Analysis of Subcellular RNA Fractions Revealed a Transcription-Independent Effect of Tumor Necrosis Factor Alpha on Splicing, Mediated by Spt5.

PubMed

Diamant, Gil; Eisenbaum, Tal; Leshkowitz, Dena; Dikstein, Rivka

2016-05-01

The proinflammatory cytokine tumor necrosis factor alpha (TNF-α) modulates the expression of many genes, primarily through activation of NF-κB. Here, we examined the global effects of the elongation factor Spt5 on nascent and mature mRNAs of TNF-α-induced cells using chromatin and cytosolic subcellular fractions. We identified several classes of TNF-α-induced genes controlled at the level of transcription, splicing, and chromatin retention. Spt5 was found to facilitate splicing and chromatin release in genes displaying high induction rates. Further analysis revealed striking effects of TNF-α on the splicing of 25% of expressed genes; the vast majority were not transcriptionally induced. Splicing enhancement of noninduced genes by TNF-α was transient and independent of NF-κB. Investigating the underlying basis, we found that Spt5 is required for the splicing facilitation of the noninduced genes. In line with this, Spt5 interacts with Sm core protein splicing factors. Furthermore, following TNF-α treatment, levels of RNA polymerase II (Pol II) but not Spt5 are reduced from the splicing-induced genes, suggesting that these genes become enriched with a Pol II-Spt5 form. Our findings revealed the Pol II-Spt5 complex as a highly competent coordinator of cotranscriptional splicing. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

U2AF1 mutations alter splice site recognition in hematological malignancies.

PubMed

Ilagan, Janine O; Ramakrishnan, Aravind; Hayes, Brian; Murphy, Michele E; Zebari, Ahmad S; Bradley, Philip; Bradley, Robert K

2015-01-01

Whole-exome sequencing studies have identified common mutations affecting genes encoding components of the RNA splicing machinery in hematological malignancies. Here, we sought to determine how mutations affecting the 3' splice site recognition factor U2AF1 alter its normal role in RNA splicing. We find that U2AF1 mutations influence the similarity of splicing programs in leukemias, but do not give rise to widespread splicing failure. U2AF1 mutations cause differential splicing of hundreds of genes, affecting biological pathways such as DNA methylation (DNMT3B), X chromosome inactivation (H2AFY), the DNA damage response (ATR, FANCA), and apoptosis (CASP8). We show that U2AF1 mutations alter the preferred 3' splice site motif in patients, in cell culture, and in vitro. Mutations affecting the first and second zinc fingers give rise to different alterations in splice site preference and largely distinct downstream splicing programs. These allele-specific effects are consistent with a computationally predicted model of U2AF1 in complex with RNA. Our findings suggest that U2AF1 mutations contribute to pathogenesis by causing quantitative changes in splicing that affect diverse cellular pathways, and give insight into the normal function of U2AF1's zinc finger domains. © 2015 Ilagan et al.; Published by Cold Spring Harbor Laboratory Press.

Mutations of RNA splicing factors in hematological malignancies.

PubMed

Shukla, Girish C; Singh, Jagjit

2017-11-28

Systematic large-scale cancer genomic studies have produced numerous significant findings. These studies have not only revealed new cancer-promoting genes, but they also have identified cancer-promoting functions of previously known "housekeeping" genes. These studies have identified numerous mutations in genes which play a fundamental role in nuclear precursor mRNA splicing. Somatic mutations and copy number variation in many of the splicing factors which participate in the formation of multiple spliceosomal complexes appear to play a role in many cancers and in particular in myelodysplastic syndromes (MDS). Mutated proteins seem to interfere with the recognition of the authentic splice sites (SS) leading to utilization of suboptimal alternative splicing sites generating aberrantly spliced mRNA isoforms. This short review is focusing on the function of the splice factors involved in the formation of splicing complexes and potential mechanisms which affect usage of the authentic splice site recognition. Copyright © 2017 Elsevier B.V. All rights reserved.

[Molecular structure and alternative splicing analysis of heat shock factors of Schistosoma japonicum].

PubMed

Yu, Xie; Hai-Yan, Liao; Shu-Jie, Chen; Ling-Yu, Shi; Li-Yan, Ou; Ping-Ying, Teng; Dan, Xia; Qi-Wei, Chen; Sinan, Zheng; Xiao-Hong, Zhou

2016-07-12

To clone and identify the heat shock factors (HSFs) of Schistosoma japonicum and analyze its molecular structure and alternative splicing pattern. The New Zealand rabbits were infected with the cercariae of Schistosoma japonicum and were killed and dissected 42 days post-infection, and the adult worms of S. japonicum and the livers of the rabbits were harvested. Then, the total RNA was extracted by using Trizol reagent. The Sj-hsf open reading frame (ORF) and the alternative splicing fragments were amplified by RT-PCR from the female, male and egg samples, then cloned and verified by enzyme digestion and sequencing. DNAMAN 8.0, InterPro, Mega 6 combined with the Internet databases were utilized to clarify the gene structure, functional domains, alternative splicing pattern, and the homology and phylogenetic tree of HSFs. Sj-hsf ORF and the alternative splicing fragments were amplified from the female, male and egg samples of S. japonicum by RT-PCR. After cloning, the positive recombinant plasmids pB Sj HSFf-F, pB Sj HSFf-M, pB Sj HSFf-E containing Sj-hsf ORF, pB Sj HSFs-F, pB Sj HSFs-M, pB Sj HSFs-E with Sj-hsf alternative splicing fragments were identified by enzyme digestion and sequencing. Three alternative splicing Sj-hsf isoforms were observed through sequence analysis: Sj-hsf -isoform1 (2 050 bp), Sj-hsf -isoform2 (2 086 bp) and Sj - hsf -isoform3 (2 111 bp); the GenBank accession numbers were KU954546, KX119143 and KX119144, respectively. All the three isoforms located in the same Contig SJC_S000780 of S. japonicum genome and all expressed at female, male and egg stages, but Sj-hsf -isoform1 with a high-level expression. Sj -HSF-isoform1 (671 aa) and Sj -HSF-isoform2 (683 aa) had DBD (DNA binding domain), HR-A/B and HR-C domains, while Sj -HSF-isoform3 (282 aa) stopped in advance without HR-C domain. Phylogenetic tree analysis of HSFs illustrated that Sj - HSFs belonged to HSF1 family, with a close phylogenetic relationship to Sm -HSFs. There are three alternative splicing isoforms of Sj -HSF existing in the female, male and egg stages of S. japonicum , but Sj -HSF-isoform1 expresses in a high-level. This study lays the foundation for further study on molecular mechanisms of Sj- HSFs in regulating the heat shock response system.

Role of epigenetic factors in the selection of the alternative splicing isoforms of human KRAS in colorectal cancer cell lines.

PubMed

Riffo-Campos, Ángela L; Gimeno-Valiente, Francisco; Rodríguez, Fernanda M; Cervantes, Andrés; López-Rodas, Gerardo; Franco, Luis; Castillo, Josefa

2018-04-17

Mutation-driven activation of KRAS is crucial to cancer development. The human gene yields four mRNA splicing isoforms, 4A and 4B being translated to protein. Their different properties and oncogenic potential have been studied, but the mechanisms deciding the ratio 4A/4B are not known. To address this issue, the expression of the four KRAS isoforms was determined in 9 human colorectal cancer cell lines. HCT116 and SW48 were further selected because they present the highest difference in the ratio 4A/4B (twice as much in HCT116 than in SW48). Chromatin structure was analysed at the exon 4A, characteristic of isoform 4A, at its intronic borders and at the two flanking exons. The low nucleosome occupancy at exon 4A in both cell lines may result in a fast transcriptional rate, which would explain the general lower abundance of isoform 4A, also found in cells and tissues by other authors, but due to its similarity between both cell lines, chromatin structure does not influence alternative splicing. DNA methylation downstream exon 4A significantly differs in HCT116 and SW48 cells, but the CCCTC-binding factor, which affects the processivity of RNA polymerase and the alternative splicing, does not bind the differentially methylated sequences. Quantitative epigenetic analysis at mononucleosomal level revealed significant differences between both cell lines in H3K4me3, H3K27me3, H3K36me3, H3K9ac, H3K27ac and H4K20me1, and the inhibition of some histone-modifying enzymes alters the ratio 4A/4B. It can be concluded that the epigenetic modification of histones has an influence on the selection of isoforms 4A and 4B.

Unique Features of Human Protein Arginine Methyltransferase 9 (PRMT9) and Its Substrate RNA Splicing Factor SF3B2*

PubMed Central

Hadjikyriacou, Andrea; Yang, Yanzhong; Espejo, Alexsandra; Bedford, Mark T.; Clarke, Steven G.

2015-01-01

Human protein arginine methyltransferase (PRMT) 9 symmetrically dimethylates arginine residues on splicing factor SF3B2 (SAP145) and has been functionally linked to the regulation of alternative splicing of pre-mRNA. Site-directed mutagenesis studies on this enzyme and its substrate had revealed essential unique residues in the double E loop and the importance of the C-terminal duplicated methyltransferase domain. In contrast to what had been observed with other PRMTs and their physiological substrates, a peptide containing the methylatable Arg-508 of SF3B2 was not recognized by PRMT9 in vitro. Although amino acid substitutions of residues surrounding Arg-508 had no great effect on PRMT9 recognition of SF3B2, moving the arginine residue within this sequence abolished methylation. PRMT9 and PRMT5 are the only known mammalian enzymes capable of forming symmetric dimethylarginine (SDMA) residues as type II PRMTs. We demonstrate here that the specificity of these enzymes for their substrates is distinct and not redundant. The loss of PRMT5 activity in mouse embryo fibroblasts results in almost complete loss of SDMA, suggesting that PRMT5 is the primary SDMA-forming enzyme in these cells. PRMT9, with its duplicated methyltransferase domain and conserved sequence in the double E loop, appears to have a unique structure and specificity among PRMTs for methylating SF3B2 and potentially other polypeptides. PMID:25979344

Biochemical and proteomic analysis of spliceosome factors interacting with intron-1 of human papillomavirus type-16.

PubMed

Martínez-Salazar, Martha; López-Urrutia, Eduardo; Arechaga-Ocampo, Elena; Bonilla-Moreno, Raul; Martínez-Castillo, Macario; Díaz-Hernández, Job; Del Moral-Hernández, Oscar; Cedillo-Barrón, Leticia; Martines-Juarez, Víctor; De Nova-Ocampo, Monica; Valdes, Jesús; Berumen, Jaime; Villegas-Sepúlveda, Nicolás

2014-12-05

The human papillomavirus type 16 (HPV-16) E6/E7 spliced transcripts are heterogeneously expressed in cervical carcinoma. The heterogeneity of the E6/E7 splicing profile might be in part due to the intrinsic variation of splicing factors in tumor cells. However, the splicing factors that bind the E6/E7 intron 1 (In-1) have not been defined. Therefore, we aimed to identify these factors; we used HeLa nuclear extracts (NE) for in vitro spliceosome assembly. The proteins were allowed to bind to an RNA/DNA hybrid formed by the In-1 transcript and a 5'-biotinylated DNA oligonucleotide complementary to the upstream exon sequence, which prevented interference in protein binding to the intron. The hybrid probes bound with the nuclear proteins were coupled to streptavidin magnetic beads for chromatography affinity purification. Proteins were eluted and identified by mass spectrometry (MS). Approximately 170 proteins were identified by MS, 80% of which were RNA binding proteins, including canonical spliceosome core components, helicases and regulatory splicing factors. The canonical factors were identified as components of the spliceosomal B-complex. Although 35-40 of the identified factors were cognate splicing factors or helicases, they have not been previously detected in spliceosome complexes that were assembled using in vivo or in vitro models. Copyright © 2014 Elsevier B.V. All rights reserved.

Expression of fibroblast growth factor receptors during development and regression of the bovine corpus luteum.

PubMed

Guerra, D M; Giometti, I C; Price, C A; Andrade, P B; Castilho, A C; Machado, M F; Ripamonte, P; Papa, P C; Buratini, J

2008-01-01

There is evidence that fibroblast growth factors (FGFs) are involved in the regulation of growth and regression of the corpus luteum (CL). However, the expression pattern of most FGF receptors (FGFRs) during CL lifespan is still unknown. The objective of the present study was to determine the pattern of expression of 'B' and 'C' splice variants of FGFRs in the bovine CL. Bovine CL were collected from an abattoir and classed as corpora hemorrhagica (Stage I), developing (Stage II), developed (Stage III) or regressed (Stage IV) CL. Expression of FGFR mRNA was measured by semiquantitative reverse transcription-polymerase chain reaction and FGFR protein was localised by immunohistochemistry. Expression of mRNA encoding the 'B' and 'C' spliced forms of FGFR1 and FGFR2 was readily detectable in the bovine CL and was accompanied by protein localisation. FGFR1C and FGFR2C mRNA expression did not vary throughout CL lifespan, whereas FGFR1B was upregulated in the developed (Stage III) CL. FGFR3B, FGFR3C and FGFR4 expression was inconsistent in the bovine CL. The present data indicate that FGFR1 and FGFR2 splice variants are the main receptors for FGF action in the bovine CL.

G-quadruplex structure at intron 2 of TFE3 and its role in Xp11.2 translocation and splicing.

PubMed

Verma, Shiv Prakash; Das, Parimal

2018-03-01

Transcription Factor E3 (TFE3) translocation is found in a group of different type of cancers and most of the translocations are located in the 5' region of TFE3 which may be considered as Breakpoint Region (BR). In our In silico study by QGRS mapper and non BdB web servers we found a Potential G-quadruplex forming Sequence (PQS) in the intron 2 of TFE3 gene. In vitro G-quadruplex formation was shown by native PAGE in presence of Pyridostatin(PDS), which with inter molecular secondary structure caused reduced mobility to migrate slower. G-quadruplex formation was mapped at single base resolution by Sanger sequencing and Circular Dichroism showed the formation of parallel G-quadruplex. FRET analysis revealed increased and decreased formation of G-quadruplex in presence of PDS and antisense oligonucleotide respectively. PCR stop assay, transcriptional and translational inhibition by PQS showed stable G-quadruplex formation affecting the biological processes. TFE3 minigene splicing study showed the involvement of this G-quadruplex in TFE3 splicing too. Therefore, G-quadruplex is evident to be the reason behind TFE3 induced oncogenesis executed by translocation and also involved in the mRNA splicing. Copyright © 2017 Elsevier B.V. All rights reserved.

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.

RNA Binding Protein RBM38 Regulates Expression of the 11-Kilodalton Protein of Parvovirus B19, Which Facilitates Viral DNA Replication.

PubMed

Ganaie, Safder S; Chen, Aaron Yun; Huang, Chun; Xu, Peng; Kleiboeker, Steve; Du, Aifang; Qiu, Jianming

2018-04-15

Human parvovirus B19 (B19V) expresses a single precursor mRNA (pre-mRNA), which undergoes alternative splicing and alternative polyadenylation to generate 12 viral mRNA transcripts that encode two structural proteins (VP1 and VP2) and three nonstructural proteins (NS1, 7.5-kDa protein, and 11-kDa protein). Splicing at the second 5' donor site (D2 site) of the B19V pre-mRNA is essential for the expression of VP2 and the 11-kDa protein. We previously identified that cis -acting intronic splicing enhancer 2 (ISE2) that lies immediately after the D2 site facilitates the recognition of the D2 donor for its efficient splicing. In this study, we report that ISE2 is critical for the expression of the 11-kDa viral nonstructural protein. We found that ISE2 harbors a consensus RNA binding motif protein 38 (RBM38) binding sequence, 5'-UGUGUG-3'. RBM38 is expressed during the middle stage of erythropoiesis. We first confirmed that RBM38 binds specifically with the ISE2 element in vitro The knockdown of RBM38 significantly decreases the level of spliced mRNA at D2 that encodes the 11-kDa protein but not that of the D2-spliced mRNA that encodes VP2. Importantly, we found that the 11-kDa protein enhances viral DNA replication and virion release. Accordingly, the knockdown of RBM38 decreases virus replication via downregulating 11-kDa protein expression. Taken together, these results suggest that the 11-kDa protein facilitates B19V DNA replication and that RBM38 is an essential host factor for B19V pre-mRNA splicing and for the expression of the 11-kDa protein. IMPORTANCE B19V is a human pathogen that can cause fifth disease, arthropathy, anemia in immunocompromised patients and sickle cell disease patients, myocarditis, and hydrops fetalis in pregnant women. Human erythroid progenitor cells (EPCs) are most susceptible to B19V infection and fully support viral DNA replication. The exclusive tropism of B19V for erythroid-lineage cells is dependent not only on the expression of viral receptors and coreceptors on the cell surface but also on the intracellular host factors that support B19V replication. Our present study shows that B19V uses a host factor, RNA binding motif protein 38 (RBM38), for the processing of its pre-mRNA during virus replication. Specifically, RBM38 interacts with the intronic splicing enhancer 2 (ISE2) element of B19V pre-mRNA and promotes 11-kDa protein expression, thereby regulating the 11-kDa protein-mediated augmentation of B19V replication. The identification of this novel host-pathogen interaction will provide mechanistic insights into B19V replication and aid in finding new targets for anti-B19V therapeutics. Copyright © 2018 American Society for Microbiology.

Methodology of splicing large air filling factor suspended core photonic crystal fibres

NASA Astrophysics Data System (ADS)

Jaroszewicz, L. R.; Murawski, M.; Nasilowski, T.; Stasiewicz, K.; Marć, P.; Szymański, M.; Mergo, P.

2011-06-01

We report the methodology of effective low-loss fusion splicing a photonic crystal fibre (PCF) to itself as well as to a standard single mode fibre (SMF). Distinctly from other papers in this area, we report on the results for splicing suspended core (SC) PCF having tiny core and non-Gaussian shape of guided beam. We show that studied splices exhibit transmission losses strongly dispersive and non-reciprocal in view of light propagation direction. Achieved splicing losses, defined as larger decrease in transmitted optical power comparing both propagation directions, are equal to 2.71 ±0.25 dB, 1.55 ±0.25 dB at 1550 nm for fibre SC PCF spliced to itself and to SMF, respectively.

Splicing factor SR34b mutation reduces cadmium tolerance in Arabidopsis by regulating iron-regulated transporter 1 gene

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

Zhang, Wentao; Du, Bojing; Liu, Di

Highlights: • Arabidopsis splicing factor SR34b gene is cadmium-inducible. • SR34b T-DNA insertion mutant is sensitive to cadmium due to high cadmium uptake. • SR34b is a regulator of cadmium transporter IRT1 at the posttranscription level. • These results highlight the roles of splicing factors in cadmium tolerance of plant. - Abstract: Serine/arginine-rich (SR) proteins are important splicing factors. However, the biological functions of plant SR proteins remain unclear especially in abiotic stresses. Cadmium (Cd) is a non-essential element that negatively affects plant growth and development. In this study, we provided clear evidence for SR gene involved in Cd tolerancemore » in planta. Systemic expression analysis of 17 Arabidopsis SR genes revealed that SR34b is the only SR gene upregulated by Cd, suggesting its potential roles in Arabidopsis Cd tolerance. Consistent with this, a SR34b T-DNA insertion mutant (sr34b) was moderately sensitive to Cd, which had higher Cd{sup 2+} uptake rate and accumulated Cd in greater amounts than wild-type. This was due to the altered expression of iron-regulated transporter 1 (IRT1) gene in sr34b mutant. Under normal growth conditions, IRT1 mRNAs highly accumulated in sr34b mutant, which was a result of increased stability of IRT1 mRNA. Under Cd stress, however, sr34b mutant plants had a splicing defect in IRT1 gene, thus reducing the IRT1 mRNA accumulation. Despite of this, sr34b mutant plants still constitutively expressed IRT1 proteins under Cd stress, thereby resulting in Cd stress-sensitive phenotype. We therefore propose the essential roles of SR34b in posttranscriptional regulation of IRT1 expression and identify it as a regulator of Arabidopsis Cd tolerance.« less

Identification of cis-Acting Elements and Splicing Factors Involved in the Regulation of BIM Pre-mRNA Splicing

PubMed Central

Juan, Wen Chun; Roca, Xavier; Ong, S. Tiong

2014-01-01

Aberrant changes in the expression of the pro-apoptotic protein, BCL-2-like 11 (BIM), can result in either impaired or excessive apoptosis, which can contribute to tumorigenesis and degenerative disorders, respectively. Altering BIM pre-mRNA splicing is an attractive approach to modulate apoptosis because BIM activity is partly determined by the alternative splicing of exons 3 or 4, whereby exon 3-containing transcripts are not apoptotic. Here we identified several cis-acting elements and splicing factors involved in BIM alternative splicing, as a step to better understand the regulation of BIM expression. We analyzed a recently discovered 2,903-bp deletion polymorphism within BIM intron 2 that biased splicing towards exon 3, and which also impaired BIM-dependent apoptosis. We found that this region harbors multiple redundant cis-acting elements that repress exon 3 inclusion. Furthermore, we have isolated a 23-nt intronic splicing silencer at the 3′ end of the deletion that is important for excluding exon 3. We also show that PTBP1 and hnRNP C repress exon 3 inclusion, and that downregulation of PTBP1 inhibited BIM-mediated apoptosis. Collectively, these findings start building our understanding of the cis-acting elements and splicing factors that regulate BIM alternative splicing, and also suggest potential approaches to alter BIM splicing for therapeutic purposes. PMID:24743263

Identification of cis-acting elements and splicing factors involved in the regulation of BIM Pre-mRNA splicing.

PubMed

Juan, Wen Chun; Roca, Xavier; Ong, S Tiong

2014-01-01

Aberrant changes in the expression of the pro-apoptotic protein, BCL-2-like 11 (BIM), can result in either impaired or excessive apoptosis, which can contribute to tumorigenesis and degenerative disorders, respectively. Altering BIM pre-mRNA splicing is an attractive approach to modulate apoptosis because BIM activity is partly determined by the alternative splicing of exons 3 or 4, whereby exon 3-containing transcripts are not apoptotic. Here we identified several cis-acting elements and splicing factors involved in BIM alternative splicing, as a step to better understand the regulation of BIM expression. We analyzed a recently discovered 2,903-bp deletion polymorphism within BIM intron 2 that biased splicing towards exon 3, and which also impaired BIM-dependent apoptosis. We found that this region harbors multiple redundant cis-acting elements that repress exon 3 inclusion. Furthermore, we have isolated a 23-nt intronic splicing silencer at the 3' end of the deletion that is important for excluding exon 3. We also show that PTBP1 and hnRNP C repress exon 3 inclusion, and that downregulation of PTBP1 inhibited BIM-mediated apoptosis. Collectively, these findings start building our understanding of the cis-acting elements and splicing factors that regulate BIM alternative splicing, and also suggest potential approaches to alter BIM splicing for therapeutic purposes.

7SK snRNP/P-TEFb couples transcription elongation with alternative splicing and is essential for vertebrate development

PubMed Central

Barboric, Matjaz; Lenasi, Tina; Chen, Hui; Johansen, Eric B.; Guo, Su; Peterlin, B. Matija

2009-01-01

Eukaryotic gene expression is commonly controlled at the level of RNA polymerase II (RNAPII) pausing subsequent to transcription initiation. Transcription elongation is stimulated by the positive transcription elongation factor b (P-TEFb) kinase, which is suppressed within the 7SK small nuclear ribonucleoprotein (7SK snRNP). However, the biogenesis and functional significance of 7SK snRNP remain poorly understood. Here, we report that LARP7, BCDIN3, and the noncoding 7SK small nuclear RNA (7SK) are vital for the formation and stability of a cell stress-resistant core 7SK snRNP. Our functional studies demonstrate that 7SK snRNP is not only critical for controlling transcription elongation, but also for regulating alternative splicing of pre-mRNAs. Using a transient expression splicing assay, we find that 7SK snRNP disintegration promotes inclusion of an alternative exon via the increased occupancy of P-TEFb, Ser2-phosphorylated (Ser2-P) RNAPII, and the splicing factor SF2/ASF at the minigene. Importantly, knockdown of larp7 or bcdin3 orthologues in zebrafish embryos destabilizes 7SK and causes severe developmental defects and aberrant splicing of analyzed transcripts. These findings reveal a key role for P-TEFb in coupling transcription elongation with alternative splicing, and suggest that maintaining core 7SK snRNP is essential for vertebrate development. PMID:19416841

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.

Alternative splicing of natriuretic peptide A and B receptor transcripts in the rat brain.

PubMed

Francoeur, F; Gossard, F; Hamet, P; Tremblay, J

1995-12-01

1. In the present study we searched for variants of alternative splicing of guanylyl cyclase A and B mRNA in rats in vivo. 2. Guanylyl cyclase A2 and guanylyl cyclase B2 isoforms of guanylyl cyclase produced by alternative splicing leading to the deletion of exon 9 of both transcripts were quantified in several rat organs. 3. Only one alternative splicing was found in the regulatory domain, encoded by exons 8-15. 4. Quantification of the guanylyl cyclase B2 isoform in different rat organs and in cultured aortic smooth muscle cells showed that this alternative splicing was tissue-specific and occurred predominantly in the central nervous system where the alternatively spliced variant represented more than 50% of the guanylyl cyclase B mRNA. 5. The same alternative splicing existed for guanylyl cyclase A mRNA but at very low levels in the organs studied. 6. Alternative splicing of guanylyl cyclase B exon 9 in the brain may play an important role in signal transduction, since the expressed protein possesses a constitutionally active guanylyl cyclase acting independently of C-type natriuretic peptide regulation.

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

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.

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

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.

Functional analysis of the isoforms of an ABI3-like factor of Pisum sativum generated by alternative splicing.

PubMed

Gagete, Andrés P; Riera, Marta; Franco, Luis; Rodrigo, M Isabel

2009-01-01

At least seven isoforms (PsABI3-1 to PsABI3-7) of a putative, pea ABI3-like factor, originated by alternative splicing, have been identified after cDNA cloning. A similar variability had previously only been described for monocot genes. The full-length isoform, PsABI3-1, contains the typical N-terminal acidic domains and C-terminal basic subdomains, B1 to B3. Reverse transcriptase-PCR analysis revealed that the gene is expressed just in seeds, starting at middle embryogenesis; no gene products are observed in embryo axes after 18 h post-imbibition although they are more persistent in cotyledons. The activity of the isoforms was studied by yeast one-hybrid assays. When yeast was transformed with the isoforms fused to the DNA binding domain of Gal4p, only the polypeptides PsABI3-2 and PsABI3-7 failed to complement the activity of Gal4p. Acidic domains A1 and A2 exhibit transactivating activity, but the former requires a small C-terminal extension to be active. Yeast two-hybrid analysis showed that PsABI3 is able to heterodimerize with Arabidopsis thaliana ABI5, thus proving that PsABI3 is functionally active. The minimum requirement for the interaction PsABI3-AtABI5 is the presence of the subdomain B1 with an extension, 81 amino acids long, at their C-terminal side. Finally, a transient onion transformation assay showed that both the active PsABI3-1 and the inactive PsABI3-2 isoforms are localized to nuclei. Considering that the major isoforms remain approximately constant in developing seeds although their relative proportion varied, the possible role of splicing in the regulatory network of ABA signalling is discussed.

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

SRSF3 maintains transcriptome integrity in oocytes by regulation of alternative splicing and transposable elements.

PubMed

Do, Dang Vinh; Strauss, Bernhard; Cukuroglu, Engin; Macaulay, Iain; Wee, Keng Boon; Hu, Tim Xiaoming; Igor, Ruiz De Los Mozos; Lee, Caroline; Harrison, Andrew; Butler, Richard; Dietmann, Sabine; Jernej, Ule; Marioni, John; Smith, Christopher W J; Göke, Jonathan; Surani, M Azim

2018-01-01

The RNA-binding protein SRSF3 (also known as SRp20) has critical roles in the regulation of pre-mRNA splicing. Zygotic knockout of Srsf3 results in embryo arrest at the blastocyst stage. However, SRSF3 is also present in oocytes, suggesting that it might be critical as a maternally inherited factor. Here we identify SRSF3 as an essential regulator of alternative splicing and of transposable elements to maintain transcriptome integrity in mouse oocyte. Using 3D time-lapse confocal live imaging, we show that conditional deletion of Srsf3 in fully grown germinal vesicle oocytes substantially compromises the capacity of germinal vesicle breakdown (GVBD), and consequently entry into meiosis. By combining single cell RNA-seq, and oocyte micromanipulation with steric blocking antisense oligonucleotides and RNAse-H inducing gapmers, we found that the GVBD defect in mutant oocytes is due to both aberrant alternative splicing and derepression of B2 SINE transposable elements. Together, our study highlights how control of transcriptional identity of the maternal transcriptome by the RNA-binding protein SRSF3 is essential to the development of fertilized-competent oocytes.

Novel down-regulatory mechanism of the surface expression of the vasopressin V2 receptor by an alternative splice receptor variant.

PubMed

Sarmiento, José M; Añazco, Carolina C; Campos, Danae M; Prado, Gregory N; Navarro, Javier; González, Carlos B

2004-11-05

In rat kidney, two alternatively spliced transcripts are generated from the V2 vasopressin receptor gene. The large transcript (1.2 kb) encodes the canonical V2 receptor, whereas the small transcript encodes a splice variant displaying a distinct sequence corresponding to the putative seventh transmembrane domain and the intracellular C terminus of the V2 receptor. This work showed that the small spliced transcript is translated in the rat kidney collecting tubules. However, the protein encoded by the small transcript (here called the V2b splice variant) is retained inside the cell, in contrast to the preferential surface distribution of the V2 receptor (here called the V2a receptor). Cells expressing the V2b splice variant do not exhibit binding to 3H-labeled vasopressin. Interestingly, we found that expression of the splice variant V2b down-regulates the surface expression of the V2a receptor, most likely via the formation of V2a.V2b heterodimers as demonstrated by co-immunoprecipitation and fluorescence resonance energy transfer experiments between the V2a receptor and the V2b splice variant. The V2b splice variant would then be acting as a dominant negative. The effect of the V2b splice variant is specific, as it does not affect the surface expression of the G protein-coupled interleukin-8 receptor (CXCR1). Furthermore, the sequence encompassing residues 242-339, corresponding to the C-terminal domain of the V2b splice variant, also down-regulates the surface expression of the V2a receptor. We suggest that some forms of nephrogenic diabetes insipidus are due to overexpression of the splice variant V2b, which could retain the wild-type V2a receptor inside the cell via the formation of V2a.V2b heterodimers.

Splice loss requirements in multi-mode fiber mode-division-multiplex transmission links.

PubMed

Warm, Stefan; Petermann, Klaus

2013-01-14

We investigate numerically the influence of fiber splices and fiber connectors to the statistics of mode dependent loss (MDL) and multiple-input multiple-output (MIMO) outage capacity in mode multiplexed multi-mode fiber links. Our results indicate required splice losses much lower than currently feasible to achieve a reasonable outage capacity in long-haul transmission systems. Splice losses as low as 0.03dB may effectively lead to an outage of MIMO channels after only a few hundred kilometers transmission length. In a first approximation, the relative capacity solely depends on the accumulated splice loss and should be less than ≈ 2dB to ensure a relative capacity of 90%. We also show that discrete mode permutation (mixing) within the transmission line may effectively increase the maximum transmission distance by a factor of 5 for conventional splice losses.

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.

Cross-talk between PRMT1-mediated methylation and ubiquitylation on RBM15 controls RNA splicing.

PubMed

Zhang, Li; Tran, Ngoc-Tung; Su, Hairui; Wang, Rui; Lu, Yuheng; Tang, Haiping; Aoyagi, Sayura; Guo, Ailan; Khodadadi-Jamayran, Alireza; Zhou, Dewang; Qian, Kun; Hricik, Todd; Côté, Jocelyn; Han, Xiaosi; Zhou, Wenping; Laha, Suparna; Abdel-Wahab, Omar; Levine, Ross L; Raffel, Glen; Liu, Yanyan; Chen, Dongquan; Li, Haitao; Townes, Tim; Wang, Hengbin; Deng, Haiteng; Zheng, Y George; Leslie, Christina; Luo, Minkui; Zhao, Xinyang

2015-11-17

RBM15, an RNA binding protein, determines cell-fate specification of many tissues including blood. We demonstrate that RBM15 is methylated by protein arginine methyltransferase 1 (PRMT1) at residue R578, leading to its degradation via ubiquitylation by an E3 ligase (CNOT4). Overexpression of PRMT1 in acute megakaryocytic leukemia cell lines blocks megakaryocyte terminal differentiation by downregulation of RBM15 protein level. Restoring RBM15 protein level rescues megakaryocyte terminal differentiation blocked by PRMT1 overexpression. At the molecular level, RBM15 binds to pre-messenger RNA intronic regions of genes important for megakaryopoiesis such as GATA1, RUNX1, TAL1 and c-MPL. Furthermore, preferential binding of RBM15 to specific intronic regions recruits the splicing factor SF3B1 to the same sites for alternative splicing. Therefore, PRMT1 regulates alternative RNA splicing via reducing RBM15 protein concentration. Targeting PRMT1 may be a curative therapy to restore megakaryocyte differentiation for acute megakaryocytic leukemia.

Fox-2 Splicing Factor Binds to a Conserved Intron Motif to PromoteInclusion of Protein 4.1R Alternative Exon 16

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

Ponthier, Julie L.; Schluepen, Christina; Chen, Weiguo

Activation of protein 4.1R exon 16 (E16) inclusion during erythropoiesis represents a physiologically important splicing switch that increases 4.1R affinity for spectrin and actin. Previous studies showed that negative regulation of E16 splicing is mediated by the binding of hnRNP A/B proteins to silencer elements in the exon and that downregulation of hnRNP A/B proteins in erythroblasts leads to activation of E16 inclusion. This paper demonstrates that positive regulation of E16 splicing can be mediated by Fox-2 or Fox-1, two closely related splicing factors that possess identical RNA recognition motifs. SELEX experiments with human Fox-1 revealed highly selective binding tomore » the hexamer UGCAUG. Both Fox-1 and Fox-2 were able to bind the conserved UGCAUG elements in the proximal intron downstream of E16, and both could activate E16 splicing in HeLa cell co-transfection assays in a UGCAUG-dependent manner. Conversely, knockdown of Fox-2 expression, achieved with two different siRNA sequences resulted in decreased E16 splicing. Moreover, immunoblot experiments demonstrate mouse erythroblasts express Fox-2, but not Fox-1. These findings suggest that Fox-2 is a physiological activator of E16 splicing in differentiating erythroid cells in vivo. Recent experiments show that UGCAUG is present in the proximal intron sequence of many tissue-specific alternative exons, and we propose that the Fox family of splicing enhancers plays an important role in alternative splicing switches during differentiation in metazoan organisms.« less

Genetic and Mechanistic Evaluation for the Mixed-Field Agglutination in B3 Blood Type with IVS3+5G>A ABO Gene Mutation

PubMed Central

Wang, Wei-Ting; Sun, Chien-Feng

2012-01-01

Background The ABO blood type B3 is the most common B subtype in the Chinese population with a frequency of 1/900. Although IVS3+5G>A (rs55852701) mutation of B gene has been shown to associate with the development of B3 blood type, genetic and mechanistic evaluation for the unique mixed-field agglutination phenotype has not yet been completely addressed. Methodology/Principal Findings In this study, we analyzed 16 cases of confirmed B3 individuals and found that IVS3+5G>A attributes to all cases of B3. RT-PCR analyses revealed the presence of at least 7 types of aberrant B3 splicing transcripts with most of the transcripts causing early termination and producing non-functional protein during translation. The splicing transcript without exon 3 that was predicted to generate functional B3 glycosyltransferase lacking 19 amino acids at the N-terminal segment constituted only 0.9% of the splicing transcripts. Expression of the B3 cDNA with exon 3 deletion in the K562 erythroleukemia cells revealed that the B3 glycosyltransferase had only 40% of B1 activity in converting H antigen to B antigen. Notably, the typical mixed-field agglutination of B3-RBCs can be mimicked by adding anti-B antibody to the K562-B3 cells. Conclusions/Significance This study thereby demonstrates that both aberrant splicing of B transcripts and the reduced B3 glycosyltransferase activity contribute to weak B expression and the mixed-field agglutination of B3, adding to the complexity for the regulatory mechanisms of ABO gene expression. PMID:22624005

Genetic and mechanistic evaluation for the mixed-field agglutination in B3 blood type with IVS3+5G>A ABO gene mutation.

