatrial node and brain function. This critically depends on their negative activation voltage range, which is further fine-tuned by alternative splicing. Shorter variants miss a C-terminal regulatory domain (CTM), which allows them to activate at even more negative potentials than C-terminally long-splice variants. It is at present unclear whether this is due to an increased voltage sensitivity of the Cav1.3 voltage-sensing domain, or an enhanced coupling of voltage-sensor conformational changes to the subsequent opening of the activation gate. We studied the voltage-dependence of voltage-sensor charge movement (QON-V) and of current activation (ICa-V) of the long (Cav1.3L) and a short Cav1.3 splice variant (Cav1.342A) expressed in tsA-201 cells using whole cell patch-clamp. Charge movement (QON) of Cav1.3L displayed a much steeper voltage-dependence and a more negative half-maximal activation voltage than Cav1.2 and Cav3.1. However, a significantly higher fraction of the total charge had to move for activation of Cav1.3 half-maximal conductance (Cav1.3: 68%; Cav1.2: 52%; Cav3.1: 22%). This indicated a weaker coupling of Cav1.3 voltage-sensor charge movement to pore opening. However, the coupling efficiency was strengthened in the absence of the CTM in Cav1.342A, thereby shifting ICa-V by 7.2 mV to potentials that were more negative without changing QON-V. We independently show that the presence of intracellular organic cations (such as n-methyl-D-glucamine) induces a pronounced negative shift of QON-V and a more negative activation of ICa-V of all three channels. These findings illustrate that the voltage sensors of Cav1.3 channels respond more sensitively to depolarization than those of Cav1.2 or Cav3.1. Weak coupling of voltage sensing to pore opening is enhanced in the absence of the CTM, allowing short Cav1.342A splice variants to activate at lower voltages without affecting QON-V. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

IRAS: High-Throughput Identification of Novel Alternative Splicing Regulators.

PubMed

Zheng, S

2016-01-01

Alternative splicing is a fundamental regulatory process of gene expression. Defects in alternative splicing can lead to various diseases, and modification of disease-causing splicing events presents great therapeutic promise. Splicing outcome is commonly affected by extracellular stimuli and signaling cascades that converge on RNA-binding splicing regulators. These trans-acting factors recognize cis-elements in pre-mRNA transcripts to affect spliceosome assembly and splice site choices. Identification of these splicing regulators and/or upstream modulators has been difficult and traditionally done by piecemeal. High-throughput screening strategies to find multiple regulators of exon splicing have great potential to accelerate the discovery process, but typically confront low sensitivity and low specificity of screening assays. Here we describe a unique screening strategy, IRAS (identifying regulators of alternative splicing), using a pair of dual-output minigene reporters to allow for sensitive detection of exon splicing changes. Each dual-output reporter produces green fluorescent protein (GFP) and red fluorescent protein (RFP) fluorescent signals to assay the two spliced isoforms exclusively. The two complementary minigene reporters alter GFP/RFP output ratios in the opposite direction in response to splicing change. Applying IRAS in cell-based high-throughput screens allows sensitive and specific identification of splicing regulators and modulators for any alternative exons of interest. In comparison to previous high-throughput screening methods, IRAS substantially enhances the specificity of the screening assay. This strategy significantly eliminates false positives without sacrificing sensitive identification of true regulators of splicing. © 2016 Elsevier Inc. All rights reserved.

Context-dependent control of alternative splicing by RNA-binding proteins

PubMed Central

Fu, Xiang-Dong; Ares, Manuel

2015-01-01

Sequence-specific RNA-binding proteins (RBPs) bind to pre-mRNA to control alternative splicing, but it is not yet possible to read the ‘splicing code’ that dictates splicing regulation on the basis of genome sequence. Each alternative splicing event is controlled by multiple RBPs, the combined action of which creates a distribution of alternatively spliced products in a given cell type. As each cell type expresses a distinct array of RBPs, the interpretation of regulatory information on a given RNA target is exceedingly dependent on the cell type. RBPs also control each other’s functions at many levels, including by mutual modulation of their binding activities on specific regulatory RNA elements. In this Review, we describe some of the emerging rules that govern the highly context-dependent and combinatorial nature of alternative splicing regulation. PMID:25112293

Estrogen anti-inflammatory activity on human monocytes is mediated through cross-talk between estrogen receptor ERα36 and GPR30/GPER1.

PubMed

Pelekanou, Vasiliki; Kampa, Marilena; Kiagiadaki, Foteini; Deli, Alexandra; Theodoropoulos, Panayiotis; Agrogiannis, George; Patsouris, Efstratios; Tsapis, Andreas; Castanas, Elias; Notas, George

2016-02-01

Estrogens are known modulators of monocyte/macrophage functions; however, the underlying mechanism has not been clearly defined. Recently, a number of estrogen receptor molecules and splice variants were identified that exert different and sometimes opposing actions. We assessed the expression of estrogen receptors and explored their role in mediating estrogenic anti-inflammatory effects on human primary monocytes. We report that the only estrogen receptors expressed are estrogen receptor-α 36-kDa splice variant and G-protein coupled receptor 30/G-protein estrogen receptor 1, in a sex-independent manner. 17-β-Estradiol inhibits the LPS-induced IL-6 inflammatory response, resulting in inhibition of NF-κB transcriptional activity. This is achieved via a direct physical interaction of ligand-activated estrogen receptor-α 36-kDa splice variant with the p65 component of NF-κB in the nucleus. G-protein coupled receptor 30/G-protein estrogen receptor 1, which also physically interacts with estrogen receptor-α 36-kDa splice variant, acts a coregulator in this process, because its inhibition blocks the effect of estrogens on IL-6 expression. However, its activation does not mimic the effect of estrogens, on neither IL-6 nor NF-κB activity. Finally, we show that the estrogen receptor profile observed in monocytes is not modified during their differentiation to macrophages or dendritic cells in vitro and is shared in vivo by macrophages present in atherosclerotic plaques. These results position estrogen receptor-α 36-kDa splice variant and G-protein coupled receptor 30 as important players and potential therapeutic targets in monocyte/macrophage-dependent inflammatory processes. © Society for Leukocyte Biology.

Potentially pathogenic germline CHEK2 c.319+2T>A among multiple early-onset cancer families.

PubMed

Dominguez-Valentin, Mev; Nakken, Sigve; Tubeuf, Hélène; Vodak, Daniel; Ekstrøm, Per Olaf; Nissen, Anke M; Morak, Monika; Holinski-Feder, Elke; Martins, Alexandra; Møller, Pål; Hovig, Eivind

2018-01-01

To study the potential contribution of genes other than BRCA1/2, PTEN, and TP53 to the biological and clinical characteristics of multiple early-onset cancers in Norwegian families, including early-onset breast cancer, Cowden-like and Li-Fraumeni-like syndromes (BC, CSL and LFL, respectively). The Hereditary Cancer Biobank from the Norwegian Radium Hospital was used to identify early-onset BC, CSL or LFL for whom no pathogenic variants in BRCA1/2, PTEN, or TP53 had been found in routine diagnostic DNA sequencing. Forty-four cancer susceptibility genes were selected and analyzed by our in-house designed TruSeq amplicon-based assay for targeted sequencing. Protein- and RNA splicing-dedicated in silico analyses were performed for all variants of unknown significance (VUS). Variants predicted as the more likely to affect splicing were experimentally analyzed by minigene assay. We identified a CSL individual carrying a variant in CHEK2 (c.319+2T>A, IVS2), here considered as likely pathogenic. Out of the five VUS (BRCA2, CDH1, CHEK2, MAP3K1, NOTCH3) tested in the minigene splicing assay, only NOTCH3 c.14090C>T (p.Ser497Leu) showed a significant effect on RNA splicing, notably by inducing partial skipping of exon 9. Among 13 early-onset BC, CSL and LFL patients, gene panel sequencing identified a potentially pathogenic variant in CHEK2 that affects a canonical RNA splicing signal. Our study provides new information on genetic loci that may affect the risk of developing cancer in these patients and their families, demonstrating that genes presently not routinely tested in molecular diagnostic settings may be important for capturing cancer predisposition in these families.

U1 small nuclear RNA variants differentially form ribonucleoprotein particles in vitro.

PubMed

Somarelli, Jason A; Mesa, Annia; Rodriguez, Carol E; Sharma, Shalini; Herrera, Rene J

2014-04-25

The U1 small nuclear (sn)RNA participates in splicing of pre-mRNAs by recognizing and binding to 5' splice sites at exon/intron boundaries. U1 snRNAs associate with 5' splice sites in the form of ribonucleoprotein particles (snRNPs) that are comprised of the U1 snRNA and 10 core components, including U1A, U1-70K, U1C and the 'Smith antigen', or Sm, heptamer. The U1 snRNA is highly conserved across a wide range of taxa; however, a number of reports have identified the presence of expressed U1-like snRNAs in multiple species, including humans. While numerous U1-like molecules have been shown to be expressed, it is unclear whether these variant snRNAs have the capacity to form snRNPs and participate in splicing. The purpose of the present study was to further characterize biochemically the ability of previously identified human U1-like variants to form snRNPs and bind to U1 snRNP proteins. A bioinformatics analysis provided support for the existence of multiple expressed variants. In vitro gel shift assays, competition assays, and immunoprecipitations (IPs) revealed that the variants formed high molecular weight assemblies to varying degrees and associated with core U1 snRNP proteins to a lesser extent than the canonical U1 snRNA. Together, these data suggest that the human U1 snRNA variants analyzed here are unable to efficiently bind U1 snRNP proteins. The current work provides additional biochemical insights into the ability of the variants to assemble into snRNPs. Copyright © 2014 Elsevier B.V. All rights reserved.

A selective splicing variant of hepcidin mRNA in hepatocellular carcinoma cell lines

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

Toki, Yasumichi; Sasaki, Katsunori, E-mail: k-sasaki@asahikawa-med.ac.jp; Tanaka, Hiroki

2016-08-05

Hepcidin is a main regulator of iron metabolism, of which abnormal expression affects intestinal absorption and reticuloendothelial sequestration of iron by interacting with ferroportin. It is also noted that abnormal iron accumulation is one of the key factors to facilitate promotion and progression of cancer including hepatoma. By RT-PCR/agarose gel electrophoresis of hepcidin mRNA in a hepatocellular carcinoma cell line HLF, a smaller mRNA band was shown in addition to the wild-type hepcidin mRNA. From sequencing analysis, this additional band was a selective splicing variant of hepcidin mRNA lacking exon 2 of HAMP gene, producing the transcript that encodes truncatedmore » peptide lacking 20 amino acids at the middle of preprohepcidin. In the present study, we used the digital PCR, because such a small amount of variant mRNA was difficult to quantitate by the conventional RT-PCR amplification. Among seven hepatoma-derived cell lines, six cell lines have significant copy numbers of this variant mRNA, but not in one cell line. In the transient transfection analysis of variant-type hepcidin cDNA, truncated preprohepcidin has a different character comparing with native preprohepcidin: its product is insensitive to digestion, and secreted into the medium as a whole preprohepcidin form without maturation. Loss or reduction of function of HAMP gene by aberrantly splicing may be a suitable phenomenon to obtain the proliferating advantage of hepatoma cells. - Highlights: • An aberrant splicing variant of hepcidin mRNA lacking exon 2 of HAMP gene. • Absolute quantification of hepcidin mRNA by digital PCR amplification. • Hepatoma-derived cell lines have significant copies of variant-type hepcidin mRNA. • Truncated preprohepcidin is secreted from cells without posttranslational cleavage.« less

Connecting the dots: chromatin and alternative splicing in EMT

PubMed Central

Warns, Jessica A.; Davie, James R.; Dhasarathy, Archana

2015-01-01

Nature has devised sophisticated cellular machinery to process mRNA transcripts produced by RNA Polymerase II, removing intronic regions and connecting exons together, to produce mature RNAs. This process, known as splicing, is very closely linked to transcription. Alternative splicing, or the ability to produce different combinations of exons that are spliced together from the same genomic template, is a fundamental means of regulating protein complexity. Similar to transcription, both constitutive and alternative splicing can be regulated by chromatin and its associated factors in response to various signal transduction pathways activated by external stimuli. This regulation can vary between different cell types, and interference with these pathways can lead to changes in splicing, often resulting in aberrant cellular states and disease. The epithelial to mesenchymal transition (EMT), which leads to cancer metastasis, is influenced by alternative splicing events of chromatin remodelers and epigenetic factors such as DNA methylation and non-coding RNAs. In this review, we will discuss the role of epigenetic factors including chromatin, chromatin remodelers, DNA methyltransferases and microRNAs in the context of alternative splicing, and discuss their potential involvement in alternative splicing during the EMT process. PMID:26291837

TGFβ1-mediated expression and alternative splicing of Fibronectin Extra Domain A in human podocyte culture.

PubMed

Madne, Tarunkumar Hemraj; Dockrell, Mark Edward Carl

2018-02-28

Alternative splicing is a fundamental phenomenon to build protein diversity in health and diseases. Extra Domain A+ Fibronectin (EDA+Fn) is an alternatively spliced form of fibronectin protein present in the extra cellular matrix (ECM) in renal fibrosis. Podocytes are spectacular cell type and play a key role in filtration and synthesise ECM proteins in renal physiology and pathology. TGFβ1 is a strong stimulator of ECM proteins in renal injury. In this study, we have investigated alternative splicing of EDA+ Fn in human podocytes in response to TGFβ1. We have performed western blotting and immunofluorescence to characterise the expression of the EDA+Fn protein, real-time PCR for RNA expression and RT-PCR to look for alternative splicing of EDA+Fn in conditionally immortalised human podocytes culture.We used TGFβ1 as a stimulator and SB431542 and SRPIN340 for inhibitory studies. In this work, for the first time we have demonstrated in human podocytes culture EDA+Fn is expressed in the basal condition and TGFβ1 2.5ng/ml induced the Fn mRNA and EDA+Fn protein expression demonstrated by real-time PCR, western blotting and immunofluorescence. TGFβ1 2.5ng/ml induced the alternative splicing of EDA+Fn shown by conventional RT-PCR. Studies with ALK5 inhibitor SB431542 and SRPIN340 show that TGFβ1 induced alternative splicing of EDA+Fn was by the ALK5 receptor and the SR proteins.  In human podocytes culture, alternative splicing of EDA+Fn occurs at basal conditions and TGFβ1 further induced the alternative splicing of EDA+Fn via ALK5 receptor activation and SR proteins. This is the first evidence of basal and TGFβ1 mediated alternative splicing of EDA+Fn in human podocytes culture.

Coordinated tissue-specific regulation of adjacent alternative 3′ splice sites in C. elegans

PubMed Central

Ragle, James Matthew; Katzman, Sol; Akers, Taylor F.; Barberan-Soler, Sergio; Zahler, Alan M.

2015-01-01

Adjacent alternative 3′ splice sites, those separated by ≤18 nucleotides, provide a unique problem in the study of alternative splicing regulation; there is overlap of the cis-elements that define the adjacent sites. Identification of the intron's 3′ end depends upon sequence elements that define the branchpoint, polypyrimidine tract, and terminal AG dinucleotide. Starting with RNA-seq data from germline-enriched and somatic cell-enriched Caenorhabditis elegans samples, we identify hundreds of introns with adjacent alternative 3′ splice sites. We identify 203 events that undergo tissue-specific alternative splicing. For these, the regulation is monodirectional, with somatic cells preferring to splice at the distal 3′ splice site (furthest from the 5′ end of the intron) and germline cells showing a distinct shift toward usage of the adjacent proximal 3′ splice site (closer to the 5′ end of the intron). Splicing patterns in somatic cells follow C. elegans consensus rules of 3′ splice site definition; a short stretch of pyrimidines preceding an AG dinucleotide. Splicing in germline cells occurs at proximal 3′ splice sites that lack a preceding polypyrimidine tract, and in three instances the germline-specific site lacks the AG dinucleotide. We provide evidence that use of germline-specific proximal 3′ splice sites is conserved across Caenorhabditis species. We propose that there are differences between germline and somatic cells in the way that the basal splicing machinery functions to determine the intron terminus. PMID:25922281

Spatio-temporal regulations and functions of neuronal alternative RNA splicing in developing and adult brains.

PubMed

Iijima, Takatoshi; Hidaka, Chiharu; Iijima, Yoko

2016-08-01

Alternative pre-mRNA splicing is a fundamental mechanism that generates molecular diversity from a single gene. In the central nervous system (CNS), key neural developmental steps are thought to be controlled by alternative splicing decisions, including the molecular diversity underlying synaptic wiring, plasticity, and remodeling. Significant progress has been made in understanding the molecular mechanisms and functions of alternative pre-mRNA splicing in neurons through studies in invertebrate systems; however, recent studies have begun to uncover the potential role of neuronal alternative splicing in the mammalian CNS. This article provides an overview of recent findings regarding the regulation and function of neuronal alternative splicing. In particular, we focus on the spatio-temporal regulation of neurexin, a synaptic adhesion molecule, by neuronal cell type-specific factors and neuronal activity, which are thought to be especially important for characterizing neural development and function within the mammalian CNS. Notably, there is increasing evidence that implicates the dysregulation of neuronal splicing events in several neurological disorders. Therefore, understanding the detailed mechanisms of neuronal alternative splicing in the mammalian CNS may provide plausible treatment strategies for these diseases. Copyright © 2016 Elsevier Ireland Ltd and Japan Neuroscience Society. All rights reserved.

Evaluation of IFITM3 rs12252 Association With Severe Pediatric Influenza Infection.

PubMed

Randolph, Adrienne G; Yip, Wai-Ki; Allen, Emma Kaitlynn; Rosenberger, Carrie M; Agan, Anna A; Ash, Stephanie A; Zhang, Yu; Bhangale, Tushar R; Finkelstein, David; Cvijanovich, Natalie Z; Mourani, Peter M; Hall, Mark W; Su, Helen C; Thomas, Paul G

2017-07-01

Interferon-induced transmembrane protein 3 (IFITM3) restricts endocytic fusion of influenza virus. IFITM3 rs12252_C, a putative alternate splice site, has been associated with influenza severity in adults. IFITM3 has not been evaluated in pediatric influenza. The Pediatric Influenza (PICFLU) study enrolled children with suspected influenza infection across 38 pediatric intensive care units during November 2008 to April 2016. IFITM3 was sequenced in patients and parents were genotyped for specific variants for family-based association testing. rs12252 was genotyped in 54 African-American pediatric outpatients with influenza (FLU09), included in the population-based comparisons with 1000 genomes. Splice site analysis of rs12252_C was performed using PICFLU and FLU09 patient RNA. In PICFLU, 358 children had influenza infection. We identified 22 rs12252_C homozygotes in 185 white non-Hispanic children. rs12252_C was not associated with influenza infection in population or family-based analyses. We did not identify the Δ21 IFITM3 isoform in RNAseq data. The rs12252 genotype was not associated with IFITM3 expression levels, nor with critical illness severity. No novel rare IFITM3 functional variants were identified. rs12252 was not associated with susceptibility to influenza-related critical illness in children or with critical illness severity. Our data also do not support it being a splice site. © The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

Intronic variants in the dopa decarboxylase (DDC) gene are associated with smoking behavior in European-Americans and African-Americans.

PubMed

Yu, Yi; Panhuysen, Carolien; Kranzler, Henry R; Hesselbrock, Victor; Rounsaville, Bruce; Weiss, Roger; Brady, Kathleen; Farrer, Lindsay A; Gelernter, Joel

2006-07-15

We report here a study considering association of alleles and haplotypes at the DOPA decarboxylase (DDC) locus with the DSM-IV diagnosis of nicotine dependence (ND) or a quantitative measure for ND using the Fagerstrom Test for Nicotine Dependence (FTND). We genotyped 18 single nucleotide polymorphisms (SNPs) spanning a region of approximately 210 kb that includes DDC and the genes immediately flanking DDC in 1,590 individuals from 621 families of African-American (AA) or European-American (EA) ancestry. Evidence of association (family-based tests) was observed with several SNPs for both traits (0.0002

The caspase-generated cleavage product of Ets-1 p51 and Ets-1 p27, Cp17, induces apoptosis.

PubMed

Choul-Li, Souhaila; Tulasne, David; Aumercier, Marc

2016-11-04

The transcription factor Ets-1 is involved in various physiological processes and invasive pathologies. Human Ets-1 exists under three isoforms: p51, the predominant full-length isoform, p42 and p27, shorter alternatively spliced isoforms. We have previously demonstrated that Ets-1 p51, but not the spliced variant Ets-1 p42, is processed by caspases in vitro and during apoptosis. However, the caspase cleavage of the second spliced variant Ets-1 p27 remains to investigate. In the present study, we demonstrate that Ets-1 p27 is a cleavage substrate of caspases. We show that Ets-1 p27 is processed in vitro by caspase-3, resulting in three C-terminal fragments Cp20, Cp17 and Cp14. Similarly, Ets-1 p27 was cleaved during apoptotic cell death induced by anisomycin, producing fragments consistent with those observed in in vitro cleavage assay. These fragments are generated by cleavage at three sites located in the exon VII-encoded region of Ets-1 p27. As a functional consequences, Cp17 fragment, the major cleavage product generated during apoptosis, induced itself apoptosis when transfected into cells. Our results show that Ets-1 p27 is cleaved in the same manner as Ets-1 p51 within the exon VII-encoded region, thus generating a stable C-terminal fragment that induces cell death by initiating apoptosis. Copyright © 2016 Elsevier Inc. All rights reserved.

In1-ghrelin, a splice variant of ghrelin gene, is associated with the evolution and aggressiveness of human neuroendocrine tumors: Evidence from clinical, cellular and molecular parameters.

PubMed

Luque, Raul M; Sampedro-Nuñez, Miguel; Gahete, Manuel D; Ramos-Levi, Ana; Ibáñez-Costa, Alejandro; Rivero-Cortés, Esther; Serrano-Somavilla, Ana; Adrados, Magdalena; Culler, Michael D; Castaño, Justo P; Marazuela, Mónica

2015-08-14

Ghrelin system comprises a complex family of peptides, receptors (GHSRs), and modifying enzymes [e.g. ghrelin-O-acyl-transferase (GOAT)] that control multiple pathophysiological processes. Aberrant alternative splicing is an emerging cancer hallmark that generates altered proteins with tumorigenic capacity. Indeed, In1-ghrelin and truncated-GHSR1b splicing variants can promote development/progression of certain endocrine-related cancers. Here, we determined the expression levels of key ghrelin system components in neuroendocrine tumor (NETs) and explored their potential functional role. Twenty-six patients with NETs were prospectively/retrospectively studied [72 samples from primary and metastatic tissues (30 normal/42 tumors)] and clinical data were obtained. The role of In1-ghrelin in aggressiveness was studied in vitro using NET cell lines (BON-1/QGP-1). In1-ghrelin, GOAT and GHSR1a/1b expression levels were elevated in tumoral compared to normal/adjacent tissues. Moreover, In1-ghrelin, GOAT, and GHSR1b expression levels were positively correlated within tumoral, but not within normal/adjacent samples, and were higher in patients with progressive vs. with stable/cured disease. Finally, In1-ghrelin increased aggressiveness (e.g. proliferation/migration) of NET cells. Altogether, our data strongly suggests a potential implication of ghrelin system in the pathogenesis and/or clinical outcome of NETs, and warrant further studies on their possible value for the future development of molecular biomarkers with diagnostic/prognostic/therapeutic value.

In1-ghrelin, a splice variant of ghrelin gene, is associated with the evolution and aggressiveness of human neuroendocrine tumors: Evidence from clinical, cellular and molecular parameters

PubMed Central

Gahete, Manuel D.; Ramos-Levi, Ana; Ibáñez-Costa, Alejandro; Rivero-Cortés, Esther; Serrano-Somavilla, Ana; Adrados, Magdalena; Culler, Michael D.; Castaño, Justo P.; Marazuela, Mónica

2015-01-01

Ghrelin system comprises a complex family of peptides, receptors (GHSRs), and modifying enzymes [e.g. ghrelin-O-acyl-transferase (GOAT)] that control multiple pathophysiological processes. Aberrant alternative splicing is an emerging cancer hallmark that generates altered proteins with tumorigenic capacity. Indeed, In1-ghrelin and truncated-GHSR1b splicing variants can promote development/progression of certain endocrine-related cancers. Here, we determined the expression levels of key ghrelin system components in neuroendocrine tumor (NETs) and explored their potential functional role. Twenty-six patients with NETs were prospectively/retrospectively studied [72 samples from primary and metastatic tissues (30 normal/42 tumors)] and clinical data were obtained. The role of In1-ghrelin in aggressiveness was studied in vitro using NET cell lines (BON-1/QGP-1). In1-ghrelin, GOAT and GHSR1a/1b expression levels were elevated in tumoral compared to normal/adjacent tissues. Moreover, In1-ghrelin, GOAT, and GHSR1b expression levels were positively correlated within tumoral, but not within normal/adjacent samples, and were higher in patients with progressive vs. with stable/cured disease. Finally, In1-ghrelin increased aggressiveness (e.g. proliferation/migration) of NET cells. Altogether, our data strongly suggests a potential implication of ghrelin system in the pathogenesis and/or clinical outcome of NETs, and warrant further studies on their possible value for the future development of molecular biomarkers with diagnostic/prognostic/therapeutic value. PMID:26124083

Neurobehavioral Differences Between Mice Receiving Distinct Neuregulin Variants as Neonates; Impact on Sensitivity to MK-801

PubMed Central

Kato, T.; Abe, Y.; Hirokawa, S.; Iwakura, Y.; Mizuno, M.; Namba, H.; Nawa, H.