PubMed

Chen, Ding-Ping; Tseng, Ching-Ping; Wang, Wei-Ting; Sun, Chien-Feng

2012-01-01

The ABO blood type B(3) is the most common B subtype in the Chinese population with a frequency of 1/900. Although IVS3+5G>A (rs55852701) mutation of B gene has been shown to associate with the development of B(3) blood type, genetic and mechanistic evaluation for the unique mixed-field agglutination phenotype has not yet been completely addressed. In this study, we analyzed 16 cases of confirmed B(3) individuals and found that IVS3+5G>A attributes to all cases of B(3). RT-PCR analyses revealed the presence of at least 7 types of aberrant B(3) splicing transcripts with most of the transcripts causing early termination and producing non-functional protein during translation. The splicing transcript without exon 3 that was predicted to generate functional B(3) glycosyltransferase lacking 19 amino acids at the N-terminal segment constituted only 0.9% of the splicing transcripts. Expression of the B(3) cDNA with exon 3 deletion in the K562 erythroleukemia cells revealed that the B(3) glycosyltransferase had only 40% of B(1) activity in converting H antigen to B antigen. Notably, the typical mixed-field agglutination of B(3)-RBCs can be mimicked by adding anti-B antibody to the K562-B(3) cells. This study thereby demonstrates that both aberrant splicing of B transcripts and the reduced B(3) glycosyltransferase activity contribute to weak B expression and the mixed-field agglutination of B(3), adding to the complexity for the regulatory mechanisms of ABO gene expression.

Protein kinase WNK3 regulates the neuronal splicing factor Fox-1.

PubMed

Lee, A-Young; Chen, Wei; Stippec, Steve; Self, Jon; Yang, Fan; Ding, Xiaojun; Chen, She; Juang, Yu-Chi; Cobb, Melanie H

2012-10-16

We report an action of the protein kinase WNK3 on the neuronal mRNA splicing factor Fox-1. Fox-1 splices mRNAs encoding proteins important in synaptic transmission and membrane excitation. WNK3, implicated in the control of neuronal excitability through actions on ion transport, binds Fox-1 and inhibits its splicing activity in a kinase activity-dependent manner. Phosphorylation of Fox-1 by WNK3 does not change its RNA binding capacity; instead, WNK3 increases the cytoplasmic localization of Fox-1, thereby suppressing Fox-1-dependent splicing. These findings demonstrate a role of WNK3 in RNA processing. Considering the implication of WNK3 and Fox-1 in disorders of neuronal development such as autism, WNK3 may offer a target for treatment of Fox-1-induced disease.

Nuclear degradation of Wilms tumor 1-associating protein and survivin splice variant switching underlie IGF-1-mediated survival.

PubMed

Small, Theodore W; Pickering, J Geoffrey

2009-09-11

WTAP (Wilms tumor 1-associating protein) is a recently identified nuclear protein that is essential for mouse embryo development. The Drosophila homolog of WTAP, Fl(2)d, regulates pre-mRNA splicing; however, the role of WTAP in mammalian cells is uncertain. To elucidate a context for WTAP action, we screened growth and survival factors for their effects on WTAP expression in vascular smooth muscle cells (SMCs), a cell type previously found to express WTAP dynamically. This revealed that insulin-like growth factor-1 (IGF-1) uniquely reduced WTAP abundance. This decline in WTAP proved to be necessary for IGF-1 to confer its antiapoptotic properties, which were blocked by transducing the WTAP gene into SMCs. WTAP down-regulation by IGF-1 was mediated by an IGF-1 receptor-phosphatidylinositol 3-kinase-Akt signaling axis that directed WTAP degradation via a nuclear 26 S proteasome. Moreover, by promoting the degradation of WTAP, IGF-1 shifted the pre-mRNA splicing program for the survival factor, survivin, to reduce expression of survivin-2B, which is proapoptotic, and increase expression of survivin, which is antiapoptotic. Knockdown of survivin-2B rescued the ability of IGF-1 to promote survival when WTAP was overexpressed. These data uncover a novel regulatory cascade for human SMC survival based on adjusting the nuclear abundance of WTAP to define the splice variant balance among survivin isoforms.

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.

Attenuation of the suppressive activity of cellular splicing factor SRSF3 by Kaposi sarcoma–associated herpesvirus ORF57 protein is required for RNA splicing

PubMed Central

Majerciak, Vladimir; Lu, Mathew; Li, Xiaofan

2014-01-01

Kaposi sarcoma–associated herpesvirus (KSHV) ORF57 is a multifunctional post-transcriptional regulator essential for viral gene expression during KSHV lytic infection. ORF57 requires interactions with various cellular proteins for its function. Here, we identified serine/arginine-rich splicing factor 3 (SRSF3, formerly known as SRp20) as a cellular cofactor involved in ORF57-mediated splicing of KSHV K8β RNA. In the absence of ORF57, SRSF3 binds to a suboptimal K8β intron and inhibits K8β splicing. Knockdown of SRSF3 promotes K8β splicing, mimicking the effect of ORF57. The N-terminal half of ORF57 binds to the RNA recognition motif of SRSF3, which prevents SRSF3 from associating with the K8β intron RNA and therefore attenuates the suppressive effect of SRSF3 on K8β splicing. ORF57 also promotes splicing of heterologous non-KSHV transcripts that are negatively regulated by SRSF3, indicating that the effect of ORF57 on SRSF3 activity is independent of RNA target. SPEN proteins, previously identified as ORF57-interacting partners, suppress ORF57 splicing activity by displacing ORF57 from SRSF3–RNA complexes. In summary, we have identified modulation of SRSF3 activity as the molecular mechanism by which ORF57 promotes RNA splicing. PMID:25234929

Attenuation of the suppressive activity of cellular splicing factor SRSF3 by Kaposi sarcoma-associated herpesvirus ORF57 protein is required for RNA splicing.

PubMed

Majerciak, Vladimir; Lu, Mathew; Li, Xiaofan; Zheng, Zhi-Ming

2014-11-01

Kaposi sarcoma-associated herpesvirus (KSHV) ORF57 is a multifunctional post-transcriptional regulator essential for viral gene expression during KSHV lytic infection. ORF57 requires interactions with various cellular proteins for its function. Here, we identified serine/arginine-rich splicing factor 3 (SRSF3, formerly known as SRp20) as a cellular cofactor involved in ORF57-mediated splicing of KSHV K8β RNA. In the absence of ORF57, SRSF3 binds to a suboptimal K8β intron and inhibits K8β splicing. Knockdown of SRSF3 promotes K8β splicing, mimicking the effect of ORF57. The N-terminal half of ORF57 binds to the RNA recognition motif of SRSF3, which prevents SRSF3 from associating with the K8β intron RNA and therefore attenuates the suppressive effect of SRSF3 on K8β splicing. ORF57 also promotes splicing of heterologous non-KSHV transcripts that are negatively regulated by SRSF3, indicating that the effect of ORF57 on SRSF3 activity is independent of RNA target. SPEN proteins, previously identified as ORF57-interacting partners, suppress ORF57 splicing activity by displacing ORF57 from SRSF3-RNA complexes. In summary, we have identified modulation of SRSF3 activity as the molecular mechanism by which ORF57 promotes RNA splicing. Published by Cold Spring Harbor Laboratory Press for the RNA Society.

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

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

The Acheta domesticus Densovirus, Isolated from the European House Cricket, Has Evolved an Expression Strategy Unique among Parvoviruses▿†

PubMed Central

Liu, Kaiyu; Li, Yi; Jousset, Françoise-Xavière; Zadori, Zoltan; Szelei, Jozsef; Yu, Qian; Pham, Hanh Thi; Lépine, François; Bergoin, Max; Tijssen, Peter

2011-01-01

The Acheta domesticus densovirus (AdDNV), isolated from crickets, has been endemic in Europe for at least 35 years. Severe epizootics have also been observed in American commercial rearings since 2009 and 2010. The AdDNV genome was cloned and sequenced for this study. The transcription map showed that splicing occurred in both the nonstructural (NS) and capsid protein (VP) multicistronic RNAs. The splicing pattern of NS mRNA predicted 3 nonstructural proteins (NS1 [576 codons], NS2 [286 codons], and NS3 [213 codons]). The VP gene cassette contained two VP open reading frames (ORFs), of 597 (ORF-A) and 268 (ORF-B) codons. The VP2 sequence was shown by N-terminal Edman degradation and mass spectrometry to correspond with ORF-A. Mass spectrometry, sequencing, and Western blotting of baculovirus-expressed VPs versus native structural proteins demonstrated that the VP1 structural protein was generated by joining ORF-A and -B via splicing (splice II), eliminating the N terminus of VP2. This splice resulted in a nested set of VP1 (816 codons), VP3 (467 codons), and VP4 (429 codons) structural proteins. In contrast, the two splices within ORF-B (Ia and Ib) removed the donor site of intron II and resulted in VP2, VP3, and VP4 expression. ORF-B may also code for several nonstructural proteins, of 268, 233, and 158 codons. The small ORF-B contains the coding sequence for a phospholipase A2 motif found in VP1, which was shown previously to be critical for cellular uptake of the virus. These splicing features are unique among parvoviruses and define a new genus of ambisense densoviruses. PMID:21775445

SF3B1 mutations constitute a novel therapeutic target in breast cancer

PubMed Central

Maguire, Sarah L; Leonidou, Andri; Wai, Patty; Marchiò, Caterina; Ng, Charlotte KY; Sapino, Anna; Salomon, Anne-Vincent; Reis-Filho, Jorge S; Weigelt, Britta; Natrajan, Rachael C

2015-01-01

Mutations in genes encoding proteins involved in RNA splicing have been found to occur at relatively high frequencies in several tumour types including myelodysplastic syndromes, chronic lymphocytic leukaemia, uveal melanoma, and pancreatic cancer, and at lower frequencies in breast cancer. To investigate whether dysfunction in RNA splicing is implicated in the pathogenesis of breast cancer, we performed a re-analysis of published exome and whole genome sequencing data. This analysis revealed that mutations in spliceosomal component genes occurred in 5.6% of unselected breast cancers, including hotspot mutations in the SF3B1 gene, which were found in 1.8% of unselected breast cancers. SF3B1 mutations were significantly associated with ER-positive disease, AKT1 mutations, and distinct copy number alterations. Additional profiling of hotspot mutations in a panel of special histological subtypes of breast cancer showed that 16% and 6% of papillary and mucinous carcinomas of the breast harboured the SF3B1 K700E mutation. RNA sequencing identified differentially spliced events expressed in tumours with SF3B1 mutations including the protein coding genes TMEM14C, RPL31, DYNL11, UQCC, and ABCC5, and the long non-coding RNA CRNDE. Moreover, SF3B1 mutant cell lines were found to be sensitive to the SF3b complex inhibitor spliceostatin A and treatment resulted in perturbation of the splicing signature. Albeit rare, SF3B1 mutations result in alternative splicing events, and may constitute drivers and a novel therapeutic target in a subset of breast cancers. © 2014 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland. PMID:25424858

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

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

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.

In vivo transcriptional profile analysis reveals RNA splicing and chromatin remodeling as prominent processes for adult neurogenesis.

PubMed

Lim, Daniel A; Suárez-Fariñas, Mayte; Naef, Felix; Hacker, Coleen R; Menn, Benedicte; Takebayashi, Hirohide; Magnasco, Marcelo; Patil, Nila; Alvarez-Buylla, Arturo

2006-01-01

Neural stem cells and neurogenesis persist in the adult mammalian brain subventricular zone (SVZ). Cells born in the rodent SVZ migrate to the olfactory bulb (Ob) where they differentiate into interneurons. To determine the gene expression and functional profile of SVZ neurogenesis, we performed three complementary sets of transcriptional analysis experiments using Affymetrix GeneChips: (1) comparison of adult mouse SVZ and Ob gene expression profiles with those of the striatum, cerebral cortex, and hippocampus; (2) profiling of SVZ stem cells and ependyma isolated by fluorescent-activated cell sorting (FACS); and (3) analysis of gene expression changes during in vivo SVZ regeneration after anti-mitotic treatment. Gene Ontology (GO) analysis of data from these three separate approaches showed that in adult SVZ neurogenesis, RNA splicing and chromatin remodeling are biological processes as statistically significant as cell proliferation, transcription, and neurogenesis. In non-neurogenic brain regions, RNA splicing and chromatin remodeling were not prominent processes. Fourteen mRNA splicing factors including Sf3b1, Sfrs2, Lsm4, and Khdrbs1/Sam68 were detected along with 9 chromatin remodeling genes including Mll, Bmi1, Smarcad1, Baf53a, and Hat1. We validated the transcriptional profile data with Northern blot analysis and in situ hybridization. The data greatly expand the catalogue of cell cycle components, transcription factors, and migration genes for adult SVZ neurogenesis and reveal RNA splicing and chromatin remodeling as prominent biological processes for these germinal cells.

Role of Bmznf-2, a Bombyx mori CCCH zinc finger gene, in masculinisation and differential splicing of Bmtra-2.

PubMed

Gopinath, Gajula; Arunkumar, Kallare P; Mita, Kazuei; Nagaraju, Javaregowda

2016-08-01

Deciphering the regulatory factors involved in Bombyx mori sex determination has been a puzzle, challenging researchers for nearly a century now. The pre-mRNA of B. mori doublesex (Bmdsx), a master regulator gene of sexual differentiation, is differentially spliced, producing Bmdsxm and Bmdsxf transcripts in males and females respectively. The putative proteins encoded by these differential transcripts orchestrate antagonistic functions, which lead to sexual differentiation. A recent study in B. mori illustrated the role of a W-derived fem piRNA in conferring femaleness. In females, the fem piRNA was shown to suppress the activity of a Z-linked CCCH type zinc finger (znf) gene, Masculiniser (masc), which indirectly promotes the Bmdsxm type of splicing. In this study, we report a novel autosomal (Chr 25) CCCH type znf motif encoding gene Bmznf-2 as one of the potential factors in the Bmdsx sex specific differential splicing, and we also provide insights into its role in the alternative splicing of Bmtra2 by using ovary derived BmN cells. Over-expression of Bmznf-2 induced Bmdsxm type of splicing (masculinisation) with a correspondingly reduced expression of Bmdsxf type isoform in BmN cells. Further, the site-directed mutational studies targeting the tandem CCCH znf motifs revealed their indispensability in the observed phenotype of masculinisation. Additionally, the dual luciferase assays in BmN cells using 5' UTR region of the Bmznf-2 strongly implied the existence of a translational repression over this gene. From these findings, we propose Bmznf-2 to be one of the potential factors of masculinisation similar to Masc. From the growing number of Bmdsx splicing regulators, we assume that the sex determination cascade of B. mori is quite intricate in nature; hence, it has to be further investigated for its comprehensive understanding. Copyright © 2016 Elsevier Ltd. All rights reserved.

Factor IX[sub Madrid 2]: A deletion/insertion in Facotr IX gene which abolishes the sequence of the donor junction at the exon IV-intron d splice site

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

Solera, J.; Magallon, M.; Martin-Villar, J.

1992-02-01

DNA from a patient with severe hemophilia B was evaluated by RFLP analysis, producing results which suggested the existence of a partial deletion within the factor IX gene. The deletion was further localized and characterized by PCR amplification and sequencing. The altered allele has a 4,442-bp deletion which removes both the donor splice site located at the 5[prime] end of intron d and the two last coding nucleotides located at the 3[prime] end of exon IV in the normal factor IX gene; this fragment has been inserted in inverted orientation. Two homologous sequences have been discovered at the ends ofmore » the deleted DNA fragment.« less

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

Base pairing between the 3' exon and an internal guide sequence increases 3' splice site specificity in the Tetrahymena self-splicing rRNA intron.

PubMed Central

Suh, E R; Waring, R B

1990-01-01

It has been proposed that recognition of the 3' splice site in many group I introns involves base pairing between the start of the 3' exon and a region of the intron known as the internal guide sequence (R. W. Davies, R. B. Waring, J. Ray, T. A. Brown, and C. Scazzocchio, Nature [London] 300:719-724, 1982). We have examined this hypothesis, using the self-splicing rRNA intron from Tetrahymena thermophila. Mutations in the 3' exon that weaken this proposed pairing increased use of a downstream cryptic 3' splice site. Compensatory mutations in the guide sequence that restore this pairing resulted in even stronger selection of the normal 3' splice site. These changes in 3' splice site usage were more pronounced in the background of a mutation (414A) which resulted in an adenine instead of a guanine being the last base of the intron. These results show that the proposed pairing (P10) plays an important role in ensuring that cryptic 3' splice sites are selected against. Surprisingly, the 414A mutation alone did not result in activation of the cryptic 3' splice site. Images PMID:2342465

Independence between pre-mRNA splicing and DNA methylation in an isogenic minigene resource.

PubMed

Nanan, Kyster K; Ocheltree, Cody; Sturgill, David; Mandler, Mariana D; Prigge, Maria; Varma, Garima; Oberdoerffer, Shalini

2017-12-15

Actively transcribed genes adopt a unique chromatin environment with characteristic patterns of enrichment. Within gene bodies, H3K36me3 and cytosine DNA methylation are elevated at exons of spliced genes and have been implicated in the regulation of pre-mRNA splicing. H3K36me3 is further responsive to splicing, wherein splicing inhibition led to a redistribution and general reduction over gene bodies. In contrast, little is known of the mechanisms supporting elevated DNA methylation at actively spliced genic locations. Recent evidence associating the de novo DNA methyltransferase Dnmt3b with H3K36me3-rich chromatin raises the possibility that genic DNA methylation is influenced by splicing-associated H3K36me3. Here, we report the generation of an isogenic resource to test the direct impact of splicing on chromatin. A panel of minigenes of varying splicing potential were integrated into a single FRT site for inducible expression. Profiling of H3K36me3 confirmed the established relationship to splicing, wherein levels were directly correlated with splicing efficiency. In contrast, DNA methylation was equivalently detected across the minigene panel, irrespective of splicing and H3K36me3 status. In addition to revealing a degree of independence between genic H3K36me3 and DNA methylation, these findings highlight the generated minigene panel as a flexible platform for the query of splicing-dependent chromatin modifications. Published by Oxford University Press on behalf of Nucleic Acids Research 2017.

Three gene-targeted mouse models of RNA splicing factor RP show late-onset RPE and retinal degeneration.

PubMed

Graziotto, John J; Farkas, Michael H; Bujakowska, Kinga; Deramaudt, Bertrand M; Zhang, Qi; Nandrot, Emeline F; Inglehearn, Chris F; Bhattacharya, Shomi S; Pierce, Eric A

2011-01-01

Mutations in genes that produce proteins involved in mRNA splicing, including pre-mRNA processing factors 3, 8, and 31 (PRPF3, 8, and 31), RP9, and SNRNP200 are common causes of the late-onset inherited blinding disorder retinitis pigmentosa (RP). It is not known how mutations in these ubiquitously expressed genes lead to retina-specific disease. To investigate the pathogenesis of the RNA splicing factor forms of RP, the authors generated and characterized the retinal phenotypes of Prpf3-T494M, Prpf8-H2309P knockin mice. The retinal ultrastructure of Prpf31-knockout mice was also investigated. The knockin mice have single codon alterations in their endogenous Prpf3 and Prpf8 genes that mimic the most common disease causing mutations in human PRPF3 and PRPF8. The Prpf31-knockout mice mimic the null alleles that result from the majority of mutations identified in PRPF31 patients. The retinal phenotypes of the gene targeted mice were evaluated by electroretinography (ERG), light, and electron microscopy. The RPE cells of heterozygous Prpf3(+/T494M) and Prpf8(+/H2309P) knockin mice exhibited loss of the basal infoldings and vacuolization, with accumulation of amorphous deposits between the RPE and Bruch[b]'s membrane at age two years. These changes were more severe in the homozygous mice, and were associated with decreased rod function in the Prpf3-T494M mice. Similar degenerative changes in the RPE were detected in Prpf31(±) mice at one year of age. The finding of similar degenerative changes in RPE cells of all three mouse models suggests that the RPE may be the primary cell type affected in the RNA splicing factor forms of RP. The relatively late-onset phenotype observed in these mice is consistent with the typical adult onset of disease in patients with RP.

Visualizing Mixed Variable-Type Multidimensional Data Using Tree Distances

DTIC Science & Technology

2015-09-01

24 1. The Regression Method .................................................. 25 2. Maximum Deviance Ratio Method... Deviance Change as a Factor of the Amount of Correlation? ................................................... 50 B. A PROPOSED SOLUTION...Splice mapping using d1 ...................... 36 Figure 18. Reduction in 2R analog ( deviance ratio-DevRat) per variable for the Splice data

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

Stepanyuk, Galina A.; Serrano, Pedro; Peralta, Eigen

RNA-binding protein 39 (RBM39) is a splicing factor and a transcriptional co-activator of estrogen receptors and Jun/AP-1, and its function has been associated with malignant progression in a number of cancers. The C-terminal RRM domain of RBM39 belongs to the U2AF homology motif family (UHM), which mediate protein–protein interactions through a short tryptophan-containing peptide known as the UHM-ligand motif (ULM). Here, crystal and solution NMR structures of the RBM39-UHM domain, and the crystal structure of its complex with U2AF65-ULM, are reported. The RBM39–U2AF65 interaction was confirmed by co-immunoprecipitation from human cell extracts, by isothermal titration calorimetry and by NMR chemicalmore » shift perturbation experiments with the purified proteins. When compared with related complexes, such as U2AF35–U2AF65 and RBM39–SF3b155, the RBM39-UHM–U2AF65-ULM complex reveals both common and discriminating recognition elements in the UHM–ULM binding interface, providing a rationale for the known specificity of UHM–ULM interactions. This study therefore establishes a structural basis for specific UHM–ULM interactions by splicing factors such as U2AF35, U2AF65, RBM39 and SF3b155, and a platform for continued studies of intermolecular interactions governing disease-related alternative splicing in eukaryotic cells.« less

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 RNA splicing and gastric cancer.

PubMed

Li, Ying; Yuan, Yuan

2017-07-01

Alternative splicing (AS) linked to diseases, especially to tumors. Recently, more and more studies focused on the relationship between AS and gastric cancer (GC). This review surveyed the hot topic from four aspects: First, the common types of AS in cancer, including exon skipping, intron retention, mutually exclusive exon, alternative 5 ' or 3' splice site, alternative first or last exon and alternative 3' untranslated regions. Second, basic mechanisms of AS and its relationship with cancer. RNA splicing in eukaryotes follows the GT-AG rule by both cis-elements and trans-acting factors regulatory. Through RNA splicing, different proteins with different forms and functions can be produced and may be associated with carcinogenesis. Third, AS types of GC-related genes and their splicing variants. In this paper, we listed 10 common genes with AS and illustrated its possible molecular mechanisms owing to genetic variation (mutation and /or polymorphism). Fourth, the splicing variants of GC-associated genes and gastric carcinogenesis, invasion and metastasis. Many studies have found that the different splicing variants of the same gene are differentially expressed in GC and its precancerous diseases, suggesting AS has important implications in GC development. Taking together, this review highlighted the role of AS and splicing variants in the process of GC. We hope that this is not only beneficial to advances in the study field of GC, but also can provide valuable information to other similar tumor research.Although we already know some gene splicing and splicing variants play an important role in the development of GC, but many phenomena and mechanisms are still unknown. For example, how the tumor microenvironment and signal transduction pathway effect the forming and function of AS? Unfortunately, this review did not cover the contents because the current study is limited. It is no doubt that clarifying the phenomena and mechanisms of these unknown may help to reveal the relationship of AS with complex tumor genetic variation and the occurrence and development of tumors. Copyright © 2016 Elsevier B.V. All rights reserved.

A 5′ Splice Site-Proximal Enhancer Binds SF1 and Activates Exon Bridging of a Microexon

PubMed Central

Carlo, Troy; Sierra, Rebecca; Berget, Susan M.

2000-01-01

Internal exon size in vertebrates occurs over a narrow size range. Experimentally, exons shorter than 50 nucleotides are poorly included in mRNA unless accompanied by strengthened splice sites or accessory sequences that act as splicing enhancers, suggesting steric interference between snRNPs and other splicing factors binding simultaneously to the 3′ and 5′ splice sites of microexons. Despite these problems, very small naturally occurring exons exist. Here we studied the factors and mechanism involved in recognizing a constitutively included six-nucleotide exon from the cardiac troponin T gene. Inclusion of this exon is dependent on an enhancer located downstream of the 5′ splice site. This enhancer contains six copies of the simple sequence GGGGCUG. The enhancer activates heterologous microexons and will work when located either upstream or downstream of the target exon, suggesting an ability to bind factors that bridge splicing units. A single copy of this sequence is sufficient for in vivo exon inclusion and is the binding site for the known bridging mammalian splicing factor 1 (SF1). The enhancer and its bound SF1 act to increase recognition of the upstream exon during exon definition, such that competition of in vitro reactions with RNAs containing the GGGGCUG repeated sequence depress splicing of the upstream intron, assembly of the spliceosome on the 3′ splice site of the exon, and cross-linking of SF1. These results suggest a model in which SF1 bridges the small exon during initial assembly, thereby effectively extending the domain of the exon. PMID:10805741

HuB/C/D, nPTB, REST4, and miR-124 regulators of neuronal cell identity are also utilized in the lens.

PubMed

Bitel, Claudine L; Perrone-Bizzozero, Nora I; Frederikse, Peter H

2010-11-04

An interlocking network of transcription factors, RNA binding proteins, and miRNAs globally regulates gene expression and alternative splicing throughout development, and ensures the coordinated mutually exclusive expression of non-neural and neuronal forms of these factors during neurogenesis. Striking similarities between lens fiber cell and neuron cell morphology led us to determine if these factors are also used in the lens. HuR and polypyrimidine tract binding protein (PTB) have been described as 'global regulators' of RNA alternative splicing, stability, and translation in non-neuronal (including ectodermal) tissues examined to date in diverse species, and REST/NRSF (RE-1 Silencing Transcription Factor/Neuron Restrictive Silencing Factor) represses>2,000 neuronal genes in all non-neuronal tissues examined to date, but has not included the lens. During neurogenesis these factors are replaced by what has been considered neuron-specific HuB/C/D, nPTB, and alternatively spliced REST (REST4), which work with miR-124 to activate this battery of genes, comprehensively reprogram neuronal alternative splicing, and maintain their exclusive expression in post-mitotic neurons. Immunoprecipitation, western blot, immunofluorescence, and immunohistochemistry were used to determine the expression and distribution of proteins in mouse and rat lenses. Mobility shift assays were used to examine lenses for REST/NRSF DNA binding activity, and RT-PCR, DNA sequencing, and northern blots were used to identify RNA expression and alternative splicing events in lenses from mouse, rat, and goldfish (N. crassa). We demonstrated that REST, HuR, and PTB proteins are expressed predominantly in epithelial cells in mouse and rat lenses, and showed these factors are also replaced by the predominant expression of REST4, HuB/C/D and nPTB in post-mitotic fiber cells, together with miR-124 expression in vertebrate lenses. REST-regulated gene products were found to be restricted to fiber cells where REST is decreased. These findings predicted nPTB- and HuB/C/D-dependent splicing reactions can also occur in lenses, and we showed Neuronal C-src and Type 1 Neurofibromatosis 1 splicing as well as calcitonin gene related peptide (CGRP) and neural cell adhesion molecule (NCAM-180) alternative transcripts in lenses. Transgenic mice with increased HuD in lens also showed increased growth associated protein 43 (GAP43) and Ca++/Calmodulin dependent kinase IIα (CamKIIα) HuD target gene expression in the lens, similar to brain. The present study provides the first evidence this fundamental set of regulatory factors, previously considered to have a unique role in governing neurogenesis are also used in the lens, and raises questions about the origins of these developmental factors and mechanisms in lens and neuronal cells that also have a basic role in determining the neuronal phenotype.

HuB/C/D, nPTB, REST4, and miR-124 regulators of neuronal cell identity are also utilized in the lens

PubMed Central

Bitel, Claudine L.; Perrone-Bizzozero, Nora I.

2010-01-01

Purpose An interlocking network of transcription factors, RNA binding proteins, and miRNAs globally regulates gene expression and alternative splicing throughout development, and ensures the coordinated mutually exclusive expression of non-neural and neuronal forms of these factors during neurogenesis. Striking similarities between lens fiber cell and neuron cell morphology led us to determine if these factors are also used in the lens. HuR and polypyrimidine tract binding protein (PTB) have been described as ‘global regulators’ of RNA alternative splicing, stability, and translation in non-neuronal (including ectodermal) tissues examined to date in diverse species, and REST/NRSF (RE-1 Silencing Transcription Factor/Neuron Restrictive Silencing Factor) represses >2,000 neuronal genes in all non-neuronal tissues examined to date, but has not included the lens. During neurogenesis these factors are replaced by what has been considered neuron-specific HuB/C/D, nPTB, and alternatively spliced REST (REST4), which work with miR-124 to activate this battery of genes, comprehensively reprogram neuronal alternative splicing, and maintain their exclusive expression in post-mitotic neurons. Methods Immunoprecipitation, western blot, immunofluorescence, and immunohistochemistry were used to determine the expression and distribution of proteins in mouse and rat lenses. Mobility shift assays were used to examine lenses for REST/NRSF DNA binding activity, and RT–PCR, DNA sequencing, and northern blots were used to identify RNA expression and alternative splicing events in lenses from mouse, rat, and goldfish (N. crassa). Results We demonstrated that REST, HuR, and PTB proteins are expressed predominantly in epithelial cells in mouse and rat lenses, and showed these factors are also replaced by the predominant expression of REST4, HuB/C/D and nPTB in post-mitotic fiber cells, together with miR-124 expression in vertebrate lenses. REST-regulated gene products were found to be restricted to fiber cells where REST is decreased. These findings predicted nPTB- and HuB/C/D-dependent splicing reactions can also occur in lenses, and we showed Neuronal C-src and Type 1 Neurofibromatosis 1 splicing as well as calcitonin gene related peptide (CGRP) and neural cell adhesion molecule (NCAM-180) alternative transcripts in lenses. Transgenic mice with increased HuD in lens also showed increased growth associated protein 43 (GAP43) and Ca++/Calmodulin dependent kinase IIα (CamKIIα) HuD target gene expression in the lens, similar to brain. Conclusions The present study provides the first evidence this fundamental set of regulatory factors, previously considered to have a unique role in governing neurogenesis are also used in the lens, and raises questions about the origins of these developmental factors and mechanisms in lens and neuronal cells that also have a basic role in determining the neuronal phenotype. PMID:21139978

Polyoma virus small tumor antigen pre-mRNA splicing requires cooperation between two 3' splice sites.

PubMed Central

Ge, H; Noble, J; Colgan, J; Manley, J L

1990-01-01

We have studied splicing of the polyoma virus early region pre-mRNA in vitro. This RNA is alternatively spliced in vivo to produce mRNA encoding the large, middle-sized (MTAg), and small (StAg) tumor antigens. Our primary interest was to learn how the 48-nucleotide StAg intron is excised, because the length of this intron is significantly less than the apparent minimum established for mammalian introns. Although the products of all three splices are detected in vitro, characterization of the pathway and sequence requirements of StAg splicing suggests that splicing factors interact with the precursor RNA in an unexpected way to catalyze removal of this intron. Specifically, StAg splicing uses either of two lariat branch points, one of which is located only 4 nucleotides from the 3' splice site. Furthermore, the StAg splice absolutely requires that the alternative MTAg 3' splice site, located 14 nucleotides downstream of the StAg 3' splice site, be intact. Insertion mutations that increase or decrease the quality of the MTAg pyrimidine stretch enhance or repress StAg as well as MTAg splicing, and a single-base change in the MTAg AG splice acceptor totally blocks both splices. These results demonstrate the ability of two 3' splice sites to cooperate with each other to bring about removal of a single intron. Images PMID:2159146

Perturbation of Chromatin Structure Globally Affects Localization and Recruitment of Splicing Factors

PubMed Central

Risso, Guillermo J.; Pawellek, Andrea; Ule, Jernej; Lamond, Angus I.; Kornblihtt, Alberto R.

2012-01-01

Chromatin structure is an important factor in the functional coupling between transcription and mRNA processing, not only by regulating alternative splicing events, but also by contributing to exon recognition during constitutive splicing. We observed that depolarization of neuroblastoma cell membrane potential, which triggers general histone acetylation and regulates alternative splicing, causes a concentration of SR proteins in nuclear speckles. This prompted us to analyze the effect of chromatin structure on splicing factor distribution and dynamics. Here, we show that induction of histone hyper-acetylation results in the accumulation in speckles of multiple splicing factors in different cell types. In addition, a similar effect is observed after depletion of the heterochromatic protein HP1α, associated with repressive chromatin. We used advanced imaging approaches to analyze in detail both the structural organization of the speckle compartment and nuclear distribution of splicing factors, as well as studying direct interactions between splicing factors and their association with chromatin in vivo. The results support a model where perturbation of normal chromatin structure decreases the recruitment efficiency of splicing factors to nascent RNAs, thus causing their accumulation in speckles, which buffer the amount of free molecules in the nucleoplasm. To test this, we analyzed the recruitment of the general splicing factor U2AF65 to nascent RNAs by iCLIP technique, as a way to monitor early spliceosome assembly. We demonstrate that indeed histone hyper-acetylation decreases recruitment of U2AF65 to bulk 3′ splice sites, coincident with the change in its localization. In addition, prior to the maximum accumulation in speckles, ∼20% of genes already show a tendency to decreased binding, while U2AF65 seems to increase its binding to the speckle-located ncRNA MALAT1. All together, the combined imaging and biochemical approaches support a model where chromatin structure is essential for efficient co-transcriptional recruitment of general and regulatory splicing factors to pre-mRNA. PMID:23152763

Modelling reveals kinetic advantages of co-transcriptional splicing.

PubMed

Aitken, Stuart; Alexander, Ross D; Beggs, Jean D

2011-10-01

Messenger RNA splicing is an essential and complex process for the removal of intron sequences. Whereas the composition of the splicing machinery is mostly known, the kinetics of splicing, the catalytic activity of splicing factors and the interdependency of transcription, splicing and mRNA 3' end formation are less well understood. We propose a stochastic model of splicing kinetics that explains data obtained from high-resolution kinetic analyses of transcription, splicing and 3' end formation during induction of an intron-containing reporter gene in budding yeast. Modelling reveals co-transcriptional splicing to be the most probable and most efficient splicing pathway for the reporter transcripts, due in part to a positive feedback mechanism for co-transcriptional second step splicing. Model comparison is used to assess the alternative representations of reactions. Modelling also indicates the functional coupling of transcription and splicing, because both the rate of initiation of transcription and the probability that step one of splicing occurs co-transcriptionally are reduced, when the second step of splicing is abolished in a mutant reporter.

C3G dynamically associates with nuclear speckles and regulates mRNA splicing.

PubMed

Shakyawar, Dhruv Kumar; Muralikrishna, Bhattiprolu; Radha, Vegesna

2018-05-01

C3G (Crk SH3 domain binding guanine nucleotide releasing factor) (Rap guanine nucleotide exchange factor 1), essential for mammalian embryonic development, is ubiquitously expressed and undergoes regulated nucleocytoplasmic exchange. Here we show that C3G localizes to SC35-positive nuclear speckles and regulates splicing activity. Reversible association of C3G with speckles was seen on inhibition of transcription and splicing. C3G shows partial colocalization with SC35 and is recruited to a chromatin and RNase-sensitive fraction of speckles. Its presence in speckles is dependent on intact cellular actin cytoskeleton and is lost on expression of the kinase Clk1. Rap1, a substrate of C3G, is also present in nuclear speckles, and inactivation of Rap signaling by expression of GFP-Rap1GAP alters speckle morphology and number. Enhanced association of C3G with speckles is seen on glycogen synthase kinase 3 beta inhibition or differentiation of C2C12 cells to myotubes. CRISPR/Cas9-mediated knockdown of C3G resulted in altered splicing activity of an artificial gene as well as endogenous CD44. C3G knockout clones of C2C12 as well as MDA-MB-231 cells showed reduced protein levels of several splicing factors compared with control cells. Our results identify C3G and Rap1 as novel components of nuclear speckles and a role for C3G in regulating cellular RNA splicing activity.

Dysferlin rescue by spliceosome-mediated pre-mRNA trans-splicing targeting introns harbouring weakly defined 3' splice sites.

PubMed

Philippi, Susanne; Lorain, Stéphanie; Beley, Cyriaque; Peccate, Cécile; Précigout, Guillaume; Spuler, Simone; Garcia, Luis

2015-07-15

The modification of the pre-mRNA cis-splicing process employing a pre-mRNA trans-splicing molecule (PTM) is an attractive strategy for the in situ correction of genes whose careful transcription regulation and full-length expression is determinative for protein function, as it is the case for the dysferlin (DYSF, Dysf) gene. Loss-of-function mutations of DYSF result in different types of muscular dystrophy mainly manifesting as limb girdle muscular dystrophy 2B (LGMD2B) and Miyoshi muscular dystrophy 1 (MMD1). We established a 3' replacement strategy for mutated DYSF pre-mRNAs induced by spliceosome-mediated pre-mRNA trans-splicing (SmaRT) by the use of a PTM. In contrast to previously established SmaRT strategies, we particularly focused on the identification of a suitable pre-mRNA target intron other than the optimization of the PTM design. By targeting DYSF pre-mRNA introns harbouring differentially defined 3' splice sites (3' SS), we found that target introns encoding weakly defined 3' SSs were trans-spliced successfully in vitro in human LGMD2B myoblasts as well as in vivo in skeletal muscle of wild-type and Dysf(-/-) mice. For the first time, we demonstrate rescue of Dysf protein by SmaRT in vivo. Moreover, we identified concordant qualities among the successfully targeted Dysf introns and targeted endogenous introns in previously reported SmaRT approaches that might facilitate a selective choice of target introns in future SmaRT strategies. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

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.

Nd:YAG-laser-based time-domain reflectometry measurements of the intrinsic reflection signature from PMMA fiber splices

NASA Astrophysics Data System (ADS)

Lawson, Christopher M.; Michael, Robert R., Jr.; Dressel, Earl M.; Harmony, David W.

1991-12-01

Optical time domain reflectometry (OTDR) measurements have been performed on polished polymethylmethacrylate (PMMA) plastic fiber splices. After the dominant splice reflection sources due to surface roughness, inexact index matching, and fiber core misalignment were eliminated, an intrinsic OTDR signature 3 - 8 dB above the Rayleigh backscatter floor remained with all tested fibers. This minimum splice reflectivity exhibits characteristics that are consistent with sub-surface polymer damage and can be used for detection of PMMA fiber splices.

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.

Effects of secondary structure on pre-mRNA splicing: hairpins sequestering the 5' but not the 3' splice site inhibit intron processing in Nicotiana plumbaginifolia.

PubMed

Liu, H X; Goodall, G J; Kole, R; Filipowicz, W

1995-01-16

We have performed a systematic study of the effect of artificial hairpins on pre-mRNA splicing in protoplasts of a dicot plant, Nicotiana plumbaginifolia. Hairpins with a potential to form 18 or 24 bp stems strongly inhibit splicing when they sequester the 5' splice site or are placed in the middle of short introns. However, similar 24 bp hairpins sequestering the 3' splice site do not prevent this site from being used as an acceptor. Utilization of the stem-located 3' site requires that the base of the stem is separated from the upstream 5' splice site by a minimum of approximately 45 nucleotides and that another 'helper' 3' splice site is present downstream of the stem. The results indicate that the spliceosome or factors associated with it may have a potential to unfold secondary structure present in the downstream portion of the intron, prior to or at the step of the 3' splice site selection. The finding that the helper 3' site is required for utilization of the stem-located acceptor confirms and extends previous observations, obtained with HeLa cell in vitro splicing systems, indicating that the 3' splice site may be recognized at least twice during spliceosome assembly.

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.

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.

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.

Transcriptomic profile analysis of mouse neural tube development by RNA-Seq.

PubMed

Yu, Juan; Mu, Jianbing; Guo, Qian; Yang, Lihong; Zhang, Juan; Liu, Zhizhen; Yu, Baofeng; Zhang, Ting; Xie, Jun

2017-09-01

The neural tube is the primordium of the central nervous system (CNS) in which its development is not entirely clear. Understanding the cellular and molecular basis of neural tube development could, therefore, provide vital clues to the mechanism of neural tube defects (NTDs). Here, we investigated the gene expression profiles of three different time points (embryonic day (E) 8.5, 9.5 and 10.5) of mouse neural tube by using RNA-seq approach. About 391 differentially expressed genes (DEGs) were screened during mouse neural tube development, including 45 DEGs involved in CNS development, among which Bmp2, Ascl1, Olig2, Lhx1, Wnt7b and Eomes might play the important roles. Of 45 DEGs, Foxp2, Eomes, Hoxb3, Gpr56, Hap1, Nkx2-1, Sez6l2, Wnt7b, Tbx20, Nfib, Cntn1 and Dcx had different isoforms, and the opposite expression pattern of different isoforms was observed for Gpr56, Nkx2-1 and Sez6l2. In addition, alternative splicing, such as mutually exclusive exon, retained intron, skipped exon and alternative 3' splice site was identified in 10 neural related differentially splicing genes, including Ngrn, Ddr1, Dctn1, Dnmt3b, Ect2, Map2, Mbnl1, Meis2, Vcan and App. Moreover, seven neural splicing factors, such as Nova1/2, nSR100/Srrm4, Elavl3/4, Celf3 and Rbfox1 were differentially expressed during mouse neural tube development. Interestingly, nine DEGs identified above were dysregulated in retinoic acid-induced NTDs model, indicating the possible important role of these genes in NTDs. Taken together, our study provides more comprehensive information on mouse neural tube development, which might provide new insights on NTDs occurrence. © 2017 IUBMB Life, 69(9):706-719, 2017. © 2017 International Union of Biochemistry and Molecular Biology.

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

PKC-Theta is a Novel SC35 Splicing Factor Regulator in Response to T Cell Activation.

PubMed

McCuaig, Robert Duncan; Dunn, Jennifer; Li, Jasmine; Masch, Antonia; Knaute, Tobias; Schutkowski, Mike; Zerweck, Johannes; Rao, Sudha

2015-01-01

Alternative splicing of nuclear pre-mRNA is essential for generating protein diversity and regulating gene expression. While many immunologically relevant genes undergo alternative splicing, the role of regulated splicing in T cell immune responses is largely unexplored, and the signaling pathways and splicing factors that regulate alternative splicing in T cells are poorly defined. Here, we show using a combination of Jurkat T cells, human primary T cells, and ex vivo naïve and effector virus-specific T cells isolated after influenza A virus infection that SC35 phosphorylation is induced in response to stimulatory signals. We show that SC35 colocalizes with RNA polymerase II in activated T cells and spatially overlaps with H3K27ac and H3K4me3, which mark transcriptionally active genes. Interestingly, SC35 remains coupled to the active histone marks in the absence of continuing stimulatory signals. We show for the first time that nuclear PKC-θ co-exists with SC35 in the context of the chromatin template and is a key regulator of SC35 in T cells, directly phosphorylating SC35 peptide residues at RNA recognition motif and RS domains. Collectively, our findings suggest that nuclear PKC-θ is a novel regulator of the key splicing factor SC35 in T cells.

β Subunits Functionally Differentiate Human Kv4.3 Potassium Channel Splice Variants

PubMed Central

Abbott, Geoffrey W.

2017-01-01

The human ventricular cardiomyocyte transient outward K+ current (Ito) mediates the initial phase of myocyte repolarization and its disruption is implicated in Brugada Syndrome and heart failure (HF). Human cardiac Ito is generated primarily by two Kv4.3 splice variants (Kv4.3L and Kv4.3S, diverging only by a C-terminal, S6-proximal, 19-residue stretch unique to Kv4.3L), which are differentially remodeled in HF, but considered functionally alike at baseline. Kv4.3 is regulated in human heart by β subunits including KChIP2b and KCNEs, but their effects were previously assumed to be Kv4.3 isoform-independent. Here, this assumption was tested experimentally using two-electrode voltage-clamp analysis of human subunits co-expressed in Xenopus laevis oocytes. Unexpectedly, Kv4.3L-KChIP2b channels exhibited up to 8-fold lower current augmentation, 40% slower inactivation, and 5 mV-shifted steady-state inactivation compared to Kv4.3S-KChIP2b. A synthetic peptide mimicking the 19-residue stretch diminished these differences, reinforcing the importance of this segment in mediating Kv4.3 regulation by KChIP2b. KCNE subunits induced further functional divergence, including a 7-fold increase in Kv4.3S-KCNE4-KChIP2b current compared to Kv4.3L-KCNE4-KChIP2b. The discovery of β-subunit-dependent functional divergence in human Kv4.3 splice variants suggests a C-terminal signaling hub is crucial to governing β-subunit effects upon Kv4.3, and demonstrates the potential significance of differential Kv4.3 gene-splicing and β subunit expression in myocyte physiology and pathobiology. PMID:28228734

β Subunits Functionally Differentiate Human Kv4.3 Potassium Channel Splice Variants.

PubMed

Abbott, Geoffrey W

2017-01-01

The human ventricular cardiomyocyte transient outward K + current ( I to ) mediates the initial phase of myocyte repolarization and its disruption is implicated in Brugada Syndrome and heart failure (HF). Human cardiac I to is generated primarily by two Kv4.3 splice variants (Kv4.3L and Kv4.3S, diverging only by a C-terminal, S6-proximal, 19-residue stretch unique to Kv4.3L), which are differentially remodeled in HF, but considered functionally alike at baseline. Kv4.3 is regulated in human heart by β subunits including KChIP2b and KCNEs, but their effects were previously assumed to be Kv4.3 isoform-independent. Here, this assumption was tested experimentally using two-electrode voltage-clamp analysis of human subunits co-expressed in Xenopus laevis oocytes. Unexpectedly, Kv4.3L-KChIP2b channels exhibited up to 8-fold lower current augmentation, 40% slower inactivation, and 5 mV-shifted steady-state inactivation compared to Kv4.3S-KChIP2b. A synthetic peptide mimicking the 19-residue stretch diminished these differences, reinforcing the importance of this segment in mediating Kv4.3 regulation by KChIP2b. KCNE subunits induced further functional divergence, including a 7-fold increase in Kv4.3S-KCNE4-KChIP2b current compared to Kv4.3L-KCNE4-KChIP2b. The discovery of β-subunit-dependent functional divergence in human Kv4.3 splice variants suggests a C-terminal signaling hub is crucial to governing β-subunit effects upon Kv4.3, and demonstrates the potential significance of differential Kv4.3 gene-splicing and β subunit expression in myocyte physiology and pathobiology.

Splicing-factor alterations in cancers

PubMed Central

Anczuków, Olga; Krainer, Adrian R.

2016-01-01

Tumor-associated alterations in RNA splicing result either from mutations in splicing-regulatory elements or changes in components of the splicing machinery. This review summarizes our current understanding of the role of splicing-factor alterations in human cancers. We describe splicing-factor alterations detected in human tumors and the resulting changes in splicing, highlighting cell-type-specific similarities and differences. We review the mechanisms of splicing-factor regulation in normal and cancer cells. Finally, we summarize recent efforts to develop novel cancer therapies, based on targeting either the oncogenic splicing events or their upstream splicing regulators. PMID:27530828

Modulation of KCNQ1 alternative splicing regulates cardiac IKs and action potential repolarization.

PubMed

Lee, Hsiang-Chun; Rudy, Yoram; Po-Yuan, Phd; Sheu, Sheng-Hsiung; Chang, Jan-Gowth; Cui, Jianmin

2013-08-01

Slow delayed-rectifier potassium current (IKs) channels, made of the pore-forming KCNQ1 and auxiliary KCNE1 subunits, play a key role in determining action potential duration (APD) in cardiac myocytes. The consequences of drug-induced KCNQ1 splice alteration remain unknown. To study the modulation of KCNQ1 alternative splicing by amiloride and the consequent changes in IKs and action potentials (APs) in ventricular myocytes. Canine endocardial, midmyocardial, and epicardial ventricular myocytes were isolated. Levels of KCNQ1a and KCNQ1b as well as a series of splicing factors were quantified by using the reverse transcriptase-polymerase chain reaction and Western blot. The effect of amiloride-induced changes in the KCNQ1b/total KCNQ1 ratio on AP was measured by using whole-cell patch clamp with and without isoproterenol. With 50 μmol/L of amiloride for 6 hours, KCNQ1a at transcriptional and translational levels increased in midmyocardial myocytes but decreased in endo- and epicardial myocytes. Likewise, changes in splicing factors in midmyocardial were opposite to that in endo- and epicardial myocytes. In midmyocardial myocytes amiloride shortened APD and decreased isoproterenol-induced early afterdepolarizations significantly. The same amiloride-induced effects were demonstrated by using human ventricular myocyte model for AP simulations under beta-adrenergic stimulation. Moreover, amiloride reduced the transmural dispersion of repolarization in pseudo-electrocardiogram. Amiloride regulates IKs and APs with transmural differences and reduces arrhythmogenicity through the modulation of KCNQ1 splicing. We suggested that the modulation of KCNQ1 splicing may help prevent arrhythmia. Copyright © 2013 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.

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.

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.

Global Profiling of hnRNP A2/B1-RNA Binding on Chromatin Highlights LncRNA Interactions.

PubMed

Nguyen, Eric D; Balas, Maggie M; Griffin, April M; Roberts, Justin T; Johnson, Aaron M

2018-06-23

Long noncoding RNAs (lncRNAs) often carry out their functions through associations with adaptor proteins. We recently identified heterogeneous ribonucleoprotein (hnRNP) A2/B1 as an adaptor of the human HOTAIR lncRNA. hnRNP A2 and B1 are splice isoforms of the same gene. The spliced version of HOTAIR preferentially associates with the B1 isoform, which we hypothesize contributes to RNA-RNA matching between HOTAIR and transcripts of target genes in breast cancer. Here we used enhanced cross-linking immunoprecipitation (eCLIP) to map the direct interactions between A2/B1 and RNA in breast cancer cells. Despite differing by only twelve amino acids, the A2 and B1 splice isoforms associate preferentially with distinct populations of RNA in vivo. Through cellular fractionation experiments we characterize the pattern of RNA association in chromatin, nucleoplasm, and cytoplasm. We find that a majority of interactions occur on chromatin, even those that do not contribute to co-transcriptional splicing. A2/B1 binding site locations on multiple RNAs hint at a contribution to the regulation and function of lncRNAs. Surprisingly, the strongest A2/B1 binding site occurs in a retained intron of HOTAIR, which interrupts an RNA-RNA interaction hotspot. In vitro eCLIP experiments highlight additional exonic B1 binding sites in HOTAIR which also surround the RNA-RNA interaction hotspot. Interestingly, a version of HOTAIR with the intron retained is still capable of making RNA-RNA interactions in vitro through the hotspot region. Our data further characterize the multiple functions of a repurposed splicing factor with isoform-biased interactions, and highlight that the majority of these functions occur on chromatin-associated RNA.

Aberrant splicing in maize rough endosperm3 reveals a conserved role for U12 splicing in eukaryotic multicellular development

PubMed Central

Barbazuk, W. Brad

2017-01-01

RNA splicing of U12-type introns functions in human cell differentiation, but it is not known whether this class of introns has a similar role in plants. The maize ROUGH ENDOSPERM3 (RGH3) protein is orthologous to the human splicing factor, ZRSR2. ZRSR2 mutations are associated with myelodysplastic syndrome (MDS) and cause U12 splicing defects. Maize rgh3 mutants have aberrant endosperm cell differentiation and proliferation. We found that most U12-type introns are retained or misspliced in rgh3. Genes affected in rgh3 and ZRSR2 mutants identify cell cycle and protein glycosylation as common pathways disrupted. Transcripts with retained U12-type introns can be found in polysomes, suggesting that splicing efficiency can alter protein isoforms. The rgh3 mutant protein disrupts colocalization with a known ZRSR2-interacting protein, U2AF2. These results indicate conserved function for RGH3/ZRSR2 in U12 splicing and a deeply conserved role for the minor spliceosome to promote cell differentiation from stem cells to terminal fates. PMID:28242684

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.

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.

A Systems-Level Analysis Reveals Circadian Regulation of Splicing in Colorectal Cancer.

PubMed

El-Athman, Rukeia; Fuhr, Luise; Relógio, Angela

2018-06-20

Accumulating evidence points to a significant role of the circadian clock in the regulation of splicing in various organisms, including mammals. Both dysregulated circadian rhythms and aberrant pre-mRNA splicing are frequently implicated in human disease, in particular in cancer. To investigate the role of the circadian clock in the regulation of splicing in a cancer progression context at the systems-level, we conducted a genome-wide analysis and compared the rhythmic transcriptional profiles of colon carcinoma cell lines SW480 and SW620, derived from primary and metastatic sites of the same patient, respectively. We identified spliceosome components and splicing factors with cell-specific circadian expression patterns including SRSF1, HNRNPLL, ESRP1, and RBM 8A, as well as altered alternative splicing events and circadian alternative splicing patterns of output genes (e.g., VEGFA, NCAM1, FGFR2, CD44) in our cellular model. Our data reveals a remarkable interplay between the circadian clock and pre-mRNA splicing with putative consequences in tumor progression and metastasis. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

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.

An Alternative Splice Product of IκB Kinase (IKKγ), IKKγ-Δ, Differentially Mediates Cytokine and Human T-Cell Leukemia Virus Type 1 Tax-Induced NF-κB Activation

PubMed Central

Hai, Tao; Yeung, Man-Lung; Wood, Thomas G.; Wei, Yuanfen; Yamaoka, Shoji; Gatalica, Zoran; Jeang, Kuan-Teh; Brasier, Allan R.

2006-01-01

NF-κB is an inducible transcription factor mediating innate immune responses whose activity is controlled by the multiprotein IκB kinase (IKK) “signalsome”. The core IKK consists of two catalytic serine kinases, IKKα and IKKβ, and a noncatalytic subunit, IKKγ. IKKγ is required for IKK activity by mediating kinase oligomerization and serving to couple the core catalytic subunits to upstream mitogen-activated protein 3-kinase cascades. We have discovered an alternatively spliced IKKγ mRNA isoform, encoding an in-frame deletion of exon 5, termed IKKγ-Δ. Using a specific reverse transcription-PCR assay, we find that IKKγ-Δ is widely expressed in cultured human cells and normal human tissues. Because IKKγ-Δ protein is lacking a critical coiled-coil domain important in protein-protein interactions, we sought to determine its signaling properties by examining its ability to self associate, couple to activators of the canonical pathway, and mediate human T-cell leukemia virus type 1 (HTLV-1) Tax-induced NF-κB activity. Coimmunoprecipitation and confocal colocalization assays indicate IKKγ-Δ has strong homo- and heterotypic association with wild-type (WT) IKKγ and, like IKKγ WT, associates with the IKKβ kinase. Similarly, IKKγ-Δ mediates IKK kinase activity and downstream NF-κB-dependent transcription in response to tumor necrosis factor (TNF) and the NF-κB-inducing kinase-IKKα signaling pathway. Surprisingly, however, in contrast to IKKγ WT, IKKγ-Δ is not able to mediate HTLV-1 Tax-induced NF-κB-dependent transcription, even though IKKγ-Δ binds and colocalizes with Tax. These observations suggest that IKKγ-Δ is a functionally distinct alternatively spliced mRNA product differentially mediating TNF-induced, but not Tax-induced, signals converging on the IKK signalsome. Differing levels of IKKγ-Δ expression, therefore, may affect signal transduction cascades coupling to IKK. PMID:16611882

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.

Both sides of the same coin: Rac1 splicing regulating by EGF signaling.

PubMed

Fu, Xiang-Dong

2017-04-01

EGF, a well-studied mitogen for cancer cells, is revealed to induce an E3 ubiquitin ligase adaptor SPSB1, which recruits the Elongin B/C-Collin complex to trigger ubiquitylation of the negative splicing regulator hnRNP A1. This event is synergized with EGF-activated SR proteins to alter alternative splicing of a key small GTPase Rac1 to enhance cell migration, highlighting converging EGF signals on both negative and positive splicing regulators to jointly promote a key cancer pathway.

Aberrant RNA splicing in cancer; expression changes and driver mutations of splicing factor genes.

PubMed

Sveen, A; Kilpinen, S; Ruusulehto, A; Lothe, R A; Skotheim, R I

2016-05-12

Alternative splicing is a widespread process contributing to structural transcript variation and proteome diversity. In cancer, the splicing process is commonly disrupted, resulting in both functional and non-functional end-products. Cancer-specific splicing events are known to contribute to disease progression; however, the dysregulated splicing patterns found on a genome-wide scale have until recently been less well-studied. In this review, we provide an overview of aberrant RNA splicing and its regulation in cancer. We then focus on the executors of the splicing process. Based on a comprehensive catalog of splicing factor encoding genes and analyses of available gene expression and somatic mutation data, we identify cancer-associated patterns of dysregulation. Splicing factor genes are shown to be significantly differentially expressed between cancer and corresponding normal samples, and to have reduced inter-individual expression variation in cancer. Furthermore, we identify enrichment of predicted cancer-critical genes among the splicing factors. In addition to previously described oncogenic splicing factor genes, we propose 24 novel cancer-critical splicing factors predicted from somatic mutations.

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.

29 CFR 1918.62 - Miscellaneous auxiliary gear.

Code of Federal Regulations, 2010 CFR

2010-07-01

... appendix II to this part. A design safety factor of at least five shall be maintained for the common sizes... cranes, designed to be used with lesser wire rope safety factors; (ii) According to design factors in... pre-slung drafts. (3) Eye splices shall consist of at least three full tucks. Short splices shall...

29 CFR 1918.62 - Miscellaneous auxiliary gear.

Code of Federal Regulations, 2011 CFR

2011-07-01

... appendix II to this part. A design safety factor of at least five shall be maintained for the common sizes... cranes, designed to be used with lesser wire rope safety factors; (ii) According to design factors in... pre-slung drafts. (3) Eye splices shall consist of at least three full tucks. Short splices shall...

Elucidation of the 14-3-3ζ interactome reveals critical roles of RNA-splicing factors during adipogenesis.

PubMed

Mugabo, Yves; Sadeghi, Mina; Fang, Nancy N; Mayor, Thibault; Lim, Gareth E

2018-05-04

Adipogenesis involves a complex signaling network requiring strict temporal and spatial organization of effector molecules. Molecular scaffolds, such as 14-3-3 proteins, facilitate such organization, and we have previously identified 14-3-3ζ as an essential scaffold in adipocyte differentiation. The interactome of 14-3-3ζ is large and diverse, and it is possible that novel adipogenic factors may be present within it, but this possibility has not yet been tested. Herein, we generated mouse embryonic fibroblasts from mice overexpressing a tandem affinity purification (TAP) epitope-tagged 14-3-3ζ molecule. After inducing adipogenesis, TAP-14-3-3ζ complexes were purified, followed by MS analysis to determine the 14-3-3ζ interactome. We observed more than 100 proteins that were unique to adipocyte differentiation, 56 of which were novel interacting partners. Among these, we were able to identify previously established regulators of adipogenesis ( i.e. Ptrf/Cavin1) within the 14-3-3ζ interactome, confirming the utility of this approach to detect adipogenic factors. We found that proteins related to RNA metabolism, processing, and splicing were enriched in the interactome. Analysis of transcriptomic data revealed that 14-3-3ζ depletion in 3T3-L1 cells affected alternative splicing of mRNA during adipocyte differentiation. siRNA-mediated depletion of RNA-splicing factors within the 14-3-3ζ interactome, that is, of Hnrpf, Hnrpk, Ddx6, and Sfpq, revealed that they have essential roles in adipogenesis and in the alternative splicing of Pparg and the adipogenesis-associated gene Lpin1 In summary, we have identified novel adipogenic factors within the 14-3-3ζ interactome. Further characterization of additional proteins within the 14-3-3ζ interactome may help identify novel targets to block obesity-associated expansion of adipose tissues. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

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.

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

PubMed Central

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

2017-01-01

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

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

PubMed

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

2017-02-17

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

Protein arginine methylation of Npl3 promotes splicing of the SUS1 intron harboring non-consensus 5' splice site and branch site.

PubMed

Muddukrishna, Bhavana; Jackson, Christopher A; Yu, Michael C

2017-06-01

Protein arginine methylation occurs on spliceosomal components and spliceosome-associated proteins, but how this modification contributes to their function in pre-mRNA splicing remains sparse. Here we provide evidence that protein arginine methylation of the yeast SR-/hnRNP-like protein Npl3 plays a role in facilitating efficient splicing of the SUS1 intron that harbors a non-consensus 5' splice site and branch site. In yeast cells lacking the major protein arginine methyltransferase HMT1, we observed a change in the co-transcriptional recruitment of the U1 snRNP subunit Snp1 and Npl3 to pre-mRNAs harboring both consensus (ECM33 and ASC1) and non-consensus (SUS1) 5' splice site and branch site. Using an Npl3 mutant that phenocopies wild-type Npl3 when expressed in Δhmt1 cells, we showed that the arginine methylation of Npl3 is responsible for this. Examination of pre-mRNA splicing efficiency in these mutants reveals the requirement of Npl3 methylation for the efficient splicing of SUS1 intron 1, but not of ECM33 or ASC1. Changing the 5' splice site and branch site in SUS1 intron 1 to the consensus form restored splicing efficiency in an Hmt1-independent manner. Results from biochemical studies show that methylation of Npl3 promotes its optimal association with the U1 snRNP through its association with the U1 snRNP subunit Mud1. Based on these data, we propose a model in which Hmt1, via arginine methylation of Npl3, facilitates U1 snRNP engagement with the pre-mRNA to promote usage of non-consensus splice sites by the splicing machinery. Published by Elsevier B.V.

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.

Serine/Arginine-rich Splicing Factor 2 Modulates Herpes Simplex Virus Type 1 Replication via Regulating Viral Gene Transcriptional Activity and Pre-mRNA Splicing.

PubMed

Wang, Ziqiang; Liu, Qing; Lu, Jinhua; Fan, Ping; Xie, Weidong; Qiu, Wei; Wang, Fan; Hu, Guangnan; Zhang, Yaou

2016-12-16

Once it enters the host cell, herpes simplex virus type 1 (HSV-1) recruits a series of host cell factors to facilitate its life cycle. Here, we demonstrate that serine/arginine-rich splicing factor 2 (SRSF2), which is an important component of the splicing speckle, mediates HSV-1 replication by regulating viral gene expression at the transcriptional and posttranscriptional levels. Our results indicate that SRSF2 functions as a transcriptional activator by directly binding to infected cell polypeptide 0 (ICP0), infected cell polypeptide 27 (ICP27), and thymidine kinase promoters. Moreover, SRSF2 participates in ICP0 pre-mRNA splicing by recognizing binding sites in ICP0 exon 3. These findings provide insight into the functions of SRSF2 in HSV-1 replication and gene expression. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Generation of fibrosarcomas in vivo by a retrovirus that expresses the normal B chain of platelet-derived growth factor and mimics the alternative splice pattern of the v-sis oncogene

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

Pech, M.; Gazit, A.; Arnstein, P.

1989-04-01

A retrovirus containing the entire human platelet-derived growth factor B-chain (PDGF-B) gene was constructed in order to investigate the in vivo biological activity of its encoded growth factor. When this virus was introduced into newborn mice, it reproducibly generated fibrosarcomas at the site of inoculation. Proviruses in each fibrosarcoma analyzed had lost 149 nucleotides downstream of the PDGF-B coding region. This deletion originated from an alternative or aberrant splice event that occurred within exon 7 of the PDGF-B gene and mimicked the v-sis oncogene. Thus, deletion of this region may be necessary for efficient retrovirus replication or for more potentmore » transforming function. Evidence that the normal growth factor coding sequence was unaltered derived from RNase protection studies and immunoprecipitation analysis. Tumors were generally polyclonal but demonstrated clonal subpopulations. Moreover, tumor-derived cell lines became monoclonal within a few tissue culture passages and rapidly formed tumors in vivo. These findings argue that overexpression of the normal human PDGF-B gene product under retrovirus control can induce the fully malignant phenotype.« less

An intronic mutation c.6430-3C>G in the F8 gene causes splicing efficiency and premature termination in hemophilia A.

PubMed

Xia, Zunjing; Lin, Jie; Lu, Lingping; Kim, Chol; Yu, Ping; Qi, Ming

2018-06-01

: Hemophilia A is a bleeding disorder caused by coagulation factor VIII protein deficiency or dysfunction, which is classified into severe, moderate, and mild according to factor clotting activity. An overwhelming majority of missense and nonsense mutations occur in exons of F8 gene, whereas mutations in introns can also be pathogenic. This study aimed to investigate the effect of an intronic mutation, c.6430-3C>G (IVS22-3C>G), on pre-mRNA splicing of the F8 gene. We applied DNA and cDNA sequencing in a Chinese boy with hemophilia A to search if any pathogenic mutation in the F8 gene. Functional analysis was performed to investigate the effect of an intronic mutation at the transcriptional level. Human Splicing Finder and PyMol were also used to predict its effect. We found the mutation c.6430-3C>G (IVS22-3C>G) in the F8 gene in the affected boy, with his mother being a carrier. cDNA from the mother and pSPL3 splicing assay showed that the mutation IVS22-3C>G results in a two-nucleotide AG inclusion at the 3' end of intron 22 and leads to a truncated coagulation factor VIII protein, with partial loss of the C1 domain and complete loss of the C2 domain. The in-silico tool predicted that the mutation induces altered pre-mRNA splicing by using a cryptic acceptor site in intron 22. The IVS22-3C>G mutation was confirmed to affect pre-mRNA splicing and produce a truncated protein, which reduces the stability of binding between the F8 protein and von Willebrand factor carrier protein due to the loss of an interaction domain.

Postnatal Expression of V2 Vasopressin Receptor Splice Variants in the Rat Cerebellum

PubMed Central

Vargas, Karina J.; Sarmiento, José M.; Ehrenfeld, Pamela; Añazco, Carolina C.; Villanueva, Carolina I.; Carmona, Pamela L.; Brenet, Marianne; Navarro, Javier; Müller-Esterl, Werner; Figueroa, Carlos D.; González, Carlos B.

2010-01-01

The V2 vasopressin receptor gene contains an alternative splice site in exon-3, which leads to the generation of two splice variants (V2a and V2b) first identified in the kidney. The open reading frame of the alternatively spliced V2b transcripten codes a truncated receptor, showing the same amino acid sequence as the canonical V2a receptor up to the 6th transmembrane segment, but displaying a distinct sequence to the corresponding 7th transmembrane segment and C-terminal domain relative to the V2a receptor. Here, we demonstrate the postnatal expression of V2a and V2b variants in the rat cerebellum. Most importantly, we showed by in situ hybridization and immunocytochemistry that both V2 splice variants were preferentially expressed in Purkinje cells, from early to late postnatal development. In addition, both variants were transiently expressed in the neuroblastic external granule cells and Bergmann fibers. These results indicate that the cellular distributions of both splice variants are developmentally regulated, and suggest that the transient expression of the V2 receptor is involved in the mechanisms of cerebellar cytodifferentiation by AVP. Finally, transfected CHO-K1 .expressing similar amounts of both V2 splice variants, as that found in the cerebellum, showed a significant reduction in the surface expression of V2a receptors, suggesting that the differential expression of the V2 splice variants regulate the vasopressin signaling in the cerebellum. PMID:19281786

Evaluation of Alternative Splicing Regulators As Targets For Selective Therapy of Triple Negative (Basal) Breast Carcinoma

DTIC Science & Technology

2016-10-01

KHDRBS3 and SRSF12 on tumor progression and metastasis (Task1). We analyzed the effect of KHDRBS3 depletion on the growth and migration properties of the...tumor growth during the second year of this project. Continued analysis of the splicing factor expression in primary tumor samples further supports...depletion on tumor initiation, growth and metastasis. Keywords Pre-mRNA splicing, breast cancer, KHDRBS3, SRPK1, SRSF12, metastasis

Low-loss polarization-maintaining fusion splicing of single-mode fibers and hollow-core photonic crystal fibers, relevant for monolithic fiber laser pulse compression.

PubMed

Kristensen, Jesper T; Houmann, Andreas; Liu, Xiaomin; Turchinovich, Dmitry

2008-06-23

We report on highly reproducible low-loss fusion splicing of polarization-maintaining single-mode fibers (PM-SMFs) and hollow-core photonic crystal fibers (HC-PCFs). The PM-SMF-to-HC-PCF splices are characterized by the loss of 0.62 +/- 0.24 dB, and polarization extinction ratio of 19 +/- 0.68 dB. The reciprocal HC-PCF-to-PM-SMF splice loss is found to be 2.19 +/- 0.33 dB, which is caused by the mode evolution in HC-PCF. The return loss in both cases was measured to be -14 dB. We show that a splice defect is caused by the HC-PCF cleave defect, and the lossy splice can be predicted at an early stage of the splicing process. We also demonstrate that the higher splice loss compromises the PM properties of the splice. Our splicing technique was successfully applied to the realization of a low-loss, environmentally stable monolithic PM fiber laser pulse compressor, enabling direct end-of-the-fiber femtosecond pulse delivery.