2015-01-01

Neuregulin-1 (NRG1) is a well-recognized risk gene for schizophrenia and is often implicated in the neurodevelopmental hypothesis of this illness. Alternative splicing and proteolytic processing of the NRG1 gene produce more than 30 structural variants; however, the neuropathological roles of individual variants remain to be characterized. On the basis of the neurodevelopmental hypothesis of schizophrenia, we administered eNRG1 (0.1~1.0 µg/g), a core epidermal growth factor-like (EGF) domain common for all splicing NRG1 variants, to neonatal mice and compared their behavioral performance with mice challenged with a full mature form of type 1 NRG1 variant. During the neonatal stage, recombinant eNRG1 protein administrated from the periphery passed the blood-brain barrier and activated its receptor (ErbB4) in the brain. In adults, the mice receiving the highest dose exhibited lower locomotor activity and deficits in prepulse inhibition and tonedependent fear learning, although the hearing reduction of the eNRG1-treated mice may explain these behavioral deficits. Neonatal eNRG1 treatment also significantly potentiated MK-801-driven locomotor activity in an eNRG1 dose-dependent manner. In parallel eNRG1 treatment enhanced MK-801-driven c-Fos induction and decreased immunoreactivity for NMDA receptor subunits in adult brain. In contrast, mice that had been treated with the same molar dose of a full mature form of type 1 NRG1 as neonates did not exhibit hypersensitivity to MK-801. However, both animal models exhibited similar hypersensitivity to methamphetamine. Collectively, our findings suggest that aberrant peripheral NRG1 signals during neurodevelopment alter later behavioral traits and auditory functions in the NRG1 subtype-dependent manner. PMID:25817857

A Functional Role for KLF6-SV1 in Lung Adenocarcinoma Prognosis and Chemotherapy Response

PubMed Central

DiFeo, Analisa; Feld, Lauren; Rodriguez, Estefania; Wang, Christine; Beer, David G.; Martignetti, John A.; Narla, Goutham

2009-01-01

Kruppel-like factor 6 (KLF6) is a tumor suppressor gene that is functionally inactivated in human cancer by loss of heterozygosity, somatic mutation, decreased expression, and increased alternative splicing into an oncogenic splice variant, KLF6-SV1. Here we show that increased expression of KLF6-SV1 is associated with decreased survival in patients with lung adenocarcinoma. In addition, KLF6-SV1 is a novel antiapoptotic protein in lung cancer cell lines, and targeted reduction of KLF6-SV1 using siRNA induces apoptosis both alone and in combination with the chemotherapeutic drug cisplatin. Together, these findings highlight a critical role for KLF6-SV1 in lung cancer, and show a potential novel therapeutic strategy for the treatment of lung cancer. PMID:18250346

Inhibition of mutant BRAF splice variant signaling by next-generation, selective RAF inhibitors.

PubMed

Basile, Kevin J; Le, Kaitlyn; Hartsough, Edward J; Aplin, Andrew E

2014-05-01

Vemurafenib and dabrafenib block MEK-ERK1/2 signaling and cause tumor regression in the majority of advanced-stage BRAF(V600E) melanoma patients; however, acquired resistance and paradoxical signaling have driven efforts for more potent and selective RAF inhibitors. Next-generation RAF inhibitors, such as PLX7904 (PB04), effectively inhibit RAF signaling in BRAF(V600E) melanoma cells without paradoxical effects in wild-type cells. Furthermore, PLX7904 blocks the growth of vemurafenib-resistant BRAF(V600E) cells that express mutant NRAS. Acquired resistance to vemurafenib and dabrafenib is also frequently driven by expression of mutation BRAF splice variants; thus, we tested the effects of PLX7904 and its clinical analog, PLX8394 (PB03), in BRAF(V600E) splice variant-mediated vemurafenib-resistant cells. We show that paradox-breaker RAF inhibitors potently block MEK-ERK1/2 signaling, G1/S cell cycle events, survival and growth of vemurafenib/PLX4720-resistant cells harboring distinct BRAF(V600E) splice variants. These data support the further investigation of paradox-breaker RAF inhibitors as a second-line treatment option for patients failing on vemurafenib or dabrafenib. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

The in vivo use of alternate 3'-splice sites in group I introns.

PubMed

Sellem, C H; Belcour, L

1994-04-11

Alternative splicing of group I introns has been postulated as a possible mechanism that would ensure the translation of proteins encoded into intronic open reading frames, discontinuous with the upstream exon and lacking an initiation signal. Alternate splice sites were previously depicted according to secondary structures of several group I introns. We present here strong evidence that, in the case of Podospora anserina nad 1-i4 and cox1-i7 mitochondrial introns, alternative splicing events do occur in vivo. Indeed, by PCR experiments we have detected molecules whose sequence is precisely that expected if the predicted alternate 3'-splice sites were used.

A Bioinformatics-Based Alternative mRNA Splicing Code that May Explain Some Disease Mutations Is Conserved in Animals.

PubMed

Qu, Wen; Cingolani, Pablo; Zeeberg, Barry R; Ruden, Douglas M

2017-01-01

Deep sequencing of cDNAs made from spliced mRNAs indicates that most coding genes in many animals and plants have pre-mRNA transcripts that are alternatively spliced. In pre-mRNAs, in addition to invariant exons that are present in almost all mature mRNA products, there are at least 6 additional types of exons, such as exons from alternative promoters or with alternative polyA sites, mutually exclusive exons, skipped exons, or exons with alternative 5' or 3' splice sites. Our bioinformatics-based hypothesis is that, in analogy to the genetic code, there is an "alternative-splicing code" in introns and flanking exon sequences, analogous to the genetic code, that directs alternative splicing of many of the 36 types of introns. In humans, we identified 42 different consensus sequences that are each present in at least 100 human introns. 37 of the 42 top consensus sequences are significantly enriched or depleted in at least one of the 36 types of introns. We further supported our hypothesis by showing that 96 out of 96 analyzed human disease mutations that affect RNA splicing, and change alternative splicing from one class to another, can be partially explained by a mutation altering a consensus sequence from one type of intron to that of another type of intron. Some of the alternative splicing consensus sequences, and presumably their small-RNA or protein targets, are evolutionarily conserved from 50 plant to animal species. We also noticed the set of introns within a gene usually share the same splicing codes, thus arguing that one sub-type of splicesosome might process all (or most) of the introns in a given gene. Our work sheds new light on a possible mechanism for generating the tremendous diversity in protein structure by alternative splicing of pre-mRNAs.

Increased Transcript Complexity in Genes Associated with Chronic Obstructive Pulmonary Disease

PubMed Central

Lackey, Lela; McArthur, Evonne; Laederach, Alain

2015-01-01

Genome-wide association studies aim to correlate genotype with phenotype. Many common diseases including Type II diabetes, Alzheimer’s, Parkinson’s and Chronic Obstructive Pulmonary Disease (COPD) are complex genetic traits with hundreds of different loci that are associated with varied disease risk. Identifying common features in the genes associated with each disease remains a challenge. Furthermore, the role of post-transcriptional regulation, and in particular alternative splicing, is still poorly understood in most multigenic diseases. We therefore compiled comprehensive lists of genes associated with Type II diabetes, Alzheimer’s, Parkinson’s and COPD in an attempt to identify common features of their corresponding mRNA transcripts within each gene set. The SERPINA1 gene is a well-recognized genetic risk factor of COPD and it produces 11 transcript variants, which is exceptional for a human gene. This led us to hypothesize that other genes associated with COPD, and complex disorders in general, are highly transcriptionally diverse. We found that COPD-associated genes have a statistically significant enrichment in transcript complexity stemming from a disproportionately high level of alternative splicing, however, Type II Diabetes, Alzheimer’s and Parkinson’s disease genes were not significantly enriched. We also identified a subset of transcriptionally complex COPD-associated genes (~40%) that are differentially expressed between mild, moderate and severe COPD. Although the genes associated with other lung diseases are not extensively documented, we found preliminary data that idiopathic pulmonary disease genes, but not cystic fibrosis modulators, are also more transcriptionally complex. Interestingly, complex COPD transcripts are more often the product of alternative acceptor site usage. To verify the biological importance of these alternative transcripts, we used RNA-sequencing analyses to determine that COPD-associated genes are frequently expressed in lung and liver tissues and are regulated in a tissue-specific manner. Additionally, many complex COPD-associated genes are spliced differently between COPD and non-COPD patients. Our analysis therefore suggests that post-transcriptional regulation, particularly alternative splicing, is an important feature specific to COPD disease etiology that warrants further investigation. PMID:26480348

Manananggal - a novel viewer for alternative splicing events.

PubMed

Barann, Matthias; Zimmer, Ralf; Birzele, Fabian

2017-02-21

Alternative splicing is an important cellular mechanism that can be analyzed by RNA sequencing. However, identification of splicing events in an automated fashion is error-prone. Thus, further validation is required to select reliable instances of alternative splicing events (ASEs). There are only few tools specifically designed for interactive inspection of ASEs and available visualization approaches can be significantly improved. Here, we present Manananggal, an application specifically designed for the identification of splicing events in next generation sequencing data. Manananggal includes a web application for visual inspection and a command line tool that allows for ASE detection. We compare the sashimi plots available in the IGV Viewer, the DEXSeq splicing plots and SpliceSeq to the Manananggal interface and discuss the advantages and drawbacks of these tools. We show that sashimi plots (such as those used by the IGV Viewer and SpliceSeq) offer a practical solution for simple ASEs, but also indicate short-comings for highly complex genes. Manananggal is an interactive web application that offers functions specifically tailored to the identification of alternative splicing events that other tools are lacking. The ability to select a subset of isoforms allows an easier interpretation of complex alternative splicing events. In contrast to SpliceSeq and the DEXSeq splicing plot, Manananggal does not obscure the gene structure by showing full transcript models that makes it easier to determine which isoforms are expressed and which are not.

Alternative Splicing Control of Abiotic Stress Responses.

PubMed

Laloum, Tom; Martín, Guiomar; Duque, Paula

2018-02-01

Alternative splicing, which generates multiple transcripts from the same gene, is an important modulator of gene expression that can increase proteome diversity and regulate mRNA levels. In plants, this post-transcriptional mechanism is markedly induced in response to environmental stress, and recent studies have identified alternative splicing events that allow rapid adjustment of the abundance and function of key stress-response components. In agreement, plant mutants defective in splicing factors are severely impaired in their response to abiotic stress. Notably, mounting evidence indicates that alternative splicing regulates stress responses largely by targeting the abscisic acid (ABA) pathway. We review here current understanding of post-transcriptional control of plant stress tolerance via alternative splicing and discuss research challenges for the near future. Copyright © 2017 Elsevier Ltd. All rights reserved.

Transcriptome analysis reveals the complexity of alternative splicing regulation in the fungus Verticillium dahliae.

PubMed

Jin, Lirong; Li, Guanglin; Yu, Dazhao; Huang, Wei; Cheng, Chao; Liao, Shengjie; Wu, Qijia; Zhang, Yi

2017-02-06

Alternative splicing (AS) regulation is extensive and shapes the functional complexity of higher organisms. However, the contribution of alternative splicing to fungal biology is not well studied. This study provides sequences of the transcriptomes of the plant wilt pathogen Verticillium dahliae, using two different strains and multiple methods for cDNA library preparations. We identified alternatively spliced mRNA isoforms in over a half of the multi-exonic fungal genes. Over one-thousand isoforms involve TopHat novel splice junction; multiple types of combinatory alternative splicing patterns were identified. We showed that one Verticillium gene could use four different 5' splice sites and two different 3' donor sites to produce up to five mature mRNAs, representing one of the most sophisticated alternative splicing model in eukaryotes other than animals. Hundreds of novel intron types involving a pair of new splice sites were identified in the V. dahliae genome. All the types of AS events were validated by using RT-PCR. Functional enrichment analysis showed that AS genes are involved in most known biological functions and enriched in ATP biosynthesis, sexual/asexual reproduction, morphogenesis, signal transduction etc., predicting that the AS regulation modulates mRNA isoform output and shapes the V. dahliae proteome plasticity of the pathogen in response to the environmental and developmental changes. These findings demonstrate the comprehensive alternative splicing mechanisms in a fungal plant pathogen, which argues the importance of this fungus in developing complicate genome regulation strategies in eukaryotes.

VAT: a computational framework to functionally annotate variants in personal genomes within a cloud-computing environment.

PubMed

Habegger, Lukas; Balasubramanian, Suganthi; Chen, David Z; Khurana, Ekta; Sboner, Andrea; Harmanci, Arif; Rozowsky, Joel; Clarke, Declan; Snyder, Michael; Gerstein, Mark

2012-09-01

The functional annotation of variants obtained through sequencing projects is generally assumed to be a simple intersection of genomic coordinates with genomic features. However, complexities arise for several reasons, including the differential effects of a variant on alternatively spliced transcripts, as well as the difficulty in assessing the impact of small insertions/deletions and large structural variants. Taking these factors into consideration, we developed the Variant Annotation Tool (VAT) to functionally annotate variants from multiple personal genomes at the transcript level as well as obtain summary statistics across genes and individuals. VAT also allows visualization of the effects of different variants, integrates allele frequencies and genotype data from the underlying individuals and facilitates comparative analysis between different groups of individuals. VAT can either be run through a command-line interface or as a web application. Finally, in order to enable on-demand access and to minimize unnecessary transfers of large data files, VAT can be run as a virtual machine in a cloud-computing environment. VAT is implemented in C and PHP. The VAT web service, Amazon Machine Image, source code and detailed documentation are available at vat.gersteinlab.org.

Cancer-associated variant expression and interaction of CIZ1 with cyclin A1 in differentiating male germ cells.

PubMed

Greaves, Erin A; Copeland, Nikki A; Coverley, Dawn; Ainscough, Justin F X

2012-05-15

CIZ1 is a nuclear-matrix-associated DNA replication factor unique to higher eukaryotes, for which alternatively spliced isoforms have been associated with a range of disorders. In vitro, the CIZ1 N-terminus interacts with cyclin E and cyclin A at distinct sites, enabling functional cooperation with cyclin-A-Cdk2 to promote replication initiation. C-terminal sequences anchor CIZ1 to fixed sites on the nuclear matrix, imposing spatial constraint on cyclin-dependent kinase activity. Here we demonstrate that CIZ1 is predominantly expressed as a predicted full-length product throughout mouse development, consistent with a ubiquitous role in cell and tissue renewal. CIZ1 is expressed in proliferating stem cells of the testis, but is notably downregulated following commitment to differentiation. Significantly, CIZ1 is re-expressed at high levels in non-proliferative spermatocytes before meiotic division. Sequence analysis identifies at least seven alternatively spliced variants, including a dominant cancer-associated form and a set of novel isoforms. Furthermore, we show that in these post-replicative cells, CIZ1 interacts with germ-cell-specific cyclin A1, which has been implicated in the repair of DNA double-strand breaks. Consistent with this role, antibody depletion of CIZ1 reduces the capacity for testis extract to repair digested plasmid DNA in vitro. Together, the data imply post-replicative roles for CIZ1 in germ cell differentiation that might include meiotic recombination - a process intrinsic to genome stability and diversification.

Identification and molecular cloning of novel transcripts of the human kallikrein-related peptidase 10 (KLK10) gene using next-generation sequencing

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

Adamopoulos, Panagiotis G.; Kontos, Christos K.; Scorilas, Andreas

Tissue kallikrein and kallikrein-related peptidases (KLKs) form the largest group of serine proteases in the human genome, sharing many structural and functional characteristics. Multiple alternative transcripts have been reported for the most human KLK genes, while many of them are aberrantly expressed in various malignancies, thus possessing significant prognostic and/or diagnostic value. Alternative splicing of cancer-related genes is a common cellular mechanism accounting for cancer cell transcriptome complexity, as it affects cell cycle control, proliferation, apoptosis, invasion, and metastasis. In this study, we describe the identification and molecular cloning of eight novel transcripts of the human KLK10 gene using 3′more » rapid amplification of cDNA ends (3′ RACE) and next-generation sequencing (NGS), as well as their expression analysis in a wide panel of cell lines, originating from several distinct cancerous and normal tissues. Bioinformatic analysis revealed that the novel KLK10 transcripts contain new alternative splicing events between already annotated exons as well as novel exons. In addition, investigation of their expression profile in a wide panel of cell lines was performed with nested RT-PCR using variant-specific pairs of primers. Since many KLK mRNA transcripts possess clinical value, these newly discovered alternatively spliced KLK10 transcripts appear as new potential biomarkers for diagnostic and/or prognostic purposes or as targets for therapeutic strategies. - Highlights: • NGS was used to identify novel transcripts of the human KLK10 gene. • 8 novel KLK10 transcripts were identified. • A novel 3′UTR was detected and characterized. • The expression profiles of all 8 novel KLK10 transcripts were identified.« less

A SIGMAR1 splice-site mutation causes distal hereditary motor neuropathy.

PubMed

Li, Xiaobo; Hu, Zhengmao; Liu, Lei; Xie, Yongzhi; Zhan, Yajing; Zi, Xiaohong; Wang, Junling; Wu, Lixiang; Xia, Kun; Tang, Beisha; Zhang, Ruxu

2015-06-16

To identify the underlying genetic cause in a consanguineous Chinese family segregating distal hereditary motor neuropathy (dHMN) in an autosomal recessive pattern. We used whole-exome sequencing and homozygosity mapping to detect the genetic variant in 2 affected individuals of the consanguineous Chinese family with dHMN. RNA analysis of peripheral blood leukocytes and immunofluorescence and immunoblotting of stable cell lines were performed to support the pathogenicity of the identified mutation. We identified 3 shared novel homozygous variants in 3 shared homozygous regions of the affected individuals. Sequencing of these 3 variants in family members revealed the c.151+1G>T mutation in SIGMAR1 gene, which located in homozygous region spanning approximately 5.3 Mb at chromosome 9p13.1-p13.3, segregated with the dHMN phenotype. The mutation causes an alternative splicing event and generates a transcript variant with an in-frame deletion of 60 base pairs in exon 1 (c.92_151del), and results in an internally shortened protein σ1R(31_50del). The proteasomal inhibitor treatment increased the intracellular amount of σ1R(31_50del) and led to the formation of nuclear aggregates. Stable expressing σ1R(31_50del) induced endoplasmic reticulum stress and enhanced apoptosis. The homozygous c.151+1G>T mutation in SIGMAR1 caused a novel form of autosomal recessive dHMN in a Chinese consanguineous family. Endoplasmic reticulum stress may have a role in the pathogenesis of dHMN. © 2015 American Academy of Neurology.

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.

Alternative Splicing of STAT3 Is Affected by RNA Editing.

PubMed

Goldberg, Lior; Abutbul-Amitai, Mor; Paret, Gideon; Nevo-Caspi, Yael

2017-05-01

A-to-I RNA editing, carried out by adenosine deaminase acting on RNA (ADAR) enzymes, is an epigenetic phenomenon of posttranscriptional modifications on pre-mRNA. RNA editing in intronic sequences may influence alternative splicing of flanking exons. We have previously shown that conditions that induce editing result in elevated expression of signal transducer and activator of transcription 3 (STAT3), preferentially the alternatively-spliced STAT3β isoform. Mechanisms regulating alternative splicing of STAT3 have not been elucidated. STAT3 undergoes A-to-I RNA editing in an intron residing in proximity to the alternatively spliced exon. We hypothesized that RNA editing plays a role in regulating alternative splicing toward STAT3β. In this study we extend our observation connecting RNA editing to the preferential induction of STAT3β expression. We study the involvement of ADAR1 in STAT3 editing and reveal the connection between editing and alternative splicing of STAT3. Deferoaxamine treatment caused the induction in STAT3 RNA editing and STAT3β expression. Silencing ADAR1 caused a decrease in STAT3 editing and expression with a preferential decrease in STAT3β. Cells transfected with a mutated minigene showed preferential splicing toward the STAT3β transcript. Editing in the STAT3 intron is performed by ADAR1 and affects STAT3 alternative splicing. These results suggest that RNA editing is one of the molecular mechanisms regulating the expression of STAT3β.

Multiple splicing events involved in regulation of human aromatase expression by a novel promoter, I.6.

PubMed

Shozu, M; Zhao, Y; Bulun, S E; Simpson, E R

1998-04-01

The expression of aromatase is regulated in a tissue-specific fashion through alternative use of multiple promoter-specific first exons. To date, eight different first exons have been reported in human aromatase, namely I.1., I.2, I.3. I.4, I.5, PII, 2a, and 1f. Recently, we have found a new putative exon I in a RACE-generated library of THP-1 cells and have conducted studies to characterize this new exon I. We confirmed that the constructs containing -1552/+17 or less flanking sequence of this exon function as a promoter in THP-1 cells, JEG-3 cells and osteoblast-like cells obtained from a human fetus. Results of transfection assays using a series of deletion constructs and mutation constructs indicate that a 1-bp mismatch of the consensus TATA-like box (TTTAAT) and the consensus sequence of the initiator site, which is located 45 bp downstream of the putative TATA box, were functioning cooperatively as a core promoter. The putative transcription site was confirmed by the results of RT-PCR southern blot analysis. We examined the regulation and the expression of this exon, I.6, in several human cells and tissues by RT-PCR Southern blot analysis. THP-1 cells (mononuclear leukemic origin) and JEG-3 cells (choriocarcinoma origin) expressed exon I.6 in serum-free media. The level of expression was increased by serum and phorbol myristyl acetate (PMA) in both cell lines. Adipose stromal cells also expressed exon I.6 in the presence of PMA. In fetal osteoblasts, the expression of exon I.6 was increased most effectively by serum and less so by dexamethasone (DEX) + IL-1beta and DEX + IL-11, whereas induction by serum was suppressed by the addition of DEX. The level of expression was low in granulosa cells in culture and did not change with forskolin. On the other hand, dibutyryl cAMP suppressed PMA-stimulated expression of exon I.6 in THP-1 cells and adipose stromal cells. This result supports the hypothesis that the expression of exon I.6 is regulated mainly via an AP-1 binding site that is found upstream of the initiator site of the promoter region. Expression of exon I.6-specific transcripts was examined in several human tissues. Testis and bone obtained from normal adults expressed exon I.6. Testicular tumor and hepatic carcinoma expressed high levels of exon I.6, whereas granulosa cell tumor did not. Fetal liver and bone also showed a significant level of exon I.6 expression, but not so much as testicular tumor and hepatic tumor. Several splicing variants of exon I.6 were detected especially in THP-1 and JEG-3 cells, and to a lesser extent in primary cultures and tissue samples. These variants were identified as an unspliced form, a form spliced at the end of exon I.4, a form spliced at the end of exon I.3 (truncated) and a form spliced 220 bp downstream of the 3' end of exon I.6. The last variant revealed a new splicing site. Because most of the splicing variants contain the sequence specific for exon I.3, RT-PCR specific for exon I.3 can coamplify these splicing variants of exon I.6 transcripts. These results suggests that it is necessary to examine the expression of I.6 in tissues that are known to express exon I.3 such as breast adipose tissue, in which promoter usage of exon I of the aromatase gene switches from exon I.4 to I.3 in the course of malignant transformation.

Co-expression networks reveal the tissue-specific regulation of transcription and splicing

PubMed Central

Saha, Ashis; Kim, Yungil; Gewirtz, Ariel D.H.; Jo, Brian; Gao, Chuan; McDowell, Ian C.; Engelhardt, Barbara E.

2017-01-01

Gene co-expression networks capture biologically important patterns in gene expression data, enabling functional analyses of genes, discovery of biomarkers, and interpretation of genetic variants. Most network analyses to date have been limited to assessing correlation between total gene expression levels in a single tissue or small sets of tissues. Here, we built networks that additionally capture the regulation of relative isoform abundance and splicing, along with tissue-specific connections unique to each of a diverse set of tissues. We used the Genotype-Tissue Expression (GTEx) project v6 RNA sequencing data across 50 tissues and 449 individuals. First, we developed a framework called Transcriptome-Wide Networks (TWNs) for combining total expression and relative isoform levels into a single sparse network, capturing the interplay between the regulation of splicing and transcription. We built TWNs for 16 tissues and found that hubs in these networks were strongly enriched for splicing and RNA binding genes, demonstrating their utility in unraveling regulation of splicing in the human transcriptome. Next, we used a Bayesian biclustering model that identifies network edges unique to a single tissue to reconstruct Tissue-Specific Networks (TSNs) for 26 distinct tissues and 10 groups of related tissues. Finally, we found genetic variants associated with pairs of adjacent nodes in our networks, supporting the estimated network structures and identifying 20 genetic variants with distant regulatory impact on transcription and splicing. Our networks provide an improved understanding of the complex relationships of the human transcriptome across tissues. PMID:29021288

Identification of novel point mutations in splicing sites integrating whole-exome and RNA-seq data in myeloproliferative diseases.

PubMed

Spinelli, Roberta; Pirola, Alessandra; Redaelli, Sara; Sharma, Nitesh; Raman, Hima; Valletta, Simona; Magistroni, Vera; Piazza, Rocco; Gambacorti-Passerini, Carlo

2013-11-01

Point mutations in intronic regions near mRNA splice junctions can affect the splicing process. To identify novel splicing variants from exome sequencing data, we developed a bioinformatics splice-site prediction procedure to analyze next-generation sequencing (NGS) data (SpliceFinder). SpliceFinder integrates two functional annotation tools for NGS, ANNOVAR and MutationTaster and two canonical splice site prediction programs for single mutation analysis, SSPNN and NetGene2. By SpliceFinder, we identified somatic mutations affecting RNA splicing in a colon cancer sample, in eight atypical chronic myeloid leukemia (aCML), and eight CML patients. A novel homozygous splicing mutation was found in APC (NM_000038.4:c.1312+5G>A) and six heterozygous in GNAQ (NM_002072.2:c.735+1C>T), ABCC 3 (NM_003786.3:c.1783-1G>A), KLHDC 1 (NM_172193.1:c.568-2A>G), HOOK 1 (NM_015888.4:c.1662-1G>A), SMAD 9 (NM_001127217.2:c.1004-1C>T), and DNAH 9 (NM_001372.3:c.10242+5G>A). Integrating whole-exome and RNA sequencing in aCML and CML, we assessed the phenotypic effect of mutations on mRNA splicing for GNAQ, ABCC 3, HOOK 1. In ABCC 3 and HOOK 1, RNA-Seq showed the presence of aberrant transcripts with activation of a cryptic splice site or intron retention, validated by the reverse transcription-polymerase chain reaction (RT-PCR) in the case of HOOK 1. In GNAQ, RNA-Seq showed 22% of wild-type transcript and 78% of mRNA skipping exon 5, resulting in a 4-6 frameshift fusion confirmed by RT-PCR. The pipeline can be useful to identify intronic variants affecting RNA sequence by complementing conventional exome analysis.

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

Alternative Splicing of hTERT Pre-mRNA: A Potential Strategy for the Regulation of Telomerase Activity.

PubMed

Liu, Xuewen; Wang, Yuchuan; Chang, Guangming; Wang, Feng; Wang, Fei; Geng, Xin

2017-03-07

The activation of telomerase is one of the key events in the malignant transition of cells, and the expression of human telomerase reverse transcriptase (hTERT) is indispensable in the process of activating telomerase. The pre-mRNA alternative splicing of hTERT at the post-transcriptional level is one of the mechanisms for the regulation of telomerase activity. Shifts in splicing patterns occur in the development, tumorigenesis, and response to diverse stimuli in a tissue-specific and cell type-specific manner. Despite the regulation of telomerase activity, the alternative splicing of hTERT pre-mRNA may play a role in other cellular functions. Modulating the mode of hTERT pre-mRNA splicing is providing a new precept of therapy for cancer and aging-related diseases. This review focuses on the patterns of hTERT pre-mRNA alternative splicing and their biological functions, describes the potential association between the alternative splicing of hTERT pre-mRNA and telomerase activity, and discusses the possible significance of the alternative splicing of the hTERT pre-mRNA in the diagnosis, therapy, and prognosis of cancer and aging-related diseases.