Oncogenes and RNA splicing of human tumor viruses

PubMed Central

Ajiro, Masahiko; Zheng, Zhi-Ming

2014-01-01

Approximately 10.8% of human cancers are associated with infection by an oncogenic virus. These viruses include human papillomavirus (HPV), Epstein–Barr virus (EBV), Merkel cell polyomavirus (MCV), human T-cell leukemia virus 1 (HTLV-1), Kaposi's sarcoma-associated herpesvirus (KSHV), hepatitis C virus (HCV) and hepatitis B virus (HBV). These oncogenic viruses, with the exception of HCV, require the host RNA splicing machinery in order to exercise their oncogenic activities, a strategy that allows the viruses to efficiently export and stabilize viral RNA and to produce spliced RNA isoforms from a bicistronic or polycistronic RNA transcript for efficient protein translation. Infection with a tumor virus affects the expression of host genes, including host RNA splicing factors, which play a key role in regulating viral RNA splicing of oncogene transcripts. A current prospective focus is to explore how alternative RNA splicing and the expression of viral oncogenes take place in a cell- or tissue-specific manner in virus-induced human carcinogenesis. PMID:26038756

Regulation of alternative VEGF-A mRNA splicing is a therapeutic target for analgesia.

PubMed

Hulse, R P; Beazley-Long, N; Hua, J; Kennedy, H; Prager, J; Bevan, H; Qiu, Y; Fernandes, E S; Gammons, M V; Ballmer-Hofer, K; Gittenberger de Groot, A C; Churchill, A J; Harper, S J; Brain, S D; Bates, D O; Donaldson, L F

2014-11-01

Vascular endothelial growth factor-A (VEGF-A) is best known as a key regulator of the formation of new blood vessels. Neutralization of VEGF-A with anti-VEGF therapy e.g. bevacizumab, can be painful, and this is hypothesized to result from a loss of VEGF-A-mediated neuroprotection. The multiple vegf-a gene products consist of two alternatively spliced families, typified by VEGF-A165a and VEGF-A165b (both contain 165 amino acids), both of which are neuroprotective. Under pathological conditions, such as in inflammation and cancer, the pro-angiogenic VEGF-A165a is upregulated and predominates over the VEGF-A165b isoform. We show here that in rats and mice VEGF-A165a and VEGF-A165b have opposing effects on pain, and that blocking the proximal splicing event - leading to the preferential expression of VEGF-A165b over VEGF165a - prevents pain in vivo. VEGF-A165a sensitizes peripheral nociceptive neurons through actions on VEGFR2 and a TRPV1-dependent mechanism, thus enhancing nociceptive signaling. VEGF-A165b blocks the effect of VEGF-A165a. After nerve injury, the endogenous balance of VEGF-A isoforms switches to greater expression of VEGF-Axxxa compared to VEGF-Axxxb, through an SRPK1-dependent pre-mRNA splicing mechanism. Pharmacological inhibition of SRPK1 after traumatic nerve injury selectively reduced VEGF-Axxxa expression and reversed associated neuropathic pain. Exogenous VEGF-A165b also ameliorated neuropathic pain. We conclude that the relative levels of alternatively spliced VEGF-A isoforms are critical for pain modulation under both normal conditions and in sensory neuropathy. Altering VEGF-Axxxa/VEGF-Axxxb balance by targeting alternative RNA splicing may be a new analgesic strategy. Copyright © 2014. Published by Elsevier Inc.

Boric acid reversibly inhibits the second step of pre-mRNA splicing.

PubMed

Shomron, Noam; Ast, Gil

2003-09-25

Several approaches have been used to identify the factors involved in mRNA splicing. None of them, however, comprises a straightforward reversible method for inhibiting the second step of splicing using an external reagent other than a chelator. This investigation demonstrates that the addition of boric acid to an in vitro pre-mRNA splicing reaction causes a dose-dependent reversible inhibition effect on the second step of splicing. The mechanism of action does not involve chelation of several metal ions; hindrance of 3' splice-site; or binding to hSlu7. This study presents a novel method for specific reversible inhibition of the second step of pre-mRNA splicing.

Splicing Factor 1 Modulates Dietary Restriction and TORC1 Pathway Longevity in C. elegans

PubMed Central

Heintz, Caroline; Escoubas, Caroline; Zhang, Yue; Weir, Heather J.; Dutta, Sneha; Silva-García, Carlos Giovanni; Bruun, Gitte Hoffmann; Morantte, Ianessa; Hoxhaj, Gerta; Manning, Brendan D.; Andresen, Brage S.; Mair, William B.

2016-01-01

Ageing is driven by a loss of transcriptional and protein homeostasis1–3 and is the key risk factor for multiple chronic diseases. Interventions that attenuate or reverse systemic dysfunction seen with age therefore have potential to reduce overall disease risk in the elderly. Pre-mRNA splicing is a fundamental link between gene expression and the proteome, and deregulation of the splicing machinery is linked to multiple age-related chronic diseases4,5. However, the role of splicing homeostasis in healthy ageing remains unclear. Here we demonstrate that pre-mRNA splicing homeostasis is a biomarker and predictor of life expectancy in Caenorhabditis elegans. Using transcriptomics and in-depth splicing analysis in young and old animals fed ad libitum or on dietary restriction (DR), we find defects in global pre-mRNA splicing with age that are reduced by DR via the branch point binding protein (BBP)/splicing factor 1 (SFA-1). We show that SFA-1 is specifically required for lifespan extension both by DR, and modulation of TORC1 pathway components AMPK, RAGA-1 and RSKS-1/S6 Kinase. Lastly, we demonstrate that overexpression of SFA-1 is sufficient to extend lifespan. Together, these data demonstrate a role for RNA splicing homeostasis in DR longevity and suggest modulation of specific spliceosome components can prolong healthy ageing. PMID:27919065

Interconnections Between RNA-Processing Pathways Revealed by a Sequencing-Based Genetic Screen for Pre-mRNA Splicing Mutants in Fission Yeast.

PubMed

Larson, Amy; Fair, Benjamin Jung; Pleiss, Jeffrey A

2016-06-01

Pre-mRNA splicing is an essential component of eukaryotic gene expression and is highly conserved from unicellular yeasts to humans. Here, we present the development and implementation of a sequencing-based reverse genetic screen designed to identify nonessential genes that impact pre-mRNA splicing in the fission yeast Schizosaccharomyces pombe, an organism that shares many of the complex features of splicing in higher eukaryotes. Using a custom-designed barcoding scheme, we simultaneously queried ∼3000 mutant strains for their impact on the splicing efficiency of two endogenous pre-mRNAs. A total of 61 nonessential genes were identified whose deletions resulted in defects in pre-mRNA splicing; enriched among these were factors encoding known or predicted components of the spliceosome. Included among the candidates identified here are genes with well-characterized roles in other RNA-processing pathways, including heterochromatic silencing and 3' end processing. Splicing-sensitive microarrays confirm broad splicing defects for many of these factors, revealing novel functional connections between these pathways. Copyright © 2016 Larson et al.

SAM68 is a physiological regulator of SMN2 splicing in spinal muscular atrophy

PubMed Central

Pagliarini, Vittoria; Pelosi, Laura; Bustamante, Maria Blaire; Nobili, Annalisa; Berardinelli, Maria Grazia; D’Amelio, Marcello; Musarò, Antonio

2015-01-01

Spinal muscular atrophy (SMA) is a neurodegenerative disease caused by loss of motor neurons in patients with null mutations in the SMN1 gene. The almost identical SMN2 gene is unable to compensate for this deficiency because of the skipping of exon 7 during pre–messenger RNA (mRNA) processing. Although several splicing factors can modulate SMN2 splicing in vitro, the physiological regulators of this disease-causing event are unknown. We found that knockout of the splicing factor SAM68 partially rescued body weight and viability of SMAΔ7 mice. Ablation of SAM68 function promoted SMN2 splicing and expression in SMAΔ7 mice, correlating with amelioration of SMA-related defects in motor neurons and skeletal muscles. Mechanistically, SAM68 binds to SMN2 pre-mRNA, favoring recruitment of the splicing repressor hnRNP A1 and interfering with that of U2AF65 at the 3′ splice site of exon 7. These findings identify SAM68 as the first physiological regulator of SMN2 splicing in an SMA mouse model. PMID:26438828

Interconnections Between RNA-Processing Pathways Revealed by a Sequencing-Based Genetic Screen for Pre-mRNA Splicing Mutants in Fission Yeast

PubMed Central

Larson, Amy; Fair, Benjamin Jung; Pleiss, Jeffrey A.

2016-01-01

Pre-mRNA splicing is an essential component of eukaryotic gene expression and is highly conserved from unicellular yeasts to humans. Here, we present the development and implementation of a sequencing-based reverse genetic screen designed to identify nonessential genes that impact pre-mRNA splicing in the fission yeast Schizosaccharomyces pombe, an organism that shares many of the complex features of splicing in higher eukaryotes. Using a custom-designed barcoding scheme, we simultaneously queried ∼3000 mutant strains for their impact on the splicing efficiency of two endogenous pre-mRNAs. A total of 61 nonessential genes were identified whose deletions resulted in defects in pre-mRNA splicing; enriched among these were factors encoding known or predicted components of the spliceosome. Included among the candidates identified here are genes with well-characterized roles in other RNA-processing pathways, including heterochromatic silencing and 3ʹ end processing. Splicing-sensitive microarrays confirm broad splicing defects for many of these factors, revealing novel functional connections between these pathways. PMID:27172183

The m6A pathway facilitates sex determination in Drosophila

PubMed Central

Kan, Lijuan; Grozhik, Anya V.; Vedanayagam, Jeffrey; Patil, Deepak P.; Pang, Nan; Lim, Kok-Seong; Huang, Yi-Chun; Joseph, Brian; Lin, Ching-Jung; Despic, Vladimir; Guo, Jian; Yan, Dong; Kondo, Shu; Deng, Wu-Min; Dedon, Peter C.; Jaffrey, Samie R.; Lai, Eric C.

2017-01-01

The conserved modification N6-methyladenosine (m6A) modulates mRNA processing and activity. Here, we establish the Drosophila system to study the m6A pathway. We first apply miCLIP to map m6A across embryogenesis, characterize its m6A ‘writer’ complex, validate its YTH ‘readers’ CG6422 and YT521-B, and generate mutants in five m6A factors. While m6A factors with additional roles in splicing are lethal, m6A-specific mutants are viable but present certain developmental and behavioural defects. Notably, m6A facilitates the master female determinant Sxl, since multiple m6A components enhance female lethality in Sxl sensitized backgrounds. The m6A pathway regulates Sxl processing directly, since miCLIP data reveal Sxl as a major intronic m6A target, and female-specific Sxl splicing is compromised in multiple m6A pathway mutants. YT521-B is a dominant m6A effector for Sxl regulation, and YT521-B overexpression can induce female-specific Sxl splicing. Overall, our transcriptomic and genetic toolkit reveals in vivo biologic function for the Drosophila m6A pathway. PMID:28675155

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

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.

Histone H3K36 methylation regulates pre-mRNA splicing in Saccharomyces cerevisiae

PubMed Central

Sorenson, Matthew R.; Jha, Deepak K.; Ucles, Stefanie A.; Flood, Danielle M.; Strahl, Brian D.; Stevens, Scott W.; Kress, Tracy L.

2016-01-01

ABSTRACT Co-transcriptional splicing takes place in the context of a highly dynamic chromatin architecture, yet the role of chromatin restructuring in coordinating transcription with RNA splicing has not been fully resolved. To further define the contribution of histone modifications to pre-mRNA splicing in Saccharomyces cerevisiae, we probed a library of histone point mutants using a reporter to monitor pre-mRNA splicing. We found that mutation of H3 lysine 36 (H3K36) – a residue methylated by Set2 during transcription elongation – exhibited phenotypes similar to those of pre-mRNA splicing mutants. We identified genetic interactions between genes encoding RNA splicing factors and genes encoding the H3K36 methyltransferase Set2 and the demethylase Jhd1 as well as point mutations of H3K36 that block methylation. Consistent with the genetic interactions, deletion of SET2, mutations modifying the catalytic activity of Set2 or H3K36 point mutations significantly altered expression of our reporter and reduced splicing of endogenous introns. These effects were dependent on the association of Set2 with RNA polymerase II and H3K36 dimethylation. Additionally, we found that deletion of SET2 reduces the association of the U2 and U5 snRNPs with chromatin. Thus, our study provides the first evidence that H3K36 methylation plays a role in co-transcriptional RNA splicing in yeast. PMID:26821844

De novo variants in EBF3 are associated with hypotonia, developmental delay, intellectual disability, and autism

PubMed Central

Tanaka, Akemi J.; Cho, Megan T.; Willaert, Rebecca; Retterer, Kyle; Zarate, Yuri A.; Bosanko, Katie; Stefans, Vikki; Oishi, Kimihiko; Williamson, Amy; Wilson, Golder N.; Basinger, Alice; Barbaro-Dieber, Tina; Ortega, Lucia; Sorrentino, Susanna; Gabriel, Melissa K.; Anderson, Ilse J.; Sacoto, Maria J. Guillen; Schnur, Rhonda E.; Chung, Wendy K.

2017-01-01

Using whole-exome sequencing, we identified seven unrelated individuals with global developmental delay, hypotonia, dysmorphic facial features, and an increased frequency of short stature, ataxia, and autism with de novo heterozygous frameshift, nonsense, splice, and missense variants in the Early B-cell Transcription Factor Family Member 3 (EBF3) gene. EBF3 is a member of the collier/olfactory-1/early B-cell factor (COE) family of proteins, which are required for central nervous system (CNS) development. COE proteins are highly evolutionarily conserved and regulate neuronal specification, migration, axon guidance, and dendritogenesis during development and are essential for maintaining neuronal identity in adult neurons. Haploinsufficiency of EBF3 may affect brain development and function, resulting in developmental delay, intellectual disability, and behavioral differences observed in individuals with a deleterious variant in EBF3. PMID:29162653

Identifying RNA splicing factors using IFT genes in Chlamydomonas reinhardtii.

PubMed

Lin, Huawen; Zhang, Zhengyan; Iomini, Carlo; Dutcher, Susan K

2018-03-01

Intraflagellar transport moves proteins in and out of flagella/cilia and it is essential for the assembly of these organelles. Using whole-genome sequencing, we identified splice site mutations in two IFT genes, IFT81 ( fla9 ) and IFT121 ( ift121-2 ), which lead to flagellar assembly defects in the unicellular green alga Chlamydomonas reinhardtii The splicing defects in these ift mutants are partially corrected by mutations in two conserved spliceosome proteins, DGR14 and FRA10. We identified a dgr14 deletion mutant, which suppresses the 3' splice site mutation in IFT81 , and a frameshift mutant of FRA10 , which suppresses the 5' splice site mutation in IFT121 Surprisingly, we found dgr14-1 and fra10 mutations suppress both splice site mutations. We suggest these two proteins are involved in facilitating splice site recognition/interaction; in their absence some splice site mutations are tolerated. Nonsense mutations in SMG1 , which is involved in nonsense-mediated decay, lead to accumulation of aberrant transcripts and partial restoration of flagellar assembly in the ift mutants. The high density of introns and the conservation of noncore splicing factors, together with the ease of scoring the ift mutant phenotype, make Chlamydomonas an attractive organism to identify new proteins involved in splicing through suppressor screening. © 2018 The Authors.

U2 small nuclear ribonucleoprotein particle (snRNP) auxiliary factor of 65 kDa, U2AF65, can promote U1 snRNP recruitment to 5' splice sites.

PubMed Central

Förch, Patrik; Merendino, Livia; Martínez, Concepción; Valcárcel, Juan

2003-01-01

The splicing factor U2AF(65), U2 small nuclear ribonucleoprotein particle (snRNP) auxillary factor of 65 kDa, binds to pyrimidine-rich sequences at 3' splice sites to recruit U2 snRNP to pre-mRNAs. We report that U2AF(65) can also promote the recruitment of U1 snRNP to weak 5' splice sites that are followed by uridine-rich sequences. The arginine- and serine-rich domain of U2AF(65) is critical for U1 recruitment, and we discuss the role of its RNA-RNA annealing activity in this novel function of U2AF(65). PMID:12558503

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.

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.

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.

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

Fusion splicing small-core photonic crystal fibers and single-mode fibers by controlled air hole collapse

NASA Astrophysics Data System (ADS)

Zhou, Xuanfeng; Chen, Zilun; Chen, Haihuan; Hou, Jing

2012-11-01

A method based on controlled air hole collapse for low-loss fusion splicing small-core photonic crystal fibers (PCFs) and single-mode fibers (SMFs) was demonstrated. A taper rig was used to control air hole collapse accurately to enlarge the MFDs of PCFs which was then spliced with SMFs using a fusion splicer. An optimum mode field match at the interface of PCF-SMF was achieved and a low-loss with 0.64 dB was obtained from 3.57 dB for a PCF with 4 μm MFD and a SMF with 10.4 μm MFD experimentally.

Activation-induced cytidine deaminase (AID)-dependent somatic hypermutation requires a splice isoform of the serine/arginine-rich (SR) protein SRSF1

PubMed Central

Kanehiro, Yuichi; Todo, Kagefumi; Negishi, Misaki; Fukuoka, Junji; Gan, Wenjian; Hikasa, Takuya; Kaga, Yoshiaki; Takemoto, Masayuki; Magari, Masaki; Li, Xialu; Manley, James L.; Ohmori, Hitoshi; Kanayama, Naoki

2012-01-01

Somatic hypermutation (SHM) of Ig variable region (IgV) genes requires both IgV transcription and the enzyme activation-induced cytidine deaminase (AID). Identification of a cofactor responsible for the fact that IgV genes are much more sensitive to AID-induced mutagenesis than other genes is a key question in immunology. Here, we describe an essential role for a splice isoform of the prototypical serine/arginine-rich (SR) protein SRSF1, termed SRSF1-3, in AID-induced SHM in a DT40 chicken B-cell line. Unexpectedly, we found that SHM does not occur in a DT40 line lacking SRSF1-3 (DT40-ASF), although it is readily detectable in parental DT40 cells. Strikingly, overexpression of AID in DT40-ASF cells led to a large increase in nonspecific (off-target) mutations. In contrast, introduction of SRSF1-3, but not SRSF1, into these cells specifically restored SHM without increasing off-target mutations. Furthermore, we found that SRSF1-3 binds preferentially to the IgV gene and inhibits processing of the Ig transcript, providing a mechanism by which SRSF1-3 makes the IgV gene available for AID-dependent SHM. SRSF1 not only acts as an essential splicing factor but also regulates diverse aspects of mRNA metabolism and maintains genome stability. Our findings, thus, define an unexpected and important role for SRSF1, particularly for its splice variant, in enabling AID to function specifically on its natural substrate during SHM. PMID:22232677

Activation-induced cytidine deaminase (AID)-dependent somatic hypermutation requires a splice isoform of the serine/arginine-rich (SR) protein SRSF1.

PubMed

Kanehiro, Yuichi; Todo, Kagefumi; Negishi, Misaki; Fukuoka, Junji; Gan, Wenjian; Hikasa, Takuya; Kaga, Yoshiaki; Takemoto, Masayuki; Magari, Masaki; Li, Xialu; Manley, James L; Ohmori, Hitoshi; Kanayama, Naoki

2012-01-24

Somatic hypermutation (SHM) of Ig variable region (IgV) genes requires both IgV transcription and the enzyme activation-induced cytidine deaminase (AID). Identification of a cofactor responsible for the fact that IgV genes are much more sensitive to AID-induced mutagenesis than other genes is a key question in immunology. Here, we describe an essential role for a splice isoform of the prototypical serine/arginine-rich (SR) protein SRSF1, termed SRSF1-3, in AID-induced SHM in a DT40 chicken B-cell line. Unexpectedly, we found that SHM does not occur in a DT40 line lacking SRSF1-3 (DT40-ASF), although it is readily detectable in parental DT40 cells. Strikingly, overexpression of AID in DT40-ASF cells led to a large increase in nonspecific (off-target) mutations. In contrast, introduction of SRSF1-3, but not SRSF1, into these cells specifically restored SHM without increasing off-target mutations. Furthermore, we found that SRSF1-3 binds preferentially to the IgV gene and inhibits processing of the Ig transcript, providing a mechanism by which SRSF1-3 makes the IgV gene available for AID-dependent SHM. SRSF1 not only acts as an essential splicing factor but also regulates diverse aspects of mRNA metabolism and maintains genome stability. Our findings, thus, define an unexpected and important role for SRSF1, particularly for its splice variant, in enabling AID to function specifically on its natural substrate during SHM.

Organellar maturases: A window into the evolution of the spliceosome.

PubMed

Schmitz-Linneweber, Christian; Lampe, Marie-Kristin; Sultan, Laure D; Ostersetzer-Biran, Oren

2015-09-01

During the evolution of eukaryotic genomes, many genes have been interrupted by intervening sequences (introns) that must be removed post-transcriptionally from RNA precursors to form mRNAs ready for translation. The origin of nuclear introns is still under debate, but one hypothesis is that the spliceosome and the intron-exon structure of genes have evolved from bacterial-type group II introns that invaded the eukaryotic genomes. The group II introns were most likely introduced into the eukaryotic genome from an α-proteobacterial predecessor of mitochondria early during the endosymbiosis event. These self-splicing and mobile introns spread through the eukaryotic genome and later degenerated. Pieces of introns became part of the general splicing machinery we know today as the spliceosome. In addition, group II introns likely brought intron maturases with them to the nucleus. Maturases are found in most bacterial introns, where they act as highly specific splicing factors for group II introns. In the spliceosome, the core protein Prp8 shows homology to group II intron-encoded maturases. While maturases are entirely intron specific, their descendant of the spliceosomal machinery, the Prp8 protein, is an extremely versatile splicing factor with multiple interacting proteins and RNAs. How could such a general player in spliceosomal splicing evolve from the monospecific bacterial maturases? Analysis of the organellar splicing machinery in plants may give clues on the evolution of nuclear splicing. Plants encode various proteins which are closely related to bacterial maturases. The organellar genomes contain one maturase each, named MatK in chloroplasts and MatR in mitochondria. In addition, several maturase genes have been found in the nucleus as well, which are acting on mitochondrial pre-RNAs. All plant maturases show sequence deviation from their progenitor bacterial maturases, and interestingly are all acting on multiple organellar group II intron targets. Moreover, they seem to function in the splicing of group II introns together with a number of additional nuclear-encoded splicing factors, possibly acting as an organellar proto-spliceosome. Together, this makes them interesting models for the early evolution of nuclear spliceosomal splicing. In this review, we summarize recent advances in our understanding of the role of plant maturases and their accessory factors in plants. This article is part of a Special Issue entitled: Chloroplast Biogenesis. Copyright © 2015 Elsevier B.V. All rights reserved.

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

Analysis of aberrant pre-messenger RNA splicing resulting from mutations in ATP8B1 and efficient in vitro rescue by adapted U1 small nuclear RNA.

PubMed

van der Woerd, Wendy L; Mulder, Johanna; Pagani, Franco; Beuers, Ulrich; Houwen, Roderick H J; van de Graaf, Stan F J

2015-04-01

ATP8B1 deficiency is a severe autosomal recessive liver disease resulting from mutations in the ATP8B1 gene characterized by a continuous phenotypical spectrum from intermittent (benign recurrent intrahepatic cholestasis; BRIC) to progressive familial intrahepatic cholestasis (PFIC). Current therapeutic options are insufficient, and elucidating the molecular consequences of mutations could lead to personalized mutation-specific therapies. We investigated the effect on pre-messenger RNA splicing of 14 ATP8B1 mutations at exon-intron boundaries using an in vitro minigene system. Eleven mutations, mostly associated with a PFIC phenotype, resulted in aberrant splicing and a complete absence of correctly spliced product. In contrast, three mutations led to partially correct splicing and were associated with a BRIC phenotype. These findings indicate an inverse correlation between the level of correctly spliced product and disease severity. Expression of modified U1 small nuclear RNAs (snRNA) complementary to the splice donor sites strongly improved or completely rescued splicing for several ATP8B1 mutations located at donor, as well as acceptor, splice sites. In one case, we also evaluated exon-specific U1 snRNAs that, by targeting nonconserved intronic sequences, might reduce possible off-target events. Although very effective in correcting exon skipping, they also induced retention of the short downstream intron. We systematically characterized the molecular consequences of 14 ATP8B1 mutations at exon-intron boundaries associated with ATP8B1 deficiency and found that the majority resulted in total exon skipping. The amount of correctly spliced product inversely correlated with disease severity. Compensatory modified U1 snRNAs, complementary to mutated donor splice sites, were able to improve exon definition very efficiently and could be a novel therapeutic strategy in ATP8B1 deficiency as well as other genetic diseases. © 2014 by the American Association for the Study of Liver Diseases.

CUS2, a Yeast Homolog of Human Tat-SF1, Rescues Function of Misfolded U2 through an Unusual RNA Recognition Motif

PubMed Central

Yan, Dong; Perriman, Rhonda; Igel, Haller; Howe, Kenneth J.; Neville, Megan; Ares, Manuel

1998-01-01

A screen for suppressors of a U2 snRNA mutation identified CUS2, an atypical member of the RNA recognition motif (RRM) family of RNA binding proteins. CUS2 protein is associated with U2 RNA in splicing extracts and interacts with PRP11, a subunit of the conserved splicing factor SF3a. Absence of CUS2 renders certain U2 RNA folding mutants lethal, arguing that a normal activity of CUS2 is to help refold U2 into a structure favorable for its binding to SF3b and SF3a prior to spliceosome assembly. Both CUS2 function in vivo and the in vitro RNA binding activity of CUS2 are disrupted by mutation of the first RRM, suggesting that rescue of misfolded U2 involves the direct binding of CUS2. Human Tat-SF1, reported to stimulate Tat-specific, transactivating region-dependent human immunodeficiency virus transcription in vitro, is structurally similar to CUS2. Anti-Tat-SF1 antibodies coimmunoprecipitate SF3a66 (SAP62), the human homolog of PRP11, suggesting that Tat-SF1 has a parallel function in splicing in human cells. PMID:9710584

Mapping the Complement Factor H-Related Protein 1 (CFHR1):C3b/C3d Interactions

PubMed Central

Laskowski, Jennifer; Thurman, Joshua M.; Hageman, Gregory S.; Holers, V. Michael

2016-01-01

Complement factor H-related protein 1 (CFHR1) is a complement regulator which has been reported to regulate complement by blocking C5 convertase activity and interfering with C5b surface association. CFHR1 also competes with complement factor H (CFH) for binding to C3b, and may act as an antagonist of CFH-directed regulation on cell surfaces. We have employed site-directed mutagenesis in conjunction with ELISA-based and functional assays to isolate the binding interaction that CFHR1 undertakes with complement components C3b and C3d to a single shared interface. The C3b/C3d:CFHR1 interface is identical to that which occurs between the two C-terminal domains (SCR19-20) of CFH and C3b. Moreover, we have been able to corroborate that dimerization of CFHR1 is necessary for this molecule to bind effectively to C3b and C3d, or compete with CFH. Finally, we have established that CFHR1 competes with complement factor H-like protein 1 (CFHL-1) for binding to C3b. CFHL-1 is a CFH gene splice variant, which is almost identical to the N-terminal 7 domains of CFH (SCR1-7). CFHR1, therefore, not only competes with the C-terminus of CFH for binding to C3b, but also sterically blocks the interaction that the N-terminus of CFH undertakes with C3b, and which is required for CFH-regulation. PMID:27814381

MYCN controls an alternative RNA splicing program in high-risk metastatic neuroblastoma

PubMed Central

Zhang, Shile; Wei, Jun S.; Li, Samuel Q.; Badgett, Tom C.; Song, Young K.; Agarwal, Saurabh; Coarfa, Cristian; Tolman, Catherine; Hurd, Laura; Liao, Hongling; He, Jianbin; Wen, Xinyu; Liu, Zhihui; Thiele, Carol J.; Westermann, Frank; Asgharzadeh, Shahab; Seeger, Robert C.; Maris, John M.; Auvil, Jamie M Guidry; Smith, Malcolm A; Kolaczyk, Eric D; Shohet, Jason; Khan, Javed

2016-01-01

The molecular mechanisms underlying the aggressive behavior of MYCN driven neuroblastoma (NBL) is under intense investigation; however, little is known about the impact of this family of transcription factors on the splicing program. Here we used high-throughput RNA sequencing to systematically study the expression of RNA isoforms in stage 4 MYCN-amplified NBL, an aggressive subtype of metastatic NBL. We show that MYCN-amplified NBL tumors display a distinct gene splicing pattern affecting multiple cancer hallmark functions. Six splicing factors displayed unique differential expression patterns in MYCN-amplified tumors and cell lines, and the binding motifs for some of these splicing factors are significantly enriched in differentially-spliced genes. Direct binding of MYCN to promoter regions of the splicing factors PTBP1 and HNRNPA1 detected by ChIP-seq demonstrates MYCN controls the splicing pattern by direct regulation of the expression of these key splicing factors. Furthermore, high expression of PTBP1 and HNRNPA1 was significantly associated with poor overall survival of stage4 NBL patients (p≤0.05). Knocking down PTBP1, HNRNPA1 and their downstream target PKM2, an isoform of pro-tumor-growth, result in repressed growth of NBL cells. Therefore, our study reveals a novel role of MYCN in controlling global splicing program through regulation of splicing factors in addition to its well-known role in the transcription program. These findings suggest a therapeutically potential to target the key splicing factors or gene isoforms in high-risk NBL with MYCN-amplification. PMID:26683771

Low-loss, robust fusion splicing of silica to chalcogenide fiber for integrated mid-infrared laser technology development.

PubMed

Thapa, Rajesh; Gattass, Rafael R; Nguyen, Vinh; Chin, Geoff; Gibson, Dan; Kim, Woohong; Shaw, L Brandon; Sanghera, Jasbinder S

2015-11-01

We demonstrate a low-loss, repeatable, and robust splice between single-mode silica fiber and single-mode chalcogenide (CHG) fiber. These splices are particularly difficult to create because of the significant difference in the two fibers' glass transition temperatures (∼1000°C) as well as the large difference in the coefficients of thermal expansion between the fibers (∼20×10(-6)/°C). With 90% light coupled through the silica-CHG fiber splice, predominantly in the fundamental circular-symmetric mode, into the core of the CHG fiber and with 0.5 dB of splice loss measured around the wavelength of 2.5 μm, after correcting only for the Fresnel loss, the silica-CHG splice offers excellent beam quality and coupling efficiency. The tensile strength of the splice is greater than 12 kpsi, and the laser damage threshold is greater than 2 W (CW) and was limited by the available laser pump power. We also utilized this splicing technique to demonstrate 2 to 4.5 μm ultrabroadband supercontinuum generation in a monolithic all-fiber system comprising a CHG fiber and a high peak power 2 μm pulsed Raman-shifted thulium fiber laser. This is a major development toward compact form factor commercial applications of soft-glass mid-IR fibers.

[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.

Structural and mechanistic insights into human splicing factor SF3b complex derived using an integrated approach guided by the cryo-EM density maps

PubMed Central

Rakesh, Ramachandran; Joseph, Agnel Praveen; Bhaskara, Ramachandra M.; Srinivasan, Narayanaswamy

2016-01-01

ABSTRACT Pre-mRNA splicing in eukaryotes is performed by the spliceosome, a highly complex macromolecular machine. SF3b is a multi-protein complex which recognizes the branch point adenosine of pre-mRNA as part of a larger U2 snRNP or U11/U12 di-snRNP in the dynamic spliceosome machinery. Although a cryo-EM map is available for human SF3b complex, the structure and relative spatial arrangement of all components in the complex are not yet known. We have recognized folds of domains in various proteins in the assembly and generated comparative models. Using an integrative approach involving structural and other experimental data, guided by the available cryo-EM density map, we deciphered a pseudo-atomic model of the closed form of SF3b which is found to be a “fuzzy complex” with highly flexible components and multiplicity of folds. Further, the model provides structural information for 5 proteins (SF3b10, SF3b155, SF3b145, SF3b130 and SF3b14b) and localization information for 4 proteins (SF3b10, SF3b145, SF3b130 and SF3b14b) in the assembly for the first time. Integration of this model with the available U11/U12 di-snRNP cryo-EM map enabled elucidation of an open form. This now provides new insights on the mechanistic features involved in the transition between closed and open forms pivoted by a hinge region in the SF3b155 protein that also harbors cancer causing mutations. Moreover, the open form guided model of the 5′ end of U12 snRNA, which includes the branch point duplex, shows that the architecture of SF3b acts as a scaffold for U12 snRNA: pre-mRNA branch point duplex formation with potential implications for branch point adenosine recognition fidelity. PMID:27618338

RtcB is the RNA ligase component of an Escherichia coli RNA repair operon.

PubMed

Tanaka, Naoko; Shuman, Stewart

2011-03-11

RNA 2',3'-cyclic phosphate ends play important roles in RNA metabolism as substrates for RNA ligases during tRNA restriction-repair and tRNA splicing. Diverse bacteria from multiple phyla encode a two-component RNA repair cassette, comprising Pnkp (polynucleotide kinase-phosphatase-ligase) and Hen1 (RNA 3'-terminal ribose 2'-O-methyltransferase), that heals and then seals broken tRNAs with 2',3'-cyclic phosphate and 5'-OH ends. The Pnkp-Hen1 repair operon is absent in the majority of bacterial species, thereby raising the prospect that other RNA repair systems might be extant. A candidate component is RNA 3'-phosphate cyclase, a widely distributed enzyme that transforms RNA 3'-monophosphate termini into 2',3'-cyclic phosphates but cannot seal the ends it produces. Escherichia coli RNA cyclase (RtcA) is encoded in a σ(54)-regulated operon with RtcB, a protein of unknown function. Taking a cue from Pnkp-Hen1, we purified E. coli RtcB and tested it for RNA ligase activity. We report that RtcB per se seals broken tRNA-like stem-loop structures with 2',3'-cyclic phosphate and 5'-OH ends to form a splice junction with a 2'-OH, 3',5'-phosphodiester. We speculate that: (i) RtcB might afford bacteria a means to recover from stress-induced RNA damage; and (ii) RtcB homologs might catalyze tRNA repair or splicing reactions in archaea and eukarya.

The Human Splicing Factor ASF/SF2 can Specifically Recognize Pre-mRNA 5' Splice Sites

NASA Astrophysics Data System (ADS)

Zuo, Ping; Manley, James L.