A novel protein factor is required for use of distal alternative 5' splice sites in vitro.

PubMed Central

Harper, J E; Manley, J L

1991-01-01

Adenovirus E1A pre-mRNA was used as a model to examine alternative 5' splice site selection during in vitro splicing reactions. Strong preference for the downstream 13S 5' splice site over the upstream 12S or 9S 5' splice sites was observed. However, the 12S 5' splice site was used efficiently when a mutant pre-mRNA lacking the 13S 5' splice site was processed, and 12S splicing from this substrate was not reduced by 13S splicing from a separate pre-mRNA, demonstrating that 13S splicing reduced 12S 5' splice site selection through a bona fide cis-competition. DEAE-cellulose chromatography of nuclear extract yielded two fractions with different splicing activities. The bound fraction contained all components required for efficient splicing of simple substrates but was unable to utilize alternative 5' splice sites. In contrast, the flow-through fraction, which by itself was inactive, contained an activity required for alternative splicing and was shown to stimulate 12S and 9S splicing, while reducing 13S splicing, when added to reactions carried out by the bound fraction. Furthermore, the activity, which we have called distal splicing factor (DSF), enhanced utilization of an upstream 5' splice site on a simian virus 40 early pre-mRNA, suggesting that the factor acts in a position-dependent, substrate-independent fashion. Several lines of evidence are presented suggesting that DSF is a non-small nuclear ribonucleoprotein protein. Finally, we describe a functional interaction between DSF and ASF, a protein that enhances use of downstream 5' splice sites. Images PMID:1658620

Survival in acute myeloid leukemia is associated with NKp44 splice variants

PubMed Central

Hadad, Uzi; Teltsh, Omri; Edri, Avishay; Rubin, Eitan; Campbell, Kerry S.; Rosental, Benyamin; Porgador, Angel

2016-01-01

NKp44 is a receptor encoded by the NCR2 gene, which is expressed by cytokine-activated natural killer (NK) cells that are involved in anti-AML immunity. NKp44 has three splice variants corresponding to NKp44ITIM+ (NKp44-1) and NKp44ITIM− (NKp44-2, and NKp44-3) isoforms. RNAseq data of AML patients revealed similar survival of NKp46+NKp44+ and NKp46+NKp44− patients. However, if grouped according to the NKp44 splice variant profile, NKp44-1 expression was significantly associated with poor survival of AML patients. Moreover, activation of PBMC from healthy controls showed co-dominant expression of NKp44-1 and NKp44-3, while primary NK clones show more diverse NKp44 splice variant profiles. Cultured primary NK cells resulted in NKp44-1 dominance and impaired function associated with PCNA over-expression by target cells. This impaired functional phenotype could be rescued by blocking of NKp44 receptor. Human NK cell lines revealed co-dominant expression of NKp44-1 and NKp44-3 and showed a functional phenotype that was not inhibited by PCNA over-expression. Furthermore, transfection-based overexpression of NKp44-1, but not NKp44-2/NKp44-3, reversed the endogenous resistance of NK-92 cells to PCNA-mediated inhibition, and resulted in poor formation of stable lytic immune synapses. This research contributes to the understanding of AML prognosis by shedding new light on the functional implications of differential splicing of NKp44. PMID:27102296

SRSF2 mutations drive oncogenesis by activating a global program of aberrant alternative splicing in hematopoietic cells.

PubMed

Liang, Yang; Tebaldi, Toma; Rejeski, Kai; Joshi, Poorval; Stefani, Giovanni; Taylor, Ashley; Song, Yuanbin; Vasic, Radovan; Maziarz, Jamie; Balasubramanian, Kunthavai; Ardasheva, Anastasia; Ding, Alicia; Quattrone, Alessandro; Halene, Stephanie

2018-06-01

Recurrent mutations in the splicing factor SRSF2 are associated with poor clinical outcomes in myelodysplastic syndromes (MDS). Their high frequency suggests these mutations drive oncogenesis, yet the molecular explanation for this process is unclear. SRSF2 mutations could directly affect pre-mRNA splicing of a vital gene product; alternatively, a whole network of gene products could be affected. Here we determine how SRSF2 mutations globally affect RNA binding and splicing in vivo using HITS-CLIP. Remarkably, the majority of differential binding events do not translate into alternative splicing of exons with SRSF2 P95H binding sites. Alternative splice alterations appear to be dominated by indirect effects. Importantly, SRSF2 P95H targets are enriched in RNA processing and splicing genes, including several members of the hnRNP and SR families of proteins, suggesting a "splicing-cascade" phenotype wherein mutation of a single splicing factor leads to widespread modifications in multiple RNA processing and splicing proteins. We show that splice alteration of HNRNPA2B1, a splicing factor differentially bound and spliced by SRSF2 P95H , impairs hematopoietic differentiation in vivo. Our data suggests a model whereby the recurrent mutations in splicing factors set off a cascade of gene regulatory events that together affect hematopoiesis and drive cancer.

Alternatively Spliced Homologous Exons Have Ancient Origins and Are Highly Expressed at the Protein Level

PubMed Central

Abascal, Federico; Ezkurdia, Iakes; Rodriguez-Rivas, Juan; Rodriguez, Jose Manuel; del Pozo, Angela; Vázquez, Jesús; Valencia, Alfonso; Tress, Michael L.

2015-01-01

Alternative splicing of messenger RNA can generate a wide variety of mature RNA transcripts, and these transcripts may produce protein isoforms with diverse cellular functions. While there is much supporting evidence for the expression of alternative transcripts, the same is not true for the alternatively spliced protein products. Large-scale mass spectroscopy experiments have identified evidence of alternative splicing at the protein level, but with conflicting results. Here we carried out a rigorous analysis of the peptide evidence from eight large-scale proteomics experiments to assess the scale of alternative splicing that is detectable by high-resolution mass spectroscopy. We find fewer splice events than would be expected: we identified peptides for almost 64% of human protein coding genes, but detected just 282 splice events. This data suggests that most genes have a single dominant isoform at the protein level. Many of the alternative isoforms that we could identify were only subtly different from the main splice isoform. Very few of the splice events identified at the protein level disrupted functional domains, in stark contrast to the two thirds of splice events annotated in the human genome that would lead to the loss or damage of functional domains. The most striking result was that more than 20% of the splice isoforms we identified were generated by substituting one homologous exon for another. This is significantly more than would be expected from the frequency of these events in the genome. These homologous exon substitution events were remarkably conserved—all the homologous exons we identified evolved over 460 million years ago—and eight of the fourteen tissue-specific splice isoforms we identified were generated from homologous exons. The combination of proteomics evidence, ancient origin and tissue-specific splicing indicates that isoforms generated from homologous exons may have important cellular roles. PMID:26061177

Splicing regulation and dysregulation of cholinergic genes expressed at the neuromuscular junction.

PubMed

Ohno, Kinji; Rahman, Mohammad Alinoor; Nazim, Mohammad; Nasrin, Farhana; Lin, Yingni; Takeda, Jun-Ichi; Masuda, Akio

2017-08-01

We humans have evolved by acquiring diversity of alternative RNA metabolisms including alternative means of splicing and transcribing non-coding genes, and not by acquiring new coding genes. Tissue-specific and developmental stage-specific alternative RNA splicing is achieved by tightly regulated spatiotemporal regulation of expressions and activations of RNA-binding proteins that recognize their cognate splicing cis-elements on nascent RNA transcripts. Genes expressed at the neuromuscular junction are also alternatively spliced. In addition, germline mutations provoke aberrant splicing by compromising binding of RNA-binding proteins, and cause congenital myasthenic syndromes (CMS). We present physiological splicing mechanisms of genes for agrin (AGRN), acetylcholinesterase (ACHE), MuSK (MUSK), acetylcholine receptor (AChR) α1 subunit (CHRNA1), and collagen Q (COLQ) in human, and their aberration in diseases. Splicing isoforms of AChE T , AChE H , and AChE R are generated by hnRNP H/F. Skipping of MUSK exon 10 makes a Wnt-insensitive MuSK isoform, which is unique to human. Skipping of exon 10 is achieved by coordinated binding of hnRNP C, YB-1, and hnRNP L to exon 10. Exon P3A of CHRNA1 is alternatively included to generate a non-functional AChR α1 subunit in human. Molecular dissection of splicing mutations in patients with CMS reveals that exon P3A is alternatively skipped by hnRNP H, polypyrimidine tract-binding protein 1, and hnRNP L. Similarly, analysis of an exonic mutation in COLQ exon 16 in a CMS patient discloses that constitutive splicing of exon 16 requires binding of serine arginine-rich splicing factor 1. Intronic and exonic splicing mutations in CMS enable us to dissect molecular mechanisms underlying alternative and constitutive splicing of genes expressed at the neuromuscular junction. This is an article for the special issue XVth International Symposium on Cholinergic Mechanisms. © 2017 International Society for Neurochemistry.

Identification of alternatively spliced isoforms of interleukin-2/15 receptor β chain in ducks.

PubMed

Jeong, Jipseol; Kim, Woo H; Yeo, Jaeseung; Fernandez, Cherry P; Kim, Suk; Lee, Youn-Jeong; Lillehoj, Hyun S; Min, Wongi

2014-12-15

Interleukin (IL)-2 and IL-15 receptor β (IL-2/15Rβ, CD122) play important roles in signal transduction for biological functions of IL-2 and IL-15. We found that ducks possess three different IL-2/15Rβ transcripts, a conventional form (duIL-2/15Rβ) and two variants. Comparisons between the cDNA and genomic sequences revealed that the two variants, duIL-2/15Rβ-d7 and duIL-2/15Rβ-d9, were novel spliced transcripts resulting from skipping exons 7 and 9, respectively. Expression profiles of duIL-2/15Rβ and its isoforms were examined in healthy tissues, concanavalin A (ConA)-stimulated splenic lymphocytes and in livers and spleens of Riemerella anatipestifer-infected ducks using quantitative real-time PCR (qRT-PCR). Generally, duIL-2/15Rβ-d9 expression was undetectable in healthy tissues, ConA-activated samples, and R. anatipestifer-infected ducks. Expression levels of duIL-2/15Rβ transcript were relatively high to moderate in all healthy tissues tested, while duIL-2/15Rβ-d7 expression was low. Compared to untreated controls, expression levels of duIL-2/15Rβ were elevated in ConA-activated splenic lymphocytes and in livers on day 7 in R. anatipestifer-infected ducks, while duIL-2/15Rβ-d7 expression was unchanged. Additionally, COS-7 cells transfected with duIL-2/15Rβ, duIL-2/15Rβ-d7, or duIL-2/15Rβ-d9 constructs generated 73 kilodalton (kDa), 31kDa, and 40kDa proteins, respectively. This study identified three different IL-2/15Rβ transcripts, including two isoforms generated by alternative splicing and their gene expression patterns in stimulated conditions. Copyright © 2014 Elsevier B.V. All rights reserved.

The neurogenetics of alternative splicing

PubMed Central

Vuong, Celine K.; Black, Douglas L.; Zheng, Sika

2016-01-01

Alternative precursor-mRNA splicing is a key mechanism for regulating gene expression in mammals and is controlled by specialized RNA-binding proteins. The misregulation of splicing is implicated in multiple neurological disorders. We describe recent mouse genetic studies of alternative splicing that reveal its critical role in both neuronal development and the function of mature neurons. We discuss the challenges in understanding the extensive genetic programmes controlled by proteins that regulate splicing, both during development and in the adult brain. PMID:27094079

A role for exon sequences in alternative splicing of the human fibronectin gene.

PubMed Central

Mardon, H J; Sebastio, G; Baralle, F E

1987-01-01

Exon EDIIIA of the fibronectin (Fn) gene is alternatively spliced via pathways which either skip or include the whole exon in the messenger RNA (mRNA). We have investigated the role of EDIIIA exon sequences in the human Fn gene in determining alternative splicing of this exon during transient expression of alpha globin/Fn minigene hybrids in HeLa cells. We demonstrate that a DNA sequence of 81bp within the central region of exon EDIIIA is required for alternative splicing during processing of the primary transcript to generate both EDIIIA+ and EDIIIA- mRNA's. Furthermore, alternative splicing of EDIIIA only occurs when this sequence is present in the correct orientation since when it is in antisense orientation splicing always occurs via exon-skipping generating EDIIIA- mRNA. Images PMID:3671064

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

CDKL5 influences RNA splicing activity by its association to the nuclear speckle molecular machinery.

PubMed

Ricciardi, Sara; Kilstrup-Nielsen, Charlotte; Bienvenu, Thierry; Jacquette, Aurélia; Landsberger, Nicoletta; Broccoli, Vania

2009-12-01

Mutations in the human X-linked cyclin-dependent kinase-like 5 (CDKL5) gene have been shown to cause severe neurodevelopmental disorders including infantile spasms, encephalopathy, West-syndrome and an early-onset variant of Rett syndrome. CDKL5 is a serine/threonine kinase whose involvement in Rett syndrome can be inferred by its ability to directly bind and mediate phosphorylation of MeCP2. However, it remains to be elucidated how CDKL5 exerts its function. Here, we report that CDKL5 localizes to specific nuclear foci referred to as nuclear speckles in both cell lines and tissues. These sub-nuclear structures are traditionally considered as storage/modification sites of pre-mRNA splicing factors. Interestingly, we provide evidence that CDKL5 regulates the dynamic behaviour of nuclear speckles. Indeed, CDKL5 overexpression leads to nuclear speckle disassembly, and this event is strictly dependent on its kinase activity. Conversely, its down-regulation affects nuclear speckle morphology leading to abnormally large and uneven speckles. Similar results were obtained for primary adult fibroblasts isolated from CDKL5-mutated patients. Altogether, these findings indicate that CDKL5 controls nuclear speckle morphology probably by regulating the phosphorylation state of splicing regulatory proteins. Nuclear speckles are dynamic sites that can continuously supply splicing factors to active transcription sites, where splicing occurs. Notably, we proved that CDKL5 influences alternative splicing, at least as proved in heterologous minigene assays. In conclusion, we provide evidence that CDKL5 is involved indirectly in pre-mRNA processing, by controlling splicing factor dynamics. These findings identify a biological process whose disregulation might affect neuronal maturation and activity in CDKL5-related disorders.

Genome-wide analysis of alternative splicing during dendritic cell response to a bacterial challenge.

PubMed

Rodrigues, Raquel; Grosso, Ana Rita; Moita, Luís

2013-01-01

The immune system relies on the plasticity of its components to produce appropriate responses to frequent environmental challenges. Dendritic cells (DCs) are critical initiators of innate immunity and orchestrate the later and more specific adaptive immunity. The generation of diversity in transcriptional programs is central for effective immune responses. Alternative splicing is widely considered a key generator of transcriptional and proteomic complexity, but its role has been rarely addressed systematically in immune cells. Here we used splicing-sensitive arrays to assess genome-wide gene- and exon-level expression profiles in human DCs in response to a bacterial challenge. We find widespread alternative splicing events and splicing factor transcriptional signatures induced by an E. coli challenge to human DCs. Alternative splicing acts in concert with transcriptional modulation, but these two mechanisms of gene regulation affect primarily distinct functional gene groups. Alternative splicing is likely to have an important role in DC immunobiology because it affects genes known to be involved in DC development, endocytosis, antigen presentation and cell cycle arrest.

Extensive alternative splicing transitions during postnatal skeletal muscle development are required for calcium handling functions

PubMed Central

Brinegar, Amy E; Xia, Zheng; Loehr, James Anthony; Li, Wei; Rodney, George Gerald

2017-01-01

Postnatal development of skeletal muscle is a highly dynamic period of tissue remodeling. Here, we used RNA-seq to identify transcriptome changes from late embryonic to adult mouse muscle and demonstrate that alternative splicing developmental transitions impact muscle physiology. The first 2 weeks after birth are particularly dynamic for differential gene expression and alternative splicing transitions, and calcium-handling functions are significantly enriched among genes that undergo alternative splicing. We focused on the postnatal splicing transitions of the three calcineurin A genes, calcium-dependent phosphatases that regulate multiple aspects of muscle biology. Redirected splicing of calcineurin A to the fetal isoforms in adult muscle and in differentiated C2C12 slows the timing of muscle relaxation, promotes nuclear localization of calcineurin target Nfatc3, and/or affects expression of Nfatc transcription targets. The results demonstrate a previously unknown specificity of calcineurin isoforms as well as the broader impact of alternative splicing during muscle postnatal development. PMID:28826478

Radiation-induced alternative transcripts as detected in total and polysome-bound mRNA.

PubMed

Wahba, Amy; Ryan, Michael C; Shankavaram, Uma T; Camphausen, Kevin; Tofilon, Philip J

2018-01-02

Alternative splicing is a critical event in the posttranscriptional regulation of gene expression. To investigate whether this process influences radiation-induced gene expression we defined the effects of ionizing radiation on the generation of alternative transcripts in total cellular mRNA (the transcriptome) and polysome-bound mRNA (the translatome) of the human glioblastoma stem-like cell line NSC11. For these studies, RNA-Seq profiles from control and irradiated cells were compared using the program SpliceSeq to identify transcripts and splice variations induced by radiation. As compared to the transcriptome (total RNA) of untreated cells, the radiation-induced transcriptome contained 92 splice events suggesting that radiation induced alternative splicing. As compared to the translatome (polysome-bound RNA) of untreated cells, the radiation-induced translatome contained 280 splice events of which only 24 were overlapping with the radiation-induced transcriptome. These results suggest that radiation not only modifies alternative splicing of precursor mRNA, but also results in the selective association of existing mRNA isoforms with polysomes. Comparison of radiation-induced alternative transcripts to radiation-induced gene expression in total RNA revealed little overlap (about 3%). In contrast, in the radiation-induced translatome, about 38% of the induced alternative transcripts corresponded to genes whose expression level was affected in the translatome. This study suggests that whereas radiation induces alternate splicing, the alternative transcripts present at the time of irradiation may play a role in the radiation-induced translational control of gene expression and thus cellular radioresponse.

Modulation of voltage- and Ca2+-dependent gating of CaV1.3 L-type calcium channels by alternative splicing of a C-terminal regulatory domain.

PubMed

Singh, Anamika; Gebhart, Mathias; Fritsch, Reinhard; Sinnegger-Brauns, Martina J; Poggiani, Chiara; Hoda, Jean-Charles; Engel, Jutta; Romanin, Christoph; Striessnig, Jörg; Koschak, Alexandra

2008-07-25

Low voltage activation of Ca(V)1.3 L-type Ca(2+) channels controls excitability in sensory cells and central neurons as well as sinoatrial node pacemaking. Ca(V)1.3-mediated pacemaking determines neuronal vulnerability of dopaminergic striatal neurons affected in Parkinson disease. We have previously found that in Ca(V)1.4 L-type Ca(2+) channels, activation, voltage, and calcium-dependent inactivation are controlled by an intrinsic distal C-terminal modulator. Because alternative splicing in the Ca(V)1.3 alpha1 subunit C terminus gives rise to a long (Ca(V)1.3(42)) and a short form (Ca(V)1.3(42A)), we investigated if a C-terminal modulatory mechanism also controls Ca(V)1.3 gating. The biophysical properties of both splice variants were compared after heterologous expression together with beta3 and alpha2delta1 subunits in HEK-293 cells. Activation of calcium current through Ca(V)1.3(42A) channels was more pronounced at negative voltages, and inactivation was faster because of enhanced calcium-dependent inactivation. By investigating several Ca(V)1.3 channel truncations, we restricted the modulator activity to the last 116 amino acids of the C terminus. The resulting Ca(V)1.3(DeltaC116) channels showed gating properties similar to Ca(V)1.3(42A) that were reverted by co-expression of the corresponding C-terminal peptide C(116). Fluorescence resonance energy transfer experiments confirmed an intramolecular protein interaction in the C terminus of Ca(V)1.3 channels that also modulates calmodulin binding. These experiments revealed a novel mechanism of channel modulation enabling cells to tightly control Ca(V)1.3 channel activity by alternative splicing. The absence of the C-terminal modulator in short splice forms facilitates Ca(V)1.3 channel activation at lower voltages expected to favor Ca(V)1.3 activity at threshold voltages as required for modulation of neuronal firing behavior and sinoatrial node pacemaking.

Kinetic and equilibrium properties of regulatory Ca(2+)-binding domains in sodium-calcium exchangers 2 and 3.

PubMed

Tal, Inbal; Kozlovsky, Tom; Brisker, Dafna; Giladi, Moshe; Khananshvili, Daniel

2016-04-01

In mammals, three sodium-calcium exchanger (NCX) protein isoforms (NCX1, NCX2, and NCX3) mediate Ca(2+) fluxes across the membrane to maintain cellular Ca(2+) homeostasis. NCX isoforms and their splice variants are expressed in a tissue-specific manner to meet physiological demands. NCX1 is ubiquitously expressed, NCX2 is expressed in the brain and spinal cord, and NCX3 is expressed in the brain and skeletal muscle. Eukaryotic NCXs contain two cytosolic regulatory Ca(2+)-binding domains, CBD1 and CBD2, which form a two-domain tandem (CBD12) through a short linker. Ca(2+) binding to the CBDs underlies allosteric regulation of NCX. Previous structural and functional studies in NCX1 have shown that the CBDs synergistically interact, where their interactions are modulated in a splice variant-specific manner by splicing segment at CBD2. Here, we analyze the equilibrium and kinetic properties of Ca(2+) binding to purified preparations of CBD1, CBD2, and CBD12 from NCX2 and from NCX3 splice variants. We show that CBD1 interacts with CBD2 in the context of the CBD12 tandem in all NCX isoforms, where these interactions specifically modulate Ca(2+) sensing at the primary sensor of CBD1 to meet the physiological requirements. For example, the rate-limiting slow dissociation of "occluded" Ca(2+) from the primary allosteric sensor of variants expressed in skeletal muscle is ∼10-fold slower than that of variants expressed in the brain. Notably, these kinetic differences between NCX variants occur while maintaining a similar Ca(2+) affinity of the primary sensor, since the resting [Ca(2+)]i levels are similar among different cell types. Copyright © 2016 Elsevier Ltd. All rights reserved.

Regulation of alternative mRNA splicing: old players and new perspectives.

PubMed

Dvinge, Heidi

2018-06-01

Nearly all human multi-exon genes are subject to alternative splicing in one or more cell types. The splicing machinery, therefore, has to select between multiple splice sites in a context-dependent manner, relying on sequence features in cis and trans-acting splicing regulators that either promote or repress splice site recognition and spliceosome assembly. However, the functional coupling between multiple gene regulatory layers signifies that splicing can also be modulated by transcriptional or epigenetic characteristics. Other, less obvious, aspects of alternative splicing have come to light in recent years, often involving core components of the spliceosome previously thought to perform a basal rather than a regulatory role in splicing. Together this paints a highly dynamic picture of splicing regulation, where the final splice site choice is governed by the entire transcriptional environment of a gene and its cellular context. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Characterization of an apparently synonymous F5 mutation causing aberrant splicing and factor V deficiency.

PubMed

Nuzzo, F; Bulato, C; Nielsen, B I; Lee, K; Wielders, S J; Simioni, P; Key, N S; Castoldi, E

2015-03-01

Coagulation factor V (FV) deficiency is a rare autosomal recessive bleeding disorder. We investigated a patient with severe FV deficiency (FV:C < 3%) and moderate bleeding symptoms. Thrombin generation experiments showed residual FV expression in the patient's plasma, which was quantified as 0.7 ± 0.3% by a sensitive prothrombinase-based assay. F5 gene sequencing identified a novel missense mutation in exon 4 (c.578G>C, p.Cys193Ser), predicting the abolition of a conserved disulphide bridge, and an apparently synonymous variant in exon 8 (c.1281C>G). The observation that half of the patient's F5 mRNA lacked the last 18 nucleotides of exon 8 prompted us to re-evaluate the c.1281C>G variant for its possible effects on splicing. Bioinformatics sequence analysis predicted that this transversion would activate a cryptic donor splice site and abolish an exonic splicing enhancer. Characterization in a F5 minigene model confirmed that the c.1281C>G variant was responsible for the patient's splicing defect, which could be partially corrected by a mutation-specific morpholino antisense oligonucleotide. The aberrantly spliced F5 mRNA, whose stability was similar to that of the normal mRNA, encoded a putative FV mutant lacking amino acids 427-432. Expression in COS-1 cells indicated that the mutant protein is poorly secreted and not functional. In conclusion, the c.1281C>G mutation, which was predicted to be translationally silent and hence neutral, causes FV deficiency by impairing pre-mRNA splicing. This finding underscores the importance of cDNA analysis for the correct assessment of exonic mutations. © 2014 John Wiley & Sons Ltd.

Interplay between Alternative Splicing and Alternative Polyadenylation Defines the Expression Outcome of the Plant Unique OXIDATIVE TOLERANT-6 Gene.

PubMed

Li, Qingshun Q; Liu, Zhaoyang; Lu, Wenjia; Liu, Man

2017-05-17

Pre-mRNA alternative splicing and alternative polyadenylation have been implicated to play important roles during eukaryotic gene expression. However, much remains unknown regarding the regulatory mechanisms and the interactions of these two processes in plants. Here we focus on an Arabidopsis gene OXT6 (Oxidative Tolerant-6) that has been demonstrated to encode two proteins through alternative splicing and alternative polyadenylation. Specifically, alternative polyadenylation at Intron-2 of OXT6 produces a transcript coding for AtCPSF30, an Arabidopsis ortholog of 30 kDa subunit of the Cleavage and Polyadenylation Specificity Factor. On the other hand, alternative splicing of Intron-2 generates a longer transcript encoding a protein named AtC30Y, a polypeptide including most part of AtCPSF30 and a YT521B domain. To investigate the expression outcome of OXT6 in plants, a set of mutations were constructed to alter the splicing and polyadenylation patterns of OXT6. Analysis of transgenic plants bearing these mutations by quantitative RT-PCR revealed a competition relationship between these two processes. Moreover, when both splice sites and poly(A) signals were mutated, polyadenylation became the preferred mode of OXT6 processing. These results demonstrate the interplay between alternative splicing and alternative polyadenylation, and it is their concerted actions that define a gene's expression outcome.

A novel splice variant of the Fas gene in patients with cutaneous T-cell lymphoma.

PubMed

van Doorn, Remco; Dijkman, Remco; Vermeer, Maarten H; Starink, Theo M; Willemze, Rein; Tensen, Cornelis P

2002-10-01

Defective apoptosis signaling has been implicated in the pathogenesis of primary cutaneous T-cell lymphomas (CTCLs), a group of malignancies derived from skin-homing T cells. An important mediator of apoptosis in T cells is the Fas receptor. We identified a novel splice variant of the Fas gene that displays retention of intron 5 and encodes a dysfunctional Fas protein in 13 of 22 patients (59%) in both early and advanced CTCL. Impairment of Fas-induced apoptosis resulting from aberrant splicing potentially contributes to the development and progression of CTCL by allowing continued clonal expansion of activated T cells and by reducing susceptibility to antitumor immune responses.

Habituation, discrimination and anxiety in transgenic mice overexpressing acetylcholinesterase splice variants.

PubMed

Kofman, Ora; Shavit, Yehoshua; Ashkenazi, Sarit; Gabay, Shai

2007-12-14

TgS and TgR transgenic mice overexpress different splice variants of acetylcholinesterase and serve as models for genetic disruption of the cholinergic system. Whereas the TgS mouse overexpresses synaptic AChE, the TgR mouse overexpresses the rare readthrough variant whose C-terminal lacks the cysteine residue which permits adherence to the membrane. The two genotypes were compared to the parent strain, FVB/N mice on locomotion, discrimination learning and anxiety behavior following two exposures to the elevated plus maze. Male TgS mice were slower to acquire a simple odor discrimination, failed to habituate to a novel environment but were not impaired on reversal or set shifting compared to the FVB/N or TgR mice. In addition, TgS mice showed less avoidance behavior on the first exposure and but less exploration on the second exposure to the EPM. TgR mice were not impaired on discrimination learning; however, the females showed excessive running in circles in the activity meter. The findings suggest that the effects of overexpression of AChE are unique to different splice variants and may be sex-dependent.

Diversification of the muscle proteome through alternative splicing.

PubMed

Nakka, Kiran; Ghigna, Claudia; Gabellini, Davide; Dilworth, F Jeffrey

2018-03-06

Skeletal muscles express a highly specialized proteome that allows the metabolism of energy sources to mediate myofiber contraction. This muscle-specific proteome is partially derived through the muscle-specific transcription of a subset of genes. Surprisingly, RNA sequencing technologies have also revealed a significant role for muscle-specific alternative splicing in generating protein isoforms that give specialized function to the muscle proteome. In this review, we discuss the current knowledge with respect to the mechanisms that allow pre-mRNA transcripts to undergo muscle-specific alternative splicing while identifying some of the key trans-acting splicing factors essential to the process. The importance of specific splicing events to specialized muscle function is presented along with examples in which dysregulated splicing contributes to myopathies. Though there is now an appreciation that alternative splicing is a major contributor to proteome diversification, the emergence of improved "targeted" proteomic methodologies for detection of specific protein isoforms will soon allow us to better appreciate the extent to which alternative splicing modifies the activity of proteins (and their ability to interact with other proteins) in the skeletal muscle. In addition, we highlight a continued need to better explore the signaling pathways that contribute to the temporal control of trans-acting splicing factor activity to ensure specific protein isoforms are expressed in the proper cellular context. An understanding of the signal-dependent and signal-independent events driving muscle-specific alternative splicing has the potential to provide us with novel therapeutic strategies to treat different myopathies.

From Genomes to Protein Models and Back

NASA Astrophysics Data System (ADS)

Tramontano, Anna; Giorgetti, Alejandro; Orsini, Massimiliano; Raimondo, Domenico

2007-12-01

The alternative splicing mechanism allows genes to generate more than one product. When the splicing events occur within protein coding regions they can modify the biological function of the protein. Alternative splicing has been suggested as one way for explaining the discrepancy between the number of human genes and functional complexity. We analysed the putative structure of the alternatively spliced gene products annotated in the ENCODE pilot project and discovered that many of the potential alternative gene products will be unlikely to produce stable functional proteins.

Alternative Splicing in the Hippo Pathway—Implications for Disease and Potential Therapeutic Targets

PubMed Central

Porazinski, Sean; Ladomery, Michael

2018-01-01

Alternative splicing is a well-studied gene regulatory mechanism that produces biological diversity by allowing the production of multiple protein isoforms from a single gene. An involvement of alternative splicing in the key biological signalling Hippo pathway is emerging and offers new therapeutic avenues. This review discusses examples of alternative splicing in the Hippo pathway, how deregulation of these processes may contribute to disease and whether these processes offer new potential therapeutic targets. PMID:29534050

High-throughput sequence analysis of Ciona intestinalis SL trans-spliced mRNAs: alternative expression modes and gene function correlates.

PubMed

Matsumoto, Jun; Dewar, Ken; Wasserscheid, Jessica; Wiley, Graham B; Macmil, Simone L; Roe, Bruce A; Zeller, Robert W; Satou, Yutaka; Hastings, Kenneth E M

2010-05-01

Pre-mRNA 5' spliced-leader (SL) trans-splicing occurs in some metazoan groups but not in others. Genome-wide characterization of the trans-spliced mRNA subpopulation has not yet been reported for any metazoan. We carried out a high-throughput analysis of the SL trans-spliced mRNA population of the ascidian tunicate Ciona intestinalis by 454 Life Sciences (Roche) pyrosequencing of SL-PCR-amplified random-primed reverse transcripts of tailbud embryo RNA. We obtained approximately 250,000 high-quality reads corresponding to 8790 genes, approximately 58% of the Ciona total gene number. The great depth of this data revealed new aspects of trans-splicing, including the existence of a significant class of "infrequently trans-spliced" genes, accounting for approximately 28% of represented genes, that generate largely non-trans-spliced mRNAs, but also produce trans-spliced mRNAs, in part through alternative promoter use. Thus, the conventional qualitative dichotomy of trans-spliced versus non-trans-spliced genes should be supplanted by a more accurate quantitative view recognizing frequently and infrequently trans-spliced gene categories. Our data include reads representing approximately 80% of Ciona frequently trans-spliced genes. Our analysis also revealed significant use of closely spaced alternative trans-splice acceptor sites which further underscores the mechanistic similarity of cis- and trans-splicing and indicates that the prevalence of +/-3-nt alternative splicing events at tandem acceptor sites, NAGNAG, is driven by spliceosomal mechanisms, and not nonsense-mediated decay, or selection at the protein level. The breadth of gene representation data enabled us to find new correlations between trans-splicing status and gene function, namely the overrepresentation in the frequently trans-spliced gene class of genes associated with plasma/endomembrane system, Ca(2+) homeostasis, and actin cytoskeleton.

Molecular characterization of a CpTRIM35-like protein and its splice variants from whitespotted bamboo shark (Chiloscyllium plagiosum)

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

Zhang, Xinshang, E-mail: sanmaosound@163.com; Zhao, Heng, E-mail: hengzhao2000@gmail.com; Chen, Yeyu, E-mail: cyyleaf@126.com

2014-10-24

Highlights: • A TRIM gene and three splice variants were firstly cloned from elasmobranch fish. • The genes were constitutively expressed with high levels in spleen and kidney. • The gene products were distributed in cytoplasm alone or cytoplasm and nucleus. • As E3 ubiquitin ligases, the proteins differed in immune responses to challenges. - Abstract: The tripartite motif (TRIM) proteins play important roles in a broad range of biological processes, including apoptosis, cell proliferation and innate immunity response. In this study, a TRIM gene and its three splice variants were cloned from an elasmobranch fish—whitespotted bamboo shark (Chiloscyllium plagiosummore » Bennett). Phylogenetic analysis indicated that the gene was closely related to TRIM35 homologs, thus termed CpTRIM35-like. Deduced CpTRIM35 has a RBCC-PRY/SPRY structure typical of TRIM proteins, and its splice variants (CpTRIM35-1–3) have different truncations at the C-terminus. The gene products were constitutively expressed in adult sharks with the highest levels in spleen and kidney. The different subcellular locations, upregulation upon LPS and poly I:C stimulation, and significant E3 ubiquitin ligase activities suggested their different roles in immune responses as an E3 ubiquitin ligase. This is the first TRIM protein ever characterized in elasmobranch fish.« less

In vivo effects on intron retention and exon skipping by the U2AF large subunit and SF1/BBP in the nematode Caenorhabditis elegans

PubMed Central

Ma, Long; Tan, Zhiping; Teng, Yanling; Hoersch, Sebastian; Horvitz, H. Robert

2011-01-01

The in vivo analysis of the roles of splicing factors in regulating alternative splicing in animals remains a challenge. Using a microarray-based screen, we identified a Caenorhabditis elegans gene, tos-1, that exhibited three of the four major types of alternative splicing: intron retention, exon skipping, and, in the presence of U2AF large subunit mutations, the use of alternative 3′ splice sites. Mutations in the splicing factors U2AF large subunit and SF1/BBP altered the splicing of tos-1. 3′ splice sites of the retained intron or before the skipped exon regulate the splicing pattern of tos-1. Our study provides in vivo evidence that intron retention and exon skipping can be regulated largely by the identities of 3′ splice sites. PMID:22033331

Regulation of alternative splicing in Drosophila by 56 RNA binding proteins

DOE PAGES

Brooks, Angela N.; Duff, Michael O.; May, Gemma; ...

2015-08-20

Alternative splicing is regulated by RNA binding proteins (RBPs) that recognize pre-mRNA sequence elements and activate or repress adjacent exons. Here, we used RNA interference and RNA-seq to identify splicing events regulated by 56 Drosophila proteins, some previously unknown to regulate splicing. Nearly all proteins affected alternative first exons, suggesting that RBPs play important roles in first exon choice. Half of the splicing events were regulated by multiple proteins, demonstrating extensive combinatorial regulation. We observed that SR and hnRNP proteins tend to act coordinately with each other, not antagonistically. We also identified a cross-regulatory network where splicing regulators affected themore » splicing of pre-mRNAs encoding other splicing regulators. In conclusion, this large-scale study substantially enhances our understanding of recent models of splicing regulation and provides a resource of thousands of exons that are regulated by 56 diverse RBPs.« less

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.

Endothelium-derived fibronectin regulates neonatal vascular morphogenesis in an autocrine fashion.

PubMed

Turner, Christopher J; Badu-Nkansah, Kwabena; Hynes, Richard O

2017-11-01

Fibronectin containing alternatively spliced EIIIA and EIIIB domains is largely absent from mature quiescent vessels in adults, but is highly expressed around blood vessels during developmental and pathological angiogenesis. The precise functions of fibronectin and its splice variants during developmental angiogenesis however remain unclear due to the presence of cardiac, somitic, mesodermal and neural defects in existing global fibronectin KO mouse models. Using a rare family of surviving EIIIA EIIIB double KO mice, as well as inducible endothelial-specific fibronectin-deficient mutant mice, we show that vascular development in the neonatal retina is regulated in an autocrine manner by endothelium-derived fibronectin, and requires both EIIIA and EIIIB domains and the RGD-binding α5 and αv integrins for its function. Exogenous sources of fibronectin do not fully substitute for the autocrine function of endothelial fibronectin, demonstrating that fibronectins from different sources contribute differentially to specific aspects of angiogenesis.

High throughput microscopy identifies bisphenol AP, a bisphenol A analog, as a novel AR down-regulator.

PubMed

Stossi, Fabio; Dandekar, Radhika D; Bolt, Michael J; Newberg, Justin Y; Mancini, Maureen G; Kaushik, Akash K; Putluri, Vasanta; Sreekumar, Arun; Mancini, Michael A

2016-03-29

Prostate cancer remains a deadly disease especially when patients become resistant to drugs that target the Androgen Receptor (AR) ligand binding domain. At this stage, patients develop recurring castrate-resistant prostate cancers (CRPCs). Interestingly, CRPC tumors maintain dependency on AR for growth; moreover, in CRPCs, constitutively active AR splice variants (e.g., AR-V7) begin to be expressed at higher levels. These splice variants lack the ligand binding domain and are rendered insensitive to current endocrine therapies. Thus, it is of paramount importance to understand what regulates the expression of AR and its splice variants to identify new therapeutic strategies in CRPCs. Here, we used high throughput microscopy and quantitative image analysis to evaluate effects of selected endocrine disruptors on AR levels in multiple breast and prostate cancer cell lines. Bisphenol AP (BPAP), which is used in chemical and medical industries, was identified as a down-regulator of both full length AR and the AR-V7 splice variant. We validated its activity by performing time-course, dose-response, Western blot and qPCR analyses. BPAP also reduced the percent of cells in S phase, which was accompanied by a ~60% loss in cell numbers and colony formation in anchorage-independent growth assays. Moreover, it affected mitochondria size and cell metabolism. In conclusion, our high content analysis-based screening platform was used to classify the effect of compounds on endogenous ARs, and identified BPAP as being capable of causing AR (both full-length and variants) down-regulation, cell cycle arrest and metabolic alterations in CRPC cell lines.

A novel AVPR2 splice site mutation leads to partial X-linked nephrogenic diabetes insipidus in two brothers.

PubMed

Schernthaner-Reiter, Marie Helene; Adams, David; Trivellin, Giampaolo; Ramnitz, Mary Scott; Raygada, Margarita; Golas, Gretchen; Faucz, Fabio R; Nilsson, Ola; Nella, Aikaterini A; Dileepan, Kavitha; Lodish, Maya; Lee, Paul; Tifft, Cynthia; Markello, Thomas; Gahl, William; Stratakis, Constantine A

2016-05-01

X-linked nephrogenic diabetes insipidus (NDI, OMIM#304800) is caused by mutations in the arginine vasopressin (AVP, OMIM*192340) receptor type 2 (AVPR2, OMIM*300538) gene. A 20-month-old boy and his 8-year-old brother presented with polyuria, polydipsia, and failure to thrive. Both boys demonstrated partial DDAVP (1-desamino-8-D AVP or desmopressin) responses; thus, NDI diagnosis was delayed. While routine sequencing of AVPR2 showed a potential splice site variant, it was not until exome sequencing confirmed the AVPR2 splice site variant and did not reveal any more likely candidates that the patients' diagnosis was made and proper treatment was instituted. Both patients were hemizygous for two AVPR2 variants predicted in silico to affect AVPR2 messenger RNA (mRNA) splicing. A minigene assay revealed that the novel AVPR2 c.276A>G mutation creates a novel splice acceptor site leading to 5' truncation of AVPR2 exon 2 in HEK293 human kidney cells. Both patients have been treated with high-dose DDAVP with a remarkable improvement of their symptoms and accelerated linear growth and weight gain. We present here a unique case of partial X-linked NDI due to an AVPR2 splice site mutation; patients with diabetes insipidus of unknown etiology may harbor splice site mutations that are initially underestimated in their pathogenicity on sequence analysis. • X-linked nephrogenic diabetes insipidus is caused by AVPR2 mutations, and disease severity can vary depending on the functional effect of the mutation. What is New: • We demonstrate here that a splice site mutation in AVPR2 leads to partial X-linked NDI in two brothers. • Treatment with high-dose DDAVP led to improvement of polyuria and polydipsia, weight gain, and growth.

TGFβ1-mediated PI3K/Akt and p38 MAP kinase dependent alternative splicing of fibronectin extra domain A in human podocyte culture.

PubMed

Madne, Tarunkumar Hemraj; Dockrell, Mark Edward Carl

2018-04-30

Alternative splicing is an important gene regulation process to distribute proteins in health and diseases. Extra Domain A+ Fibronectin (EDA+Fn) is an alternatively spliced form of fibronectin (Fn) protein, present in the extra cellular matrix (ECM) and a recognised marker of various pathologies. TGFβ1 has been shown to induce alternative splicing of EDA+Fn in many cell types. Podocytes are spectacular cell type and play a key role in filtration and synthesise ECM proteins in renal physiology and pathology. In our previous study we have demonstrated expression and alternative splicing of EDA+Fn in basal condition in human podocytes culture. TGFβ1 further induced the basal expression and alternative splicing of EDA+Fn through Alk5 receptor and SR proteins. In this study, we have investigated TGFβ1 mediated signalling involved in alternative splicing of EDA+Fn in human podocytes. We have performed western blotting to characterise the expression of the EDA+Fn protein and other signalling proteins and RT-PCR to look for signalling pathways involved in regulation of alternative splicing of EDA+Fn in conditionally immortalised human podocytes culture.We have used TGFβ1 as a stimulator and SB431542, SB202190 and LY294002 for inhibitory studies. In this work, we have demonstrated in human podocytes culture TGFβ1 2.5ng/ml induced phosphorylation of Smad1/5/8, Smad2 and Smad3 via the ALK5 receptor. TGFβ1 significantly induced the PI3K/Akt pathway and the PI3K/Akt pathway inhibitor LY294002 significantly downregulated basal as well as TGFβ1 induced alternative splicing of EDA+Fn in human podocytes. In addition to this, TGFβ1 significantly induced the p38 MAP kinase signalling pathway and p38 MAP kinase signalling pathway inhibitor SB202190 downregulated the TGFβ1-mediated alternative splicing of EDA+Fn in human podocytes. The results with PI3K and p38 MAP kinase signalling pathway suggest that inhibiting PI3K signalling pathway downregulated the basal alternative splicing of EDA+Fn in human podocytes and its the inhibition of p38 Map Kinase signalling pathway which had specifically downregulated the TGFβ1 mediated alternative splicing of EDA+Fn in human podocytes culture. Activation of TGFβ1-mediated Smad1/5/8 via Alk5 receptor suggests that TGFβ1 signalling pathway involved Alk5/Alk1 receptor axis signalling in human podocytes.

Effects of prenatal stress and monoaminergic perturbations on the expression of serotonin 5-HT₄ and adrenergic β₂ receptors in the embryonic mouse telencephalon.

PubMed

Chen, Angela; Kelley, Lauren D S; Janušonis, Skirmantas

2012-06-12

The serotonin 5-HT(4) receptor (5-HT(4)R) is coded by a complex gene that produces four mRNA splice variants in mice (5-HT(4(a))R, 5-HT(4(b))R, 5-HT(4(e))R, 5-HT(4(f))R). This receptor has highly dynamic expression in brain development and its splice variants differ in their developmental trajectories. Since 5-HT(4)Rs are important in forebrain function (including forebrain control of serotonergic activity in the brainstem), we investigated the susceptibility of 5-HT(4)R expression in the mouse embryonic telencephalon to prenatal maternal stress and altered serotonin (5-hydroxytryptamine, 5-HT) levels. Because the gene coding the adrenergic β(2) receptor (β(2)AR) is embedded in the 5-HT(4)R gene, we also investigated whether 5-HT(4)R mRNA levels were modulated by selective β(2)AR agents. Timed-pregnant C57BL/6 mice were treated beginning at embryonic day (E) 14 and quantitative reverse-transcription polymerase chain reaction (qRT-PCR) was used to assess the mRNA levels of all 5-HT(4)R splice variants and β(2)AR in the embryonic telencephalon at E17. Maternal prenatal stress and 5-HT depletion with pCPA, a tryptophan hydroxylase inhibitor, reduced the levels of the 5-HT(4(b))R splice variant. Terbutaline (a selective β(2)AR agonist) and ICI 118,551 (a selective β(2)AR antagonist) had no effect on β(2)AR and 5-HT(4)R mRNA levels. These results show that prenatal stress and reduced 5-HT levels can alter 5-HT(4)R expression in the developing forebrain and that some 5-HT(4)R splice variants may be more susceptible than others. Copyright © 2012 Elsevier B.V. All rights reserved.

Peptidic tools applied to redirect alternative splicing events.

PubMed

Nancy, Martínez-Montiel; Nora, Rosas-Murrieta; Rebeca, Martínez-Contreras

2015-05-01

Peptides are versatile and attractive biomolecules that can be applied to modulate genetic mechanisms like alternative splicing. In this process, a single transcript yields different mature RNAs leading to the production of protein isoforms with diverse or even antagonistic functions. During splicing events, errors can be caused either by mutations present in the genome or by defects or imbalances in regulatory protein factors. In any case, defects in alternative splicing have been related to several genetic diseases including muscular dystrophy, Alzheimer's disease and cancer from almost every origin. One of the most effective approaches to redirect alternative splicing events has been to attach cell-penetrating peptides to oligonucleotides that can modulate a single splicing event and restore correct gene expression. Here, we summarize how natural existing and bioengineered peptides have been applied over the last few years to regulate alternative splicing and genetic expression. Under different genetic and cellular backgrounds, peptides have been shown to function as potent vehicles for splice correction, and their therapeutic benefits have reached clinical trials and patenting stages, emphasizing the use of regulatory peptides as an exciting therapeutic tool for the treatment of different genetic diseases. Copyright © 2015 Elsevier Inc. All rights reserved.

[AR-V7 Androgen Receptor Variant as a Predictor of Response to Androgen-receptor Targeting Agents Used to Treat Castration-refractory Metastatic Prostate Cancer].

PubMed

Büchler, Tomáš; Bobek, Vladimír; Kološtová, Katarína

2017-01-01

Several systemic treatment options are currently available for patients with metastatic castration-refractory prostate cancer (mCRPC), including the androgen-receptor targeting agents (ARTA) enzalutamide and abiraterone, the taxanes docetaxel and cabazitaxel, and the radioisotope drug 223-radium dichloride. In some patients with mCRCP, alternative splicing of androgen receptor (AR) mRNA occurs, resulting in the formation of a truncated AR lacking the androgen-binding domain. These receptors activate downstream signalling pathways even without the ligand. Recent studies show that the presence of the AR-V7 (ARV - AR variants) splicing variant is associated with resistance to ARTA. Bec>ause the presence of AR-V7 does not affect the efficacy of other systemic therapies used in mCRCPs, particularly taxanes, AR-V7 is a candidate predictive biomarker for the individualisation of mCRCP treatment. Two types of assays based on mRNA or abnormal protein detection are used to detect AR-V7 in circulating tumour cells. To describe the current status of AR-V7 testing in mCRPC and possible applications of this method for predicting outcomes of ARTA therapy. The percentage of CTC AR-V7+ in ARTA-naive men is relatively low at baseline, but in patients pretreated with ARTA, the prevalence of AR-V7 increases to 19-34%. Given the relatively high expected prevalence, AR-V7 testing may be economically feasible in this population. The proportion of AR-V7+ patients responding to ARTA retreatment appears to be very low, at only 4.8%. AR-V7 testing could thus be useful if an ARTA switch is considered in a patient progressing onto an ARTA drug. Both protein-based tests and mRNA-based tests are currently undergoing clinical validation in prospective studies, with results expected within a year.Key words: prostate cancer - abiraterone - enzalutamide - alternative splicing - drug resistanceSubmitted: 30. 8. 2017Accepted: 5. 11. 2017 doc. MUDr. Tomáš Büchler, Ph.D. received honorary lectures and publications from Astellas and Janssen and a travel grant from Janssen. Supported by Ministry of Health, Czech Republic - conceptual development of research organization Thomayer Hospital - TN 0064190. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers.

Co-expression networks reveal the tissue-specific regulation of transcription and splicing.

PubMed

Saha, Ashis; Kim, Yungil; Gewirtz, Ariel D H; Jo, Brian; Gao, Chuan; McDowell, Ian C; Engelhardt, Barbara E; Battle, Alexis

2017-11-01

Gene co-expression networks capture biologically important patterns in gene expression data, enabling functional analyses of genes, discovery of biomarkers, and interpretation of genetic variants. Most network analyses to date have been limited to assessing correlation between total gene expression levels in a single tissue or small sets of tissues. Here, we built networks that additionally capture the regulation of relative isoform abundance and splicing, along with tissue-specific connections unique to each of a diverse set of tissues. We used the Genotype-Tissue Expression (GTEx) project v6 RNA sequencing data across 50 tissues and 449 individuals. First, we developed a framework called Transcriptome-Wide Networks (TWNs) for combining total expression and relative isoform levels into a single sparse network, capturing the interplay between the regulation of splicing and transcription. We built TWNs for 16 tissues and found that hubs in these networks were strongly enriched for splicing and RNA binding genes, demonstrating their utility in unraveling regulation of splicing in the human transcriptome. Next, we used a Bayesian biclustering model that identifies network edges unique to a single tissue to reconstruct Tissue-Specific Networks (TSNs) for 26 distinct tissues and 10 groups of related tissues. Finally, we found genetic variants associated with pairs of adjacent nodes in our networks, supporting the estimated network structures and identifying 20 genetic variants with distant regulatory impact on transcription and splicing. Our networks provide an improved understanding of the complex relationships of the human transcriptome across tissues. © 2017 Saha et al.; Published by Cold Spring Harbor Laboratory Press.

Adaptive Significance of ERα Splice Variants in Killifish ...

EPA Pesticide Factsheets

The possibility that chronic, multigenerational exposure to environmental estrogens selects for adaptive hormone response phenotypes is a critical unanswered question. Embryos/larvae of killifish from an estrogenic polluted environment (New Bedford Harbor, NBH), as compared to those from a reference site, overexpress estrogen receptor a (ERa) mRNA but are hypo-responsive to estradiol (E2). Analysis of ERa mRNAs in the two populations revealed differences in splicing of the gene encoding ERa (esr1). Here we tested the transactivation functions of four differentially expressed ERa mRNAs and tracked their association with the hypo-responsive phenotype for three generations after transfer of NBH parents to a clean environment. Deletion variants ERaΔ6 and ERaΔ6 – 8 were specific to NBH killifish; had dominant negative functions in an in vitro reporter assay; and were heritable. Morpholino-mediated induction of ERaΔ6 mRNA in zebrafish embryos verified its role as a dominant negative ER on natural estrogen-responsive promoters. Alternate long (ERaL) and short (ERaS) 5'-variants were similar transcriptionally but differed in estrogen responsiveness (ERaS >> ERaL). ERaS accounted for high total ERa expression in F1 NBH embryos/ larvae but this trait was abolished by transfer to clean water. By contrast, the hypo-responsive phenotype of F1 NBH embryos/larvae persisted after long term lab holding but reverted to a normal or hyper-responsive phenotype after two or thre

Exploiting differential RNA splicing patterns: a potential new group of therapeutic targets in cancer.

PubMed

Jyotsana, Nidhi; Heuser, Michael

2018-02-01

Mutations in genes associated with splicing have been found in hematologic malignancies, but also in solid cancers. Aberrant cancer specific RNA splicing either results from mutations or misexpression of the spliceosome genes directly, or from mutations in splice sites of oncogenes or tumor suppressors. Areas covered: In this review, we present molecular targets of aberrant splicing in various malignancies, information on existing and emerging therapeutics against such targets, and strategies for future drug development. Expert opinion: Alternative splicing is an important mechanism that controls gene expression, and hence pharmacologic and genetic control of aberrant alternative RNA splicing has been proposed as a potential therapy in cancer. To identify and validate aberrant RNA splicing patterns as therapeutic targets we need to (1) characterize the most common genetic aberrations of the spliceosome and of splice sites, (2) understand the dysregulated downstream pathways and (3) exploit in-vivo disease models of aberrant splicing. Antisense oligonucleotides show promising activity, but will benefit from improved delivery tools. Inhibitors of mutated splicing factors require improved specificity, as alternative and aberrant splicing are often intertwined like two sides of the same coin. In summary, targeting aberrant splicing is an early but emerging field in cancer treatment.