1994-04-01

ASF/SF2 is a human protein previously shown to function in in vitro pre-mRNA splicing as an essential factor necessary for all splices and also as an alternative splicing factor, capable of switching selection of 5' splice sites. To begin to study the protein's mechanism of action, we have investigated the RNA binding properties of purified recombinant ASF/SF2. Using UV crosslinking and gel shift assays, we demonstrate that the RNA binding region of ASF/SF2 can interact with RNA in a sequence-specific manner, recognizing the 5' splice site in each of two different pre-mRNAs. Point mutations in the 5' splice site consensus can reduce binding by as much as a factor of 100, with the largest effects observed in competition assays. These findings support a model in which ASF/SF2 aids in the recognition of pre-mRNA 5' splice sites.

De novo variants in EBF3 are associated with hypotonia, developmental delay, intellectual disability, and autism.

PubMed

Tanaka, Akemi J; Cho, Megan T; Willaert, Rebecca; Retterer, Kyle; Zarate, Yuri A; Bosanko, Katie; Stefans, Vikki; Oishi, Kimihiko; Williamson, Amy; Wilson, Golder N; Basinger, Alice; Barbaro-Dieber, Tina; Ortega, Lucia; Sorrentino, Susanna; Gabriel, Melissa K; Anderson, Ilse J; Sacoto, Maria J Guillen; Schnur, Rhonda E; Chung, Wendy K

2017-11-01

Using whole-exome sequencing, we identified seven unrelated individuals with global developmental delay, hypotonia, dysmorphic facial features, and an increased frequency of short stature, ataxia, and autism with de novo heterozygous frameshift, nonsense, splice, and missense variants in the Early B-cell Transcription Factor Family Member 3 ( EBF3 ) gene. EBF3 is a member of the collier/olfactory-1/early B-cell factor (COE) family of proteins, which are required for central nervous system (CNS) development. COE proteins are highly evolutionarily conserved and regulate neuronal specification, migration, axon guidance, and dendritogenesis during development and are essential for maintaining neuronal identity in adult neurons. Haploinsufficiency of EBF3 may affect brain development and function, resulting in developmental delay, intellectual disability, and behavioral differences observed in individuals with a deleterious variant in EBF3 . © 2017 Tanaka et al.; Published by Cold Spring Harbor Laboratory Press.

A Conserved Nuclear Cyclophilin Is Required for Both RNA Polymerase II Elongation and Co-transcriptional Splicing in Caenorhabditis elegans

PubMed Central

Ahn, Jeong H.; Rechsteiner, Andreas; Strome, Susan; Kelly, William G.

2016-01-01

The elongation phase of transcription by RNA Polymerase II (Pol II) involves numerous events that are tightly coordinated, including RNA processing, histone modification, and chromatin remodeling. RNA splicing factors are associated with elongating Pol II, and the interdependent coupling of splicing and elongation has been documented in several systems. Here we identify a conserved, multi-domain cyclophilin family member, SIG-7, as an essential factor for both normal transcription elongation and co-transcriptional splicing. In embryos depleted for SIG-7, RNA levels for over a thousand zygotically expressed genes are substantially reduced, Pol II becomes significantly reduced at the 3’ end of genes, marks of transcription elongation are reduced, and unspliced mRNAs accumulate. Our findings suggest that SIG-7 plays a central role in both Pol II elongation and co-transcriptional splicing and may provide an important link for their coordination and regulation. PMID:27541139

Competition between pre-mRNAs for the splicing machinery drives global regulation of splicing

PubMed Central

Munding, Elizabeth M.; Shiue, Lily; Katzman, Sol; Donohue, John Paul; Ares, Manuel

2013-01-01

Summary During meiosis in yeast, global splicing efficiency increases and then decreases. Here we provide evidence that splicing improves due to reduced competition for the splicing machinery. The timing of this regulation corresponds to repression and reactivation of ribosomal protein genes (RPGs) during meiosis. In vegetative cells RPG repression by rapamycin treatment also increases splicing efficiency. Down-regulation of the RPG-dedicated transcription factor gene IFH1 genetically suppresses two spliceosome mutations prp11-1 and prp4-1, and globally restores splicing efficiency in prp4-1 cells. We conclude that the splicing apparatus is limiting and pre-mRNAs compete. Splicing efficiency of a pre-mRNA therefore depends not just on its own concentration and affinity for limiting splicing factor(s) but also on those of competing pre-mRNAs. Competition between RNAs for limiting RNA processing factors appears to be a general condition in eukaryotic cells important for function of a variety of post-transcriptional control mechanisms including miRNA repression, polyadenylation and splicing. PMID:23891561

Effects of RNAi-Mediated Knockdown of Histone Methyltransferases on the Sex-Specific mRNA Expression of Imp in the Silkworm Bombyx mori

PubMed Central

Suzuki, Masataka G.; Ito, Haruka; Aoki, Fugaku

2014-01-01

Sexual differentiation in Bombyx mori is controlled by sex-specific splicing of Bmdsx, which results in the omission of exons 3 and 4 in a male-specific manner. In B. mori, insulin-like growth factor II mRNA-binding protein (Imp) is a male-specific factor involved in male-specific splicing of Bmdsx. Male-specific Imp mRNA results from the male-specific inclusion of exon 8. To verify the link between histone methylation and alternative RNA processing in Imp, we examined the effects of RNAi-mediated knockdown of several histone methyltransferases on the sex-specific mRNA expression of Imp. As a result, male-specific expression of Imp mRNA was completely abolished when expression of the H3K79 methyltransferase DOT1L was repressed to <10% of that in control males. Chromatin immunoprecipitation-quantitative PCR analysis revealed a higher distribution of H3K79me2 in normal males than in normal females across Imp. RNA polymerase II (RNAP II) processivity assays indicated that RNAi knockdown of DOT1L in males caused a twofold decrease in RNAP II processivity compared to that in control males, with almost equivalent levels to those observed in normal females. Inhibition of RNAP II-mediated elongation in male cells repressed the male-specific splicing of Imp. Our data suggest the possibility that H3K79me2 accumulation along Imp is associated with the male-specific alternative processing of Imp mRNA that results from increased RNAP II processivity. PMID:24758924

MYCN controls an alternative RNA splicing program in high-risk metastatic neuroblastoma.

PubMed

Zhang, Shile; Wei, Jun S; Li, Samuel Q; Badgett, Tom C; Song, Young K; Agarwal, Saurabh; Coarfa, Cristian; Tolman, Catherine; Hurd, Laura; Liao, Hongling; He, Jianbin; Wen, Xinyu; Liu, Zhihui; Thiele, Carol J; Westermann, Frank; Asgharzadeh, Shahab; Seeger, Robert C; Maris, John M; Guidry Auvil, Jamie M; Smith, Malcolm A; Kolaczyk, Eric D; Shohet, Jason; Khan, Javed

2016-02-28

The molecular mechanisms underlying the aggressive behavior of MYCN driven neuroblastoma (NBL) is under intense investigation; however, little is known about the impact of this family of transcription factors on the splicing program. Here we used high-throughput RNA sequencing to systematically study the expression of RNA isoforms in stage 4 MYCN-amplified NBL, an aggressive subtype of metastatic NBL. We show that MYCN-amplified NBL tumors display a distinct gene splicing pattern affecting multiple cancer hallmark functions. Six splicing factors displayed unique differential expression patterns in MYCN-amplified tumors and cell lines, and the binding motifs for some of these splicing factors are significantly enriched in differentially-spliced genes. Direct binding of MYCN to promoter regions of the splicing factors PTBP1 and HNRNPA1 detected by ChIP-seq demonstrates that MYCN controls the splicing pattern by direct regulation of the expression of these key splicing factors. Furthermore, high expression of PTBP1 and HNRNPA1 was significantly associated with poor overall survival of stage4 NBL patients (p ≤ 0.05). Knocking down PTBP1, HNRNPA1 and their downstream target PKM2, an isoform of pro-tumor-growth, result in repressed growth of NBL cells. Therefore, our study reveals a novel role of MYCN in controlling global splicing program through regulation of splicing factors in addition to its well-known role in the transcription program. These findings suggest a therapeutically potential to target the key splicing factors or gene isoforms in high-risk NBL with MYCN-amplification. Published by Elsevier Ireland Ltd.

RNA splicing process analysis for identifying antisense oligonucleotide inhibitors with padlock probe-based isothermal amplification.

PubMed

Ren, Xiaojun; Deng, Ruijie; Wang, Lida; Zhang, Kaixiang; Li, Jinghong

2017-08-01

RNA splicing, which mainly involves two transesterification steps, is a fundamental process of gene expression and its abnormal regulation contributes to serious genetic diseases. Antisense oligonucleotides (ASOs) are genetic control tools that can be used to specifically control genes through alteration of the RNA splicing pathway. Despite intensive research, how ASOs or various other factors influence the multiple processes of RNA splicing still remains obscure. This is largely due to an inability to analyze the splicing efficiency of each step in the RNA splicing process with high sensitivity. We addressed this limitation by introducing a padlock probe-based isothermal amplification assay to achieve quantification of the specific products in different splicing steps. With this amplified assay, the roles that ASOs play in RNA splicing inhibition in the first and second steps could be distinguished. We identified that 5'-ASO could block RNA splicing by inhibiting the first step, while 3'-ASO could block RNA splicing by inhibiting the second step. This method provides a versatile tool for assisting efficient ASO design and discovering new splicing modulators and therapeutic drugs.

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.

SON and its alternatively spliced isoforms control MLL complex-mediated H3K4me3 and transcription of leukemia-associated genes

PubMed Central

Kim, Jung-Hyun; Baddoo, Melody C.; Park, Eun Young; Stone, Joshua K.; Park, Hyeonsoo; Butler, Thomas W.; Huang, Gang; Yan, Xiaomei; Pauli-Behn, Florencia; Myers, Richard M.; Tan, Ming; Flemington, Erik K.; Lim, Ssang-Taek; Erin Ahn, Eun-Young

2016-01-01

SUMMARY Dysregulation of MLL complex-mediated histone methylation plays a pivotal role in gene expression associated with diseases, but little is known about cellular factors modulating MLL complex activity. Here, we report that SON, previously known as an RNA splicing factor, controls MLL complex-mediated transcriptional initiation. SON binds to DNA near transcription start sites, interacts with menin, and inhibits MLL complex assembly, resulting in decreased H3K4me3 and transcriptional repression. Importantly, alternatively spliced short isoforms of SON are markedly upregulated in acute myeloid leukemia. The short isoforms compete with full-length SON for chromatin occupancy, but lack the menin-binding ability, thereby antagonizing full-length SON function in transcriptional repression while not impairing full-length SON-mediated RNA splicing. Furthermore, overexpression of a short isoform of SON enhances replating potential of hematopoietic progenitors. Our findings define SON as a fine-tuner of the MLL-menin interaction and reveal short SON overexpression as a marker indicating aberrant transcriptional initiation in leukemia. PMID:26990989

Control of alternative splicing by forskolin through hnRNP K during neuronal differentiation.

PubMed

Cao, Wenguang; Razanau, Aleh; Feng, Dairong; Lobo, Vincent G; Xie, Jiuyong

2012-09-01

The molecular basis of cell signal-regulated alternative splicing at the 3' splice site remains largely unknown. We isolated a protein kinase A-responsive ribonucleic acid (RNA) element from a 3' splice site of the synaptosomal-associated protein 25 (Snap25) gene for forskolin-inhibited splicing during neuronal differentiation of rat pheochromocytoma PC12 cells. The element binds specifically to heterogeneous nuclear ribonucleo protein (hnRNP) K in a phosphatase-sensitive way, which directly competes with the U2 auxiliary factor U2AF65, an essential component of early spliceosomes. Transcripts with similarly localized hnRNP K target motifs upstream of alternative exons are enriched in genes often associated with neurological diseases. We show that such motifs upstream of the Runx1 exon 6 also bind hnRNP K, and importantly, hnRNP K is required for forskolin-induced repression of the exon. Interestingly, this exon encodes the peptide domain that determines the switch of the transcriptional repressor/activator activity of Runx1, a change known to be critical in specifying neuron lineages. Consistent with an important role of the target genes in neurons, knocking down hnRNP K severely disrupts forskolin-induced neurite growth. Thus, through hnRNP K, the neuronal differentiation stimulus forskolin targets a critical 3' splice site component of the splicing machinery to control alternative splicing of crucial genes. This also provides a regulated direct competitor of U2AF65 for cell signal control of 3' splice site usage.

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.

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.

Identification of U2AF(35)-dependent exons by RNA-Seq reveals a link between 3′ splice-site organization and activity of U2AF-related proteins

PubMed Central

Kralovicova, Jana; Knut, Marcin; Cross, Nicholas C. P.; Vorechovsky, Igor

2015-01-01

The auxiliary factor of U2 small nuclear RNA (U2AF) is a heterodimer consisting of 65- and 35-kD proteins that bind the polypyrimidine tract (PPT) and AG dinucleotides at the 3′ splice site (3′ss). The gene encoding U2AF35 (U2AF1) is alternatively spliced, giving rise to two isoforms U2AF35a and U2AF35b. Here, we knocked down U2AF35 and each isoform and characterized transcriptomes of HEK293 cells with varying U2AF35/U2AF65 and U2AF35a/b ratios. Depletion of both isoforms preferentially modified alternative RNA processing events without widespread failure to recognize 3′ss or constitutive exons. Over a third of differentially used exons were terminal, resulting largely from the use of known alternative polyadenylation (APA) sites. Intronic APA sites activated in depleted cultures were mostly proximal whereas tandem 3′UTR APA was biased toward distal sites. Exons upregulated in depleted cells were preceded by longer AG exclusion zones and PPTs than downregulated or control exons and were largely activated by PUF60 and repressed by CAPERα. The U2AF(35) repression and activation was associated with a significant interchange in the average probabilities to form single-stranded RNA in the optimal PPT and branch site locations and sequences further upstream. Although most differentially used exons were responsive to both U2AF subunits and their inclusion correlated with U2AF levels, a small number of transcripts exhibited distinct responses to U2AF35a and U2AF35b, supporting the existence of isoform-specific interactions. These results provide new insights into function of U2AF and U2AF35 in alternative RNA processing. PMID:25779042

Switchable multi-wavelength erbium-doped fiber ring laser based on a tapered in-line Mach–Zehnder interferometer

NASA Astrophysics Data System (ADS)

Zhou, Yuxin; Wang, Xin; Tang, Zijuan; Lou, Shuqin

2018-05-01

In this paper, a switchable multi-wavelength erbium-doped fiber ring laser based on a tapered in-line Mach–Zehnder interferometer is proposed. The in-line Mach–Zehnder interferometer is fabricated by splicing a large-core fiber between two segments of single mode fibers, in which the first splicing point is tapered and the second splicing point is connected directly. By carefully rotating the polarization controller, switchable single-, dual-, triple- and quad-wavelength lasing outputs can be obtained with a side mode suppression ratio higher than 50 dB. The maximal peak power difference of multi-wavelength lasing is 3.67 dB, demonstrating a good power equalization performance. Furthermore, the proposed laser is proven to be very stable at room temperature. The wavelength shifts and peak power fluctuations are less than 0.02 nm and 1.3 dB over half an hour. In addition, stable quintuple-wavelength lasing with a side mode suppression ratio higher than 50 dB can also be realized when the filter length is changed.

SRSF3 represses the expression of PDCD4 protein by coordinated regulation of alternative splicing, export and translation

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

Park, Seung Kuk; Jeong, Sunjoo, E-mail: sjsj@dankook.ac.kr

2016-02-05

Gene expression is regulated at multiple steps, such as transcription, splicing, export, degradation and translation. Considering diverse roles of SR proteins, we determined whether the tumor-related splicing factor SRSF3 regulates the expression of the tumor-suppressor protein, PDCD4, at multiple steps. As we have reported previously, knockdown of SRSF3 increased the PDCD4 protein level in SW480 colon cancer cells. More interestingly, here we showed that the alternative splicing and the nuclear export of minor isoforms of pdcd4 mRNA were repressed by SRSF3, but the translation step was unaffected. In contrast, only the translation step of the major isoform of pdcd4 mRNAmore » was repressed by SRSF3. Therefore, overexpression of SRSF3 might be relevant to the repression of all isoforms of PDCD4 protein levels in most types of cancer cell. We propose that SRSF3 could act as a coordinator of the expression of PDCD4 protein via two mechanisms on two alternatively spliced mRNA isoforms.« less

Quaking and PTB control overlapping splicing regulatory networks during muscle cell differentiation

PubMed Central

Hall, Megan P.; Nagel, Roland J.; Fagg, W. Samuel; Shiue, Lily; Cline, Melissa S.; Perriman, Rhonda J.; Donohue, John Paul; Ares, Manuel

2013-01-01

Alternative splicing contributes to muscle development, but a complete set of muscle-splicing factors and their combinatorial interactions are unknown. Previous work identified ACUAA (“STAR” motif) as an enriched intron sequence near muscle-specific alternative exons such as Capzb exon 9. Mass spectrometry of myoblast proteins selected by the Capzb exon 9 intron via RNA affinity chromatography identifies Quaking (QK), a protein known to regulate mRNA function through ACUAA motifs in 3′ UTRs. We find that QK promotes inclusion of Capzb exon 9 in opposition to repression by polypyrimidine tract-binding protein (PTB). QK depletion alters inclusion of 406 cassette exons whose adjacent intron sequences are also enriched in ACUAA motifs. During differentiation of myoblasts to myotubes, QK levels increase two- to threefold, suggesting a mechanism for QK-responsive exon regulation. Combined analysis of the PTB- and QK-splicing regulatory networks during myogenesis suggests that 39% of regulated exons are under the control of one or both of these splicing factors. This work provides the first evidence that QK is a global regulator of splicing during muscle development in vertebrates and shows how overlapping splicing regulatory networks contribute to gene expression programs during differentiation. PMID:23525800

The RNA Splicing Response to DNA Damage.

PubMed

Shkreta, Lulzim; Chabot, Benoit

2015-10-29

The number of factors known to participate in the DNA damage response (DDR) has expanded considerably in recent years to include splicing and alternative splicing factors. While the binding of splicing proteins and ribonucleoprotein complexes to nascent transcripts prevents genomic instability by deterring the formation of RNA/DNA duplexes, splicing factors are also recruited to, or removed from, sites of DNA damage. The first steps of the DDR promote the post-translational modification of splicing factors to affect their localization and activity, while more downstream DDR events alter their expression. Although descriptions of molecular mechanisms remain limited, an emerging trend is that DNA damage disrupts the coupling of constitutive and alternative splicing with the transcription of genes involved in DNA repair, cell-cycle control and apoptosis. A better understanding of how changes in splice site selection are integrated into the DDR may provide new avenues to combat cancer and delay aging.

The RNA Splicing Response to DNA Damage

PubMed Central

Shkreta, Lulzim; Chabot, Benoit

2015-01-01

The number of factors known to participate in the DNA damage response (DDR) has expanded considerably in recent years to include splicing and alternative splicing factors. While the binding of splicing proteins and ribonucleoprotein complexes to nascent transcripts prevents genomic instability by deterring the formation of RNA/DNA duplexes, splicing factors are also recruited to, or removed from, sites of DNA damage. The first steps of the DDR promote the post-translational modification of splicing factors to affect their localization and activity, while more downstream DDR events alter their expression. Although descriptions of molecular mechanisms remain limited, an emerging trend is that DNA damage disrupts the coupling of constitutive and alternative splicing with the transcription of genes involved in DNA repair, cell-cycle control and apoptosis. A better understanding of how changes in splice site selection are integrated into the DDR may provide new avenues to combat cancer and delay aging. PMID:26529031

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.

Differential expression profile of CXCR3 splicing variants is associated with thyroid neoplasia. Potential role in papillary thyroid carcinoma oncogenesis?

PubMed Central

Urra, Soledad; Fischer, Martin C.; Martínez, José R.; Véliz, Loreto; Orellana, Paulina; Solar, Antonieta; Bohmwald, Karen; Kalergis, Alexis; Riedel, Claudia; Corvalán, Alejandro H.; Roa, Juan C.; Fuentealba, Rodrigo; Cáceres, C. Joaquin; López-Lastra, Marcelo; León, Augusto; Droppelmann, Nicolás; González, Hernán E.

2018-01-01

Papillary thyroid cancer (PTC) is the most prevalent endocrine neoplasia. The increased incidence of PTC in patients with thyroiditis and the frequent immune infiltrate found in PTC suggest that inflammation might be a risk factor for PTC development. The CXCR3-ligand system is involved in thyroid inflammation and CXCR3 has been found upregulated in many tumors, suggesting its pro-tumorigenic role under the inflammatory microenvironment. CXCR3 ligands (CXCL4, CXCL9, CXCL10 and CXCL11) trigger antagonistic responses partly due to the presence of two splice variants, CXCR3A and CXCR3B. Whereas CXCR3A promotes cell proliferation, CXCR3B induces apoptosis. However, the relation between CXCR3 variant expression with chronic inflammation and PTC development remains unknown. Here, we characterized the expression pattern of CXCR3 variants and their ligands in benign tumors and PTC. We found that CXCR3A and CXCL10 mRNA levels were increased in non-metastatic PTC when compared to non-neoplastic tissue. This increment was also observed in a PTC epithelial cell line (TPC-1). Although elevated protein levels of both isoforms were detected in benign and malignant tumors, the CXCR3A expression remained greater than CXCR3B and promoted proliferation in Nthy-ori-3-1 cells. In non-metastatic PTC, inflammation was conditioning for the CXCR3 ligands increased availability. Consistently, CXCL10 was strongly induced by interferon gamma in normal and tumor thyrocytes. Our results suggest that persistent inflammation upregulates CXCL10 expression favoring tumor development via enhanced CXCR3A-CXCL10 signaling. These findings may help to further understand the contribution of inflammation as a risk factor in PTC development and set the basis for potential therapeutic studies. PMID:29416784

KH-type splicing regulatory protein is regulated by nuclear factor-κB signaling to mediate innate immunity in Caco-2 cells infected by Salmonella enteritidis.

PubMed

Nie, Yuanyang; Cao, Mei; Wu, Daoyan; Li, Ningzhe; Peng, Jingshan; Yi, Sijun; Yang, Xiaofan; Zhang, Mao; Hu, Guoku; Zhao, Jian

2018-05-04

Salmonella enteritidis infection occurs in enterogenous diseases, such as gastroenteritis and parenteral focal infection, which often involve inflammation of intestinal epithelial cells. The nuclear factor kappa B (NF-κB) pathway participates in the innate immune response to many gram-negative pathogenic bacteria and initiates inflammation in epithelial cells. KH-type splicing regulatory protein (KSRP) is a multi-domain RNA-binding protein that recruits the exosome-containing mRNA degradation complex to mRNAs coding for inflammatory response factors. However, it remains unclear whether KSRP is regulated by NF-κB signaling pathway in response to S. enteritidis infection and affects the development of inflammation. Accordingly, in this study, we investigated the role of KSRP in mediating the response to S. enteritidis in Caco-2 cells. The data revealed that S. enteritidis infection decreased KSRP expression, which was suppressed by blocking the NF-κB pathway. Additionally, S. enteritidis infection significantly increased the expression of inducible nitric oxide synthase and cyclooxygenase-2. Overexpression of KSRP reduced the expression levels of inflammatory factors in Caco-2 cells. KSRP was regulated by the NF-κB signaling pathway and participated in mediating the innate immune response to S. enteritidis infection in Caco-2 cells, and KSRP acted as a negative regulator of inflammatory gene expression.

U.S. Navy Wire-Rope Handbook. Volume 1. Design and Engineering of Wire-Rope Systems

DTIC Science & Technology

1976-01-01

by " braiding " or "weaving" one rope end into another. Splices may be made at the end of a single rope after forming a loop (an eye splice) or between...rags, large pieces of old hemp rope and fire hose are other popular types of chafing gear for semi-fixed position ropes (see Figure 7-3(b)). 7.4. LINKS...Figure 7-3. Chafing Gear 7-6 Links 7.4. SWOOD PLANK FIRE HOSE ., ’ _APPLY GREASE CANJVAS, LEATHER, COPPER (b) For Semi-Fixed Position Ropes lo- Figcre

Functional domains of the human splicing factor ASF/SF2.

PubMed Central

Zuo, P; Manley, J L

1993-01-01

The human splicing factor ASF/SF2 displays two predominant activities in in vitro splicing assays: (i) it is an essential factor apparently required for all splices and (ii) it is able to switch utilization of alternative 5' splice sites in a concentration-dependent manner. ASF/SF2 is the prototype of a family of proteins typified by the presence of one or two RNP-type RNA binding domains (RBDs) and a region highly enriched in repeating arginine-serine dipeptides (RS regions). Here we describe a functional analysis of ASF/SF2, which defines several regions essential for one, or both, of its two principal activities, and provides insights into how this type of protein functions in splicing. Two isoforms of the protein, which arise from alternative splicing, are by themselves inactive, but each can block the activity of ASF/SF2, thereby functioning as splicing repressors. Some, but not all, mutations in the RS region prevent ASF/SF2 from functioning as an essential splicing factor. However, the entire RS region can be deleted without reducing splice site switching activity, indicating that it is not absolutely required for interaction with other splicing factors. Experiments with deletion and substitution mutants reveal that the protein contains two related, but highly diverged, RBDs, and that both are essential for activity. Each RBD by itself retains the ability to bind RNA, although optimal binding requires both domains. Images PMID:8223481

Structural Basis by Which Alternative Splicing Modulates the Organizer Activity of FGF8 in the Brain

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

Olsen,S.; Li, J.; Eliseenkova, A.

2006-01-01

Two of the four human FGF8 splice isoforms, FGF8a and FGF8b, are expressed in the mid-hindbrain region during development. Although the only difference between these isoforms is the presence of an additional 11 amino acids at the N terminus of FGF8b, these isoforms possess remarkably different abilities to pattern the midbrain and anterior hindbrain. To reveal the structural basis by which alternative splicing modulates the organizing activity of FGF8, we solved the crystal structure of FGF8b in complex with the 'c' splice isoform of FGF receptor 2 (FGFR2c). Using surface plasmon resonance (SPR), we also characterized the receptor-binding specificity ofmore » FGF8a and FGF8b, the 'b' isoform of FGF17 (FGF17b), and FGF18. The FGF8b-FGFR2c structure shows that alternative splicing permits a single additional contact between phenylalanine 32 (F32) of FGF8b and a hydrophobic groove within Ig domain 3 of the receptor that is also present in FGFR1c, FGFR3c, and FGFR4. Consistent with the structure, mutation of F32 to alanine reduces the affinity of FGF8b toward all these receptors to levels characteristic of FGF8a. More importantly, analysis of the mid-hindbrain patterning ability of the FGF8b{sup F32A} mutant in chick embryos and murine midbrain explants shows that this mutation functionally converts FGF8b to FGF8a. Moreover, our data suggest that the intermediate receptor-binding affinities of FGF17b and FGF18, relative to FGF8a and FGF8b, also account for the distinct patterning abilities of these two ligands. We also show that the mode of FGF8 receptor-binding specificity is distinct from that of other FGFs and provide the first biochemical evidence for a physiological FGF8b-FGFR1c interaction during mid-hindbrain development. Consistent with the indispensable role of FGF8 in embryonic development, we show that the FGF8 mode of receptor binding appeared as early as in nematodes and has been preserved throughout evolution.« less

Pre-mRNA Splicing in Plants: In Vivo Functions of RNA-Binding Proteins Implicated in the Splicing Process

PubMed Central

Meyer, Katja; Koester, Tino; Staiger, Dorothee

2015-01-01

Alternative pre-messenger RNA splicing in higher plants emerges as an important layer of regulation upon exposure to exogenous and endogenous cues. Accordingly, mutants defective in RNA-binding proteins predicted to function in the splicing process show severe phenotypic alterations. Among those are developmental defects, impaired responses to pathogen threat or abiotic stress factors, and misregulation of the circadian timing system. A suite of splicing factors has been identified in the model plant Arabidopsis thaliana. Here we summarize recent insights on how defects in these splicing factors impair plant performance. PMID:26213982

A genome landscape of SRSF3-regulated splicing events and gene expression in human osteosarcoma U2OS cells

PubMed Central

Ajiro, Masahiko; Jia, Rong; Yang, Yanqin; Zhu, Jun; Zheng, Zhi-Ming

2016-01-01

Alternative RNA splicing is an essential process to yield proteomic diversity in eukaryotic cells, and aberrant splicing is often associated with numerous human diseases and cancers. We recently described serine/arginine-rich splicing factor 3 (SRSF3 or SRp20) being a proto-oncogene. However, the SRSF3-regulated splicing events responsible for its oncogenic activities remain largely unknown. By global profiling of the SRSF3-regulated splicing events in human osteosarcoma U2OS cells, we found that SRSF3 regulates the expression of 60 genes including ERRFI1, ANXA1 and TGFB2, and 182 splicing events in 164 genes, including EP300, PUS3, CLINT1, PKP4, KIF23, CHK1, SMC2, CKLF, MAP4, MBNL1, MELK, DDX5, PABPC1, MAP4K4, Sp1 and SRSF1, which are primarily associated with cell proliferation or cell cycle. Two SRSF3-binding motifs, CCAGC(G)C and A(G)CAGCA, are enriched to the alternative exons. An SRSF3-binding site in the EP300 exon 14 is essential for exon 14 inclusion. We found that the expression of SRSF1 and SRSF3 are mutually dependent and coexpressed in normal and tumor tissues/cells. SRSF3 also significantly regulates the expression of at least 20 miRNAs, including a subset of oncogenic or tumor suppressive miRNAs. These data indicate that SRSF3 affects a global change of gene expression to maintain cell homeostasis. PMID:26704980

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.

Control of alternative splicing by forskolin through hnRNP K during neuronal differentiation

PubMed Central

Cao, Wenguang; Razanau, Aleh; Feng, Dairong; Lobo, Vincent G.; Xie, Jiuyong

2012-01-01

The molecular basis of cell signal-regulated alternative splicing at the 3′ splice site remains largely unknown. We isolated a protein kinase A-responsive ribonucleic acid (RNA) element from a 3′ splice site of the synaptosomal-associated protein 25 (Snap25) gene for forskolin-inhibited splicing during neuronal differentiation of rat pheochromocytoma PC12 cells. The element binds specifically to heterogeneous nuclear ribonucleo protein (hnRNP) K in a phosphatase-sensitive way, which directly competes with the U2 auxiliary factor U2AF65, an essential component of early spliceosomes. Transcripts with similarly localized hnRNP K target motifs upstream of alternative exons are enriched in genes often associated with neurological diseases. We show that such motifs upstream of the Runx1 exon 6 also bind hnRNP K, and importantly, hnRNP K is required for forskolin-induced repression of the exon. Interestingly, this exon encodes the peptide domain that determines the switch of the transcriptional repressor/activator activity of Runx1, a change known to be critical in specifying neuron lineages. Consistent with an important role of the target genes in neurons, knocking down hnRNP K severely disrupts forskolin-induced neurite growth. Thus, through hnRNP K, the neuronal differentiation stimulus forskolin targets a critical 3′ splice site component of the splicing machinery to control alternative splicing of crucial genes. This also provides a regulated direct competitor of U2AF65 for cell signal control of 3′ splice site usage. PMID:22684629

Evolution of a tissue-specific splicing network

PubMed Central

Taliaferro, J. Matthew; Alvarez, Nehemiah; Green, Richard E.; Blanchette, Marco; Rio, Donald C.

2011-01-01

Alternative splicing of precursor mRNA (pre-mRNA) is a strategy employed by most eukaryotes to increase transcript and proteomic diversity. Many metazoan splicing factors are members of multigene families, with each member having different functions. How these highly related proteins evolve unique properties has been unclear. Here we characterize the evolution and function of a new Drosophila splicing factor, termed LS2 (Large Subunit 2), that arose from a gene duplication event of dU2AF50, the large subunit of the highly conserved heterodimeric general splicing factor U2AF (U2-associated factor). The quickly evolving LS2 gene has diverged from the splicing-promoting, ubiquitously expressed dU2AF50 such that it binds a markedly different RNA sequence, acts as a splicing repressor, and is preferentially expressed in testes. Target transcripts of LS2 are also enriched for performing testes-related functions. We therefore propose a path for the evolution of a new splicing factor in Drosophila that regulates specific pre-mRNAs and contributes to transcript diversity in a tissue-specific manner. PMID:21406555

An exonic missense mutation c.28G>A is associated with weak B blood group by affecting RNA splicing of the ABO gene.

PubMed

Cai, Xiaohong; Qian, Chengrui; Wu, Wenman; Lei, Hang; Ding, Qiulan; Zou, Wei; Xiang, Dong; Wang, Xuefeng

2017-09-01

The amino acid substitutions caused by ABO gene mutations are usually predicted to impact glycosyltransferase's function or its biosynthesis. Here we report an ABO exonic missense mutation that affects B-antigen expression by decreasing the mRNA level of the ABO gene rather than the amino acid change. Serologic studies including plasma total GTB transfer capacity were performed. The exon sequences of the ABO gene were analyzed by Sanger sequencing. B 310 cDNA with c.28G>A (p.G10R) mutation was expressed in HeLa cells and total GTB transfer capacity in cell supernatant was measured. Flow cytometry was performed on these HeLa cells after transfection, and agglutination of Hela-B weak cells was also examined. The mRNA of the ABO gene was analyzed by direct sequencing and real-time reverse transcriptase-polymerase chain reaction. A minigene construct was prepared to evaluate the potential of splicing. While plasma total GTB transfer capacity was undetectable in this B 3 -like individual, the relative percentage of antigen-expressing cells and mean fluorescence index of the B weak red blood cells (RBCs) were 19 and 14% of normal B RBCs, respectively. There was no significant difference of total GTB transfer capacity in cell supernatant and B-antigen expression on cell surfaces between HeLa cells transfected with B 310 cDNA and B cDNA. The mRNA expression level of B 310 in peripheral whole blood was significantly reduced. The amount of splicing is significantly lower in c.28G>A construct compared to that in wild-type construct after transfection in K562 cells. ABO c.28G>A mutation may cause B 3 -like subgroup by affecting RNA splicing of the ABO gene. © 2017 AABB.

Alterations in expression pattern of splicing factors in epithelial ovarian cancer and its clinical impact.

PubMed

Iborra, Severine; Hirschfeld, Marc; Jaeger, Markus; Zur Hausen, Axel; Braicu, Iona; Sehouli, Jalid; Gitsch, Gerald; Stickeler, Elmar

2013-07-01

Alternative splicing represents an important nuclear mechanism in the posttranscriptional regulation of gene expression, which is frequently altered during tumorigenesis. Previously, we described marked changes in alternative splicing of the CD44 gene in ovarian and breast cancer as well as specific induction of distinct splicing factors during tumor development. The present study was focused on the expression profiles of different splicing factors, including classical serine-arginine (SR) proteins including ASF/SF2, hTra2β1, hTra2α, and Y-box-binding protein (YB-1) in physiological and malignant epithelial ovarian tissue to evaluate their expression pattern with regard to tumor development and disease progression. Expression levels of the different splicing factors were analyzed in physiological epithelial ovarian tissue samples, primary tumors, and metastatic samples of patients with a diagnosis of epithelial ovarian cancer using quantified reverse transcription polymerase chain reaction analysis. We examined more closely the splicing factor hTra2β1 using Western blot analysis and immunohistochemistry. The analysis revealed a marked and specific induction of ASF/SF2, SRp20, hTra2β1, and YB-1 in primary tumors as well as in their metastatic sites. However, in our patient cohort, no induction was seen for the other investigated splicing factors SRp55, SRp40, and hTra2α. Our results suggest a specific induction of distinct splicing factors in ovarian cancer tumorigenesis. The involvement of hTra2β1, YB-1, SRp20, and ASF/SF2 in exon recognition and alternative splicing may be important for gene regulation of alternatively spliced genes like CD44 with potential functional consequences in this tumor type leading to progression and metastasis.