A Novel Human Ghrelin Variant (In1-Ghrelin) and Ghrelin-O-Acyltransferase Are Overexpressed in Breast Cancer: Potential Pathophysiological Relevance

PubMed Central

Gahete, Manuel D.; Córdoba-Chacón, José; Hergueta-Redondo, Marta; Martínez-Fuentes, Antonio J.; Kineman, Rhonda D.; Moreno-Bueno, Gema

2011-01-01

The human ghrelin gene, which encodes the ghrelin and obestatin peptides, contains 5 exons (Ex), with Ex1-Ex4 encoding a 117 amino-acid (aa) preproprotein that is known to be processed to yield a 28-aa (ghrelin) and/or a 23-aa (obestatin) mature peptides, which possess biological activities in multiple tissues. However, the ghrelin gene also encodes additional peptides through alternative splicing or post-translational modifications. Indeed, we previously identified a spliced mRNA ghrelin variant in mouse (In2-ghrelin-variant), which is regulated in a tissue-dependent manner by metabolic status and may thus be of biological relevance. Here, we have characterized a new human ghrelin variant that contains Ex0-1, intron (In) 1, and Ex2 and lacks Ex3-4. This human In1-ghrelin variant would encode a new prepropeptide that conserves the first 12aa of native-ghrelin (including the Ser3-potential octanoylation site) but has a different C-terminal tail. Expression of In1-variant was detected in 22 human tissues and its levels were positively correlated with those of ghrelin-O-acyltransferase (GOAT; p = 0.0001) but not with native-ghrelin expression, suggesting that In1-ghrelin could be a primary substrate for GOAT in human tissues. Interestingly, levels of In1-ghrelin variant expression in breast cancer samples were 8-times higher than those of normal mammary tissue, and showed a strong correlation in breast tumors with GOAT (p = 0.0001), ghrelin receptor-type 1b (GHSR1b; p = 0.049) and cyclin-D3 (a cell-cycle inducer/proliferation marker; p = 0.009), but not with native-ghrelin or GHSR1a expression. Interestingly, In1-ghrelin variant overexpression increased basal proliferation of MDA-MB-231 breast cancer cells. Taken together, our results provide evidence that In1-ghrelin is a novel element of the ghrelin family with a potential pathophysiological role in breast cancer. PMID:21829727

Functions for fission yeast splicing factors SpSlu7 and SpPrp18 in alternative splice-site choice and stress-specific regulated splicing.

PubMed

Melangath, Geetha; Sen, Titash; Kumar, Rakesh; Bawa, Pushpinder; Srinivasan, Subha; Vijayraghavan, Usha

2017-01-01

Budding yeast spliceosomal factors ScSlu7 and ScPrp18 interact and mediate intron 3'ss choice during second step pre-mRNA splicing. The fission yeast genome with abundant multi-intronic transcripts, degenerate splice signals and SR proteins is an apt unicellular fungal model to deduce roles for core spliceosomal factors in alternative splice-site choice, intron retention and to study the cellular implications of regulated splicing. From our custom microarray data we deduce a stringent reproducible subset of S. pombe alternative events. We examined the role of factors SpSlu7 or SpPrp18 for these splice events and investigated the relationship to growth phase and stress. Wild-type log and stationary phase cells showed ats1+ exon 3 skipped and intron 3 retained transcripts. Interestingly the non-consensus 5'ss in ats1+ intron 3 caused SpSlu7 and SpPrp18 dependent intron retention. We validated the use of an alternative 5'ss in dtd1+ intron 1 and of an upstream alternative 3'ss in DUF3074 intron 1. The dtd1+ intron 1 non-canonical 5'ss yielded an alternative mRNA whose levels increased in stationary phase. Utilization of dtd1+ intron 1 sub-optimal 5' ss required functional SpPrp18 and SpSlu7 while compromise in SpSlu7 function alone hampered the selection of the DUF3074 intron 1 non canonical 3'ss. We analysed the relative abundance of these splice isoforms during mild thermal, oxidative and heavy metal stress and found stress-specific splice patterns for ats1+ and DUF3074 intron 1 some of which were SpSlu7 and SpPrp18 dependent. By studying ats1+ splice isoforms during compromised transcription elongation rates in wild-type, spslu7-2 and spprp18-5 mutant cells we found dynamic and intron context-specific effects in splice-site choice. Our work thus shows the combinatorial effects of splice site strength, core splicing factor functions and transcription elongation kinetics to dictate alternative splice patterns which in turn serve as an additional recourse of gene regulation in fission yeast.

Functions for fission yeast splicing factors SpSlu7 and SpPrp18 in alternative splice-site choice and stress-specific regulated splicing

PubMed Central

Kumar, Rakesh; Bawa, Pushpinder; Srinivasan, Subha

2017-01-01

Budding yeast spliceosomal factors ScSlu7 and ScPrp18 interact and mediate intron 3’ss choice during second step pre-mRNA splicing. The fission yeast genome with abundant multi-intronic transcripts, degenerate splice signals and SR proteins is an apt unicellular fungal model to deduce roles for core spliceosomal factors in alternative splice-site choice, intron retention and to study the cellular implications of regulated splicing. From our custom microarray data we deduce a stringent reproducible subset of S. pombe alternative events. We examined the role of factors SpSlu7 or SpPrp18 for these splice events and investigated the relationship to growth phase and stress. Wild-type log and stationary phase cells showed ats1+ exon 3 skipped and intron 3 retained transcripts. Interestingly the non-consensus 5’ss in ats1+ intron 3 caused SpSlu7 and SpPrp18 dependent intron retention. We validated the use of an alternative 5’ss in dtd1+ intron 1 and of an upstream alternative 3’ss in DUF3074 intron 1. The dtd1+ intron 1 non-canonical 5’ss yielded an alternative mRNA whose levels increased in stationary phase. Utilization of dtd1+ intron 1 sub-optimal 5’ ss required functional SpPrp18 and SpSlu7 while compromise in SpSlu7 function alone hampered the selection of the DUF3074 intron 1 non canonical 3’ss. We analysed the relative abundance of these splice isoforms during mild thermal, oxidative and heavy metal stress and found stress-specific splice patterns for ats1+ and DUF3074 intron 1 some of which were SpSlu7 and SpPrp18 dependent. By studying ats1+ splice isoforms during compromised transcription elongation rates in wild-type, spslu7-2 and spprp18-5 mutant cells we found dynamic and intron context-specific effects in splice-site choice. Our work thus shows the combinatorial effects of splice site strength, core splicing factor functions and transcription elongation kinetics to dictate alternative splice patterns which in turn serve as an additional recourse of gene regulation in fission yeast. PMID:29236736

Functional characterization and molecular mechanism exploration of three granulin epithelin precursor splice variants in biomineralization of the pearl oyster Pinctada fucata.

PubMed

Zhao, Mi; He, Maoxian; Huang, Xiande; Wang, Qi; Shi, Yu

2016-02-01

The granulin/epithelin precursor (GEP) encodes a glycoprotein precursor which exhibits pleiotropic tissue growth factor activity with multiple functions. Here, GEP was isolated and its role in the shell biomineralization process of the pearl oyster Pinctada fucata was investigated. Three forms of GEP mRNA were isolated from the pearl oyster (designated PfGEP-1, PfGEP-2 and PfGEP-3). Genomic DNA flanking the splicing region of the PfGEP variants was sequenced and it was found that PfGEP-2 splices out Exon 4, whereas PfGEP-3 splices out Exon 3 compared to PfGEP-1. PfGEP-1 (1505 amino acids) consists of 18 granulin domains, whereas PfGEP-2 (1459 amino acids) and PfGEP-3 (1471 amino acids) consist of 17.5 granulin domains, respectively. Analyses of PfGEP-1 and PfGEP-3 mRNA showed differential patterns in the tissues and developmental stages. Western blotting results showed that the three splice variants can translate to proteins in HEK293T cells. A knockdown experiment using PfGEP dsRNA showed decreased PfGEP-1/PfGEP-3 and PfMSX mRNA, and irregular crystallization of the nacreous layer using scanning electron microscopy. In luciferase assays, co-transfection of PfGEP-1 could activate as well as repress luciferase expression of the reporter plasmid driven by the PfMSX promoter, whereas PfGEP-3 stimulated the expression, elucidating the molecular mechanisms involved in the correlation between PfGEP and PfMSX. These results suggested that GEP variants might function differently during the biomineralization process, which provides new knowledge on the mechanism regulating nacre formation.

Dominant-Negative Regulation of Cell Surface Expression by a Pentapeptide Motif at the Extreme COOH Terminus of an Slo1 Calcium-Activated Potassium Channel Splice Variant

PubMed Central

Chiu, Yu-Hsin; Alvarez-Baron, Claudia; Kim, Eun Young

2010-01-01

Large-conductance Ca2+-activated K+ (BKCa) channels regulate the physiology of many cell types. A single vertebrate gene variously known as Slo1, KCa1.1, or KCNMA1 encodes the pore-forming subunits of BKCa channel but is expressed in a potentially very large number of alternative splice variants. Two splice variants of Slo1, Slo1VEDEC and Slo1QEERL, which differ at the extreme COOH terminus, show markedly different steady-state expression levels on the cell surface. Here we show that Slo1VEDEC and Slo1QEERL can reciprocally coimmunoprecipitate, indicating that they form heteromeric complexes. Moreover, coexpression of even small amounts of Slo1VEDEC markedly reduces surface expression of Slo1QEERL and total Slo1 as indicated by cell-surface biotinylation assays. The effects of Slo1VEDEC on steady-state surface expression can be attributed primarily to the last five residues of the protein based on surface expression of motif-swapped constructs of Slo1 in human embryonic kidney (HEK) 293T cells. In addition, the presence of the VEDEC motif at the COOH terminus of Slo1 channels is sufficient to confer a dominant-negative effect on cell surface expression of itself or other types of Slo1 subunits. Treating cells with short peptides containing the VEDEC motif increased surface expression of Slo1VEDEC channels transiently expressed in HEK293T cells and increased current through endogenous BKCa channels in mouse podocytes. Slo1VEDEC and Slo1QEERL channels are removed from the HEK293T cell surface with similar kinetics and to a similar extent, which suggests that the inhibitory effect of the VEDEC motif is exerted primarily on forward trafficking into the plasma membrane. PMID:20051533

FULL-GENOME ANALYSIS OF ALTERNATIVE SPLICING IN MOUSE LIVER AFTER HEPATOTOXICANT EXPOSURE

EPA Science Inventory

Alternative splicing plays a role in determining gene function and protein diversity. We have employed whole genome exon profiling using Affymetrix Mouse Exon 1.0 ST arrays to understand the significance of alternative splicing on a genome-wide scale in response to multiple toxic...

Non-coding functions of alternative pre-mRNA splicing in development

PubMed Central

Mockenhaupt, Stefan; Makeyev, Eugene V.

2015-01-01

A majority of messenger RNA precursors (pre-mRNAs) in the higher eukaryotes undergo alternative splicing to generate more than one mature product. By targeting the open reading frame region this process increases diversity of protein isoforms beyond the nominal coding capacity of the genome. However, alternative splicing also frequently controls output levels and spatiotemporal features of cellular and organismal gene expression programs. Here we discuss how these non-coding functions of alternative splicing contribute to development through regulation of mRNA stability, translational efficiency and cellular localization. PMID:26493705

The evolution of Dscam genes across the arthropods.

PubMed

Armitage, Sophie A O; Freiburg, Rebecca Y; Kurtz, Joachim; Bravo, Ignacio G

2012-04-13

One way of creating phenotypic diversity is through alternative splicing of precursor mRNAs. A gene that has evolved a hypervariable form is Down syndrome cell adhesion molecule (Dscam-hv), which in Drosophila melanogaster can produce thousands of isoforms via mutually exclusive alternative splicing. The extracellular region of this protein is encoded by three variable exon clusters, each containing multiple exon variants. The protein is vital for neuronal wiring where the extreme variability at the somatic level is required for axonal guidance, and it plays a role in immunity where the variability has been hypothesised to relate to recognition of different antigens. Dscam-hv has been found across the Pancrustacea. Additionally, three paralogous non-hypervariable Dscam-like genes have also been described for D. melanogaster. Here we took a bioinformatics approach, building profile Hidden Markov Models to search across species for putative orthologs to the Dscam genes and for hypervariable alternatively spliced exons, and inferring the phylogenetic relationships among them. Our aims were to examine whether Dscam orthologs exist outside the Bilateria, whether the origin of Dscam-hv could lie outside the Pancrustacea, when the Dscam-like orthologs arose, how many alternatively spliced exons of each exon cluster were present in the most common recent ancestor, and how these clusters evolved. Our results suggest that the origin of Dscam genes may lie after the split between the Cnidaria and the Bilateria and supports the hypothesis that Dscam-hv originated in the common ancestor of the Pancrustacea. Our phylogeny of Dscam gene family members shows six well-supported clades: five containing Dscam-like genes and one containing all the Dscam-hv genes, a seventh clade contains arachnid putative Dscam genes. Furthermore, the exon clusters appear to have experienced different evolutionary histories. Dscam genes have undergone independent duplication events in the insects and in an arachnid genome, which adds to the more well-known tandem duplications that have taken place within Dscam-hv genes. Therefore, two forms of gene expansion seem to be active within this gene family. The evolutionary history of this dynamic gene family will be further unfolded as genomes of species from more disparate groups become available.

The evolution of Dscam genes across the arthropods

PubMed Central

2012-01-01

Background One way of creating phenotypic diversity is through alternative splicing of precursor mRNAs. A gene that has evolved a hypervariable form is Down syndrome cell adhesion molecule (Dscam-hv), which in Drosophila melanogaster can produce thousands of isoforms via mutually exclusive alternative splicing. The extracellular region of this protein is encoded by three variable exon clusters, each containing multiple exon variants. The protein is vital for neuronal wiring where the extreme variability at the somatic level is required for axonal guidance, and it plays a role in immunity where the variability has been hypothesised to relate to recognition of different antigens. Dscam-hv has been found across the Pancrustacea. Additionally, three paralogous non-hypervariable Dscam-like genes have also been described for D. melanogaster. Here we took a bioinformatics approach, building profile Hidden Markov Models to search across species for putative orthologs to the Dscam genes and for hypervariable alternatively spliced exons, and inferring the phylogenetic relationships among them. Our aims were to examine whether Dscam orthologs exist outside the Bilateria, whether the origin of Dscam-hv could lie outside the Pancrustacea, when the Dscam-like orthologs arose, how many alternatively spliced exons of each exon cluster were present in the most common recent ancestor, and how these clusters evolved. Results Our results suggest that the origin of Dscam genes may lie after the split between the Cnidaria and the Bilateria and supports the hypothesis that Dscam-hv originated in the common ancestor of the Pancrustacea. Our phylogeny of Dscam gene family members shows six well-supported clades: five containing Dscam-like genes and one containing all the Dscam-hv genes, a seventh clade contains arachnid putative Dscam genes. Furthermore, the exon clusters appear to have experienced different evolutionary histories. Conclusions Dscam genes have undergone independent duplication events in the insects and in an arachnid genome, which adds to the more well-known tandem duplications that have taken place within Dscam-hv genes. Therefore, two forms of gene expansion seem to be active within this gene family. The evolutionary history of this dynamic gene family will be further unfolded as genomes of species from more disparate groups become available. PMID:22500922

Muscle-Specific Mis-Splicing and Heart Disease Exemplified by RBM20.

PubMed

Rexiati, Maimaiti; Sun, Mingming; Guo, Wei

2018-01-05

Alternative splicing is an essential post-transcriptional process to generate multiple functional RNAs or proteins from a single transcript. Progress in RNA biology has led to a better understanding of muscle-specific RNA splicing in heart disease. The recent discovery of the muscle-specific splicing factor RNA-binding motif 20 (RBM20) not only provided great insights into the general alternative splicing mechanism but also demonstrated molecular mechanism of how this splicing factor is associated with dilated cardiomyopathy. Here, we review our current knowledge of muscle-specific splicing factors and heart disease, with an emphasis on RBM20 and its targets, RBM20-dependent alternative splicing mechanism, RBM20 disease origin in induced Pluripotent Stem Cells (iPSCs), and RBM20 mutations in dilated cardiomyopathy. In the end, we will discuss the multifunctional role of RBM20 and manipulation of RBM20 as a potential therapeutic target for heart disease.

Specific-detection of clinical samples, systematic functional investigations, and transcriptome analysis reveals that splice variant MUC4/Y contributes to the malignant progression of pancreatic cancer by triggering malignancy-related positive feedback loops signaling.

PubMed

Zhu, Yi; Zhang, Jing-Jing; Xie, Kun-Ling; Tang, Jie; Liang, Wen-Biao; Zhu, Rong; Zhu, Yan; Wang, Bin; Tao, Jin-Qiu; Zhi, Xiao-Fei; Li, Zheng; Gao, Wen-Tao; Jiang, Kui-Rong; Miao, Yi; Xu, Ze-Kuan

2014-11-04

MUC4 plays important roles in the malignant progression of human pancreatic cancer. But the huge length of MUC4 gene fragment restricts its functional and mechanism research. As one of its splice variants, MUC4/Y with coding sequence is most similar to that of the full-length MUC4 (FL-MUC4), together with alternative splicing of the MUC4 transcript has been observed in pancreatic carcinomas but not in normal pancreas. So we speculated that MUC4/Y might be involved in malignant progression similarly to FL-MUC4, and as a research model of MUC4 in pancreatic cancer. The conjecture was confirmed in the present study. MUC4/Y expression was detected by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) using gene-specific probe in the clinic samples. The effects of MUC4/Y were observed by serial in vitro and in vivo experiments based on stable over-expressed cell model. The underlying mechanisms were investigated by sequence-based transcriptome analysis and verified by qRT-PCR, Western blot and enzyme-linked immunosorbent assays. The detection of clinical samples indicates that MUC4/Y is significantly positive-correlated with tumor invasion and distant metastases. Based on stable forced-expressed pancreatic cancer PANC-1 cell model, functional studies show that MUC4/Y enhances malignant activity in vitro and in vivo, including proliferation under low-nutritional-pressure, resistance to apoptosis, motility, invasiveness, angiogenesis, and distant metastasis. Mechanism studies indicate the novel finding that MUC4/Y triggers malignancy-related positive feedback loops for concomitantly up-regulating the expression of survival factors to resist adverse microenvironment and increasing the expression of an array of cytokines and adhesion molecules to affect the tumor milieu. In light of the enormity of the potential regulatory circuitry in cancer afforded by MUC4 and/or MUC4/Y, repressing MUC4 transcription, inhibiting post-transcriptional regulation, including alternative splicing, or blocking various pathways simultaneously may be helpful for controlling malignant progression. MUC4/Y- expression model is proven to a valuable tool for the further dissection of MUC4-mediated functions and mechanisms.

Intragenic DNA methylation and BORIS-mediated cancer-specific splicing contribute to the Warburg effect

PubMed Central

Singh, Smriti; Narayanan, Sathiya Pandi; Biswas, Kajal; Gupta, Amit; Ahuja, Neha; Yadav, Sandhya; Panday, Rajendra Kumar; Samaiya, Atul; Sharan, Shyam K.

2017-01-01

Aberrant alternative splicing and epigenetic changes are both associated with various cancers, but epigenetic regulation of alternative splicing in cancer is largely unknown. Here we report that the intragenic DNA methylation-mediated binding of Brother of Regulator of Imprinted Sites (BORIS) at the alternative exon of Pyruvate Kinase (PKM) is associated with cancer-specific splicing that promotes the Warburg effect and breast cancer progression. Interestingly, the inhibition of DNA methylation, BORIS depletion, or CRISPR/Cas9-mediated deletion of the BORIS binding site leads to a splicing switch from cancer-specific PKM2 to normal PKM1 isoform. This results in the reversal of the Warburg effect and the inhibition of breast cancer cell growth, which may serve as a useful approach to inhibit the growth of breast cancer cells. Importantly, our results show that in addition to PKM splicing, BORIS also regulates the alternative splicing of several genes in a DNA methylation-dependent manner. Our findings highlight the role of intragenic DNA methylation and DNA binding protein BORIS in cancer-specific splicing and its role in tumorigenesis. PMID:29073069

Population- and individual-specific regulatory variation in Sardinia.

PubMed

Pala, Mauro; Zappala, Zachary; Marongiu, Mara; Li, Xin; Davis, Joe R; Cusano, Roberto; Crobu, Francesca; Kukurba, Kimberly R; Gloudemans, Michael J; Reinier, Frederic; Berutti, Riccardo; Piras, Maria G; Mulas, Antonella; Zoledziewska, Magdalena; Marongiu, Michele; Sorokin, Elena P; Hess, Gaelen T; Smith, Kevin S; Busonero, Fabio; Maschio, Andrea; Steri, Maristella; Sidore, Carlo; Sanna, Serena; Fiorillo, Edoardo; Bassik, Michael C; Sawcer, Stephen J; Battle, Alexis; Novembre, John; Jones, Chris; Angius, Andrea; Abecasis, Gonçalo R; Schlessinger, David; Cucca, Francesco; Montgomery, Stephen B

2017-05-01

Genetic studies of complex traits have mainly identified associations with noncoding variants. To further determine the contribution of regulatory variation, we combined whole-genome and transcriptome data for 624 individuals from Sardinia to identify common and rare variants that influence gene expression and splicing. We identified 21,183 expression quantitative trait loci (eQTLs) and 6,768 splicing quantitative trait loci (sQTLs), including 619 new QTLs. We identified high-frequency QTLs and found evidence of selection near genes involved in malarial resistance and increased multiple sclerosis risk, reflecting the epidemiological history of Sardinia. Using family relationships, we identified 809 segregating expression outliers (median z score of 2.97), averaging 13.3 genes per individual. Outlier genes were enriched for proximal rare variants, providing a new approach to study large-effect regulatory variants and their relevance to traits. Our results provide insight into the effects of regulatory variants and their relationship to population history and individual genetic risk.

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

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

VAT: a computational framework to functionally annotate variants in personal genomes within a cloud-computing environment

PubMed Central

Habegger, Lukas; Balasubramanian, Suganthi; Chen, David Z.; Khurana, Ekta; Sboner, Andrea; Harmanci, Arif; Rozowsky, Joel; Clarke, Declan; Snyder, Michael; Gerstein, Mark

2012-01-01

Summary: The functional annotation of variants obtained through sequencing projects is generally assumed to be a simple intersection of genomic coordinates with genomic features. However, complexities arise for several reasons, including the differential effects of a variant on alternatively spliced transcripts, as well as the difficulty in assessing the impact of small insertions/deletions and large structural variants. Taking these factors into consideration, we developed the Variant Annotation Tool (VAT) to functionally annotate variants from multiple personal genomes at the transcript level as well as obtain summary statistics across genes and individuals. VAT also allows visualization of the effects of different variants, integrates allele frequencies and genotype data from the underlying individuals and facilitates comparative analysis between different groups of individuals. VAT can either be run through a command-line interface or as a web application. Finally, in order to enable on-demand access and to minimize unnecessary transfers of large data files, VAT can be run as a virtual machine in a cloud-computing environment. Availability and Implementation: VAT is implemented in C and PHP. The VAT web service, Amazon Machine Image, source code and detailed documentation are available at vat.gersteinlab.org. Contact: lukas.habegger@yale.edu or mark.gerstein@yale.edu Supplementary Information: Supplementary data are available at Bioinformatics online. PMID:22743228

Splicing regulatory factors, ageing and age-related disease.

PubMed

Latorre, Eva; Harries, Lorna W

2017-07-01

Alternative splicing is a co-transcriptional process, which allows for the production of multiple transcripts from a single gene and is emerging as an important control point for gene expression. Alternatively expressed isoforms often have antagonistic function and differential temporal or spatial expression patterns, yielding enormous plasticity and adaptability to cells and increasing their ability to respond to environmental challenge. The regulation of alternative splicing is critical for numerous cellular functions in both pathological and physiological conditions, and deregulated alternative splicing is a key feature of common chronic diseases. Isoform choice is controlled by a battery of splicing regulatory proteins, which include the serine arginine rich (SRSF) proteins and the heterogeneous ribonucleoprotein (hnRNP) classes of genes. These important splicing regulators have been implicated in age-related disease, and in the ageing process itself. This review will outline the important contribution of splicing regulator proteins to ageing and age-related disease. Copyright © 2017 Elsevier B.V. All rights reserved.

Osteopontin-a Alters Glucose Homeostasis in Anchorage-Independent Breast Cancer Cells*

PubMed Central

Wang, Bo; Kennedy, Michael A.; Weber, Georg F.