Functional analyses of interacting factors involved in both pre-mRNA splicing and cell cycle progression in Saccharomyces cerevisiae.

PubMed Central

Russell, C S; Ben-Yehuda, S; Dix, I; Kupiec, M; Beggs, J D

2000-01-01

Through a genetic screen to search for factors that interact with Prp17/Cdc40p, a protein involved in both cell cycle progression and pre-mRNA splicing, we identify three novel factors, which we call Syf1p, Syf2p, and Syf3 (SYnthetic lethal with cdc Forty). Here we present evidence that all three proteins are spliceosome associated, that they associate weakly or transiently with U6 and U5 snRNAs, and that Syf1p and Syf3p (also known as Clf1p) are required for pre-mRNA splicing. In addition we show that depletion of Syf1p or Syf3p results in cell cycle arrest at the G2/M transition. Thus, like Prp17/Cdc40p, Syf1p and Syf3p are involved in two distinct cellular processes. We discuss the likelihood that Syf1p, Syf2p, and Syf3p are components of a protein complex that assembles into spliceosomes and also regulates cell cycle progression. PMID:11105756

Functional analyses of interacting factors involved in both pre-mRNA splicing and cell cycle progression in Saccharomyces cerevisiae.

PubMed

Russell, C S; Ben-Yehuda, S; Dix, I; Kupiec, M; Beggs, J D

2000-11-01

Through a genetic screen to search for factors that interact with Prp17/Cdc40p, a protein involved in both cell cycle progression and pre-mRNA splicing, we identify three novel factors, which we call Syf1p, Syf2p, and Syf3 (SYnthetic lethal with cdc Forty). Here we present evidence that all three proteins are spliceosome associated, that they associate weakly or transiently with U6 and U5 snRNAs, and that Syf1p and Syf3p (also known as Clf1p) are required for pre-mRNA splicing. In addition we show that depletion of Syf1p or Syf3p results in cell cycle arrest at the G2/M transition. Thus, like Prp17/Cdc40p, Syf1p and Syf3p are involved in two distinct cellular processes. We discuss the likelihood that Syf1p, Syf2p, and Syf3p are components of a protein complex that assembles into spliceosomes and also regulates cell cycle progression.

The chromatin-binding protein HMGN3 stimulates histone acetylation and transcription across the Glyt1 gene

PubMed Central

Barkess, Gráinne; Postnikov, Yuri; Campos, Chrisanne D.; Mishra, Shivam; Mohan, Gokula; Verma, Sakshi; Bustin, Michael; West, Katherine L.

2013-01-01

HMGNs are nucleosome-binding proteins that alter the pattern of histone modifications and modulate the binding of linker histones to chromatin. The HMGN3 family member exists as two splice forms, HMGN3a which is full-length and HMGN3b which lacks the C-terminal RD (regulatory domain). In the present study, we have used the Glyt1 (glycine transporter 1) gene as a model system to investigate where HMGN proteins are bound across the locus in vivo, and to study how the two HMGN3 splice variants affect histone modifications and gene expression. We demonstrate that HMGN1, HMGN2, HMGN3a and HMGN3b are bound across the Glyt1 gene locus and surrounding regions, and are not enriched more highly at the promoter or putative enhancer. We conclude that the peaks of H3K4me3 (trimethylated Lys4 of histone H3) and H3K9ac (acetylated Lys9 of histone H3) at the active Glyt1a promoter do not play a major role in recruiting HMGN proteins. HMGN3a/b binding leads to increased H3K14 (Lys14 of histone H3) acetylation and stimulates Glyt1a expression, but does not alter the levels of H3K4me3 or H3K9ac enrichment. Acetylation assays show that HMGN3a stimulates the ability of PCAF [p300/CREB (cAMP-response-element-binding protein)-binding protein-associated factor] to acetylate nucleosomal H3 in vitro, whereas HMGN3b does not. We propose a model where HMGN3a/b-stimulated H3K14 acetylation across the bodies of large genes such as Glyt1 can lead to more efficient transcription elongation and increased mRNA production. PMID:22150271

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

APP mRNA splicing is upregulated in the brain of biglycan transgenic mice.

PubMed

Bjelik, Annamária; Pákáski, Magdolna; Bereczki, Erika; Gonda, Szilvia; Juhász, Anna; Rimanóczy, Agnes; Zana, Marianna; Janka, Zoltán; Sántha, Miklós; Kálmán, János

2007-01-01

Many of the risk factors for cerebrovascular disease and atherosclerosis also increase the risk of Alzheimer's disease, characterized by the cerebral deposition of beta-amyloid plaques resulting from the abnormal processing of the transmembrane amyloid precursor protein (APP). The initiating event of cholesterol-induced atherosclerosis is the retention and accumulation of atherogenic apolipoprotein B (apoB) together with low-density lipoproteins in the vascular intima. Biglycan, a member of the small leucine-rich protein family, was suspected of contributing to this process. The individual and combined overexpressions of biglycan and apoB-100 were therefore examined on the cortical APP mRNA levels of transgenic mice by means of semiquantitative PCR. As compared with the control littermates, transgenic biglycan mice had significantly increased cortical APP695 (122%) and APP770 (157%) mRNA levels, while the double transgenic (apoB(+/-)xbiglycan(+/-)) mice did not exhibit any changes. These results provide the first experimental evidence that the atherogenic risk factor biglycan alters APP splicing and may participate in the pathogenesis of both Alzheimer and vascular dementias.

Redirecting RNA splicing by SMAD3 turns TGF-β into a tumor promoter.

PubMed

Tripathi, Veenu; Zhang, Ying E

2017-01-01

Transforming growth factor β (TGF-β) is a well-known growth inhibitor of normal epithelial cells, but it is also secreted by solid tumors to promote cancer progression. Our recent discovery of SMAD3-PCBP1 complex with direct RNA-binding properties has shed light on how this conversion is implemented by controlling pre-mRNA splicing patterns.

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

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.

Cwf16p Associating with the Nineteen Complex Ensures Ordered Exon Joining in Constitutive Pre-mRNA Splicing in Fission Yeast

PubMed Central

Sasaki-Haraguchi, Noriko; Ikuyama, Takeshi; Yoshii, Shogo; Takeuchi-Andoh, Tomoko; Frendewey, David; Tani, Tokio

2015-01-01

Exons are ligated in an ordered manner without the skipping of exons in the constitutive splicing of pre-mRNAs with multiple introns. To identify factors ensuring ordered exon joining in constitutive pre-mRNA splicing, we previously screened for exon skipping mutants in Schizosaccharomyces pombe using a reporter plasmid, and characterized three exon skipping mutants named ods1 (ordered splicing 1), ods2, and ods3, the responsible genes of which encode Prp2/U2AF59, U2AF23, and SF1, respectively. They form an SF1-U2AF59-U2AF23 complex involved in recognition of the branch and 3′ splice sites in pre-mRNA. In the present study, we identified a fourth ods mutant, ods4, which was isolated in an exon-skipping screen. The ods4 + gene encodes Cwf16p, which interacts with the NineTeen Complex (NTC), a complex thought to be involved in the first catalytic step of the splicing reaction. We isolated two multi-copy suppressors for the ods4-1 mutation, Srp2p, an SR protein essential for pre-mRNA splicing, and Tif213p, a translation initiation factor, in S. pombe. The overexpression of Srp2p suppressed the exon-skipping phenotype of all ods mutants, whereas Tif213p suppressed only ods4-1, which has a mutation in the translational start codon of the cwf16 gene. We also showed that the decrease in the transcriptional elongation rate induced by drug treatment suppressed exon skipping in ods4-1. We propose that Cwf16p/NTC participates in the early recognition of the branch and 3′ splice sites and cooperates with the SF1-U2AF59-U2AF23 complex to maintain ordered exon joining. PMID:26302002

Systematic Profiling of Poly(A)+ Transcripts Modulated by Core 3’ End Processing and Splicing Factors Reveals Regulatory Rules of Alternative Cleavage and Polyadenylation

PubMed Central

Li, Wencheng; You, Bei; Hoque, Mainul; Zheng, Dinghai; Luo, Wenting; Ji, Zhe; Park, Ji Yeon; Gunderson, Samuel I.; Kalsotra, Auinash; Manley, James L.; Tian, Bin

2015-01-01

Alternative cleavage and polyadenylation (APA) results in mRNA isoforms containing different 3’ untranslated regions (3’UTRs) and/or coding sequences. How core cleavage/polyadenylation (C/P) factors regulate APA is not well understood. Using siRNA knockdown coupled with deep sequencing, we found that several C/P factors can play significant roles in 3’UTR-APA. Whereas Pcf11 and Fip1 enhance usage of proximal poly(A) sites (pAs), CFI-25/68, PABPN1 and PABPC1 promote usage of distal pAs. Strong cis element biases were found for pAs regulated by CFI-25/68 or Fip1, and the distance between pAs plays an important role in APA regulation. In addition, intronic pAs are substantially regulated by splicing factors, with U1 mostly inhibiting C/P events in introns near the 5’ end of gene and U2 suppressing those in introns with features for efficient splicing. Furthermore, PABPN1 inhibits expression of transcripts with pAs near the transcription start site (TSS), a property possibly related to its role in RNA degradation. Finally, we found that groups of APA events regulated by C/P factors are also modulated in cell differentiation and development with distinct trends. Together, our results support an APA code where an APA event in a given cellular context is regulated by a number of parameters, including relative location to the TSS, splicing context, distance between competing pAs, surrounding cis elements and concentrations of core C/P factors. PMID:25906188

The splicing of tiny introns of Paramecium is controlled by MAGO.

PubMed

Contreras, Julia; Begley, Victoria; Marsella, Laura; Villalobo, Eduardo

2018-07-15

The exon junction complex (EJC) is a key element of the splicing machinery. The EJC core is composed of eIF4A3, MAGO, Y14 and MLN51. Few accessory proteins, such as CWC22 or UPF3, bind transiently to the EJC. The EJC has been implicated in the control of the splicing of long introns. To ascertain whether the EJC controls the splicing of short introns, we used Paramecium tetraurelia as a model organism, since it has thousands of very tiny introns. To elucidate whether EJC affects intron splicing in P. tetraurelia, we searched for EJC protein-coding genes, and silenced those genes coding for eIF4A3, MAGO and CWC22. We found that P. tetraurelia likely assembles an active EJC with only three of the core proteins, since MLN51 is lacking. Silencing of eIF4A3 or CWC22 genes, but not that of MAGO, caused lethality. Silencing of the MAGO gene caused either an increase, decrease, or no change in intron retention levels of some intron-containing mRNAs used as reporters. We suggest that a fine-tuning expression of EJC genes is required for steady intron removal in P. tetraurelia. Taking into consideration our results and those published by others, we conclude that the EJC controls splicing independently of the intron size. Copyright © 2018 Elsevier B.V. All rights reserved.

Structure–function analysis and genetic interactions of the SmG, SmE, and SmF subunits of the yeast Sm protein ring

PubMed Central

Schwer, Beate; Kruchten, Joshua; Shuman, Stewart

2016-01-01

A seven-subunit Sm protein ring forms a core scaffold of the U1, U2, U4, and U5 snRNPs that direct pre-mRNA splicing. Using human snRNP structures to guide mutagenesis in Saccharomyces cerevisiae, we gained new insights into structure–function relationships of the SmG, SmE, and SmF subunits. An alanine scan of 19 conserved amino acids of these three proteins, comprising the Sm RNA binding sites or inter-subunit interfaces, revealed that, with the exception of Arg74 in SmF, none are essential for yeast growth. Yet, for SmG, SmE, and SmF, as for many components of the yeast spliceosome, the effects of perturbing protein–RNA and protein–protein interactions are masked by built-in functional redundancies of the splicing machine. For example, tests for genetic interactions with non-Sm splicing factors showed that many benign mutations of SmG, SmE, and SmF (and of SmB and SmD3) were synthetically lethal with null alleles of U2 snRNP subunits Lea1 and Msl1. Tests of pairwise combinations of SmG, SmE, SmF, SmB, and SmD3 alleles highlighted the inherent redundancies within the Sm ring, whereby simultaneous mutations of the RNA binding sites of any two of the Sm subunits are lethal. Our results suggest that six intact RNA binding sites in the Sm ring suffice for function but five sites may not. PMID:27417296

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

A contracted DNA repeat in LHX3 intron 5 is associated with aberrant splicing and pituitary dwarfism in German shepherd dogs.

PubMed

Voorbij, Annemarie M W Y; van Steenbeek, Frank G; Vos-Loohuis, Manon; Martens, Ellen E C P; Hanson-Nilsson, Jeanette M; van Oost, Bernard A; Kooistra, Hans S; Leegwater, Peter A

2011-01-01

Dwarfism in German shepherd dogs is due to combined pituitary hormone deficiency of unknown genetic cause. We localized the recessively inherited defect by a genome wide approach to a region on chromosome 9 with a lod score of 9.8. The region contains LHX3, which codes for a transcription factor essential for pituitary development. Dwarfs have a deletion of one of six 7 bp repeats in intron 5 of LHX3, reducing the intron size to 68 bp. One dwarf was compound heterozygous for the deletion and an insertion of an asparagine residue in the DNA-binding homeodomain of LHX3, suggesting involvement of the gene in the disorder. An exon trapping assay indicated that the shortened intron is not spliced efficiently, probably because it is too small. We applied bisulfite conversion of cytosine to uracil in RNA followed by RT-PCR to analyze the splicing products. The aberrantly spliced RNA molecules resulted from either skipping of exon 5 or retention of intron 5. The same splicing defects were observed in cDNA derived from the pituitary of dwarfs. A survey of similarly mutated introns suggests that there is a minimal distance requirement between the splice donor and branch site of 50 nucleotides. In conclusion, a contraction of a DNA repeat in intron 5 of canine LHX3 leads to deficient splicing and is associated with pituitary dwarfism.

A Contracted DNA Repeat in LHX3 Intron 5 Is Associated with Aberrant Splicing and Pituitary Dwarfism in German Shepherd Dogs

PubMed Central

Voorbij, Annemarie M. W. Y.; van Steenbeek, Frank G.; Vos-Loohuis, Manon; Martens, Ellen E. C. P.; Hanson-Nilsson, Jeanette M.; van Oost, Bernard A.; Kooistra, Hans S.; Leegwater, Peter A.

2011-01-01

Dwarfism in German shepherd dogs is due to combined pituitary hormone deficiency of unknown genetic cause. We localized the recessively inherited defect by a genome wide approach to a region on chromosome 9 with a lod score of 9.8. The region contains LHX3, which codes for a transcription factor essential for pituitary development. Dwarfs have a deletion of one of six 7 bp repeats in intron 5 of LHX3, reducing the intron size to 68 bp. One dwarf was compound heterozygous for the deletion and an insertion of an asparagine residue in the DNA-binding homeodomain of LHX3, suggesting involvement of the gene in the disorder. An exon trapping assay indicated that the shortened intron is not spliced efficiently, probably because it is too small. We applied bisulfite conversion of cytosine to uracil in RNA followed by RT-PCR to analyze the splicing products. The aberrantly spliced RNA molecules resulted from either skipping of exon 5 or retention of intron 5. The same splicing defects were observed in cDNA derived from the pituitary of dwarfs. A survey of similarly mutated introns suggests that there is a minimal distance requirement between the splice donor and branch site of 50 nucleotides. In conclusion, a contraction of a DNA repeat in intron 5 of canine LHX3 leads to deficient splicing and is associated with pituitary dwarfism. PMID:22132174

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.

RNA splicing process analysis for identifying antisense oligonucleotide inhibitors with padlock probe-based isothermal amplification† †Electronic supplementary information (ESI) available: Additional experimental materials, methods, DNA sequences and supplementary figures and tables. See DOI: 10.1039/c7sc01336a Click here for additional data file.

PubMed Central

Ren, Xiaojun; Deng, Ruijie; Wang, Lida; Zhang, Kaixiang

2017-01-01

RNA splicing, which mainly involves two transesterification steps, is a fundamental process of gene expression and its abnormal regulation contributes to serious genetic diseases. Antisense oligonucleotides (ASOs) are genetic control tools that can be used to specifically control genes through alteration of the RNA splicing pathway. Despite intensive research, how ASOs or various other factors influence the multiple processes of RNA splicing still remains obscure. This is largely due to an inability to analyze the splicing efficiency of each step in the RNA splicing process with high sensitivity. We addressed this limitation by introducing a padlock probe-based isothermal amplification assay to achieve quantification of the specific products in different splicing steps. With this amplified assay, the roles that ASOs play in RNA splicing inhibition in the first and second steps could be distinguished. We identified that 5′-ASO could block RNA splicing by inhibiting the first step, while 3′-ASO could block RNA splicing by inhibiting the second step. This method provides a versatile tool for assisting efficient ASO design and discovering new splicing modulators and therapeutic drugs. PMID:28989608

Cyclin D1b splice variant promotes αvβ3-mediated adhesion and invasive migration of breast cancer cells.

PubMed

Wu, Feng-Hua; Luo, Li-Qiong; Liu, Yi; Zhan, Qiu-Xiao; Luo, Chao; Luo, Jing; Zhang, Gui-Mei; Feng, Zuo-Hua

2014-12-01

Cyclin D1b, a splice variant of the cell cycle regulator cyclin D1, holds oncogenic functions in human cancer. However, the mechanisms underlying cyclin D1b function remain poorly understood. Here we introduced wild-type cyclin D1a or cyclin D1b variant into non-metastatic MCF-7 cells. Our results show that ectopic expression of cyclin D1b promotes invasiveness of the cancer cells in a cyclin D1a independent manner. Specifically, cyclin D1b is found to modulate the expression of αvβ3, which characterizes the metastatic phenotype, and enhance tumor cell invasive potential in cooperating with HoxD3. Notably, cyclin D1b promotes αvβ3-mediated adhesion and invasive migration, which are associated with invasive potential of breast cancer cells. Further exploration indicates that cyclin D1b makes breast cancer cells more sensitive to toll-like receptor 4 ligand released from damaged tumor cells. These findings reveal a role of cyclin D1b as a possible mediator of αvβ3 transcription to promote tumor metastasis. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

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.

Characterization of Ovine A3Z1 Restriction Properties against Small Ruminant Lentiviruses (SRLVs)

PubMed Central

de Pablo-Maiso, Lorena; Glaria, Idoia; Crespo, Helena; Nistal-Villán, Estanislao; Andrésdóttir, Valgerdur; de Andrés, Damián; Amorena, Beatriz

2017-01-01

Intrinsic factors of the innate immune system include the apolipoprotein B editing enzyme catalytic polypeptide-like 3 (APOBEC3) protein family. APOBEC3 inhibits replication of different virus families by cytosine deamination of viral DNA and a not fully characterized cytosine deamination-independent mechanism. Sheep are susceptible to small ruminant lentivirus (SRLVs) infection and contain three APOBEC3 genes encoding four proteins (A3Z1, Z2, Z3 and Z2-Z3) with yet not deeply described antiviral properties. Using sheep blood monocytes and in vitro-derived macrophages, we found that A3Z1 expression is associated with lower viral replication in this cellular type. A3Z1 transcripts may also contain spliced variants (A3Z1Tr) lacking the cytidine deaminase motif. A3Z1 exogenous expression in fully permissive fibroblast-like cells restricted SRLVs infection while A3Z1Tr allowed infection. A3Z1Tr was induced after SRLVs infection or stimulation of blood-derived macrophages with interferon gamma (IFN-γ). Interaction between truncated isoform and native A3Z1 protein was detected as well as incorporation of both proteins into virions. A3Z1 and A3Z1Tr interacted with SRLVs Vif, but this interaction was not associated with degradative properties. Similar A3Z1 truncated isoforms were also present in human and monkey cells suggesting a conserved alternative splicing regulation in primates. A3Z1-mediated retroviral restriction could be constrained by different means, including gene expression and specific alternative splicing regulation, leading to truncated protein isoforms lacking a cytidine-deaminase motif. PMID:29149056

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.

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.

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.

Full-length single-cell RNA-seq applied to a viral human cancer: applications to HPV expression and splicing analysis in HeLa S3 cells.

PubMed

Wu, Liang; Zhang, Xiaolong; Zhao, Zhikun; Wang, Ling; Li, Bo; Li, Guibo; Dean, Michael; Yu, Qichao; Wang, Yanhui; Lin, Xinxin; Rao, Weijian; Mei, Zhanlong; Li, Yang; Jiang, Runze; Yang, Huan; Li, Fuqiang; Xie, Guoyun; Xu, Liqin; Wu, Kui; Zhang, Jie; Chen, Jianghao; Wang, Ting; Kristiansen, Karsten; Zhang, Xiuqing; Li, Yingrui; Yang, Huanming; Wang, Jian; Hou, Yong; Xu, Xun

2015-01-01

Viral infection causes multiple forms of human cancer, and HPV infection is the primary factor in cervical carcinomas. Recent single-cell RNA-seq studies highlight the tumor heterogeneity present in most cancers, but virally induced tumors have not been studied. HeLa is a well characterized HPV+ cervical cancer cell line. We developed a new high throughput platform to prepare single-cell RNA on a nanoliter scale based on a customized microwell chip. Using this method, we successfully amplified full-length transcripts of 669 single HeLa S3 cells and 40 of them were randomly selected to perform single-cell RNA sequencing. Based on these data, we obtained a comprehensive understanding of the heterogeneity of HeLa S3 cells in gene expression, alternative splicing and fusions. Furthermore, we identified a high diversity of HPV-18 expression and splicing at the single-cell level. By co-expression analysis we identified 283 E6, E7 co-regulated genes, including CDC25, PCNA, PLK4, BUB1B and IRF1 known to interact with HPV viral proteins. Our results reveal the heterogeneity of a virus-infected cell line. It not only provides a transcriptome characterization of HeLa S3 cells at the single cell level, but is a demonstration of the power of single cell RNA-seq analysis of virally infected cells and cancers.

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.

Nuclear poly(A)-binding protein aggregates misplace a pre-mRNA outside of SC35 speckle causing its abnormal splicing

PubMed Central

Klein, Pierre; Oloko, Martine; Roth, Fanny; Montel, Valérie; Malerba, Alberto; Jarmin, Susan; Gidaro, Teresa; Popplewell, Linda; Perie, Sophie; Lacau St Guily, Jean; de la Grange, Pierre; Antoniou, Michael N.; Dickson, George; Butler-Browne, Gillian; Bastide, Bruno; Mouly, Vincent; Trollet, Capucine

2016-01-01

A short abnormal polyalanine expansion in the polyadenylate-binding protein nuclear-1 (PABPN1) protein causes oculopharyngeal muscular dystrophy (OPMD). Mutated PABPN1 proteins accumulate as insoluble intranuclear aggregates in muscles of OPMD patients. While the roles of PABPN1 in nuclear polyadenylation and regulation of alternative poly(A) site choice have been established, the molecular mechanisms which trigger pathological defects in OPMD and the role of aggregates remain to be determined. Using exon array, for the first time we have identified several splicing defects in OPMD. In particular, we have demonstrated a defect in the splicing regulation of the muscle-specific Troponin T3 (TNNT3) mutually exclusive exons 16 and 17 in OPMD samples compared to controls. This splicing defect is directly linked to the SC35 (SRSF2) splicing factor and to the presence of nuclear aggregates. As reported here, PABPN1 aggregates are able to trap TNNT3 pre-mRNA, driving it outside nuclear speckles, leading to an altered SC35-mediated splicing. This results in a decreased calcium sensitivity of muscle fibers, which could in turn plays a role in muscle pathology. We thus report a novel mechanism of alternative splicing deregulation that may play a role in various other diseases with nuclear inclusions or foci containing an RNA binding protein. PMID:27507886

Sequential recognition of the pre-mRNA branch point by U2AF65 and a novel spliceosome-associated 28-kDa protein.

PubMed Central

Gaur, R K; Valcárcel, J; Green, M R

1995-01-01

Splicing of pre-mRNAs occurs via a lariat intermediate in which an intronic adenosine, embedded within a branch point sequence, forms a 2',5'-phosphodiester bond (RNA branch) with the 5' end of the intron. How the branch point is recognized and activated remains largely unknown. Using site-specific photochemical cross-linking, we have identified two proteins that specifically interact with the branch point during the splicing reaction. U2AF65, an essential splicing factor that binds to the adjacent polypyrimidine tract, crosslinks to the branch point at the earliest stage of spliceosome formation in an ATP-independent manner. A novel 28-kDa protein, which is a constituent of the mature spliceosome, contacts the branch point after the first catalytic step. Our results indicate that the branch point is sequentially recognized by distinct splicing factors in the course of the splicing reaction. Images FIGURE 1 FIGURE 2 FIGURE 3 FIGURE 4 FIGURE 5 FIGURE 6 FIGURE 7 FIGURE 8 FIGURE 9 PMID:7493318

Genetic and physical interactions between factors involved in both cell cycle progression and pre-mRNA splicing in Saccharomyces cerevisiae.

PubMed Central

Ben-Yehuda, S; Dix, I; Russell, C S; McGarvey, M; Beggs, J D; Kupiec, M

2000-01-01

The PRP17/CDC40 gene of Saccharomyces cerevisiae functions in two different cellular processes: pre-mRNA splicing and cell cycle progression. The Prp17/Cdc40 protein participates in the second step of the splicing reaction and, in addition, prp17/cdc40 mutant cells held at the restrictive temperature arrest in the G2 phase of the cell cycle. Here we describe the identification of nine genes that, when mutated, show synthetic lethality with the prp17/cdc40Delta allele. Six of these encode known splicing factors: Prp8p, Slu7p, Prp16p, Prp22p, Slt11p, and U2 snRNA. The other three, SYF1, SYF2, and SYF3, represent genes also involved in cell cycle progression and in pre-mRNA splicing. Syf1p and Syf3p are highly conserved proteins containing several copies of a repeated motif, which we term RTPR. This newly defined motif is shared by proteins involved in RNA processing and represents a subfamily of the known TPR (tetratricopeptide repeat) motif. Using two-hybrid interaction screens and biochemical analysis, we show that the SYF gene products interact with each other and with four other proteins: Isy1p, Cef1p, Prp22p, and Ntc20p. We discuss the role played by these proteins in splicing and cell cycle progression. PMID:11102353

Genetic and physical interactions between factors involved in both cell cycle progression and pre-mRNA splicing in Saccharomyces cerevisiae.

PubMed

Ben-Yehuda, S; Dix, I; Russell, C S; McGarvey, M; Beggs, J D; Kupiec, M

2000-12-01

The PRP17/CDC40 gene of Saccharomyces cerevisiae functions in two different cellular processes: pre-mRNA splicing and cell cycle progression. The Prp17/Cdc40 protein participates in the second step of the splicing reaction and, in addition, prp17/cdc40 mutant cells held at the restrictive temperature arrest in the G2 phase of the cell cycle. Here we describe the identification of nine genes that, when mutated, show synthetic lethality with the prp17/cdc40Delta allele. Six of these encode known splicing factors: Prp8p, Slu7p, Prp16p, Prp22p, Slt11p, and U2 snRNA. The other three, SYF1, SYF2, and SYF3, represent genes also involved in cell cycle progression and in pre-mRNA splicing. Syf1p and Syf3p are highly conserved proteins containing several copies of a repeated motif, which we term RTPR. This newly defined motif is shared by proteins involved in RNA processing and represents a subfamily of the known TPR (tetratricopeptide repeat) motif. Using two-hybrid interaction screens and biochemical analysis, we show that the SYF gene products interact with each other and with four other proteins: Isy1p, Cef1p, Prp22p, and Ntc20p. We discuss the role played by these proteins in splicing and cell cycle progression.

Human type II pneumocyte chemotactic responses to CXCR3 activation are mediated by splice variant A.

PubMed

Ji, Rong; Lee, Clement M; Gonzales, Linda W; Yang, Yi; Aksoy, Mark O; Wang, Ping; Brailoiu, Eugen; Dun, Nae; Hurford, Matthew T; Kelsen, Steven G

2008-06-01

Chemokine receptors control several fundamental cellular processes in both hematopoietic and structural cells, including directed cell movement, i.e., chemotaxis, cell differentiation, and proliferation. We have previously demonstrated that CXCR3, the chemokine receptor expressed by Th1/Tc1 inflammatory cells present in the lung, is also expressed by human airway epithelial cells. In airway epithelial cells, activation of CXCR3 induces airway epithelial cell movement and proliferation, processes that underlie lung repair. The present study examined the expression and function of CXCR3 in human alveolar type II pneumocytes, whose destruction causes emphysema. CXCR3 was present in human fetal and adult type II pneumocytes as assessed by immunocytochemistry, immunohistochemistry, and Western blotting. CXCR3-A and -B splice variant mRNA was present constitutively in cultured type II cells, but levels of CXCR3-B greatly exceeded CXCR3-A mRNA. In cultured type II cells, I-TAC, IP-10, and Mig induced chemotaxis. Overexpression of CXCR3-A in the A549 pneumocyte cell line produced robust chemotactic responses to I-TAC and IP-10. In contrast, I-TAC did not induce chemotactic responses in CXCR3-B and mock-transfected cells. Finally, I-TAC increased cytosolic Ca(2+) and activated the extracellular signal-regulated kinase, p38, and phosphatidylinositol 3-kinase (PI 3-kinase)/protein kinase B kinases only in CXCR3-A-transfected cells. These data indicate that the CXCR3 receptor is expressed by human type II pneumocytes, and the CXCR3-A splice variant mediates chemotactic responses possibly through Ca(2+) activation of both mitogen-activated protein kinase and PI 3-kinase signaling pathways. Expression of CXCR3 in alveolar epithelial cells may be important in pneumocyte repair from injury.

When SUMO met splicing.

PubMed

Pozzi, Berta; Mammi, Pablo; Bragado, Laureano; Giono, Luciana E; Srebrow, Anabella

2018-05-09

Spliceosomal proteins have been revealed as SUMO conjugation targets. Moreover, we have reported that many of these are in a SUMO-conjugated form when bound to a pre-mRNA substrate during a splicing reaction. We demonstrated that SUMOylation of Prp3 (PRPF3), a component of the U4/U6 di-snRNP, is required for U4/U6•U5 tri-snRNP formation and/or recruitment to active spliceosomes. Expanding upon our previous results, we have shown that the splicing factor SRSF1 stimulates SUMO conjugation to several spliceosomal proteins. Given the relevance of the splicing process, as well as the complex and dynamic nature of its governing machinery, the spliceosome, the molecular mechanisms that modulate its function represent an attractive topic of research. We posit that SUMO conjugation could represent a way of modulating spliceosome assembly and thus, splicing efficiency. How cycles of SUMOylation/de-SUMOylation of spliceosomal proteins become integrated throughout the highly choreographed spliceosomal cycle awaits further investigation.

Inactive DNMT3B Splice Variants Modulate De Novo DNA Methylation

PubMed Central

Gordon, Catherine A.; Hartono, Stella R.; Chédin, Frédéric

2013-01-01

Inactive DNA methyltransferase (DNMT) 3B splice isoforms are associated with changes in DNA methylation, yet the mechanisms by which they act remain largely unknown. Using biochemical and cell culture assays, we show here that the inactive DNMT3B3 and DNMT3B4 isoforms bind to and regulate the activity of catalytically competent DNMT3A or DNMT3B molecules. DNMT3B3 modestly stimulated the de novo methylation activity of DNMT3A and also counteracted the stimulatory effects of DNMT3L, therefore leading to subtle and contrasting effects on activity. DNMT3B4, by contrast, significantly inhibited de novo DNA methylation by active DNMT3 molecules, most likely due to its ability to reduce the DNA binding affinity of co-complexes, thereby sequestering them away from their substrate. Immunocytochemistry experiments revealed that in addition to their effects on the intrinsic catalytic function of active DNMT3 enzymes, DNMT3B3 and DNMT34 drive distinct types of chromatin compaction and patterns of histone 3 lysine 9 tri-methylation (H3K9me3) deposition. Our findings suggest that regulation of active DNMT3 members through the formation of co-complexes with inactive DNMT3 variants is a general mechanism by which DNMT3 variants function. This may account for some of the changes in DNA methylation patterns observed during development and disease. PMID:23894490

Drug discovery with an RBM20 dependent titin splice reporter identifies cardenolides as lead structures to improve cardiac filling.

PubMed

Liss, Martin; Radke, Michael H; Eckhard, Jamina; Neuenschwander, Martin; Dauksaite, Vita; von Kries, Jens-Peter; Gotthardt, Michael

2018-01-01

Diastolic dysfunction is increasingly prevalent in our ageing society and an important contributor to heart failure. The giant protein titin could serve as a therapeutic target, as its elastic properties are a main determinant of cardiac filling in diastole. This study aimed to develop a high throughput pharmacological screen to identify small molecules that affect titin isoform expression through differential inclusion of exons encoding the elastic PEVK domains. We used a dual luciferase splice reporter assay that builds on the titin splice factor RBM20 to screen ~34,000 small molecules and identified several compounds that inhibit the exclusion of PEVK exons. These compounds belong to the class of cardenolides and affect RBM20 dependent titin exon exclusion but did not affect RBFOX1 mediated splicing of FMNL3. We provide evidence that cardenolides do not bind to the RNA interacting domain of RBM20, but reduce RBM20 protein levels and alter transcription of select splicing factors that interact with RBM20. Cardenolides affect titin isoform expression. Understanding their mode of action and harnessing the splice effects through chemical modifications that suppress the effects on ion homeostasis and more selectively affect cardiac splicing has the potential to improve cardiac filling and thus help patients with diastolic heart failure, for which currently no targeted therapy exists.

Splicing factor SRSF3 is crucial for hepatocyte differentiation and metabolic function

PubMed Central

Sen, Supriya; Jumaa, Hassan; Webster, Nicholas J.G.