2013-01-01

Invasive breast tumor cells generate three splice variants of the metastasis gene osteopontin, while non-invasive breast cells express only the unspliced form or no osteopontin at all. One role for osteopontin in tumor progression is the support of anchorage-independence. Here we show that the full-length gene product, osteopontin-a, induces a gene expression profile that is associated with tissue remodeling and directed movement/sprouting. This occurs via signals through STAT1 and STAT3 to snglycero-3-phosphocholine. Osteopontin-a upregulates the levels of glucose in breast cancer cells, likely through STAT3 and its transcriptional targets apolipoprotein D and IGFBP5. The splice variants osteopontin-a and osteopontin-c may synergize, with each form activating signal transduction pathways that are distinct from the other. The elevated glucose is used by osteopontin-c dependent signals to generate chemical energy (Shi et al. manuscript submitted). The splice variant-specific metabolic effects of osteopontin add a novel aspect to the pro-metastatic functions of this molecule. PMID:24157812

PUF60 variants cause a syndrome of ID, short stature, microcephaly, coloboma, craniofacial, cardiac, renal and spinal features

PubMed Central

Low, Karen J; Ansari, Morad; Abou Jamra, Rami; Clarke, Angus; El Chehadeh, Salima; FitzPatrick, David R; Greenslade, Mark; Henderson, Alex; Hurst, Jane; Keller, Kory; Kuentz, Paul; Prescott, Trine; Roessler, Franziska; Selmer, Kaja K; Schneider, Michael C; Stewart, Fiona; Tatton-Brown, Katrina; Thevenon, Julien; Vigeland, Magnus D; Vogt, Julie; Willems, Marjolaine; Zonana, Jonathan; Study, D D D; Smithson, Sarah F

2017-01-01

PUF60 encodes a nucleic acid-binding protein, a component of multimeric complexes regulating RNA splicing and transcription. In 2013, patients with microdeletions of chromosome 8q24.3 including PUF60 were found to have developmental delay, microcephaly, craniofacial, renal and cardiac defects. Very similar phenotypes have been described in six patients with variants in PUF60, suggesting that it underlies the syndrome. We report 12 additional patients with PUF60 variants who were ascertained using exome sequencing: six through the Deciphering Developmental Disorders Study and six through similar projects. Detailed phenotypic analysis of all patients was undertaken. All 12 patients had de novo heterozygous PUF60 variants on exome analysis, each confirmed by Sanger sequencing: four frameshift variants resulting in premature stop codons, three missense variants that clustered within the RNA recognition motif of PUF60 and five essential splice-site (ESS) variant. Analysis of cDNA from a fibroblast cell line derived from one of the patients with an ESS variants revealed aberrant splicing. The consistent feature was developmental delay and most patients had short stature. The phenotypic variability was striking; however, we observed similarities including spinal segmentation anomalies, congenital heart disease, ocular colobomata, hand anomalies and (in two patients) unilateral renal agenesis/horseshoe kidney. Characteristic facial features included micrognathia, a thin upper lip and long philtrum, narrow almond-shaped palpebral fissures, synophrys, flared eyebrows and facial hypertrichosis. Heterozygote loss-of-function variants in PUF60 cause a phenotype comprising growth/developmental delay and craniofacial, cardiac, renal, ocular and spinal anomalies, adding to disorders of human development resulting from aberrant RNA processing/spliceosomal function. PMID:28327570

Unusual Intron Conservation near Tissue-Regulated Exons Found by Splicing Microarrays

PubMed Central

Sugnet, Charles W; Srinivasan, Karpagam; Clark, Tyson A; O'Brien, Georgeann; Cline, Melissa S; Wang, Hui; Williams, Alan; Kulp, David; Blume, John E; Haussler, David; Ares, Manuel

2006-01-01

Alternative splicing contributes to both gene regulation and protein diversity. To discover broad relationships between regulation of alternative splicing and sequence conservation, we applied a systems approach, using oligonucleotide microarrays designed to capture splicing information across the mouse genome. In a set of 22 adult tissues, we observe differential expression of RNA containing at least two alternative splice junctions for about 40% of the 6,216 alternative events we could detect. Statistical comparisons identify 171 cassette exons whose inclusion or skipping is different in brain relative to other tissues and another 28 exons whose splicing is different in muscle. A subset of these exons is associated with unusual blocks of intron sequence whose conservation in vertebrates rivals that of protein-coding exons. By focusing on sets of exons with similar regulatory patterns, we have identified new sequence motifs implicated in brain and muscle splicing regulation. Of note is a motif that is strikingly similar to the branchpoint consensus but is located downstream of the 5′ splice site of exons included in muscle. Analysis of three paralogous membrane-associated guanylate kinase genes reveals that each contains a paralogous tissue-regulated exon with a similar tissue inclusion pattern. While the intron sequences flanking these exons remain highly conserved among mammalian orthologs, the paralogous flanking intron sequences have diverged considerably, suggesting unusually complex evolution of the regulation of alternative splicing in multigene families. PMID:16424921

Variant-specific quantification of factor H in plasma reveals null alleles associated with atypical hemolytic uremic syndrome

PubMed Central

Hakobyan, Svetlana; Tortajada, Agustín; Harris, Claire L.; de Córdoba, Santiago Rodríguez; Morgan, B. Paul

2011-01-01

Atypical hemolytic uremic syndrome (aHUS) associates with complement alternative pathway defects in over 50% of cases. Mutations in factor H (fH) are most common, usually point mutations affecting complement surface regulation and sometimes null mutations in heterozygosity. The latter are difficult to identify; although consistently low plasma fH concentration is suggestive, definitive proof has required the demonstration that the mutant sequence does not express in vitro. Here, novel reagents and assays that distinguish and individually quantify the common fH-Y402H polymorphic variants were used to identify alleles of the CFH gene resulting in low or no (‘null’) expression of full-length fH, but normal or increased expression of the alternative splice product FHL-1, also detected in these assays. Their use in an aHUS cohort identified three Y402H heterozygotes with low or absent fH-H402 but normal or increased FHL-1 levels. Novel mutations in heterozygosis explained the null phenotype in two cases, confirmed by family studies in one. In the third case, family studies showed that a known mutation was present on the Y allele; the cause of the reduced expression of H allele was not found, although data suggested altered fH/FHL-1 splicing. In each family, inheritance of “low expression” or “null” alleles for fH strongly associated with aHUS. These assays provide a rapid means to identify fH expression defects in aHUS without resorting to gene sequencing or expression analysis. PMID:20703214

Zinc-induced modulation of SRSF6 activity alters Bim splicing to promote generation of the most potent apoptotic isoform BimS.

PubMed

Hara, Hirokazu; Takeda, Tatsuya; Yamamoto, Nozomi; Furuya, Keisuke; Hirose, Kazuya; Kamiya, Tetsuro; Adachi, Tetsuo

2013-07-01

Bim is a member of the pro-apoptotic BH3-only Bcl-2 family of proteins. Bim gene undergoes alternative splicing to produce three predominant splicing variants (BimEL, BimL and BimS). The smallest variant BimS is the most potent inducer of apoptosis. Zinc (Zn(2+)) has been reported to stimulate apoptosis in various cell types. In this study, we examined whether Zn(2+) affects the expression of Bim in human neuroblastoma SH-SY5Y cells. Zn(2+) triggered alterations in Bim splicing and induced preferential generation of BimS, but not BimEL and BimL, in a dose- and time-dependent manner. Other metals (cadmium, cobalt and copper) and stresses (oxidative, endoplasmic reticulum and genotoxic stresses) had little or no effect on the expression of BimS. To address the mechanism of Zn(2+)-induced preferential generation of BimS, which lacks exon 4, we developed a Bim mini-gene construct. Deletion analysis using the Bim mini-gene revealed that predicted binding sites of the SR protein SRSF6, also known as SRp55, are located in the intronic region adjacent to exon 4. We also found that mutations in the predicted SRSF6-binding sites abolished generation of BimS mRNA from the mutated Bim mini-gene. In addition, a UV cross-linking assay followed by Western blotting showed that SRSF6 directly bound to the predicted binding site and Zn(2+) suppressed this binding. Moreover, Zn(2+) stimulated SRSF6 hyper-phosphorylation. TG003, a cdc2-like kinase inhibitor, partially prevented Zn(2+)-induced generation of BimS and SRSF6 hyper-phosphorylation. Taken together, our findings suggest that Zn(2+) inhibits the activity of SRSF6 and promotes elimination of exon 4, leading to preferential generation of BimS. © 2013 FEBS.

Expressed sequence tag analysis of human RPE/choroid for the NEIBank Project: over 6000 non-redundant transcripts, novel genes and splice variants.

PubMed

Wistow, Graeme; Bernstein, Steven L; Wyatt, M Keith; Fariss, Robert N; Behal, Amita; Touchman, Jeffrey W; Bouffard, Gerald; Smith, Don; Peterson, Katherine

2002-06-15

The retinal pigment epithelium (RPE) and choroid comprise a functional unit of the eye that is essential to normal retinal health and function. Here we describe expressed sequence tag (EST) analysis of human RPE/choroid as part of a project for ocular bioinformatics. A cDNA library (cs) was made from human RPE/choroid and sequenced. Data were analyzed and assembled using the program GRIST (GRouping and Identification of Sequence Tags). Complete sequencing, Northern and Western blots, RH mapping, peptide antibody synthesis and immunofluorescence (IF) have been used to examine expression patterns and genome location for selected transcripts and proteins. Ten thousand individual sequence reads yield over 6300 unique gene clusters of which almost half have no matches with named genes. One of the most abundant transcripts is from a gene (named "alpha") that maps to the BBS1 region of chromosome 11. A number of tissue preferred transcripts are common to both RPE/choroid and iris. These include oculoglycan/opticin, for which an alternative splice form is detected in RPE/choroid, and "oculospanin" (Ocsp), a novel tetraspanin that maps to chromosome 17q. Antiserum to Ocsp detects expression in RPE, iris, ciliary body, and retinal ganglion cells by IF. A newly identified gene for a zinc-finger protein (TIRC) maps to 19q13.4. Variant transcripts of several genes were also detected. Most notably, the predominant form of Bestrophin represented in cs contains a longer open reading frame as a result of splice junction skipping. The unamplified cs library gives a view of the transcriptional repertoire of the adult RPE/choroid. A large number of potentially novel genes and splice forms and candidates for genetic diseases are revealed. Clones from this collection are being included in a large, nonredundant set for cDNA microarray construction.

Identification of novel point mutations in splicing sites integrating whole-exome and RNA-seq data in myeloproliferative diseases

PubMed Central

Spinelli, Roberta; Pirola, Alessandra; Redaelli, Sara; Sharma, Nitesh; Raman, Hima; Valletta, Simona; Magistroni, Vera; Piazza, Rocco; Gambacorti-Passerini, Carlo

2013-01-01

Point mutations in intronic regions near mRNA splice junctions can affect the splicing process. To identify novel splicing variants from exome sequencing data, we developed a bioinformatics splice-site prediction procedure to analyze next-generation sequencing (NGS) data (SpliceFinder). SpliceFinder integrates two functional annotation tools for NGS, ANNOVAR and MutationTaster and two canonical splice site prediction programs for single mutation analysis, SSPNN and NetGene2. By SpliceFinder, we identified somatic mutations affecting RNA splicing in a colon cancer sample, in eight atypical chronic myeloid leukemia (aCML), and eight CML patients. A novel homozygous splicing mutation was found in APC (NM_000038.4:c.1312+5G>A) and six heterozygous in GNAQ (NM_002072.2:c.735+1C>T), ABCC3 (NM_003786.3:c.1783-1G>A), KLHDC1 (NM_172193.1:c.568-2A>G), HOOK1 (NM_015888.4:c.1662-1G>A), SMAD9 (NM_001127217.2:c.1004-1C>T), and DNAH9 (NM_001372.3:c.10242+5G>A). Integrating whole-exome and RNA sequencing in aCML and CML, we assessed the phenotypic effect of mutations on mRNA splicing for GNAQ, ABCC3, HOOK1. In ABCC3 and HOOK1, RNA-Seq showed the presence of aberrant transcripts with activation of a cryptic splice site or intron retention, validated by the reverse transcription-polymerase chain reaction (RT-PCR) in the case of HOOK1. In GNAQ, RNA-Seq showed 22% of wild-type transcript and 78% of mRNA skipping exon 5, resulting in a 4–6 frameshift fusion confirmed by RT-PCR. The pipeline can be useful to identify intronic variants affecting RNA sequence by complementing conventional exome analysis. PMID:24498620

Genetics of alternative splicing evolution during sunflower domestication.

PubMed

Smith, Chris C R; Tittes, Silas; Mendieta, J Paul; Collier-Zans, Erin; Rowe, Heather C; Rieseberg, Loren H; Kane, Nolan C

2018-06-11

Alternative splicing enables organisms to produce the diversity of proteins necessary for multicellular life by using relatively few protein-coding genes. Although differences in splicing have been identified among divergent taxa, the shorter-term evolution of splicing is understudied. The origins of novel splice forms, and the contributions of alternative splicing to major evolutionary transitions, are largely unknown. This study used transcriptomes of wild and domesticated sunflowers to examine splice differentiation and regulation during domestication. We identified substantial splicing divergence between wild and domesticated sunflowers, mainly in the form of intron retention. Transcripts with divergent splicing were enriched for seed-development functions, suggesting that artificial selection impacted splicing patterns. Mapping of quantitative trait loci (QTLs) associated with 144 differential splicing cases revealed primarily trans -acting variation affecting splicing patterns. A large proportion of identified QTLs contain known spliceosome proteins and are associated with splicing variation in multiple genes. Examining a broader set of wild and domesticated sunflower genotypes revealed that most differential splicing patterns in domesticated sunflowers likely arose from standing variation in wild Helianthus annuus and gained frequency during the domestication process. However, several domesticate-associated splicing patterns appear to be introgressed from other Helianthus species. These results suggest that sunflower domestication involved selection on pleiotropic regulatory alleles. More generally, our findings indicate that substantial differences in isoform abundances arose rapidly during a recent evolutionary transition and appear to contribute to adaptation and population divergence.

Spliceman2: a computational web server that predicts defects in pre-mRNA splicing.

PubMed

Cygan, Kamil Jan; Sanford, Clayton Hendrick; Fairbrother, William Guy

2017-09-15

Most pre-mRNA transcripts in eukaryotic cells must undergo splicing to remove introns and join exons, and splicing elements present a large mutational target for disease-causing mutations. Splicing elements are strongly position dependent with respect to the transcript annotations. In 2012, we presented Spliceman, an online tool that used positional dependence to predict how likely distant mutations around annotated splice sites were to disrupt splicing. Here, we present an improved version of the previous tool that will be more useful for predicting the likelihood of splicing mutations. We have added industry-standard input options (i.e. Spliceman now accepts variant call format files), which allow much larger inputs than previously available. The tool also can visualize the locations-within exons and introns-of sequence variants to be analyzed and the predicted effects on splicing of the pre-mRNA transcript. In addition, Spliceman2 integrates with RNAcompete motif libraries to provide a prediction of which trans -acting factors binding sites are disrupted/created and links out to the UCSC genome browser. In summary, the new features in Spliceman2 will allow scientists and physicians to better understand the effects of single nucleotide variations on splicing. Freely available on the web at http://fairbrother.biomed.brown.edu/spliceman2 . Website implemented in PHP framework-Laravel 5, PostgreSQL, Apache, and Perl, with all major browsers supported. william_fairbrother@brown.edu. Supplementary data are available at Bioinformatics online. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com

Shark class II invariant chain reveals ancient conserved relationships with cathepsins and MHC class II.

PubMed

Criscitiello, Michael F; Ohta, Yuko; Graham, Matthew D; Eubanks, Jeannine O; Chen, Patricia L; Flajnik, Martin F

2012-03-01

The invariant chain (Ii) is the critical third chain required for the MHC class II heterodimer to be properly guided through the cell, loaded with peptide, and expressed on the surface of antigen presenting cells. Here, we report the isolation of the nurse shark Ii gene, and the comparative analysis of Ii splice variants, expression, genomic organization, predicted structure, and function throughout vertebrate evolution. Alternative splicing to yield Ii with and without the putative protease-protective, thyroglobulin-like domain is as ancient as the MHC-based adaptive immune system, as our analyses in shark and lizard further show conservation of this mechanism in all vertebrate classes except bony fish. Remarkable coordinate expression of Ii and class II was found in shark tissues. Conserved Ii residues and cathepsin L orthologs suggest their long co-evolution in the antigen presentation pathway, and genomic analyses suggest 450 million years of conserved Ii exon/intron structure. Other than an extended linker preceding the thyroglobulin-like domain in cartilaginous fish, the Ii gene and protein are predicted to have largely similar physiology from shark to man. Duplicated Ii genes found only in teleosts appear to have become sub-functionalized, as one form is predicted to play the same role as that mediated by Ii mRNA alternative splicing in all other vertebrate classes. No Ii homologs or potential ancestors of any of the functional Ii domains were found in the jawless fish or lower chordates. Copyright © 2011 Elsevier Ltd. All rights reserved.

Non-coding functions of alternative pre-mRNA splicing in development.

PubMed

Mockenhaupt, Stefan; Makeyev, Eugene V

2015-12-01

A majority of messenger RNA precursors (pre-mRNAs) in the higher eukaryotes undergo alternative splicing to generate more than one mature product. By targeting the open reading frame region this process increases diversity of protein isoforms beyond the nominal coding capacity of the genome. However, alternative splicing also frequently controls output levels and spatiotemporal features of cellular and organismal gene expression programs. Here we discuss how these non-coding functions of alternative splicing contribute to development through regulation of mRNA stability, translational efficiency and cellular localization. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Identification of the thiamin pyrophosphokinase gene in rainbow trout: Characteristic structure and expression of seven splice variants in tissues and cell lines and during embryo development

USGS Publications Warehouse

Yuge, Shinya; Richter, Catherine A.; Wright-Osment, Maureen K.; Nicks, Diane; Saloka, Stephanie K.; Tillitt, Donald E.; Li, Weiming

2012-01-01

Thiamin pyrophosphokinase (TPK) converts thiamin to its active form, thiamin diphosphate. In humans, TPK expression is down-regulated in some thiamin deficiency related syndrome, and enhanced during pregnancy. Rainbow trout are also vulnerable to thiamin deficiency in wild life and are useful models for thiamin metabolism research. We identified the tpk gene transcript including seven splice variants in the rainbow trout. Almost all cell lines and tissues examined showed co-expression of several tpk splice variants including a potentially major one at both mRNA and protein levels. However, relative to other tissues, the longest variant mRNA expression was predominant in the ovary and abundant in embryos. During embryogenesis, total tpk transcripts increased abruptly in early development, and decreased to about half of the peak shortly after hatching. In rainbow trout, the tpk transcript complex is ubiquitously expressed for all tissues and cells examined, and its increase in expression could be important in the early-middle embryonic stages. Moreover, decimated tpk expression in a hepatoma cell line relative to hepatic and gonadal cell lines appears to be consistent with previously reported down-regulation of thiamin metabolism in cancer.

Identification and characterization of an alternative splice variant of Mpl with a high affinity for TPO and its activation of ERK1/2 signaling.

PubMed

Wang, Qiong; Sun, Rui; Wu, Leyan; Huang, Junfeng; Wang, Ping; Yuan, Hailong; Qiu, Feifei; Xu, Xiaohong; Wu, Di; Yu, Ying; Liu, Xin; Zhang, Qing

2013-12-01

The thrombopoietin receptor is a crucial element in thrombopoietin-initiated signaling pathways, which stimulates the differentiation of normal hematopoietic progenitor cells, the maturation of megakaryocytes, and the generation of platelets. In this study, we identified a novel activating variant of thrombopoietin receptor, termed Mpl-D, in human megakaryoblastic leukemia Dami cells and demonstrated that the binding affinity of the Mpl-D receptor for thrombopoietin is enhanced. Cell cycle analysis revealed that in the presence of thrombopoietin, most Mpl-D expressing NIH3T3 (NIH3T3/Mpl-D) cells were prevalent in G1 phase while the S and G2/M populations were less frequently observed. Unexpectedly, thrombopoietin induced strong and prolonged ERK1/2 signaling in NIH3T3/Mpl-D cells compared with its receptor wild-type expressing NIH3T3 (NIH3T3/Mpl-F) cells. Further analysis of the mRNA levels of cyclin D1/D2 in NIH3T3/Mpl-D cells demonstrated markedly down-regulated expression compared to NIH3T3/Mpl-F cells in the presence of thrombopoietin. Thus, the prolonged activation of ERK1/2 by Mpl-D might lead to G1 cell cycle arrest through a profound reduction of cyclin D1/D2 in order to support cell survival without proliferation. We also provided tertiary structural basis for the Mpl-D and thrombopoietin interaction, which might provide insights into how Mpl-D effectively increases binding to thrombopoietin and significantly contributes to its specific signaling pathway. These results suggest a new paradigm for the regulation of cytokine receptor expression and function through the alternative splicing variant of Mpl in Dami cells, which may play a role in the pathogenesis of megakaryoblastic leukemia. Copyright © 2013 Elsevier Ltd. All rights reserved.

Male-specific expression of Sox9 during gonad development of crocodile and mouse is mediated by alternative splicing of its proline-glutamine-alanine rich domain.

PubMed

Agrawal, Raman; Wessely, Oliver; Anand, Amit; Singh, Lalji; Aggarwal, Ramesh K

2009-08-01

The initial trigger for sexual differentiation is regulated by multiple ways during embryonic development. In vertebrates, chromosome-based mechanisms generally known as genetic sex determination are prevalent; however, some species, such as many reptilians, display temperature-dependent sex determination. The Sry-related transcription factor, Sox9, which is expressed by an evolutionary conserved gene, has been shown to be a key player in the process of sex determination. In the present study, we report the identification and expression of crocodile homolog of Sox9 (cpSox9) from the Indian Mugger, Crocodylus palustris. We show that cpSox9 undergoes extensive alternative splicing around the proline-glutamine-alanine rich transactivation domain that results in cpSox9 variants with presumably impaired or reduced transactivation potential. The multiple isoforms were also detected in various embryonic tissues, with some of them displaying a differential expression profile. With respect to sex differentiation, a putative unspliced full-length cpSox9 could be detected only in the genital ridge-adrenal-mesonephros complex of male, but not female embryos during the temperature-sensitive period. Importantly, we further show that this phenomenon was not restricted to the temperature-dependent sex determination species C. palustris, but was also observed in the mouse, a species exhibiting genetic sex determination. Thus, the present study describes, for the first time, a complete coding locus of Sox9 homolog from a temperature-dependent sex determination species. More importantly, we demonstrate an evolutionarily conserved role of alternative splicing resulting in transcriptional diversity and male-sex specific expression of Sox9 during testis development in vertebrates (i.e. irrespective of their underlying sex-determination mechanisms).

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.

PXR (NR1I2): splice variants in human tissues, including brain, and identification of neurosteroids and nicotine as PXR activators.

PubMed

Lamba, Vishal; Yasuda, Kazuto; Lamba, Jatinder K; Assem, Mahfoud; Davila, Julio; Strom, Stephen; Schuetz, Erin G

2004-09-15

To gain insight on the expression of pregnane X receptor (PXR), we analyzed PXR.1 and PXR alternatively spliced transcripts in a panel of 36 human tissues. PXR.1 was expressed in many more tissues than previously determined, including human bone marrow and select regions of the human brain. In each of these tissues, we observed alternative splicing of various exons of PXR that generated multiple distinct PXR isoforms. The most abundant PXR alternative mRNA transcripts lacked 111 nucleotides, deleting 37 amino acids from the PXR LBD (PXR.2), or lacked 123 nt, deleting 41 amino acids from the PXR LBD (PXR.3). CYP3A4, a gene transcriptionally regulated by PXR, showed incomplete overlap with PXR in its tissue distribution. Quantitation of PXR mRNAs in human liver demonstrated that PXR.2 and PXR.3 represented 6.7% and 0.32% of total PXR mRNA transcripts. Brain expression of PXR prompted analysis of whether some brain acting chemicals were PXR ligands. The neurosteroids allopregnanolone and pregnanolone activated PXR and induced transcription of a CYP3A4-luciferase reporter. Nicotine, the psychoactive and addictive chemical in cigarettes, and a known inducer of brain CYP2B6, was an efficacious activator of PXR and inducer of CYP3A4 transcription. Because nicotine activation of PXR will enhance metabolism of nicotine to the non-psychoactive cotinine, these results provide one molecular mechanism for the development of tolerance to nicotine. Moreover, the identification of PXR in many human tissues, such as brain, and activation by tissue specific ligands (such as neurosteroids) suggests additional biological roles for this receptor in these tissues.

Regulation of alternative splicing by local histone modifications: potential roles for RNA-guided mechanisms

PubMed Central

Zhou, Hua-Lin; Luo, Guangbin; Wise, Jo Ann; Lou, Hua

2014-01-01

The molecular mechanisms through which alternative splicing and histone modifications regulate gene expression are now understood in considerable detail. Here, we discuss recent studies that connect these two previously separate avenues of investigation, beginning with the unexpected discoveries that nucleosomes are preferentially positioned over exons and DNA methylation and certain histone modifications also show exonic enrichment. These findings have profound implications linking chromatin structure, histone modification and splicing regulation. Complementary single gene studies provided insight into the mechanisms through which DNA methylation and histones modifications modulate alternative splicing patterns. Here, we review an emerging theme resulting from these studies: RNA-guided mechanisms integrating chromatin modification and splicing. Several groundbreaking papers reported that small noncoding RNAs affect alternative exon usage by targeting histone methyltransferase complexes to form localized facultative heterochromatin. More recent studies provided evidence that pre-messenger RNA itself can serve as a guide to enable precise alternative splicing regulation via local recruitment of histone-modifying enzymes, and emerging evidence points to a similar role for long noncoding RNAs. An exciting challenge for the future is to understand the impact of local modulation of transcription elongation rates on the dynamic interplay between histone modifications, alternative splicing and other processes occurring on chromatin. PMID:24081581

miR-200/375 control epithelial plasticity-associated alternative splicing by repressing the RNA-binding protein Quaking.

PubMed

Pillman, Katherine A; Phillips, Caroline A; Roslan, Suraya; Toubia, John; Dredge, B Kate; Bert, Andrew G; Lumb, Rachael; Neumann, Daniel P; Li, Xiaochun; Conn, Simon J; Liu, Dawei; Bracken, Cameron P; Lawrence, David M; Stylianou, Nataly; Schreiber, Andreas W; Tilley, Wayne D; Hollier, Brett G; Khew-Goodall, Yeesim; Selth, Luke A; Goodall, Gregory J; Gregory, Philip A

2018-06-05

Members of the miR-200 family are critical gatekeepers of the epithelial state, restraining expression of pro-mesenchymal genes that drive epithelial-mesenchymal transition (EMT) and contribute to metastatic cancer progression. Here, we show that miR-200c and another epithelial-enriched miRNA, miR-375, exert widespread control of alternative splicing in cancer cells by suppressing the RNA-binding protein Quaking (QKI). During EMT, QKI-5 directly binds to and regulates hundreds of alternative splicing targets and exerts pleiotropic effects, such as increasing cell migration and invasion and restraining tumour growth, without appreciably affecting mRNA levels. QKI-5 is both necessary and sufficient to direct EMT-associated alternative splicing changes, and this splicing signature is broadly conserved across many epithelial-derived cancer types. Importantly, several actin cytoskeleton-associated genes are directly targeted by both QKI and miR-200c, revealing coordinated control of alternative splicing and mRNA abundance during EMT These findings demonstrate the existence of a miR-200/miR-375/QKI axis that impacts cancer-associated epithelial cell plasticity through widespread control of alternative splicing. © 2018 The Authors. Published under the terms of the CC BY 4.0 license.

Dietary Fat Quantity and Type Induce Transcriptome-Wide Effects on Alternative Splicing of Pre-mRNA in Rat Skeletal Muscle.