2015-01-01

SR family RNA binding proteins regulate splicing of nascent RNAs in vitro but their physiological role in vivo is largely unexplored, as genetic deletion of many SR protein genes results in embryonic lethality. Here we show that SRSF3HKO mice carrying a hepatocyte-specific deletion of Srsf3 (homologous to human SRSF3/SRp20) have a disrupted hepatic architecture and show pre- and postnatal growth retardation. SRSF3HKO mice exhibit impaired hepatocyte maturation with alterations in glucose and lipid homeostasis characterized by reduced glycogen storage, fasting hypoglycemia, increased insulin sensitivity and reduced cholesterol synthesis. We identify various splicing alterations in the SRSF3HKO liver that explain the in vivo phenotype. In particular, loss of SRSF3 causes aberrant splicing of Hnf1α, Ern1, Hmgcs1, Dhcr7 and Scap genes, which are critical regulators of glucose and lipid metabolism. Our study provides the first evidence for a SRSF3-driven genetic programme required for morphological and functional differentiation of hepatocytes that may have relevance for human liver disease and metabolic dysregulation. PMID:23299886

Low-loss fusion splicing of single-mode fiber and a photonic crystal fiber suitable for construction of a patch cord for measurement devices

NASA Astrophysics Data System (ADS)

Jaroszewicz, Leszek R.; Murawski, Michal; Stasiewicz, Karol; Marc, Pawel

2009-10-01

The optimization of the fused splice between two identical PCFs as well as SMF•28 with different MFD PCFs made using the filament fusion with continuum laser illumination is reported. For identical PCFs splice loss of 0.15+/-0.04 dB @ 1.55 μm has been obtained. The SMF with PCF (MFD = 6.0 μm) splice losses are lower than 0.40 dB in comparison with the reported dependences in spectral range 1.51-1.63μm. Moreover, the splice SMF with extremely small core PCF is also presented. The discussed data has shown that such SMF-PCF splice is suitable for construction of a patch cord for measurement devices.

Binding of hnRNP H and U2AF65 to Respective G-codes and a Poly-Uridine Tract Collaborate in the N50-5'ss Selection of the REST N Exon in H69 Cells

PubMed Central

Ortuño-Pineda, Carlos; Galindo-Rosales, José Manuel; Calderón-Salinas, José Victor; Villegas-Sepúlveda, Nicolás; Saucedo-Cárdenas, Odila; De Nova-Ocampo, Mónica; Valdés, Jesús

2012-01-01

The splicing of the N exon in the pre-mRNA coding for the RE1-silencing transcription factor (REST) results in a truncated protein that modifies the expression pattern of some of its target genes. A weak 3'ss, three alternative 5'ss (N4-, N50-, and N62-5'ss) and a variety of putative target sites for splicing regulatory proteins are found around the N exon; two GGGG codes (G2-G3) and a poly-Uridine tract (N-PU) are found in front of the N50-5'ss. In this work we analyzed some of the regulatory factors and elements involved in the preferred selection of the N50-5'ss (N50 activation) in the small cell lung cancer cell line H69. Wild type and mutant N exon/β-globin minigenes recapitulated N50 exon splicing in H69 cells, and showed that the N-PU and the G2-G3 elements are required for N50 exon splicing. Biochemical and knockdown experiments identified these elements as U2AF65 and hnRNP H targets, respectively, and that they are also required for N50 exon activation. Compared to normal MRC5 cells, and in keeping with N50 exon activation, U2AF65, hnRNP H and other splicing factors were highly expressed in H69 cells. CLIP experiments revealed that hnRNP H RNA-binding occurs first and is a prerequisite for U2AF65 RNA binding, and EMSA and CLIP experiments suggest that U2AF65-RNA recognition displaces hnRNP H and helps to recruit other splicing factors (at least U1 70K) to the N50-5'ss. Our results evidenced novel hnRNP H and U2AF65 functions: respectively, U2AF65-recruiting to a 5'ss in humans and the hnRNP H-displacing function from two juxtaposed GGGG codes. PMID:22792276

Wild-Type U2AF1 Antagonizes the Splicing Program Characteristic of U2AF1-Mutant Tumors and Is Required for Cell Survival

PubMed Central

Fei, Dennis Liang; Motowski, Hayley; Chatrikhi, Rakesh; Gao, Shaojian; Kielkopf, Clara L.; Varmus, Harold

2016-01-01

We have asked how the common S34F mutation in the splicing factor U2AF1 regulates alternative splicing in lung cancer, and why wild-type U2AF1 is retained in cancers with this mutation. A human lung epithelial cell line was genetically modified so that U2AF1S34F is expressed from one of the two endogenous U2AF1 loci. By altering levels of mutant or wild-type U2AF1 in this cell line and by analyzing published data on human lung adenocarcinomas, we show that S34F-associated changes in alternative splicing are proportional to the ratio of S34F:wild-type gene products and not to absolute levels of either the mutant or wild-type factor. Preferential recognition of specific 3′ splice sites in S34F-expressing cells is largely explained by differential in vitro RNA-binding affinities of mutant versus wild-type U2AF1 for those same 3′ splice sites. Finally, we show that lung adenocarcinoma cell lines bearing U2AF1 mutations do not require the mutant protein for growth in vitro or in vivo. In contrast, wild-type U2AF1 is required for survival, regardless of whether cells carry the U2AF1S34F allele. Our results provide mechanistic explanations of the magnitude of splicing changes observed in U2AF1-mutant cells and why tumors harboring U2AF1 mutations always retain an expressed copy of the wild-type allele. PMID:27776121

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.

Novel splice mutation in microthalmia-associated transcription factor in Waardenburg Syndrome.

PubMed

Brenner, Laura; Burke, Kelly; Leduc, Charles A; Guha, Saurav; Guo, Jiancheng; Chung, Wendy K

2011-01-01

Waardenburg Syndrome (WS) is a syndromic form of hearing loss associated with mutations in six different genes. We identified a large family with WS that had previously undergone clinical testing, with no reported pathogenic mutation. Using linkage analysis, a region on 3p14.1 with an LOD score of 6.6 was identified. Microthalmia-Associated Transcription Factor, a gene known to cause WS, is located within this region of linkage. Sequencing of Microthalmia-Associated Transcription Factor demonstrated a c.1212 G>A synonymous variant that segregated with the WS in the family and was predicted to cause a novel splicing site that was confirmed with expression analysis of the mRNA. This case illustrates the need to computationally analyze novel synonymous sequence variants for possible effects on splicing to maximize the clinical sensitivity of sequence-based genetic testing.

Zebrafish pax8 is required for otic placode induction and plays a redundant role with Pax2 genes in the maintenance of the otic placode.

PubMed

Mackereth, Melinda D; Kwak, Su-Jin; Fritz, Andreas; Riley, Bruce B

2005-01-01

Vertebrate Pax2 and Pax8 proteins are closely related transcription factors hypothesized to regulate early aspects of inner ear development. In zebrafish and mouse, Pax8 expression is the earliest known marker of otic induction, and Pax2 homologs are expressed at slightly later stages of placodal development. Analysis of compound mutants has not been reported. To facilitate analysis of zebrafish pax8, we completed sequencing of the entire gene, including the 5' and 3' UTRs. pax8 transcripts undergo complex alternative splicing to generate at least ten distinct isoforms. Two different subclasses of pax8 splice isoforms encode different translation initiation sites. Antisense morpholinos (MOs) were designed to block translation from both start sites, and four additional MOs were designed to target different exon-intron boundaries to block splicing. Injection of MOs, individually and in various combinations, generated similar phenotypes. Otic induction was impaired, and otic vesicles were small. Regional ear markers were expressed correctly, but hair cell production was significantly reduced. This phenotype was strongly enhanced by simultaneously disrupting either of the co-inducers fgf3 or fgf8, or another early regulator, dlx3b, which is thought to act in a parallel pathway. In contrast, the phenotype caused by disrupting foxi1, which is required for pax8 expression, was not enhanced by simultaneously disrupting pax8. Disrupting pax8, pax2a and pax2b did not further impair otic induction relative to loss of pax8 alone. However, the amount of otic tissue gradually decreased in pax8-pax2a-pax2b-deficient embryos such that no otic tissue was detectable by 24 hours post-fertilization. Loss of otic tissue did not correlate with increased cell death, suggesting that otic cells dedifferentiate or redifferentiate as other cell type(s). These data show that pax8 is initially required for normal otic induction, and subsequently pax8, pax2a and pax2b act redundantly to maintain otic fate.

Disturbed expression of type 1 iodothyronine deiodinase splice variants in human renal cancer.

PubMed

Piekielko-Witkowska, Agnieszka; Master, Adam; Wojcicka, Anna; Boguslawska, Joanna; Brozda, Izabela; Tanski, Zbigniew; Nauman, Alicja

2009-10-01

Alternative splicing, one of the sources of protein diversity, is often disturbed in cancer. Type 1 iodothyronine deiodinase (DIO1) catalyzes deiodination of thyroxine generating triiodothyronine, an important regulator of cell proliferation and differentiation. The expression of DIO1 is disturbed in different types of cancer. The aim of the study was to analyze the alternative splicing of DIO1 and its possible disturbance in renal cancer. Using real-time PCR, we analyzed 19 tissue samples (T) of renal cancer and 19 matched control samples (C) of the opposite pole of the kidney, not infiltrated by tumor, and 6 control samples (N) (nonneoplastic kidney abnormalities). Cloning of DIO1 mRNA isoforms revealed 11 different transcripts, among them 7 new splice variants, not previously reported. The expression of all variants of DIO1 was dramatically (>90%) and significantly (p < or = 0.0003) lowered in samples T compared to control samples C. The ratio of mRNA isoforms encoding DIO1 protein variants possessing or lacking the active center was lowered in samples T compared with control samples C, suggesting disturbed alternative splicing of DIO1. The expression of mRNA of splicing factors SF2/ASF (splicing factor-2/alternative-splicing factor) and hnRNPA1 (heterogeneous ribonucleoprotein A1), regulating 5'-splice site selection, was significantly but not proportionally lowered in samples T compared to samples C. The mRNA ratio of splicing factors SF2/ASF and hnRNPA1 correlated with the ratio of mRNA isoforms encoding DIO1 protein variants possessing or lacking the active center in controls C but not in samples T. Our results show that the expression and alternative splicing of DIO1 mRNA is disturbed in renal cancer, possibly due to changes in expression of splicing factors SF2/ASF and hnRNPA1.

30 CFR 77.602 - Permanent splicing of trailing cables.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 30 Mineral Resources 1 2010-07-01 2010-07-01 false Permanent splicing of trailing cables. 77.602... COAL MINES Trailing Cables § 77.602 Permanent splicing of trailing cables. When permanent splices in trailing cables are made, they shall be: (a) Mechanically strong with adequate electrical conductivity; (b...

Interplay between DMD Point Mutations and Splicing Signals in Dystrophinopathy Phenotypes

PubMed Central

Juan-Mateu, Jonàs; González-Quereda, Lidia; Rodríguez, Maria José; Verdura, Edgard; Lázaro, Kira; Jou, Cristina; Nascimento, Andrés; Jiménez-Mallebrera, Cecilia; Colomer, Jaume; Monges, Soledad; Lubieniecki, Fabiana; Foncuberta, Maria Eugenia; Pascual-Pascual, Samuel Ignacio; Molano, Jesús; Baiget, Montserrat; Gallano, Pia

2013-01-01

DMD nonsense and frameshift mutations lead to severe Duchenne muscular dystrophy while in-frame mutations lead to milder Becker muscular dystrophy. Exceptions are found in 10% of cases and the production of alternatively spliced transcripts is considered a key modifier of disease severity. Several exonic mutations have been shown to induce exon-skipping, while splice site mutations result in exon-skipping or activation of cryptic splice sites. However, factors determining the splicing pathway are still unclear. Point mutations provide valuable information regarding the regulation of pre-mRNA splicing and elements defining exon identity in the DMD gene. Here we provide a comprehensive analysis of 98 point mutations related to clinical phenotype and their effect on muscle mRNA and dystrophin expression. Aberrant splicing was found in 27 mutations due to alteration of splice sites or splicing regulatory elements. Bioinformatics analysis was performed to test the ability of the available algorithms to predict consequences on mRNA and to investigate the major factors that determine the splicing pathway in mutations affecting splicing signals. Our findings suggest that the splicing pathway is highly dependent on the interplay between splice site strength and density of regulatory elements. PMID:23536893

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.

Global analysis of pre-mRNA subcellular localization following splicing inhibition by spliceostatin A

PubMed Central

Yoshimoto, Rei; Kaida, Daisuke; Furuno, Masaaki; Burroughs, A. Maxwell; Noma, Shohei; Suzuki, Harukazu; Kawamura, Yumi; Hayashizaki, Yoshihide; Mayeda, Akila; Yoshida, Minoru

2017-01-01

Spliceostatin A (SSA) is a methyl ketal derivative of FR901464, a potent antitumor compound isolated from a culture broth of Pseudomonas sp. no. 2663. These compounds selectively bind to the essential spliceosome component SF3b, a subcomplex of the U2 snRNP, to inhibit pre-mRNA splicing. However, the mechanism of SSA's antitumor activity is unknown. It is noteworthy that SSA causes accumulation of a truncated form of the CDK inhibitor protein p27 translated from CDKN1B pre-mRNA, which is involved in SSA-induced cell-cycle arrest. However, it is still unclear whether pre-mRNAs are uniformly exported from the nucleus following SSA treatment. We performed RNA-seq analysis on nuclear and cytoplasmic fractions of SSA-treated cells. Our statistical analyses showed that intron retention is the major consequence of SSA treatment, and a small number of intron-containing pre-mRNAs leak into the cytoplasm. Using a series of reporter plasmids to investigate the roles of intronic sequences in the pre-mRNA leakage, we showed that the strength of the 5′ splice site affects pre-mRNA leakage. Additionally, we found that the level of pre-mRNA leakage is related to transcript length. These results suggest that the strength of the 5′ splice site and the length of the transcripts are determinants of the pre-mRNA leakage induced by SF3b inhibitors. PMID:27754875

RNA splicing factors as oncoproteins and tumor suppressors

PubMed Central

Dvinge, Heidi; Kim, Eunhee; Abdel-Wahab, Omar; Bradley, Robert K.

2016-01-01

Preface The recent genomic characterization of cancers has revealed recurrent somatic point mutations and copy number changes affecting genes encoding RNA splicing factors. Initial studies of these ‘spliceosomal mutations’ suggest that the proteins bearing these mutations exhibit altered splice site and/or exon recognition preferences relative to their wild-type counterparts, resulting in cancer-specific mis-splicing. Such changes in the splicing machinery may create novel vulnerabilities in cancer cells that can be therapeutically exploited using compounds that can influence the splicing process. Further studies to dissect the biochemical, genomic, and biological effects of spliceosomal mutations are critical for the development of cancer therapies targeted to these mutations. PMID:27282250

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

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.

Deciphering Transcriptome and Complex Alternative Splicing Transcripts in Mammary Gland Tissues from Cows Naturally Infected with Staphylococcus aureus Mastitis

PubMed Central

Jiang, Qiang; Yang, Chun Hong; Zhang, Yan; Sun, Yan; Li, Rong Ling; Wang, Chang Fa; Zhong, Ji Feng; Huang, Jin Ming

2016-01-01

Alternative splicing (AS) contributes to the complexity of the mammalian proteome and plays an important role in diseases, including infectious diseases. The differential AS patterns of these transcript sequences between the healthy (HS3A) and mastitic (HS8A) cows naturally infected by Staphylococcus aureus were compared to understand the molecular mechanisms underlying mastitis resistance and susceptibility. In this study, using the Illumina paired-end RNA sequencing method, 1352 differentially expressed genes (DEGs) with higher than twofold changes were found in the HS3A and HS8A mammary gland tissues. Gene ontology and KEGG pathway analyses revealed that the cytokine–cytokine receptor interaction pathway is the most significantly enriched pathway. Approximately 16k annotated unigenes were respectively identified in two libraries, based on the bovine Bos taurus UMD3.1 sequence assembly and search. A total of 52.62% and 51.24% annotated unigenes were alternatively spliced in term of exon skipping, intron retention, alternative 5′ splicing and alternative 3ʹ splicing. Additionally, 1,317 AS unigenes were HS3A-specific, whereas 1,093 AS unigenes were HS8A-specific. Some immune-related genes, such as ITGB6, MYD88, ADA, ACKR1, and TNFRSF1B, and their potential relationships with mastitis were highlighted. From Chromosome 2, 4, 6, 7, 10, 13, 14, 17, and 20, 3.66% (HS3A) and 5.4% (HS8A) novel transcripts, which harbor known quantitative trait locus associated with clinical mastitis, were identified. Many DEGs in the healthy and mastitic mammary glands are involved in immune, defense, and inflammation responses. These DEGs, which exhibit diverse and specific splicing patterns and events, can endow dairy cattle with the potential complex genetic resistance against mastitis. PMID:27459697

Structure-function analysis and genetic interactions of the SmG, SmE, and SmF subunits of the yeast Sm protein ring.

PubMed

Schwer, Beate; Kruchten, Joshua; Shuman, Stewart

2016-09-01

A seven-subunit Sm protein ring forms a core scaffold of the U1, U2, U4, and U5 snRNPs that direct pre-mRNA splicing. Using human snRNP structures to guide mutagenesis in Saccharomyces cerevisiae, we gained new insights into structure-function relationships of the SmG, SmE, and SmF subunits. An alanine scan of 19 conserved amino acids of these three proteins, comprising the Sm RNA binding sites or inter-subunit interfaces, revealed that, with the exception of Arg74 in SmF, none are essential for yeast growth. Yet, for SmG, SmE, and SmF, as for many components of the yeast spliceosome, the effects of perturbing protein-RNA and protein-protein interactions are masked by built-in functional redundancies of the splicing machine. For example, tests for genetic interactions with non-Sm splicing factors showed that many benign mutations of SmG, SmE, and SmF (and of SmB and SmD3) were synthetically lethal with null alleles of U2 snRNP subunits Lea1 and Msl1. Tests of pairwise combinations of SmG, SmE, SmF, SmB, and SmD3 alleles highlighted the inherent redundancies within the Sm ring, whereby simultaneous mutations of the RNA binding sites of any two of the Sm subunits are lethal. Our results suggest that six intact RNA binding sites in the Sm ring suffice for function but five sites may not. © 2016 Schwer et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

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

Physiological Srsf2 P95H expression causes impaired hematopoietic stem cell functions and aberrant RNA splicing in mice.

PubMed

Kon, Ayana; Yamazaki, Satoshi; Nannya, Yasuhito; Kataoka, Keisuke; Ota, Yasunori; Nakagawa, Masahiro Marshall; Yoshida, Kenichi; Shiozawa, Yusuke; Morita, Maiko; Yoshizato, Tetsuichi; Sanada, Masashi; Nakayama, Manabu; Koseki, Haruhiko; Nakauchi, Hiromitsu; Ogawa, Seishi

2018-02-08

Splicing factor mutations are characteristic of myelodysplastic syndromes (MDS) and related myeloid neoplasms and implicated in their pathogenesis, but their roles in the development of MDS have not been fully elucidated. In the present study, we investigated the consequence of mutant Srsf2 expression using newly generated Vav1-Cre -mediated conditional knockin mice. Mice carrying a heterozygous Srsf2 P95H mutation showed significantly reduced numbers of hematopoietic stem and progenitor cells (HSPCs) and differentiation defects both in the steady-state condition and transplantation settings. Srsf2 -mutated hematopoietic stem cells (HSCs) showed impaired long-term reconstitution compared with control mice in competitive repopulation assays. Although the Srsf2 mutant mice did not develop MDS under the steady-state condition, when their stem cells were transplanted into lethally irradiated mice, the recipients developed anemia, leukopenia, and erythroid dysplasia, which suggests the role of replicative stress in the development of an MDS-like phenotype in Srsf2 -mutated mice. RNA sequencing of the Srsf2 -mutated HSPCs revealed a number of abnormal splicing events and differentially expressed genes, including several potential targets implicated in the pathogenesis of hematopoietic malignancies, such as Csf3r , Fyn , Gnas , Nsd1 , Hnrnpa2b1 , and Trp53bp1 Among the mutant Srsf2 -associated splicing events, most commonly observed were the enhanced inclusion and/or exclusion of cassette exons, which were caused by the altered consensus motifs for the recognition of exonic splicing enhancers. Our findings suggest that the mutant Srsf2 leads to a compromised HSC function by causing abnormal RNA splicing and expression, contributing to the deregulated hematopoiesis that recapitulates the MDS phenotypes, possibly as a result of additional genetic and/or environmental insults. © 2018 by The American Society of Hematology.

A Novel Alternative Splicing Isoform of Human T-Cell Leukemia Virus Type 1 bZIP Factor (HBZ-SI) Targets Distinct Subnuclear Localization

PubMed Central

Murata, Ken; Hayashibara, Toshihisa; Sugahara, Kazuyuki; Uemura, Akiko; Yamaguchi, Taku; Harasawa, Hitomi; Hasegawa, Hiroo; Tsuruda, Kazuto; Okazaki, Toshiro; Koji, Takehiko; Miyanishi, Takayuki; Yamada, Yasuaki; Kamihira, Shimeru

2006-01-01

Adult T-cell leukemia (ATL) is associated with prior infection with human T-cell leukemia virus type 1 (HTLV-1); however, the mechanism by which HTLV-1 causes adult T-cell leukemia has not been fully elucidated. Recently, a functional basic leucine zipper (bZIP) protein coded in the minus strand of HTLV-1 genome (HBZ) was identified. We report here a novel isoform of the HTLV-1 bZIP factor (HBZ), HBZ-SI, identified by means of reverse transcription-PCR (RT-PCR) in conjunction with 5′ and 3′ rapid amplification of cDNA ends (RACE). HBZ-SI is a 206-amino-acid-long protein and is generated by alternative splicing between part of the HBZ gene and a novel exon located in the 3′ long terminal repeat of the HTLV-1 genome. Consequently, these isoforms share >95% amino acid sequence identity, and differ only at their N termini, indicating that HBZ-SI is also a functional protein. Duplex RT-PCR and real-time quantitative RT-PCR analyses showed that the mRNAs of these isoforms were expressed at equivalent levels in all ATL cell samples examined. Nonetheless, we found by Western blotting that the HBZ-SI protein was preferentially expressed in some ATL cell lines examined. A key finding was obtained from the subcellular localization analyses of these isoforms. Despite their high sequence similarity, each isoform was targeted to distinguishable subnuclear structures. These data show the presence of a novel isoform of HBZ in ATL cells, and in addition, shed new light on the possibility that each isoform may play a unique role in distinct regions in the cell nucleus. PMID:16474156

Novel splice site mutation in the growth hormone receptor gene in Turkish patients with Laron-type dwarfism.

PubMed

Arman, Ahmet; Ozon, Alev; Isguven, Pinar S; Coker, Ajda; Peker, Ismail; Yordam, Nursen

2008-01-01

Growth hormone (GH) is involved in growth, and fat and carbohydrate metabolism. Interaction of GH with the GH receptor (GHR) is necessary for systemic and local production of insulin-like growth factor-I (IGF-I) which mediates GH actions. Mutations in the GHR cause severe postnatal growth failure; the disorder is an autosomal recessive genetic disease resulting in GH insensitivity, called Laron syndrome. It is characterized by dwarfism with elevated serum GH and low levels of IGF-I. We analyzed the GHR gene for mutations and polymorphisms in eight patients with Laron-type dwarfism from six families. We found three missense mutations (S40L, V125A, I526L), one nonsense mutation (W157X), and one splice site mutation in the extracellular domain of GHR. Furthermore, G168G and exon 3 deletion polymorphisms were detected in patients with Laron syndrome. The splice site mutation, which is a novel mutation, was located at the donor splice site of exon 2/ intron 2 within GHR. Although this mutation changed the highly conserved donor splice site consensus sequence GT to GGT by insertion of a G residue, the intron splicing between exon 2 and exon 3 was detected in the patient. These results imply that the splicing occurs arthe GT site in intron 2, leaving the extra inserted G residue at the end of exon 2, thus changing the open reading frame of GHR resulting in a premature termination codon in exon 3.

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

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.

SKIP Is a Component of the Spliceosome Linking Alternative Splicing and the Circadian Clock in Arabidopsis[W

PubMed Central

Wang, Xiaoxue; Wu, Fangming; Xie, Qiguang; Wang, Huamei; Wang, Ying; Yue, Yanling; Gahura, Ondrej; Ma, Shuangshuang; Liu, Lei; Cao, Ying; Jiao, Yuling; Puta, Frantisek; McClung, C. Robertson; Xu, Xiaodong; Ma, Ligeng

2012-01-01

Circadian clocks generate endogenous rhythms in most organisms from cyanobacteria to humans and facilitate entrainment to environmental diurnal cycles, thus conferring a fitness advantage. Both transcriptional and posttranslational mechanisms are prominent in the basic network architecture of circadian systems. Posttranscriptional regulation, including mRNA processing, is emerging as a critical step for clock function. However, little is known about the molecular mechanisms linking RNA metabolism to the circadian clock network. Here, we report that a conserved SNW/Ski-interacting protein (SKIP) domain protein, SKIP, a splicing factor and component of the spliceosome, is involved in posttranscriptional regulation of circadian clock genes in Arabidopsis thaliana. Mutation in SKIP lengthens the circadian period in a temperature-sensitive manner and affects light input and the sensitivity of the clock to light resetting. SKIP physically interacts with the spliceosomal splicing factor Ser/Arg-rich protein45 and associates with the pre-mRNA of clock genes, such as PSEUDORESPONSE REGULATOR7 (PRR7) and PRR9, and is necessary for the regulation of their alternative splicing and mRNA maturation. Genome-wide investigations reveal that SKIP functions in regulating alternative splicing of many genes, presumably through modulating recognition or cleavage of 5′ and 3′ splice donor and acceptor sites. Our study addresses a fundamental question on how the mRNA splicing machinery contributes to circadian clock function at a posttranscriptional level. PMID:22942380

Ott1 (Rbm15) regulates thrombopoietin response in hematopoietic stem cells through alternative splicing of c-Mpl

PubMed Central

Xiao, Nan; Laha, Suparna; Das, Shankar P.; Morlock, Kayla; Jesneck, Jonathan L.

2015-01-01

Thrombopoietin (Thpo) signaling through the c-Mpl receptor promotes either quiescence or proliferation of hematopoietic stem cells (HSCs) in a concentration-dependent manner; however, in vivo Thpo serum levels are responsive to platelet mass rather than HSC demands, suggesting additional regulation exists. Ott1 (Rbm15), a spliceosomal component originally identified as a fusion partner in t(1;22)-associated acute megakaryocytic leukemia, is also essential for maintaining HSC quiescence under stress. Ott1 controls the alternative splicing of a dominant negative isoform, Mpl-TR, capable of inhibiting HSC engraftment and attenuating Thpo signaling. Ott1, which associates with Hdac3 and the histone methyltransferase, Setd1b, binds to both c-Mpl RNA and chromatin and regulates H4 acetylation and H3K4me3 marks. Histone deacetylase or histone methyltransferase inhibition also increases Mpl-TR levels, suggesting that Ott1 uses an underlying epigenetic mechanism to control alternative splicing of c-Mpl. Manipulation of Ott1-dependent alternative splicing may therefore provide a novel pharmacologic avenue for regulating HSC quiescence and proliferation in response to Thpo. PMID:25468569

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.

A Sub-Element in PRE enhances nuclear export of intronless mRNAs by recruiting the TREX complex via ZC3H18

PubMed Central

Chi, Binkai; Wang, Ke; Du, Yanhua; Gui, Bin; Chang, Xingya; Wang, Lantian; Fan, Jing; Chen, She; Wu, Xudong; Li, Guohui; Cheng, Hong

2014-01-01

Viral RNA elements that facilitate mRNA export are useful tools for identifying cellular RNA export factors. Here we show that hepatitis B virus post-transcriptional element (PRE) is one such element, and using PRE several new cellular mRNA export factors were identified. We found that PRE drastically enhances the cytoplasmic accumulation of cDNA transcripts independent of any viral protein. Systematic deletion analysis revealed the existence of a 116 nt functional Sub-Element of PRE (SEP1). The RNP that forms on the SEP1 RNA was affinity purified, in which TREX components as well as several other proteins were identified. TREX components and the SEP1-associating protein ZC3H18 are required for SEP1-mediated mRNA export. Significantly, ZC3H18 directly binds to the SEP1 RNA, interacts with TREX and is required for stable association of TREX with the SEP1-containing mRNA. Requirements for SEP1-mediated mRNA export are similar to those for splicing-dependent mRNA export. Consistent with these similarities, several SEP1-interacting proteins, including ZC3H18, ARS2, Acinus and Brr2, are required for efficient nuclear export of polyA RNAs. Together, our data indicate that SEP1 enhances mRNA export by recruiting TREX via ZC3H18. The new mRNA export factors that we identified might be involved in cap- and splicing-dependent TREX recruitment to cellular mRNAs. PMID:24782531

Genome-Wide Identification and Characterization of Warming-Related Genes in Brassica rapa ssp. pekinensis.

PubMed

Song, Hayoung; Dong, Xiangshu; Yi, Hankuil; Ahn, Ju Young; Yun, Keunho; Song, Myungchul; Han, Ching-Tack; Hur, Yoonkang

2018-06-11

For sustainable crop cultivation in the face of global warming, it is important to unravel the genetic mechanisms underlying plant adaptation to a warming climate and apply this information to breeding. Thermomorphogenesis and ambient temperature signaling pathways have been well studied in model plants, but little information is available for vegetable crops. Here, we investigated genes responsive to warming conditions from two Brassica rapa inbred lines with different geographic origins: subtropical (Kenshin) and temperate (Chiifu). Genes in Gene Ontology categories “response to heat”, “heat acclimation”, “response to light intensity”, “response to oxidative stress”, and “response to temperature stimulus” were upregulated under warming treatment in both lines, but genes involved in “response to auxin stimulus” were upregulated only in Kenshin under both warming and minor-warming conditions. We identified 16 putative high temperature (HT) adaptation-related genes, including 10 heat-shock response genes, 2 transcription factor genes, 1 splicing factor gene, and 3 others. BrPIF4 , BrROF2 , and BrMPSR1 are candidate genes that might function in HT adaptation. Auxin response, alternative splicing of BrHSFA2 , and heat shock memory appear to be indispensable for HT adaptation in B. rapa . These results lay the foundation for molecular breeding and marker development to improve warming tolerance in B. rapa .

The IRE1/bZIP60 pathway and Bax inhibitor 1 suppress systemic accumulation of potyviruses and potexviruses in Arabidopsis and Nicotiana benthamiana plants

USDA-ARS?s Scientific Manuscript database

The inositol requiring enzyme (IRE1) is an endoplasmic reticulum (ER) stress sensor and when activated it splices the bZIP60 mRNA producing a truncated transcription factor that upregulates expression of genes involved in the unfolded protein response (UPR). Bax inhibitor 1 (BI-1) is another ER stre...

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

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.

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

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.

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

Methylation of eukaryotic elongation factor 2 induced by basic fibroblast growth factor via mitogen-activated protein kinase.

PubMed

Jung, Gyung Ah; Shin, Bong Shik; Jang, Yeon Sue; Sohn, Jae Bum; Woo, Seon Rang; Kim, Jung Eun; Choi, Go; Lee, Kyung Mi; Min, Bon Hong; Lee, Kee Ho; Park, Gil Hong

2011-10-31

Protein arginine methylation is important for a variety of cellular processes including transcriptional regulation, mRNA splicing, DNA repair, nuclear/cytoplasmic shuttling and various signal transduction pathways. However, the role of arginine methylation in protein biosynthesis and the extracellular signals that control arginine methylation are not fully understood. Basic fibroblast growth factor (bFGF) has been identified as a potent stimulator of myofibroblast dedifferentiation into fibroblasts. We demonstrated that symmetric arginine dimethylation of eukaryotic elongation factor 2 (eEF2) is induced by bFGF without the change in the expression level of eEF2 in mouse embryo fibroblast NIH3T3 cells. The eEF2 methylation is preceded by ras-raf-mitogen-activated protein kinase kinase (MEK)-extracellular signal-regulated kinase (ERK1/2)- p21Cip/WAF1 activation, and suppressed by the mitogenactivated protein kinase (MAPK) inhibitor PD98059 and p21Cip/WAF1 short interfering RNA (siRNA). We determined that protein arginine methyltransferase 7 (PRMT7) is responsible for the methylation, and that PRMT5 acts as a coordinator. Collectively, we demonstrated that eEF2, a key factor involved in protein translational elongation is symmetrically arginine-methylated in a reversible manner, being regulated by bFGF through MAPK signaling pathway.

Methylation of eukaryotic elongation factor 2 induced by basic fibroblast growth factor via mitogen-activated protein kinase

PubMed Central

Jung, Gyung Ah; Shin, Bong Shik; Jang, Yeon Sue; Sohn, Jae Bum; Woo, Seon Rang; Kim, Jung Eun; Choi, Go; Lee, Kyung-Mi; Min, Bon Hong

2011-01-01

Protein arginine methylation is important for a variety of cellular processes including transcriptional regulation, mRNA splicing, DNA repair, nuclear/cytoplasmic shuttling and various signal transduction pathways. However, the role of arginine methylation in protein biosynthesis and the extracellular signals that control arginine methylation are not fully understood. Basic fibroblast growth factor (bFGF) has been identified as a potent stimulator of myofibroblast dedifferentiation into fibroblasts. We demonstrated that symmetric arginine dimethylation of eukaryotic elongation factor 2 (eEF2) is induced by bFGF without the change in the expression level of eEF2 in mouse embryo fibroblast NIH3T3 cells. The eEF2 methylation is preceded by ras-raf-mitogen-activated protein kinase kinase (MEK)-extracellular signal-regulated kinase (ERK1/2)-p21Cip/WAF1 activation, and suppressed by the mitogen-activated protein kinase (MAPK) inhibitor PD98059 and p21Cip/WAF1 short interfering RNA (siRNA). We determined that protein arginine methyltransferase 7 (PRMT7) is responsible for the methylation, and that PRMT5 acts as a coordinator. Collectively, we demonstrated that eEF2, a key factor involved in protein translational elongation is symmetrically arginine-methylated in a reversible manner, being regulated by bFGF through MAPK signaling pathway. PMID:21778808

VEGF111b, a new member of VEGFxxxb isoforms and induced by mitomycin C, inhibits angiogenesis

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

Gu, Fang; Li, Xiuli; Kong, Jian

2013-11-08

Highlights: •We discovered a new member of VEGFxxxb family-VEGF111b. •We found VEGF111b mRNA and protein can be induced by mitomycin C. •We confirmed VEGF111b over-expression inhibits angiogenesis. •VEGF111b inhibits angiogenesis through inhibiting VEGF-R2/PI3K/Akt and VEGF-R2/ERK1/2 phosphorylation. -- Abstract: Vascular endothelial growth factor (VEGF-A) stimulating angiogenesis is required for tumor growth and progression. The conventional VEGF-A isoforms have been considered as pro-angiogenic factors. Another family of VEGF-A isoforms generated by alternative splicing, termed VEGFxxxb isoforms, has anti-angiogenic property, exemplified by VEGF165b. Here, we identify a new number of VEGFxxx family-VEGF111b induced by mitomycin C, although not detected in mitomycin C-unexposed ovarianmore » cancer cells. SKOV3 cells were transfected with pcDNA{sub 3.1} empty vector, pcDNA{sub 3.1}-VEGF111b or pcDNA{sub 3.1}-VEGF165b to collect conditioned mediums respectively. VEGF111b overexpression inhibits proliferation, migration and tube formation of endothelial cell by inhibiting VEGF-R2 phosphorylation and its downstream signaling, similar to VEGF165b but slightly lower than VEGF165b. The anti-angiogenic property depends on the six amino acids of exon 8b of the VEGFxxxb isoforms. Our results show that VEGF111b is a novel potent anti-angiogenic agent that can target the VEGF-R2 and its signaling pathway to inhibit ovarian tumor growth.« less

A short conserved motif in ALYREF directs cap- and EJC-dependent assembly of export complexes on spliced mRNAs

PubMed Central

Gromadzka, Agnieszka M.; Steckelberg, Anna-Lena; Singh, Kusum K.; Hofmann, Kay; Gehring, Niels H.