PubMed

Black, Adam J; Ravi, Suhana; Jefferson, Leonard S; Kimball, Scot R; Schilder, Rudolf J

2017-09-01

Background: Fat-enriched diets produce metabolic changes in skeletal muscle, which in turn can mediate changes in gene regulation. Objective: We examined the high-fat-diet-induced changes in skeletal muscle gene expression by characterizing variations in pre-mRNA alternative splicing. Methods: Affymetrix Exon Array analysis was performed on the transcriptome of the gastrocnemius/plantaris complex of male obesity-prone Sprague-Dawley rats fed a 10% or 60% fat (lard) diet for 2 or 8 wk. The validation of exon array results was focused on troponin T ( Tnnt3 ). Tnnt3 splice form analyses were extended in studies of rats fed 10% or 30% fat diets across 1- to 8-wk treatment periods and rats fed 10% or 45% fat diets with fat sources from lard or mono- or polyunsaturated fats for 2 wk. Nuclear magnetic resonance (NMR) was used to measure body composition. Results: Consumption of a 60% fat diet for 2 or 8 wk resulted in alternative splicing of 668 and 726 pre-mRNAs, respectively, compared with rats fed a 10% fat diet. Tnnt3 transcripts were alternatively spliced in rats fed a 60% fat diet for either 2 or 8 wk. The high-fat-diet-induced changes in Tnnt3 alternative splicing were observed in rats fed a 30% fat diet across 1- to 8-wk treatment periods. Moreover, this effect depended on fat type, because Tnnt3 alternative splicing occurred in response to 45% fat diets enriched with lard but not in response to diets enriched with mono- or polyunsaturated fatty acids. Fat mass (a proxy for obesity as measured by NMR) did not differ between groups in any study. Conclusions: Rat skeletal muscle responds to overconsumption of dietary fat by modifying gene expression through pre-mRNA alternative splicing. Variations in Tnnt3 alternative splicing occur independently of obesity and are dependent on dietary fat quantity and suggest a role for saturated fatty acids in the high-fat-diet-induced modifications in Tnnt3 alternative splicing. © 2017 American Society for Nutrition.

Tools to covisualize and coanalyze proteomic data with genomes and transcriptomes: validation of genes and alternative mRNA splicing.

PubMed

Pang, Chi Nam Ignatius; Tay, Aidan P; Aya, Carlos; Twine, Natalie A; Harkness, Linda; Hart-Smith, Gene; Chia, Samantha Z; Chen, Zhiliang; Deshpande, Nandan P; Kaakoush, Nadeem O; Mitchell, Hazel M; Kassem, Moustapha; Wilkins, Marc R

2014-01-03

Direct links between proteomic and genomic/transcriptomic data are not frequently made, partly because of lack of appropriate bioinformatics tools. To help address this, we have developed the PG Nexus pipeline. The PG Nexus allows users to covisualize peptides in the context of genomes or genomic contigs, along with RNA-seq reads. This is done in the Integrated Genome Viewer (IGV). A Results Analyzer reports the precise base position where LC-MS/MS-derived peptides cover genes or gene isoforms, on the chromosomes or contigs where this occurs. In prokaryotes, the PG Nexus pipeline facilitates the validation of genes, where annotation or gene prediction is available, or the discovery of genes using a "virtual protein"-based unbiased approach. We illustrate this with a comprehensive proteogenomics analysis of two strains of Campylobacter concisus . For higher eukaryotes, the PG Nexus facilitates gene validation and supports the identification of mRNA splice junction boundaries and splice variants that are protein-coding. This is illustrated with an analysis of splice junctions covered by human phosphopeptides, and other examples of relevance to the Chromosome-Centric Human Proteome Project. The PG Nexus is open-source and available from https://github.com/IntersectAustralia/ap11_Samifier. It has been integrated into Galaxy and made available in the Galaxy tool shed.

RNA splicing during terminal erythropoiesis.

PubMed

Conboy, John G

2017-05-01

Erythroid progenitors must accurately and efficiently splice thousands of pre-mRNAs as the cells undergo extensive changes in gene expression and cellular remodeling during terminal erythropoiesis. Alternative splicing choices are governed by interactions between RNA binding proteins and cis-regulatory binding motifs in the RNA. This review will focus on recent studies that define the genome-wide scope of splicing in erythroblasts and discuss what is known about its regulation. RNA-seq analysis of highly purified erythroblast populations has revealed an extensive program of alternative splicing of both exons and introns. During normal erythropoiesis, stage-specific splicing transitions alter the structure and abundance of protein isoforms required for optimized red cell production. Mutation or deficiency of splicing regulators underlies hematopoietic disease in myelopdysplasia syndrome patients via disrupting the splicing program. Erythroid progenitors execute an elaborate alternative splicing program that modulates gene expression posttranscriptionally, ultimately regulating the structure and function of the proteome in a differentiation stage-specific manner during terminal erythropoiesis. This program helps drive differentiation and ensure synthesis of the proper protein isoforms required to produce mechanically stable red cells. Mutation or deficiency of key splicing regulatory proteins disrupts the splicing program to cause disease.

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.

CTCF, a Novel Regulator of Alternative Splicing | Center for Cancer Research

Cancer.gov

Alternative splicing, or the inclusion of different patterns of exons from the same gene, plays an important role in expanding the coding possibilities of a limited genome. The immune system is an ideal system to study this since alternative splicing is used to generate an almost unlimited number of antibodies against any pathogen we might encounter.

Jacalin Lectin At5g28520 Is Regulated By ABA and miR846

PubMed Central

Jia, Fan; Rock, Christopher D.

2013-01-01

Plant microRNAs (miRNAs) are important regulators of development and stress responses and are oftentimes under transcriptional regulation by stresses and plant hormones. We recently showed that polycistronic MIR842 and MIR846 are expressed from the same primary transcript which is subject to alternative splicing. ABA treatment affects the alternative splicing of the primary cistronic transcript which results in differential expression of the two miRNAs that are predicted to target the same family of jacalin lectin genes. One variant of miR846 in roots can direct the cleavage of AT5G28520, which is also highly upregulated by ABA in roots. In this addendum, we present additional results further supporting the regulation of AT5G28520 by MIR846 using a T-DNA insertion line mapping upstream of MIR842 and MIR846. We also show that AT5G28520 is transcriptionally induced by ABA and this induction is subject to ABA signaling effectors in seedlings. Based on previous results and data presented in this paper, we propose an interaction loop between MIR846, AT5G28520 and ABA in roots. PMID:23603955

Genomic structure and expression of STM2, the chromosome 1 familial Alzheimer disease gene

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

Levy-Lahad, E.; Wang, Kai; Fu, Ying Hui

1996-06-01

Mutations in the gene STM2 result in autosomal dominant familial Alzheimer disease. To screen for mutations and to identify regulatory elements for this gene, the genomic DNA sequence and intron-exon structure were determined. Twelve exons including 10 coding exons were identified in a genomic region spanning 23, 737 bp. The first 2 exons encode the 5{prime}-untranslated region. Expression analysis of STM2 indicates that two transcripts of 2.4 and 2.8 kb are found in skeletal muscle, pancreas, and heart. In addition, a splice variant of the 2.4-kb transcript was identified that is the result of the use of an alternative splicemore » acceptor site located in exon 10. The use of this site results in a transcript lacking a single glutamate. The promotor for this gene and the alternatively spliced exons leading to the 2.8-kb form of the gene remain to be identified. Expression of STM2 was high in skeletal muscle and pancreas, with comparatively low levels observed in brain. This expression pattern is intriguing since in Alzheimer disease, pathology and degeneration are observed only in the central nervous system. 19 refs., 2 figs., 3 tabs.« less

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

Chemotherapy induces alternative transcription and splicing: Facts and hopes for cancer treatment.

PubMed

Lambert, Charles A; Garbacki, Nancy; Colige, Alain C

2017-10-01

Alternative promoter usage, alternative splicing and alternative cleavage/polyadenylation (referred here as to alternative transcription and splicing) are main instruments to diversify the transcriptome from a limited set of genes. There is a good deal of evidence that chemotherapeutic drugs affect these processes, but the therapeutic incidence of these effects is poorly documented. The scope of this study is to review the impact of chemotherapy on alternative transcription and splicing and to discuss potential implications in cancer therapy. A literature survey identified >2200 events induced by chemotherapeutic drugs. The molecular pathways involved in these regulations are briefly discussed. The GO terms associated with the alternative transcripts are mainly related to cell cycle/division, mRNA processing, DNA repair, macromolecules catabolism and chromatin. A large fraction (43%) of transcripts are also related to the new hallmarks of cancer, mostly genetic instability and replicative immortality. Finally, we ask the question of the impact of alternative transcription and splicing on drug efficacy and of the possible curative benefit of combining chemotherapy and pharmaceutical regulation of this process. Copyright © 2017 Elsevier Ltd. All rights reserved.

Serine/Arginine-Rich Splicing Factor 3 and Heterogeneous Nuclear Ribonucleoprotein A1 Regulate Alternative RNA Splicing and Gene Expression of Human Papillomavirus 18 through Two Functionally Distinguishable cis Elements.

PubMed

Ajiro, Masahiko; Tang, Shuang; Doorbar, John; Zheng, Zhi-Ming

2016-10-15

Human papillomavirus 18 (HPV18) is the second most common oncogenic HPV type associated with cervical, anogenital, and oropharyngeal cancers. Like other oncogenic HPVs, HPV18 encodes two major (one early and one late) polycistronic pre-mRNAs that are regulated by alternative RNA splicing to produce a repertoire of viral transcripts for the expression of individual viral genes. However, RNA cis-regulatory elements and trans-acting factors contributing to HPV18 alternative RNA splicing remain unknown. In this study, an exonic splicing enhancer (ESE) in the nucleotide (nt) 3520 to 3550 region in the HPV18 genome was identified and characterized for promotion of HPV18 929^3434 splicing and E1^E4 production through interaction with SRSF3, a host oncogenic splicing factor differentially expressed in epithelial cells and keratinocytes. Introduction of point mutations in the SRSF3-binding site or knockdown of SRSF3 expression in cells reduces 929^3434 splicing and E1^E4 production but activates other, minor 929^3465 and 929^3506 splicing. Knockdown of SRSF3 expression also enhances the expression of E2 and L1 mRNAs. An exonic splicing silencer (ESS) in the HPV18 nt 612 to 639 region was identified as being inhibitory to the 233^416 splicing of HPV18 E6E7 pre-mRNAs via binding to hnRNP A1, a well-characterized, abundantly and ubiquitously expressed RNA-binding protein. Introduction of point mutations into the hnRNP A1-binding site or knockdown of hnRNP A1 expression promoted 233^416 splicing and reduced E6 expression. These data provide the first evidence that the alternative RNA splicing of HPV18 pre-mRNAs is subject to regulation by viral RNA cis elements and host trans-acting splicing factors. Expression of HPV18 genes is regulated by alternative RNA splicing of viral polycistronic pre-mRNAs to produce a repertoire of viral early and late transcripts. RNA cis elements and trans-acting factors contributing to HPV18 alternative RNA splicing have been discovered in this study for the first time. The identified ESS at the E7 open reading frame (ORF) prevents HPV18 233^416 splicing in the E6 ORF through interaction with a host splicing factor, hnRNP A1, and regulates E6 and E7 expression of the early E6E7 polycistronic pre-mRNA. The identified ESE at the E1^E4 ORF promotes HPV18 929^3434 splicing of both viral early and late pre-mRNAs and E1^E4 production through interaction with SRSF3. This study provides important observations on how alternative RNA splicing of HPV18 pre-mRNAs is subject to regulation by viral RNA cis elements and host splicing factors and offers potential therapeutic targets to overcome HPV-related cancer. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Serine/Arginine-Rich Splicing Factor 3 and Heterogeneous Nuclear Ribonucleoprotein A1 Regulate Alternative RNA Splicing and Gene Expression of Human Papillomavirus 18 through Two Functionally Distinguishable cis Elements

PubMed Central

Ajiro, Masahiko; Tang, Shuang; Doorbar, John

2016-01-01

ABSTRACT Human papillomavirus 18 (HPV18) is the second most common oncogenic HPV type associated with cervical, anogenital, and oropharyngeal cancers. Like other oncogenic HPVs, HPV18 encodes two major (one early and one late) polycistronic pre-mRNAs that are regulated by alternative RNA splicing to produce a repertoire of viral transcripts for the expression of individual viral genes. However, RNA cis-regulatory elements and trans-acting factors contributing to HPV18 alternative RNA splicing remain unknown. In this study, an exonic splicing enhancer (ESE) in the nucleotide (nt) 3520 to 3550 region in the HPV18 genome was identified and characterized for promotion of HPV18 929^3434 splicing and E1^E4 production through interaction with SRSF3, a host oncogenic splicing factor differentially expressed in epithelial cells and keratinocytes. Introduction of point mutations in the SRSF3-binding site or knockdown of SRSF3 expression in cells reduces 929^3434 splicing and E1^E4 production but activates other, minor 929^3465 and 929^3506 splicing. Knockdown of SRSF3 expression also enhances the expression of E2 and L1 mRNAs. An exonic splicing silencer (ESS) in the HPV18 nt 612 to 639 region was identified as being inhibitory to the 233^416 splicing of HPV18 E6E7 pre-mRNAs via binding to hnRNP A1, a well-characterized, abundantly and ubiquitously expressed RNA-binding protein. Introduction of point mutations into the hnRNP A1-binding site or knockdown of hnRNP A1 expression promoted 233^416 splicing and reduced E6 expression. These data provide the first evidence that the alternative RNA splicing of HPV18 pre-mRNAs is subject to regulation by viral RNA cis elements and host trans-acting splicing factors. IMPORTANCE Expression of HPV18 genes is regulated by alternative RNA splicing of viral polycistronic pre-mRNAs to produce a repertoire of viral early and late transcripts. RNA cis elements and trans-acting factors contributing to HPV18 alternative RNA splicing have been discovered in this study for the first time. The identified ESS at the E7 open reading frame (ORF) prevents HPV18 233^416 splicing in the E6 ORF through interaction with a host splicing factor, hnRNP A1, and regulates E6 and E7 expression of the early E6E7 polycistronic pre-mRNA. The identified ESE at the E1^E4 ORF promotes HPV18 929^3434 splicing of both viral early and late pre-mRNAs and E1^E4 production through interaction with SRSF3. This study provides important observations on how alternative RNA splicing of HPV18 pre-mRNAs is subject to regulation by viral RNA cis elements and host splicing factors and offers potential therapeutic targets to overcome HPV-related cancer. PMID:27489271

Aberrant Splicing Promotes Proteasomal Degradation of L-type CaV1.2 Calcium Channels by Competitive Binding for CaVβ Subunits in Cardiac Hypertrophy.

PubMed

Hu, Zhenyu; Wang, Jiong-Wei; Yu, Dejie; Soon, Jia Lin; de Kleijn, Dominique P V; Foo, Roger; Liao, Ping; Colecraft, Henry M; Soong, Tuck Wah

2016-10-12

Decreased expression and activity of Ca V 1.2 calcium channels has been reported in pressure overload-induced cardiac hypertrophy and heart failure. However, the underlying mechanisms remain unknown. Here we identified in rodents a splice variant of Ca V 1.2 channel, named Ca V 1.2 e21+22 , that contained the pair of mutually exclusive exons 21 and 22. This variant was highly expressed in neonatal hearts. The abundance of this variant was gradually increased by 12.5-folds within 14 days of transverse aortic banding that induced cardiac hypertrophy in adult mouse hearts and was also elevated in left ventricles from patients with dilated cardiomyopathy. Although this variant did not conduct Ca 2+ ions, it reduced the cell-surface expression of wild-type Ca V 1.2 channels and consequently decreased the whole-cell Ca 2+ influx via the Ca V 1.2 channels. In addition, the Ca V 1.2 e21+22 variant interacted with Ca V β subunits significantly more than wild-type Ca V 1.2 channels, and competition of Ca V β subunits by Ca V 1.2 e21+22 consequently enhanced ubiquitination and subsequent proteasomal degradation of the wild-type Ca V 1.2 channels. Our findings show that the resurgence of a specific neonatal splice variant of Ca V 1.2 channels in adult heart under stress may contribute to heart failure.

Novel variants in PAX6 gene caused congenital aniridia in two Chinese families.

PubMed

Zhang, R; Linpeng, S; Wei, X; Li, H; Huang, Y; Guo, J; Wu, Q; Liang, D; Wu, L

2017-06-01

PurposeTo reveal the underlying genetic defect in two four-generation Chinese families with aniridia and explore the pathologic mechanism.MethodsFull ophthalmic examinations were performed in two families with aniridia. The PAX6 gene was directly sequenced in patients of two families, and the detected variants were screened in unaffected family members and two hundred unrelated healthy controls. Real-time quantitative PCR was used to explore pathologic mechanisms of the two variants.ResultsAniridia, cataract, and oscillatory nystagmus were observed in patients of the two families. In addition, we observed corneal opacity and microphthalmus in family 1, and strabismus, left ectopia lentis, microphthalmus, and microcornea in family 2. Sanger sequencing detected a novel 1-bp duplication (c.50dupA) in family 1 and a novel 2-bp splice site deletion (c.765+1_765+2delGT) in family 2. Sequencing of cDNA indicated skipping of exon 9 caused by the splice site deletion, being predicted to cause a premature stop codon, as well as the duplication. The PAX6 mRNA significantly lower in patients with aniridia than in unaffected family members in both families, suggesting that the duplication and splice site deletion caused nonsense-mediated mRNA decay.ConclusionsOur study identified two novel PAX6 variants in two families with aniridia and revealed the pathogenicity of the variants; this would expand the variant spectrum of PAX6 and help us better understand the molecular basis of aniridia, thus facilitating genetic counseling.

Analysis of Alternative Pre-RNA Splicing in the Mouse Retina Using a Fluorescent Reporter.

PubMed

Murphy, Daniel; Kolandaivelu, Saravanan; Ramamurthy, Visvanathan; Stoilov, Peter

2016-01-01

In vivo alternative splicing is controlled in a tissue and cell type specific manner. Often individual cellular components of complex tissues will express different splicing programs. Thus, when studying splicing in multicellular organisms it is critical to determine the exon inclusion levels in individual cells positioned in the context of their native tissue or organ. Here we describe how a fluorescent splicing reporter in combination with in vivo electroporation can be used to visualize alternative splicing in individual cells within mature tissues. In a test case we show how the splicing of a photoreceptor specific exon can be visualized within the mouse retina. The retina was chosen as an example of a complex tissue that is fragile and whose cells cannot be studied in culture. With minor modifications to the injection and electroporation procedure, the protocol we outline can be applied to other tissues and organs.

Identification of a chemical inhibitor for nuclear speckle formation: Implications for the function of nuclear speckles in regulation of alternative pre-mRNA splicing

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

Kurogi, Yutaro; Matsuo, Yota; Mihara, Yuki

2014-03-28

Highlights: • We identified tubercidin as a compound inducing aberrant formation of the speckles. • Tubercidin causes delocalization of poly (A){sup +}RNAs from nuclear speckles. • Tubercidin induces dispersion of splicing factors from nuclear speckles. • Tubercidin affects alternative pre-mRNA splicing. • Nuclear speckles play a role in regulation of alternative pre-mRNA splicing. - Abstract: Nuclear speckles are subnuclear structures enriched with RNA processing factors and poly (A){sup +} RNAs comprising mRNAs and poly (A){sup +} non-coding RNAs (ncRNAs). Nuclear speckles are thought to be involved in post-transcriptional regulation of gene expression, such as pre-mRNA splicing. By screening 3585 culturemore » extracts of actinomycetes with in situ hybridization using an oligo dT probe, we identified tubercidin, an analogue of adenosine, as an inhibitor of speckle formation, which induces the delocalization of poly (A){sup +} RNA and dispersion of splicing factor SRSF1/SF2 from nuclear speckles in HeLa cells. Treatment with tubercidin also decreased steady-state MALAT1 long ncRNA, thought to be involved in the retention of SRSF1/SF2 in nuclear speckles. In addition, we found that tubercidin treatment promoted exon skipping in the alternative splicing of Clk1 pre-mRNA. These results suggest that nuclear speckles play a role in modulating the concentration of splicing factors in the nucleoplasm to regulate alternative pre-mRNA splicing.« less

Structure and Function of the Splice Variants of TMPRSS2-ERG, a Prevalent Genomic Alteration in Prostate Cancer

DTIC Science & Technology

2009-09-01

binding ETS domain) and five type II (without ETS domain). Fusion-positive type I– and type II–containing phages were amplified with T3 and T7 primers...will be performed to identify the authentic 3’ UTRs from the mRNA pool from CaP patient specimens. Using phage excision strategy, we will use to... phage DNA sequences plasmids (cDNA) clones were generated by using phage excision strategy. Figure 1. ERG splice variants in prostate cancer

Alternative splicing of mutually exclusive exons--a review.

PubMed

Pohl, Martin; Bortfeldt, Ralf H; Grützmann, Konrad; Schuster, Stefan

2013-10-01

Alternative splicing (AS) of pre-mRNAs in higher eukaryotes and several viruses is one major source of protein diversity. Usually, the following major subtypes of AS are distinguished: exon skipping, intron retention, and alternative 3' and 5' splice sites. Moreover, mutually exclusive exons (MXEs) represent a rare subtype. In the splicing of MXEs, two (or more) splicing events are not independent anymore, but are executed or disabled in a coordinated manner. In this review, several bioinformatics approaches for analyzing MXEs are presented and discussed. In particular, we revisit suitable definitions and nomenclatures, and bioinformatics tools for finding MXEs, adjacent and non-adjacent MXEs, clustered and grouped MXEs. Moreover, the molecular mechanisms for splicing MXEs proposed in the literature are reviewed and discussed. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Comprehensive analysis of alternative splicing and functionality in neuronal differentiation of P19 cells.

PubMed

Suzuki, Hitoshi; Osaki, Ken; Sano, Kaori; Alam, A H M Khurshid; Nakamura, Yuichiro; Ishigaki, Yasuhito; Kawahara, Kozo; Tsukahara, Toshifumi

2011-02-18

Alternative splicing, which produces multiple mRNAs from a single gene, occurs in most human genes and contributes to protein diversity. Many alternative isoforms are expressed in a spatio-temporal manner, and function in diverse processes, including in the neural system. The purpose of the present study was to comprehensively investigate neural-splicing using P19 cells. GeneChip Exon Array analysis was performed using total RNAs purified from cells during neuronal cell differentiation. To efficiently and readily extract the alternative exon candidates, 9 filtering conditions were prepared, yielding 262 candidate exons (236 genes). Semiquantitative RT-PCR results in 30 randomly selected candidates suggested that 87% of the candidates were differentially alternatively spliced in neuronal cells compared to undifferentiated cells. Gene ontology and pathway analyses suggested that many of the candidate genes were associated with neural events. Together with 66 genes whose functions in neural cells or organs were reported previously, 47 candidate genes were found to be linked to 189 events in the gene-level profile of neural differentiation. By text-mining for the alternative isoform, distinct functions of the isoforms of 9 candidate genes indicated by the result of Exon Array were confirmed. Alternative exons were successfully extracted. Results from the informatics analyses suggested that neural events were primarily governed by genes whose expression was increased and whose transcripts were differentially alternatively spliced in the neuronal cells. In addition to known functions in neural cells or organs, the uninvestigated alternative splicing events of 11 genes among 47 candidate genes suggested that cell cycle events are also potentially important. These genes may help researchers to differentiate the roles of alternative splicing in cell differentiation and cell proliferation.

[Deregulation of pre-messenger RNA splicing and rare diseases].

PubMed

de la Grange, Pierre

2016-12-01

Most of protein-coding human genes are subjected to alternative pre-mRNA splicing. This mechanism is highly regulated to precisely modulate detection of specific splice sites. This regulation is under control of the spliceosome and several splicing factors are also required to modulate the alternative usage of splice sites. Splicing factors and spliceosome components recognize splicing signals and regulatory sequences of the pre-mRNAs. These splicing sequences make splicing susceptible to polymorphisms and mutations. Examples of associations between human rare diseases and defects in pre-messenger RNA splicing are accumulating. Although many alterations are caused by mutations in splicing sequence (i.e., cis acting mutations), recent studies described the disruptive impact of mutations within spliceosome components or splicing factors (i.e., trans acting mutations). Following growing of knowledge regarding splicing regulation, several approaches have been developed to compensate for the effect of deleterious mutations and to restore sufficient amounts of functional protein. © 2016 médecine/sciences – Inserm.

Short linear motif acquisition, exon formation and alternative splicing determine a pathway to diversity for NCoR-family co-repressors

PubMed Central

Short, Stephen; Peterkin, Tessa; Guille, Matthew; Patient, Roger; Sharpe, Colin

2015-01-01

Vertebrate NCoR-family co-repressors play central roles in the timing of embryo and stem cell differentiation by repressing the activity of a range of transcription factors. They interact with nuclear receptors using short linear motifs (SLiMs) termed co-repressor for nuclear receptor (CoRNR) boxes. Here, we identify the pathway leading to increasing co-repressor diversity across the deuterostomes. The final complement of CoRNR boxes arose in an ancestral cephalochordate, and was encoded in one large exon; the urochordates and vertebrates then split this region between 10 and 12 exons. In Xenopus, alternative splicing is prevalent in NCoR2, but absent in NCoR1. We show for one NCoR1 exon that alternative splicing can be recovered by a single point mutation, suggesting NCoR1 lost the capacity for alternative splicing. Analyses in Xenopus and zebrafish identify that cellular context, rather than gene sequence, predominantly determines species differences in alternative splicing. We identify a pathway to diversity for the NCoR family beginning with the addition of a SLiM, followed by gene duplication, the generation of alternatively spliced isoforms and their differential deployment. PMID:26289800

Identification and functional analysis of two alternatively spliced transcripts of ABSCISIC ACID INSENSITIVE3 (ABI3) in linseed flax (Linum usitatissimum L.).

PubMed

Wang, Yanyan; Zhang, Tianbao; Song, Xiaxia; Zhang, Jianping; Dang, Zhanhai; Pei, Xinwu; Long, Yan

2018-01-01

Alternative splicing is a popular phenomenon in different types of plants. It can produce alternative spliced transcripts that encode proteins with altered functions. Previous studies have shown that one transcription factor, ABSCISIC ACID INSENSITIVE3 (ABI3), which encodes an important component in abscisic acid (ABA) signaling, is subjected to alternative splicing in both mono- and dicotyledons. In the current study, we identified two homologs of ABI3 in the genome of linseed flax. We screened two alternatively spliced flax LuABI3 transcripts, LuABI3-2 and LuABI3-3, and one normal flax LuABI3 transcript, LuABI3-1. Sequence analysis revealed that one of the alternatively spliced transcripts, LuABI3-3, retained a 6 bp intron. RNA accumulation analysis showed that all three transcripts were expressed during seed development, while subcellular localization and transgene experiments showed that LuABI3-3 had no biological function. The two normal transcripts, LuABI3-1 and LuABI3-2, are the important functional isoforms in flax and play significant roles in the ABA regulatory pathway during seed development, germination, and maturation.

Molecular outcomes, clinical consequences, and genetic diagnosis of Oculocutaneous Albinism in Pakistani population.

PubMed

Shahzad, Mohsin; Yousaf, Sairah; Waryah, Yar M; Gul, Hadia; Kausar, Tasleem; Tariq, Nabeela; Mahmood, Umair; Ali, Muhammad; Khan, Muzammil A; Waryah, Ali M; Shaikh, Rehan S; Riazuddin, Saima; Ahmed, Zubair M

2017-03-07

Nonsyndromic oculocutaneous Albinism (nsOCA) is clinically characterized by the loss of pigmentation in the skin, hair, and iris. OCA is amongst the most common causes of vision impairment in children. To date, pathogenic variants in six genes have been identified in individuals with nsOCA. Here, we determined the identities, frequencies, and clinical consequences of OCA alleles in 94 previously unreported Pakistani families. Combination of Sanger and Exome sequencing revealed 38 alleles, including 22 novel variants, segregating with nsOCA phenotype in 80 families. Variants of TYR and OCA2 genes were the most common cause of nsOCA, occurring in 43 and 30 families, respectively. Twenty-two novel variants include nine missense, four splice site, two non-sense, one insertion and six gross deletions. In vitro studies revealed retention of OCA proteins harboring novel missense alleles in the endoplasmic reticulum (ER) of transfected cells. Exon-trapping assays with constructs containing splice site alleles revealed errors in splicing. As eight alleles account for approximately 56% (95% CI: 46.52-65.24%) of nsOCA cases, primarily enrolled from Punjab province of Pakistan, hierarchical strategies for variant detection would be feasible and cost-efficient genetic tests for OCA in families with similar origin. Thus, we developed Tetra-primer ARMS assays for rapid, reliable, reproducible and economical screening of most of these common alleles.

Alternative Pre-mRNA Splicing in Mammals and Teleost Fish: A Effective Strategy for the Regulation of Immune Responses Against Pathogen Infection.

PubMed

Chang, Ming Xian; Zhang, Jie

2017-07-15

Pre-mRNA splicing is the process by which introns are removed and the protein coding elements assembled into mature mRNAs. Alternative pre-mRNA splicing provides an important source of transcriptome and proteome complexity through selectively joining different coding elements to form mRNAs, which encode proteins with similar or distinct functions. In mammals, previous studies have shown the role of alternative splicing in regulating the function of the immune system, especially in the regulation of T-cell activation and function. As lower vertebrates, teleost fish mainly rely on a large family of pattern recognition receptors (PRRs) to recognize pathogen-associated molecular patterns (PAMPs) from various invading pathogens. In this review, we summarize recent advances in our understanding of alternative splicing of piscine PRRs including peptidoglycan recognition proteins (PGRPs), nucleotide binding and oligomerization domain (NOD)-like receptors (NLRs), retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs) and their downstream signaling molecules, compared to splicing in mammals. We also discuss what is known and unknown about the function of splicing isoforms in the innate immune responses against pathogens infection in mammals and teleost fish. Finally, we highlight the consequences of alternative splicing in the innate immune system and give our view of important directions for future studies.

Intron-mediated alternative splicing of WOOD-ASSOCIATED NAC TRANSCRIPTION FACTOR1B regulates cell wall thickening during fiber development in Populus species.

PubMed

Zhao, Yunjun; Sun, Jiayan; Xu, Peng; Zhang, Rui; Li, Laigeng

2014-02-01

Alternative splicing is an important mechanism involved in regulating the development of multicellular organisms. Although many genes in plants undergo alternative splicing, little is understood of its significance in regulating plant growth and development. In this study, alternative splicing of black cottonwood (Populus trichocarpa) wood-associated NAC domain transcription factor (PtrWNDs), PtrWND1B, is shown to occur exclusively in secondary xylem fiber cells. PtrWND1B is expressed with a normal short-transcript PtrWND1B-s as well as its alternative long-transcript PtrWND1B-l. The intron 2 structure of the PtrWND1B gene was identified as a critical sequence that causes PtrWND1B alternative splicing. Suppression of PtrWND1B expression specifically inhibited fiber cell wall thickening. The two PtrWND1B isoforms play antagonistic roles in regulating cell wall thickening during fiber cell differentiation in Populus spp. PtrWND1B-s overexpression enhanced fiber cell wall thickening, while overexpression of PtrWND1B-l repressed fiber cell wall thickening. Alternative splicing may enable more specific regulation of processes such as fiber cell wall thickening during wood formation.

Intron-Mediated Alternative Splicing of WOOD-ASSOCIATED NAC TRANSCRIPTION FACTOR1B Regulates Cell Wall Thickening during Fiber Development in Populus Species1[W

PubMed Central

Zhao, Yunjun; Sun, Jiayan; Xu, Peng; Zhang, Rui; Li, Laigeng

2014-01-01

Alternative splicing is an important mechanism involved in regulating the development of multicellular organisms. Although many genes in plants undergo alternative splicing, little is understood of its significance in regulating plant growth and development. In this study, alternative splicing of black cottonwood (Populus trichocarpa) wood-associated NAC domain transcription factor (PtrWNDs), PtrWND1B, is shown to occur exclusively in secondary xylem fiber cells. PtrWND1B is expressed with a normal short-transcript PtrWND1B-s as well as its alternative long-transcript PtrWND1B-l. The intron 2 structure of the PtrWND1B gene was identified as a critical sequence that causes PtrWND1B alternative splicing. Suppression of PtrWND1B expression specifically inhibited fiber cell wall thickening. The two PtrWND1B isoforms play antagonistic roles in regulating cell wall thickening during fiber cell differentiation in Populus spp. PtrWND1B-s overexpression enhanced fiber cell wall thickening, while overexpression of PtrWND1B-l repressed fiber cell wall thickening. Alternative splicing may enable more specific regulation of processes such as fiber cell wall thickening during wood formation. PMID:24394777

Lineage-specific splicing of a brain-enriched alternative exon promotes glioblastoma progression

PubMed Central

Ferrarese, Roberto; Harsh, Griffith R.; Yadav, Ajay K.; Bug, Eva; Maticzka, Daniel; Reichardt, Wilfried; Dombrowski, Stephen M.; Miller, Tyler E.; Masilamani, Anie P.; Dai, Fangping; Kim, Hyunsoo; Hadler, Michael; Scholtens, Denise M.; Yu, Irene L.Y.; Beck, Jürgen; Srinivasasainagendra, Vinodh; Costa, Fabrizio; Baxan, Nicoleta; Pfeifer, Dietmar; von Elverfeldt, Dominik; Backofen, Rolf; Weyerbrock, Astrid; Duarte, Christine W.; He, Xiaolin; Prinz, Marco; Chandler, James P.; Vogel, Hannes; Chakravarti, Arnab; Rich, Jeremy N.; Carro, Maria S.; Bredel, Markus

2014-01-01

Tissue-specific alternative splicing is critical for the emergence of tissue identity during development, yet the role of this process in malignant transformation is undefined. Tissue-specific splicing involves evolutionarily conserved, alternative exons that represent only a minority of the total alternative exons identified. Many of these conserved exons have functional features that influence signaling pathways to profound biological effect. Here, we determined that lineage-specific splicing of a brain-enriched cassette exon in the membrane-binding tumor suppressor annexin A7 (ANXA7) diminishes endosomal targeting of the EGFR oncoprotein, consequently enhancing EGFR signaling during brain tumor progression. ANXA7 exon splicing was mediated by the ribonucleoprotein PTBP1, which is normally repressed during neuronal development. PTBP1 was highly expressed in glioblastomas due to loss of a brain-enriched microRNA (miR-124) and to PTBP1 amplification. The alternative ANXA7 splicing trait was present in precursor cells, suggesting that glioblastoma cells inherit the trait from a potential tumor-initiating ancestor and that these cells exploit this trait through accumulation of mutations that enhance EGFR signaling. Our data illustrate that lineage-specific splicing of a tissue-regulated alternative exon in a constituent of an oncogenic pathway eliminates tumor suppressor functions and promotes glioblastoma progression. This paradigm may offer a general model as to how tissue-specific regulatory mechanisms can reprogram normal developmental processes into oncogenic ones. PMID:24865424

Cloning and characterization of an alternative splicing transcript of the gene coding for human cytidine deaminase.

PubMed

Lisboa, Bianca Cristina Garcia; Machado, Tamara da Rocha; Pimenta, Daniel Carvalho; Han, Sang Won

2007-02-01

Human cytidine deaminase (HCD) catalyzes the deamination of cytidine or deoxycytidine to uridine or deoxyuridine, respectively. The genomic sequence of HCD is formed by 31 kb with 4 exons and several alternative splicing signals, but an alternative form of HCD has yet to be reported. Here we describe the cloning and characterization of a small form of HCD, HSCD, and it is likely to be a product of alternative splicing of HCD. The alignment of DNA sequences shows that the HSCD matches HCD in 2 parts, except for a deletion of 170 bp. Based on the HCD genome organization, exons 1 and 4 should be joined and all sequences of introns and exons 2 and 3 should be deleted by splicing. This alternative splicing shifted the translation of the reading frame from the point of splicing. The estimated molecular mass is 9.8 kDa, and this value was confirmed by Western blot and mass spectroscopy after expressing the gene fused with glutathionine-S-transferase in the pGEX vector. The deletion and shift of the reading frame caused a loss of HCD activity, which was confirmed by enzyme assay and also with NIH3T3 cells modified to express HSCD and challenged against cytosine arabinoside. In this work we describe the identification and characterization of HSCD, which is the product of alternative splicing of the HCD gene.

Identification of a Novel Transcript and Regulatory Mechanism for Microsomal Triglyceride Transfer Protein

PubMed Central

Suzuki, Takashi; Brown, Judy J.; Swift, Larry L.

2016-01-01

Microsomal triglyceride transfer protein (MTP) is essential for the assembly of triglyceride-rich apolipoprotein B-containing lipoproteins. Previous studies in our laboratory identified a novel splice variant of MTP in mice that we named MTP-B. MTP-B has a unique first exon (1B) located 2.7 kB upstream of the first exon (1A) for canonical MTP (MTP-A). The two mature isoforms, though nearly identical in sequence and function, have different tissue expression patterns. In this study we report the identification of a second MTP splice variant (MTP-C), which contains both exons 1B and 1A. MTP-C is expressed in all the tissues we tested. In cells transfected with MTP-C, protein expression was less than 15% of that found when the cells were transfected with MTP-A or MTP-B. In silico analysis of the 5’-UTR of MTP-C revealed seven ATGs upstream of the start site for MTP-A, which is the only viable start site in frame with the main coding sequence. One of those ATGs was located in the 5’-UTR for MTP-A. We generated reporter constructs in which the 5’-UTRs of MTP-A or MTP-C were inserted between an SV40 promoter and the coding sequence of the luciferase gene and transfected these constructs into HEK 293 cells. Luciferase activity was significantly reduced by the MTP-C 5’-UTR, but not by the MTP-A 5’-UTR. We conclude that alternative splicing plays a key role in regulating MTP expression by introducing unique 5’-UTRs, which contain elements that alter translation efficiency, enabling the cell to optimize MTP levels and activity. PMID:26771188

Axed MUC4 (MUC4/X) aggravates pancreatic malignant phenotype by activating integrin-β1/FAK/ERK pathway.

PubMed

Jahan, Rahat; Macha, Muzafar A; Rachagani, Satyanarayana; Das, Srustidhar; Smith, Lynette M; Kaur, Sukhwinder; Batra, Surinder K

2018-08-01

Alternative splicing is evolving as an eminent player of oncogenic signaling for tumor development and progression. Mucin 4 (MUC4), a type I membrane-bound mucin, is differentially expressed in pancreatic cancer (PC) and plays a critical role in its progression and metastasis. However, the molecular implications of MUC4 splice variants during disease pathogenesis remain obscure. The present study delineates the pathological and molecular significance of a unique splice variant of MUC4, MUC4/X, which lacks the largest exon 2, along with exon 3. Exon 2 encodes for the highly glycosylated tandem repeat (TR) domain of MUC4 and its absence creates MUC4/X, which is devoid of TR. Expression analysis from PC clinical samples revealed significant upregulation of MUC4/X in PC tissues with most differential expression in poorly differentiated tumors. In vitro studies suggest that overexpression of MUC4/X in wild-type-MUC4 (WT-MUC4) null PC cell lines markedly enhanced PC cell proliferation, invasion, and adhesion to extracellular matrix (ECM) proteins. Furthermore, MUC4/X overexpression leads to an increase in the tumorigenic potential of PC cells in orthotopic transplantation studies. In line with these findings, doxycycline-induced expression of MUC4/X in an endogenous WT-MUC4 expressing PC cell line (Capan-1) also displayed enhanced cell proliferation, invasion, and adhesion to ECM, compared to WT-MUC4 alone, emphasizing its direct involvement in the aggressive behavior of PC cells. Investigation into the molecular mechanism suggested that MUC4/X facilitated PC tumorigenesis via integrin-β1/FAK/ERK signaling pathway. Overall, these findings revealed the novel role of MUC4/X in promoting and sustaining the oncogenic features of PC. Copyright © 2018 Elsevier B.V. All rights reserved.

Evolution of Nova-Dependent Splicing Regulation in the Brain

PubMed Central

Živin, Marko; Darnell, Robert B

2007-01-01

A large number of alternative exons are spliced with tissue-specific patterns, but little is known about how such patterns have evolved. Here, we study the conservation of the neuron-specific splicing factors Nova1 and Nova2 and of the alternatively spliced exons they regulate in mouse brain. Whereas Nova RNA binding domains are 94% identical across vertebrate species, Nova-dependent splicing silencer and enhancer elements (YCAY clusters) show much greater divergence, as less than 50% of mouse YCAY clusters are conserved at orthologous positions in the zebrafish genome. To study the relation between the evolution of tissue-specific splicing and YCAY clusters, we compared the brain-specific splicing of Nova-regulated exons in zebrafish, chicken, and mouse. The presence of YCAY clusters in lower vertebrates invariably predicted conservation of brain-specific splicing across species, whereas their absence in lower vertebrates correlated with a loss of alternative splicing. We hypothesize that evolution of Nova-regulated splicing in higher vertebrates proceeds mainly through changes in cis-acting elements, that tissue-specific splicing might in some cases evolve in a single step corresponding to evolution of a YCAY cluster, and that the conservation level of YCAY clusters relates to the functions encoded by the regulated RNAs. PMID:17937501

Can the HIV-1 splicing machinery be targeted for drug discovery?

PubMed Central

Dlamini, Zodwa; Hull, Rodney

2017-01-01

HIV-1 is able to express multiple protein types and isoforms from a single 9 kb mRNA transcript. These proteins are also expressed at particular stages of viral development, and this is achieved through the control of alternative splicing and the export of these transcripts from the nucleus. The nuclear export is controlled by the HIV protein Rev being required to transport incompletely spliced and partially spliced mRNA from the nucleus where they are normally retained. This implies a close relationship between the control of alternate splicing and the nuclear export of mRNA in the control of HIV-1 viral proliferation. This review discusses both the processes. The specificity and regulation of splicing in HIV-1 is controlled by the use of specific splice sites as well as exonic splicing enhancer and exonic splicing silencer sequences. The use of these silencer and enhancer sequences is dependent on the serine arginine family of proteins as well as the heterogeneous nuclear ribonucleoprotein family of proteins that bind to these sequences and increase or decrease splicing. Since alternative splicing is such a critical factor in viral development, it presents itself as a promising drug target. This review aims to discuss the inhibition of splicing, which would stall viral development, as an anti-HIV therapeutic strategy. In this review, the most recent knowledge of splicing in human immunodeficiency viral development and the latest therapeutic strategies targeting human immunodeficiency viral splicing are discussed. PMID:28331370

Alternative splicing of the tyrosinase gene transcript in normal human melanocytes and lymphocytes.

PubMed

Fryer, J P; Oetting, W S; Brott, M J; King, R A

2001-11-01

We have identified and isolated ectopically expressed tyrosinase transcripts in normal human melanocytes and lymphocytes and in a human melanoma (MNT-1) cell line to establish a baseline for the expression pattern of this gene in normal tissue. Tyrosinase mRNA from human lymphoblastoid cell lines was reverse transcribed and amplified using specific "nested" primers. This amplification yielded eight identifiable transcripts; five that resulted from alternative splicing patterns arising from the utilization of normal and alternative splice sequences. Identical splicing patterns were found in transcripts from human primary melanocytes in culture and a melanoma cell line, indicating that lymphoblastoid cell lines provide an accurate reflection of transcript processing in melanocytes. Similar splicing patterns have also been found with murine melanocyte tyrosinase transcripts. Our results demonstrate that alternative splicing of human tyrosinase gene transcript produces a number of predictable and identifiable transcripts, and that human lymphoblastoid cell lines provide a source of ectopically expressed transcripts that can be used to study the biology of tyrosinase gene expression in humans.

Novel association of familial testicular germ cell tumor and autosomal dominant polycystic kidney disease with PKD1 mutation.

PubMed

Truscott, Laurel; Gell, Joanna; Chang, Vivian Y; Lee, Hane; Strom, Samuel P; Pillai, Rex; Sisk, Anthony; Martinez-Agosto, Julian A; Anderson, Martin; Federman, Noah

2017-01-01

Adolescent brothers were diagnosed with testicular germ cell tumors within the same month. Both were found to have multiple renal cysts on pretreatment imaging done for staging. The proband, his brother, and their mother, were all found to have a novel splice variant in intron 8 of the PKD1 gene by clinical exome sequencing. This is the second family reported with both familial testicular germ cell tumor (FTGCT) and autosomal dominant polycystic kidney disease (ADPKD), and the first described association of FTGCT with a splice variant in PKD1. We suggest that this novel variant in PKD1 may convey increased risk for FTGCT in addition to causing ADPKD. © 2016 Wiley Periodicals, Inc.

Crystal structure of p44, a constitutively active splice variant of visual arrestin.

PubMed

Granzin, Joachim; Cousin, Anneliese; Weirauch, Moritz; Schlesinger, Ramona; Büldt, Georg; Batra-Safferling, Renu

2012-03-09

Visual arrestin specifically binds to photoactivated and phosphorylated rhodopsin and inactivates phototransduction. In contrast, the p44 splice variant can terminate phototransduction by binding to nonphosphorylated light-activated rhodopsin. Here we report the crystal structure of bovine p44 at a resolution of 1.85 Å. Compared to native arrestin, the p44 structure reveals significant differences in regions crucial for receptor binding, namely flexible loop V-VI and polar core regions. Additionally, electrostatic potential is remarkably positive on the N-domain and the C-domain. The p44 structure represents an active conformation that serves as a model to explain the 'constitutive activity' found in arrestin variants. Copyright © 2012 Elsevier Ltd. All rights reserved.

Growth Inhibition by Testosterone in an Androgen Receptor Splice Variant-Driven Prostate Cancer Model.

PubMed

Nakata, Daisuke; Nakayama, Kazuhide; Masaki, Tsuneo; Tanaka, Akira; Kusaka, Masami; Watanabe, Tatsuya

2016-12-01

Castration resistance creates a significant problem in the treatment of prostate cancer. Constitutively active splice variants of androgen receptor (AR) have emerged as drivers for resistance to androgen deprivation therapy, including the next-generation androgen-AR axis inhibitors abiraterone and enzalutamide. In this study, we describe the characteristics of a novel castration-resistant prostate cancer (CRPC) model, designated JDCaP-hr (hormone refractory). JDCaP-hr was established from an androgen-dependent JDCaP xenograft model after surgical castration. The expression of AR and its splice variants in JDCaP-hr was evaluated by immunoblotting and quantitative reverse transcription-polymerase chain reaction. The effects of AR antagonists and testosterone on JDCaP-hr were evaluated in vivo and in vitro. The roles of full-length AR (AR-FL) and AR-V7 in JDCaP-hr cell growth were evaluated using RNA interference. JDCaP-hr acquired a C-terminally truncated AR protein during progression from the parental JDCaP. The expression of AR-FL and AR-V7 mRNA was upregulated by 10-fold in JDCaP-hr compared with that in JDCaP, indicating that the JDCaP and JDCaP-hr models simulate castration resistance with some clinical features, such as overexpression of AR and its splice variants. The AR antagonist bicalutamide did not affect JDCaP-hr xenograft growth, and importantly, testosterone induced tumor regression. In vitro analysis demonstrated that androgen-independent prostate-specific antigen secretion and cell proliferation of JDCaP-hr were predominantly mediated by AR-V7. JDCaP-hr cell growth displayed a bell-shaped dependence on testosterone, and it was suppressed by physiological concentrations of testosterone. Testosterone induced rapid downregulation of both AR-FL and AR-V7 expression at physiological concentrations and suppressed expression of the AR target gene KLK3. Our findings support the clinical value of testosterone therapy, including bipolar androgen therapy, in the treatment of AR-overexpressed CRPC driven by AR splice variants that are not clinically actionable at present. Prostate 76:1536-1545, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

A Novel Intra-U1 snRNP Cross-Regulation Mechanism: Alternative Splicing Switch Links U1C and U1-70K Expression

PubMed Central

Rösel-Hillgärtner, Tanja Dorothe; Hung, Lee-Hsueh; Khrameeva, Ekaterina; Le Querrec, Patrick; Gelfand, Mikhail S.; Bindereif, Albrecht

2013-01-01

The U1 small nuclear ribonucleoprotein (snRNP)-specific U1C protein participates in 5′ splice site recognition and regulation of pre-mRNA splicing. Based on an RNA-Seq analysis in HeLa cells after U1C knockdown, we found a conserved, intra-U1 snRNP cross-regulation that links U1C and U1-70K expression through alternative splicing and U1 snRNP assembly. To investigate the underlying regulatory mechanism, we combined mutational minigene analysis, in vivo splice-site blocking by antisense morpholinos, and in vitro binding experiments. Alternative splicing of U1-70K pre-mRNA creates the normal (exons 7–8) and a non-productive mRNA isoform, whose balance is determined by U1C protein levels. The non-productive isoform is generated through a U1C-dependent alternative 3′ splice site, which requires an adjacent cluster of regulatory 5′ splice sites and binding of intact U1 snRNPs. As a result of nonsense-mediated decay (NMD) of the non-productive isoform, U1-70K mRNA and protein levels are down-regulated, and U1C incorporation into the U1 snRNP is impaired. U1-70K/U1C-deficient particles are assembled, shifting the alternative splicing balance back towards productive U1-70K splicing, and restoring assembly of intact U1 snRNPs. Taken together, we established a novel feedback regulation that controls U1-70K/U1C homeostasis and ensures correct U1 snRNP assembly and function. PMID:24146627

Consequences of germline variation disrupting the constitutional translational initiation codon start sites of MLH1 and BRCA2: use of potential alternative start sites and implications for predicting variant pathogenicity

PubMed Central

Parsons, Michael T.; Whiley, Phillip J.; Beesley, Jonathan; Drost, Mark; de Wind, Niels; Thompson, Bryony A.; Marquart, Louise; Hopper, John L.; Jenkins, Mark A.; Brown, Melissa A.; Tucker, Kathy; Warwick, Linda; Buchanan, Daniel D.; Spurdle, Amanda B.

2014-01-01

Variants that disrupt the translation initiation sequences in cancer predisposition genes are generally assumed to be deleterious. However few studies have validated these assumptions with functional and clinical data. Two cancer syndrome gene variants likely to affect native translation initiation were identified by clinical genetic testing: MLH1:c.1A>G p.(Met1?) and BRCA2:c.67+3A>G. In vitro GFP-reporter assays were conducted to assess the consequences of translation initiation disruption on alternative downstream initiation codon usage. Analysis of MLH1:c.1A>G p.(Met1?) showed that translation was mostly initiated at an in-frame position 103 nucleotides downstream, but also at two ATG sequences downstream. The protein product encoded by the in-frame transcript initiating from position c.103 showed loss of in vitro mismatch repair activity comparable to known pathogenic mutations. BRCA2:c.67+3A>G was shown by mRNA analysis to result in an aberrantly spliced transcript deleting exon 2 and the consensus ATG site. In the absence of exon 2, translation initiated mostly at an out-of-frame ATG 323 nucleotides downstream, and to a lesser extent at an in-frame ATG 370 nucleotides downstream. Initiation from any of the downstream alternative sites tested in both genes would lead to loss of protein function, but further clinical data is required to confirm if these variants are associated with a high cancer risk. Importantly, our results highlight the need for caution in interpreting the functional and clinical consequences of variation that leads to disruption of the initiation codon, since translation may not necessarily occur from the first downstream alternative start site, or from a single alternative start site. PMID:24302565

SplicePlot: a utility for visualizing splicing quantitative trait loci.

PubMed

Wu, Eric; Nance, Tracy; Montgomery, Stephen B

2014-04-01

RNA sequencing has provided unprecedented resolution of alternative splicing and splicing quantitative trait loci (sQTL). However, there are few tools available for visualizing the genotype-dependent effects of splicing at a population level. SplicePlot is a simple command line utility that produces intuitive visualization of sQTLs and their effects. SplicePlot takes mapped RNA sequencing reads in BAM format and genotype data in VCF format as input and outputs publication-quality Sashimi plots, hive plots and structure plots, enabling better investigation and understanding of the role of genetics on alternative splicing and transcript structure. Source code and detailed documentation are available at http://montgomerylab.stanford.edu/spliceplot/index.html under Resources and at Github. SplicePlot is implemented in Python and is supported on Linux and Mac OS. A VirtualBox virtual machine running Ubuntu with SplicePlot already installed is also available.

Mapping of oxidative stress responses of human tumor cells following photodynamic therapy using hexaminolevulinate

PubMed Central

Cekaite, Lina; Peng, Qian; Reiner, Andrew; Shahzidi, Susan; Tveito, Siri; Furre, Ingegerd E; Hovig, Eivind

2007-01-01

Background Photodynamic therapy (PDT) involves systemic or topical administration of a lesion-localizing photosensitizer or its precursor, followed by irradiation of visible light to cause singlet oxygen-induced damage to the affected tissue. A number of mechanisms seem to be involved in the protective responses to PDT, including activation of transcription factors, heat shock proteins, antioxidant enzymes and apoptotic pathways. Results In this study, we address the effects of a destructive/lethal hexaminolevulinate (HAL) mediated PDT dose on the transcriptome by using transcriptional exon evidence oligo microarrays. Here, we confirm deviations in the steady state expression levels of previously identified early defence response genes and extend this to include unreported PDT inducible gene groups, most notably the metallothioneins and histones. HAL-PDT mediated stress also altered expression of genes encoded by mitochondrial DNA (mtDNA). Further, we report PDT stress induced alternative splicing. Specifically, the ATF3 alternative isoform (deltaZip2) was up-regulated, while the full-length variant was not changed by the treatment. Results were independently verified by two different technological microarray platforms. Good microarray, RT-PCR and Western immunoblotting correlation for selected genes support these findings. Conclusion Here, we report new insights into how destructive/lethal PDT alters the transcriptome not only at the transcriptional level but also at post-transcriptional level via alternative splicing. PMID:17692132