2016-01-01

The export of messenger RNAs (mRNAs) is the final of several nuclear posttranscriptional steps of gene expression. The formation of export-competent mRNPs involves the recruitment of export factors that are assumed to facilitate transport of the mature mRNAs. Using in vitro splicing assays, we show that a core set of export factors, including ALYREF, UAP56 and DDX39, readily associate with the spliced RNAs in an EJC (exon junction complex)- and cap-dependent manner. In order to elucidate how ALYREF and other export adaptors mediate mRNA export, we conducted a computational analysis and discovered four short, conserved, linear motifs present in RNA-binding proteins. We show that mutation in one of the new motifs (WxHD) in an unstructured region of ALYREF reduced RNA binding and abolished the interaction with eIF4A3 and CBP80. Additionally, the mutation impaired proper localization to nuclear speckles and export of a spliced reporter mRNA. Our results reveal important details of the orchestrated recruitment of export factors during the formation of export competent mRNPs. PMID:26773052

Crystal Structure of the Extracellular Cholinesterase-Like Domain from Neuroligin-2

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

Koehnke,J.; Jin, X.; Budreck, E.

Neuroligins (NLs) are catalytically inactive members of a family of cholinesterase-like transmembrane proteins that mediate cell adhesion at neuronal synapses. Postsynaptic neuroligins engage in Ca2+-dependent transsynaptic interactions via their extracellular cholinesterase domain with presynaptic neurexins (NRXs). These interactions may be regulated by two short splice insertions (termed A and B) in the NL cholinesterase domain. Here, we present the 3.3- Angstroms crystal structure of the ectodomain from NL2 containing splice insertion A (NL2A). The overall structure of NL2A resembles that of cholinesterases, but several structural features are unique to the NL proteins. First, structural elements surrounding the esterase active-site regionmore » differ significantly between active esterases and NL2A. On the opposite surface of the NL2A molecule, the positions of the A and B splice insertions identify a candidate NRX interaction site of the NL protein. Finally, sequence comparisons of NL isoforms allow for mapping the location of residues of previously identified mutations in NL3 and NL4 found in patients with autism spectrum disorders. Overall, the NL2 structure promises to provide a valuable model for dissecting NL isoform- and synapse-specific functions.« less

Crystal structure of the extracellular cholinesterase-like domain from neuroligin-2

PubMed Central

Koehnke, Jesko; Jin, Xiangshu; Budreck, Elaine C.; Posy, Shoshana; Scheiffele, Peter; Honig, Barry; Shapiro, Lawrence

2008-01-01

Neuroligins (NLs) are catalytically inactive members of a family of cholinesterase-like transmembrane proteins that mediate cell adhesion at neuronal synapses. Postsynaptic neuroligins engage in Ca2+-dependent transsynaptic interactions via their extracellular cholinesterase domain with presynaptic neurexins (NRXs). These interactions may be regulated by two short splice insertions (termed A and B) in the NL cholinesterase domain. Here, we present the 3.3-Å crystal structure of the ectodomain from NL2 containing splice insertion A (NL2A). The overall structure of NL2A resembles that of cholinesterases, but several structural features are unique to the NL proteins. First, structural elements surrounding the esterase active-site region differ significantly between active esterases and NL2A. On the opposite surface of the NL2A molecule, the positions of the A and B splice insertions identify a candidate NRX interaction site of the NL protein. Finally, sequence comparisons of NL isoforms allow for mapping the location of residues of previously identified mutations in NL3 and NL4 found in patients with autism spectrum disorders. Overall, the NL2 structure promises to provide a valuable model for dissecting NL isoform- and synapse-specific functions. PMID:18250328

Antisense oligonucleotide-induced alternative splicing of the APOB mRNA generates a novel isoform of APOB.

PubMed

Khoo, Bernard; Roca, Xavier; Chew, Shern L; Krainer, Adrian R

2007-01-17

Apolipoprotein B (APOB) is an integral part of the LDL, VLDL, IDL, Lp(a) and chylomicron lipoprotein particles. The APOB pre-mRNA consists of 29 constitutively-spliced exons. APOB exists as two natural isoforms: the full-length APOB100 isoform, assembled into LDL, VLDL, IDL and Lp(a) and secreted by the liver in humans; and the C-terminally truncated APOB48, assembled into chylomicrons and secreted by the intestine in humans. Down-regulation of APOB100 is a potential therapy to lower circulating LDL and cholesterol levels. We investigated the ability of 2'O-methyl RNA antisense oligonucleotides (ASOs) to induce the skipping of exon 27 in endogenous APOB mRNA in HepG2 cells. These ASOs are directed towards the 5' and 3' splice-sites of exon 27, the branch-point sequence (BPS) of intron 26-27 and several predicted exonic splicing enhancers within exon 27. ASOs targeting either the 5' or 3' splice-site, in combination with the BPS, are the most effective. The splicing of other alternatively spliced genes are not influenced by these ASOs, suggesting that the effects seen are not due to non-specific changes in alternative splicing. The skip 27 mRNA is translated into a truncated isoform, APOB87SKIP27. The induction of APOB87SKIP27 expression in vivo should lead to decreased LDL and cholesterol levels, by analogy to patients with hypobetalipoproteinemia. As intestinal APOB mRNA editing and APOB48 expression rely on sequences within exon 26, exon 27 skipping should not affect APOB48 expression unlike other methods of down-regulating APOB100 expression which also down-regulate APOB48.

Incorporating significant amino acid pairs and protein domains to predict RNA splicing-related proteins with functional roles

NASA Astrophysics Data System (ADS)

Hsu, Justin Bo-Kai; Huang, Kai-Yao; Weng, Tzu-Ya; Huang, Chien-Hsun; Lee, Tzong-Yi

2014-01-01

Machinery of pre-mRNA splicing is carried out through the interaction of RNA sequence elements and a variety of RNA splicing-related proteins (SRPs) (e.g. spliceosome and splicing factors). Alternative splicing, which is an important post-transcriptional regulation in eukaryotes, gives rise to multiple mature mRNA isoforms, which encodes proteins with functional diversities. However, the regulation of RNA splicing is not yet fully elucidated, partly because SRPs have not yet been exhaustively identified and the experimental identification is labor-intensive. Therefore, we are motivated to design a new method for identifying SRPs with their functional roles in the regulation of RNA splicing. The experimentally verified SRPs were manually curated from research articles. According to the functional annotation of Splicing Related Gene Database, the collected SRPs were further categorized into four functional groups including small nuclear Ribonucleoprotein, Splicing Factor, Splicing Regulation Factor and Novel Spliceosome Protein. The composition of amino acid pairs indicates that there are remarkable differences among four functional groups of SRPs. Then, support vector machines (SVMs) were utilized to learn the predictive models for identifying SRPs as well as their functional roles. The cross-validation evaluation presents that the SVM models trained with significant amino acid pairs and functional domains could provide a better predictive performance. In addition, the independent testing demonstrates that the proposed method could accurately identify SRPs in mammals/plants as well as effectively distinguish between SRPs and RNA-binding proteins. This investigation provides a practical means to identifying potential SRPs and a perspective for exploring the regulation of RNA splicing.

Incorporating significant amino acid pairs and protein domains to predict RNA splicing-related proteins with functional roles.

PubMed

Hsu, Justin Bo-Kai; Huang, Kai-Yao; Weng, Tzu-Ya; Huang, Chien-Hsun; Lee, Tzong-Yi

2014-01-01

Machinery of pre-mRNA splicing is carried out through the interaction of RNA sequence elements and a variety of RNA splicing-related proteins (SRPs) (e.g. spliceosome and splicing factors). Alternative splicing, which is an important post-transcriptional regulation in eukaryotes, gives rise to multiple mature mRNA isoforms, which encodes proteins with functional diversities. However, the regulation of RNA splicing is not yet fully elucidated, partly because SRPs have not yet been exhaustively identified and the experimental identification is labor-intensive. Therefore, we are motivated to design a new method for identifying SRPs with their functional roles in the regulation of RNA splicing. The experimentally verified SRPs were manually curated from research articles. According to the functional annotation of Splicing Related Gene Database, the collected SRPs were further categorized into four functional groups including small nuclear Ribonucleoprotein, Splicing Factor, Splicing Regulation Factor and Novel Spliceosome Protein. The composition of amino acid pairs indicates that there are remarkable differences among four functional groups of SRPs. Then, support vector machines (SVMs) were utilized to learn the predictive models for identifying SRPs as well as their functional roles. The cross-validation evaluation presents that the SVM models trained with significant amino acid pairs and functional domains could provide a better predictive performance. In addition, the independent testing demonstrates that the proposed method could accurately identify SRPs in mammals/plants as well as effectively distinguish between SRPs and RNA-binding proteins. This investigation provides a practical means to identifying potential SRPs and a perspective for exploring the regulation of RNA splicing.

Functional Genomic Screening Reveals Splicing of the EWS-FLI1 Fusion Transcript as a Vulnerability in Ewing Sarcoma.

PubMed

Grohar, Patrick J; Kim, Suntae; Rangel Rivera, Guillermo O; Sen, Nirmalya; Haddock, Sara; Harlow, Matt L; Maloney, Nichole K; Zhu, Jack; O'Neill, Maura; Jones, Tamara L; Huppi, Konrad; Grandin, Magdalena; Gehlhaus, Kristen; Klumpp-Thomas, Carleen A; Buehler, Eugen; Helman, Lee J; Martin, Scott E; Caplen, Natasha J

2016-01-26

Ewing sarcoma cells depend on the EWS-FLI1 fusion transcription factor for cell survival. Using an assay of EWS-FLI1 activity and genome-wide RNAi screening, we have identified proteins required for the processing of the EWS-FLI1 pre-mRNA. We show that Ewing sarcoma cells harboring a genomic breakpoint that retains exon 8 of EWSR1 require the RNA-binding protein HNRNPH1 to express in-frame EWS-FLI1. We also demonstrate the sensitivity of EWS-FLI1 fusion transcripts to the loss of function of the U2 snRNP component, SF3B1. Disrupted splicing of the EWS-FLI1 transcript alters EWS-FLI1 protein expression and EWS-FLI1-driven expression. Our results show that the processing of the EWS-FLI1 fusion RNA is a potentially targetable vulnerability in Ewing sarcoma cells. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Structure, dynamics and RNA binding of the multi-domain splicing factor TIA-1

PubMed Central

Wang, Iren; Hennig, Janosch; Jagtap, Pravin Kumar Ankush; Sonntag, Miriam; Valcárcel, Juan; Sattler, Michael

2014-01-01

Alternative pre-messenger ribonucleic acid (pre-mRNA) splicing is an essential process in eukaryotic gene regulation. The T-cell intracellular antigen-1 (TIA-1) is an apoptosis-promoting factor that modulates alternative splicing of transcripts, including the pre-mRNA encoding the membrane receptor Fas. TIA-1 is a multi-domain ribonucleic acid (RNA) binding protein that recognizes poly-uridine tract RNA sequences to facilitate 5′ splice site recognition by the U1 small nuclear ribonucleoprotein (snRNP). Here, we characterize the RNA interaction and conformational dynamics of TIA-1 by nuclear magnetic resonance (NMR), isothermal titration calorimetry (ITC) and small angle X-ray scattering (SAXS). Our NMR-derived solution structure of TIA-1 RRM2–RRM3 (RRM2,3) reveals that RRM2 adopts a canonical RNA recognition motif (RRM) fold, while RRM3 is preceded by an non-canonical helix α0. NMR and SAXS data show that all three RRMs are largely independent structural modules in the absence of RNA, while RNA binding induces a compact arrangement. RRM2,3 binds to pyrimidine-rich FAS pre-mRNA or poly-uridine (U9) RNA with nanomolar affinities. RRM1 has little intrinsic RNA binding affinity and does not strongly contribute to RNA binding in the context of RRM1,2,3. Our data unravel the role of binding avidity and the contributions of the TIA-1 RRMs for recognition of pyrimidine-rich RNAs. PMID:24682828

Comprehensive analysis of titin protein isoform and alternative splicing in normal and mutant rats.

PubMed

Li, Shijun; Guo, Wei; Schmitt, Benjamin M; Greaser, Marion L

2012-04-01

Titin is a giant protein with multiple functions in cardiac and skeletal muscles. Rat cardiac titin undergoes developmental isoform transition from the neonatal 3.7 MDa N2BA isoform to primarily the adult 2.97 MDa N2B isoform. An autosomal dominant mutation dramatically altered this transformation. Titins from eight skeletal muscles: Tibialis Anterior (TA), Longissimus Dorsi (LD) and Gastrocnemius (GA), Extensor Digitorum Longus (ED), Soleus (SO), Psoas (PS), Extensor Oblique (EO), and Diaphram (DI) were characterized in wild type and in homozygous mutant (Hm) rats with a titin splicing defect. Results showed that the developmental reduction in titin size is eliminated in the mutant rat so that the titins in all investigated skeletal muscles remain large in the adult. The alternative splicing of titin mRNA was found repressed by this mutation, a result consistent with the large titin isoform in the mutant. The developmental pattern of titin mRNA alternative splicing differs between heart and skeletal muscles. The retention of intron 49 reveals a possible mechanism for the absence of the N2B unique region in the expressed titin protein of skeletal muscle. © 2011 Wiley Periodicals, Inc.

Spliced RNA of woodchuck hepatitis virus.

PubMed

Ogston, C W; Razman, D G

1992-07-01

Polymerase chain reaction was used to investigate RNA splicing in liver of woodchucks infected with woodchuck hepatitis virus (WHV). Two spliced species were detected, and the splice junctions were sequenced. The larger spliced RNA has an intron of 1300 nucleotides, and the smaller spliced sequence shows an additional downstream intron of 1104 nucleotides. We did not detect singly spliced sequences from which the smaller intron alone was removed. Control experiments showed that spliced sequences are present in both RNA and DNA in infected liver, showing that the viral reverse transcriptase can use spliced RNA as template. Spliced sequences were detected also in virion DNA prepared from serum. The upstream intron produces a reading frame that fuses the core to the polymerase polypeptide, while the downstream intron causes an inframe deletion in the polymerase open reading frame. Whereas the splicing patterns in WHV are superficially similar to those reported recently in hepatitis B virus, we detected no obvious homology in the coding capacity of spliced RNAs from these two viruses.

Unusual splice site mutations disrupt FANCA exon 8 definition.

PubMed

Mattioli, Chiara; Pianigiani, Giulia; De Rocco, Daniela; Bianco, Anna Monica Rosaria; Cappelli, Enrico; Savoia, Anna; Pagani, Franco

2014-07-01

The pathological role of mutations that affect not conserved splicing regulatory sequences can be difficult to determine. In a patient with Fanconi anemia, we identified two unpredictable splicing mutations that act on either sides of FANCA exon 8. In patients-derived cells and in minigene splicing assay, we showed that both an apparently benign intronic c.710-5T>C transition and the nonsense c.790C>T substitution induce almost complete exon 8 skipping. Site-directed mutagenesis experiments indicated that the c.710-5T>C transition affects a polypyrimidine tract where most of the thymidines cannot be compensated by cytidines. The c.790C>T mutation located in position -3 relative to the donor site induce exon 8 skipping in an NMD-independent manner and complementation experiments with modified U1 snRNAs showed that U1 snRNP is only partially involved in the splicing defect. Our results highlight the importance of performing splicing functional assay for correct identification of disease-causing mechanism of genomic variants and provide mechanistic insights on how these two FANCA mutations affect exon 8 definition. Copyright © 2014 Elsevier B.V. All rights reserved.

Reflections on the history of pre-mRNA processing and highlights of current knowledge: A unified picture

PubMed Central

Darnell, James E.

2013-01-01

Several strong conclusions emerge concerning pre-mRNA processing from both old and newer experiments. The RNAPII complex is involved with pre-mRNA processing through binding of processing proteins to the CTD (carboxyl terminal domain) of the largest RNAPII subunit. These interactions are necessary for efficient processing, but whether factor binding to the CTD and delivery to splicing sites is obligatory or facilitatory is unsettled. Capping, addition of an m7Gppp residue (cap) to the initial transcribed residue of a pre-mRNA, occurs within seconds. Splicing of pre-mRNA by spliceosomes at particular sites is most likely committed during transcription by the binding of initiating processing factors and ∼50% of the time is completed in mammalian cells before completion of the primary transcript. This fact has led to an outpouring in the literature about “cotranscriptional splicing.” However splicing requires several minutes for completion and can take longer. The RNAPII complex moves through very long introns and also through regions dense with alternating exons and introns at an average rate of ∼3 kb per min and is, therefore, not likely detained at each splice site for more than a few seconds, if at all. Cleavage of the primary transcript at the 3′ end and polyadenylation occurs within 30 sec or less at recognized polyA sites, and the majority of newly polyadenylated pre-mRNA molecules are much larger than the average mRNA. Finally, it seems quite likely that the nascent RNA most often remains associated with the chromosomal locus being transcribed until processing is complete, possibly acquiring factors related to the transport of the new mRNA to the cytoplasm. PMID:23440351

Factors influencing alternative splice site utilization in vivo.

PubMed Central

Fu, X Y; Manley, J L

1987-01-01

To study factors that influence the choice of alternative pre-mRNA splicing pathways, we introduced plasmids expressing either wild-type or mutated simian virus 40 (SV40) early regions into tissue culture cells and then measured the quantities of small-t and large-T RNAs produced. One important element controlling splice site selection was found to be the size of the intron removed in the production of small-t mRNA; expansion of this intron (from 66 to 77 or more nucleotides) resulted in a substantial increase in the amount of small-t mRNA produced relative to large-T mRNA. This suggests that in the normal course of SV40 early pre-mRNA processing, large-T splicing is at a competitive advantage relative to small-t splicing because of the small size of the latter intron. Several additional features of the pre-mRNA that can influence splice site selection were also identified by analyzing the effects of mutations containing splice site duplications. These include the strengths of competing 5' splice sites and the relative positions of splice sites in the pre-mRNA. Finally, we showed that the ratio of small-t to large-T mRNA was 10 to 15-fold greater in human 293 cells than in HeLa cells or other mammalian cell types. These results suggest the existence of cell-specific trans-acting factors that can dramatically alter the pattern of splice site selection in a pre-mRNA. Images PMID:3029566

Pre-mRNA splicing in cancer: the relevance in oncogenesis, treatment and drug resistance.

PubMed

Wojtuszkiewicz, Anna; Assaraf, Yehuda G; Maas, Marielle J P; Kaspers, Gertjan J L; Jansen, Gerrit; Cloos, Jacqueline

2015-05-01

Aberrant pre-mRNA splicing in cancer is emerging as an important determinant of oncogenesis, response to treatment and anticancer drug resistance. At the same time, the spliceosome has become a target for a novel class of pre-clinical chemotherapeutics with a potential future application in cancer treatment. Taken together, these findings offer novel opportunities for the enhancement of the efficacy of cancer therapy. This review presents a comprehensive overview of the molecular mechanisms involved in splicing and current developments regarding splicing aberrations in relation to several aspects of cancer formation and therapy. Identified mutations in the various components of the spliceosome and their implications for cancer prognosis are delineated. Moreover, the contribution of abnormal splicing patterns as well as deregulated splicing factors to chemoresistance is discussed, along with novel splicing-based therapeutic approaches. Significant progress has been made in deciphering the role of splicing factors in cancer including carcinogenesis and drug resistance. Splicing-based prognostic tools as well as therapeutic options hold great potential towards improvements in cancer therapy. However, gaining more in-depth molecular insight into the consequences of mutations in various components of the splicing machinery as well as of cellular effects of spliceosome inhibition is a prerequisite to establish the role of splicing in tumor progression and treatment options, respectively.

Mutations in PRPF31 Inhibit Pre-mRNA Splicing of Rhodopsin Gene and Cause Apoptosis of Retinal Cells

PubMed Central

Yuan, Liya; Kawada, Mariko; Havlioglu, Necat; Tang, Hao; Wu, Jane Y.

2007-01-01

Mutations in human PRPF31 gene have been identified in patients with autosomal dominant retinitis pigmentosa (adRP). To begin to understand mechanisms by which defects in this general splicing factor cause retinal degeneration, we examined the relationship between PRPF31 and pre-mRNA splicing of photoreceptor-specific genes. We used a specific anti-PRPF31 antibody to immunoprecipitate splicing complexes from retinal cells and identified the transcript of rhodopsin gene (RHO) among RNA species associated with PRPF31-containing complexes. Mutant PRPF31 proteins significantly inhibited pre-mRNA splicing of intron 3 in RHO gene. In primary retinal cell cultures, expression of the mutant PRPF31 proteins reduced rhodopsin expression and caused apoptosis of rhodopsin-positive retinal cells. This primary retinal culture assay provides an in vitro model to study photoreceptor cell death caused by PRPF31 mutations. Our results demonstrate that mutations in PRPF31 gene affect RHO pre-mRNA splicing and reveal a link between PRPF31 and RHO, two major adRP genes. PMID:15659613

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

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

Splicing Ge-doped photonic crystal fibers using commercial fusion splicer with default discharge parameters.

PubMed

Wang, Yiping; Bartelt, Hartmut; Brueckner, Sven; Kobelke, Jens; Rothhardt, Manfred; Mörl, Klaus; Ecke, Wolfgang; Willsch, Reinhardt

2008-05-12

A novel technique for splicing a small core Ge-doped photonic crystal fiber (PCF) was demonstrated using a commercial fusion splicer with default discharge parameters for the splicing of two standard single mode fibers (SMFs). Additional discharge parameter adjustments are not required to splice the PCF to several different SMFs. A low splice loss of 1.0 approximately 1.4 dB is achieved. Low or no light reflection is expected at the splice joint due to the complete fusion of the two fiber ends. The splice joint has a high bending strength and does not break when the bending radius is decreased to 4 mm.

HBV X Protein induces overexpression of HERV-W env through NF-κB in HepG2 cells.

PubMed

Liu, Cong; Liu, Lijuan; Wang, Xiuling; Liu, Youyi; Wang, Miao; Zhu, Fan

2017-12-01

Human endogenous retrovirus W family (HERV-W) envelope (env) at chromosome 7 is highly expressed in the placenta and possesses fusogenic activity in trophoblast development. HERV-W env has been found to be overexpressed in some cancers and immune diseases. Viral transactivators can induce the overexpression of HERV-W env in human cell lines. Hepatitis B virus X protein (HBx) is believed to be a multifunctional oncogenic protein. Here, we reported that HBx could increase the promoter activity of HERV-W env and upregulate the mRNA levels of non-spliced and spliced HERV-W env and also its protein in human hepatoma HepG2 cells. Interestingly, we found that the inhibition of nuclear factor κB (NF-κB) using shRNA targeting NF-κB/p65 or PDTC (an inhibitor of NF-κB) could attenuate the upregulation of HERV-W env induced by HBx. These suggested that HBx might upregulate the expression of HERV-W env through NF-κB in HepG2 cells. This study might provide a new insight in HBV-associated liver diseases including HCC.

A Comprehensive Analysis of Alternative Splicing in Paleopolyploid Maize.

PubMed

Mei, Wenbin; Liu, Sanzhen; Schnable, James C; Yeh, Cheng-Ting; Springer, Nathan M; Schnable, Patrick S; Barbazuk, William B

2017-01-01

Identifying and characterizing alternative splicing (AS) enables our understanding of the biological role of transcript isoform diversity. This study describes the use of publicly available RNA-Seq data to identify and characterize the global diversity of AS isoforms in maize using the inbred lines B73 and Mo17, and a related species, sorghum. Identification and characterization of AS within maize tissues revealed that genes expressed in seed exhibit the largest differential AS relative to other tissues examined. Additionally, differences in AS between the two genotypes B73 and Mo17 are greatest within genes expressed in seed. We demonstrate that changes in the level of alternatively spliced transcripts (intron retention and exon skipping) do not solely reflect differences in total transcript abundance, and we present evidence that intron retention may act to fine-tune gene expression across seed development stages. Furthermore, we have identified temperature sensitive AS in maize and demonstrate that drought-induced changes in AS involve distinct sets of genes in reproductive and vegetative tissues. Examining our identified AS isoforms within B73 × Mo17 recombinant inbred lines (RILs) identified splicing QTL (sQTL). The 43.3% of cis- sQTL regulated junctions are actually identified as alternatively spliced junctions in our analysis, while 10 Mb windows on each side of 48.2% of trans -sQTLs overlap with splicing related genes. Using sorghum as an out-group enabled direct examination of loss or conservation of AS between homeologous genes representing the two subgenomes of maize. We identify several instances where AS isoforms that are conserved between one maize homeolog and its sorghum ortholog are absent from the second maize homeolog, suggesting that these AS isoforms may have been lost after the maize whole genome duplication event. This comprehensive analysis provides new insights into the complexity of AS in maize.

Ott1 (Rbm15) regulates thrombopoietin response in hematopoietic stem cells through alternative splicing of c-Mpl.

PubMed

Xiao, Nan; Laha, Suparna; Das, Shankar P; Morlock, Kayla; Jesneck, Jonathan L; Raffel, Glen D

2015-02-05

Thrombopoietin (Thpo) signaling through the c-Mpl receptor promotes either quiescence or proliferation of hematopoietic stem cells (HSCs) in a concentration-dependent manner; however, in vivo Thpo serum levels are responsive to platelet mass rather than HSC demands, suggesting additional regulation exists. Ott1 (Rbm15), a spliceosomal component originally identified as a fusion partner in t(1;22)-associated acute megakaryocytic leukemia, is also essential for maintaining HSC quiescence under stress. Ott1 controls the alternative splicing of a dominant negative isoform, Mpl-TR, capable of inhibiting HSC engraftment and attenuating Thpo signaling. Ott1, which associates with Hdac3 and the histone methyltransferase, Setd1b, binds to both c-Mpl RNA and chromatin and regulates H4 acetylation and H3K4me3 marks. Histone deacetylase or histone methyltransferase inhibition also increases Mpl-TR levels, suggesting that Ott1 uses an underlying epigenetic mechanism to control alternative splicing of c-Mpl. Manipulation of Ott1-dependent alternative splicing may therefore provide a novel pharmacologic avenue for regulating HSC quiescence and proliferation in response to Thpo. © 2015 by The American Society of Hematology.

Regulation of insulin preRNA splicing by glucose

PubMed Central

Wang, Juehu; Shen, Luping; Najafi, Habiba; Kolberg, Janice; Matschinsky, Franz M.; Urdea, Mickey; German, Michael

1997-01-01

Glucose tightly regulates the synthesis and secretion of insulin by β cells in the pancreatic islets of Langerhans. To investigate whether glucose regulates insulin synthesis at the level of insulin RNA splicing, we developed a method to detect and quantify a small amount of RNA by using the branched DNA (bDNA) signal-amplification technique. This assay is both sensitive and highly specific: mouse insulin II mRNA can be detected from a single β cell (βTC3 cells or mouse islets), whereas 1 million non-insulin-producing α cells (αTC1.6 cells) give no signal. By using intron and exon sequences, oligonucleotide probes were designed to distinguish the various unspliced and partially spliced insulin preRNAs from mature insulin mRNA. Insulin RNA splicing rates were estimated from the rate of disappearance of insulin preRNA signal from β cells treated with actinomycin D to block transcription. We found that the two introns in mouse insulin II are not spliced with the same efficiency. Intron 2 is spliced out more efficiently than intron 1. As a result, some mRNA retaining intron 1 enters the cytoplasm, making up ≈2-10% of insulin mRNA in the cell. This partially spliced cytoplasmic mRNA is quite stable, with a half-life similar to the completely spliced form. When islets grown in high glucose are shifted to low glucose medium, the level of insulin preRNA and the rate of splicing fall significantly. We conclude that glucose stimulates insulin gene transcription and insulin preRNA splicing. Previous estimates of insulin transcription rates based on insulin preRNA levels that did not consider the rate of splicing may have underestimated the effect of glucose on insulin gene transcription. PMID:9113994

Tumor suppressive microRNA-1 mediated novel apoptosis pathways through direct inhibition of splicing factor serine/arginine-rich 9 (SRSF9/SRp30c) in bladder cancer

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

Yoshino, Hirofumi; Enokida, Hideki, E-mail: enokida@m.kufm.kagoshima-u.ac.jp; Chiyomaru, Takeshi

2012-01-06

Highlights: Black-Right-Pointing-Pointer Tumor suppressive miRNA-1 directly inhibits splicing factor serine/arginine-rich 9 (SRSF9). Black-Right-Pointing-Pointer SRSF9 mRNA expression was up-regulated in bladder cancer specimens compared to normal tissues. Black-Right-Pointing-Pointer Cell viability (proliferation, migration, and invasion) was reduced in SRSF9 knockdown cells. Black-Right-Pointing-Pointer SRSF9 knockdown by miR-1 induced cell apoptosis through caspase-3/7 activation in BC cell lines. -- Abstract: We have previously found that restoration of tumor suppressive microRNA-1 (miR-1), induced cell apoptosis in bladder cancer (BC) cell lines. However, the apoptosis mechanism induced by miR-1 was not fully elucidated. Alternative splicing of mRNA precursors provides cancer cells with opportunities to translate manymore » oncogenic protein variants, which promote cell proliferation and survival under unpreferable condition for cancer development. Serine/arginine-rich (SR) protein family, which involved in alternative pre-mRNA splicing, plays a critical role for regulating apoptosis by splicing apoptosis-related genes. However, transcriptional regulation of SR proteins, themselves, has not been elucidated. In this study, we focused on splicing factor serine/arginine-rich 9 (SRSF9/SRp30c) on the basis of our previous genome-wide gene expression analysis using miR-1-transfected BC cell lines because putative target sites of miR-1 are existed in 3 Prime -untranslated region (UTR) of SRSF9 mRNA. The expression levels of mRNA of SRSF9 were extremely reduced in the miR-1 transfectants. A luciferase activity significantly decreased in the transfectants suggesting that actual binding occurred between miR-1 and 3 Prime UTR of SRSF9 mRNA. Loss-of-function assays demonstrated that significant inhibitions of cell proliferation, migration, and invasion were observed in the si-SRSF9 transfectants. Apoptosis assays demonstrated that cell apoptosis fraction increased and that caspase-3/7 was activated in the si-SRSF9 transfectants. Our data indicated that tumor suppressive miR-1 induces apoptosis through direct inhibition of SRSF9 in BC. The identification of molecular mechanisms between miRNAs and SR proteins could provide novel apoptosis pathways and their epigenetic regulations and offer new strategies for BC treatment.« less

Mammalian splicing factor SF1 interacts with SURP domains of U2 snRNP-associated proteins

PubMed Central

Crisci, Angela; Raleff, Flore; Bagdiul, Ivona; Raabe, Monika; Urlaub, Henning; Rain, Jean-Christophe; Krämer, Angela

2015-01-01

Splicing factor 1 (SF1) recognizes the branch point sequence (BPS) at the 3′ splice site during the formation of early complex E, thereby pre-bulging the BPS adenosine, thought to facilitate subsequent base-pairing of the U2 snRNA with the BPS. The 65-kDa subunit of U2 snRNP auxiliary factor (U2AF65) interacts with SF1 and was shown to recruit the U2 snRNP to the spliceosome. Co-immunoprecipitation experiments of SF1-interacting proteins from HeLa cell extracts shown here are consistent with the presence of SF1 in early splicing complexes. Surprisingly almost all U2 snRNP proteins were found associated with SF1. Yeast two-hybrid screens identified two SURP domain-containing U2 snRNP proteins as partners of SF1. A short, evolutionarily conserved region of SF1 interacts with the SURP domains, stressing their role in protein–protein interactions. A reduction of A complex formation in SF1-depleted extracts could be rescued with recombinant SF1 containing the SURP-interaction domain, but only partial rescue was observed with SF1 lacking this sequence. Thus, SF1 can initially recruit the U2 snRNP to the spliceosome during E complex formation, whereas U2AF65 may stabilize the association of the U2 snRNP with the spliceosome at later times. In addition, these findings may have implications for alternative splicing decisions. PMID:26420826

Two Genetic Determinants Acquired Late in Mus Evolution Regulate the Inclusion of Exon 5, which Alters Mouse APOBEC3 Translation Efficiency

PubMed Central

Li, Jun; Hakata, Yoshiyuki; Takeda, Eri; Liu, Qingping; Iwatani, Yasumasa; Kozak, Christine A.; Miyazawa, Masaaki

2012-01-01

Mouse apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like editing complex 3 (mA3), an intracellular antiviral factor, has 2 allelic variations that are linked with different susceptibilities to beta- and gammaretrovirus infections among various mouse strains. In virus-resistant C57BL/6 (B6) mice, mA3 transcripts are more abundant than those in susceptible BALB/c mice both in the spleen and bone marrow. These strains of mice also express mA3 transcripts with different splicing patterns: B6 mice preferentially express exon 5-deficient (Δ5) mA3 mRNA, while BALB/c mice produce exon 5-containing full-length mA3 mRNA as the major transcript. Although the protein product of the Δ5 mRNA exerts stronger antiretroviral activities than the full-length protein, how exon 5 affects mA3 antiviral activity, as well as the genetic mechanisms regulating exon 5 inclusion into the mA3 transcripts, remains largely uncharacterized. Here we show that mA3 exon 5 is indeed a functional element that influences protein synthesis at a post-transcriptional level. We further employed in vitro splicing assays using genomic DNA clones to identify two critical polymorphisms affecting the inclusion of exon 5 into mA3 transcripts: the number of TCCT repeats upstream of exon 5 and the single nucleotide polymorphism within exon 5 located 12 bases upstream of the exon 5/intron 5 boundary. Distribution of the above polymorphisms among different Mus species indicates that the inclusion of exon 5 into mA3 mRNA is a relatively recent event in the evolution of mice. The widespread geographic distribution of this exon 5-including genetic variant suggests that in some Mus populations the cost of maintaining an effective but mutagenic enzyme may outweigh its antiviral function. PMID:22275865

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.

A splice junction-targeted CRISPR approach (spJCRISPR) reveals human FOXO3B to be a protein-coding gene.

PubMed

Santo, Evan E; Paik, Jihye

2018-06-17

The rapid development of CRISPR technology is revolutionizing molecular approaches to the dissection of complex biological phenomena. Here we describe an alternative generally applicable implementation of the CRISPR-Cas9 system that allows for selective knockdown of extremely homologous genes. This strategy employs the lentiviral delivery of paired sgRNAs and nickase Cas9 (Cas9D10A) to achieve targeted deletion of splice junctions. This general strategy offers several advantages over standard single-guide exon-targeting CRISPR-Cas9 such as greatly reduced off-target effects, more restricted genomic editing, routine disruption of target gene mRNA expression and the ability to differentiate between closely related genes. Here we demonstrate the utility of this strategy by achieving selective knockdown of the highly homologous human genes FOXO3A and suspected pseudogene FOXO3B. We find the spJCRISPR strategy to efficiently and selectively disrupt FOXO3A and FOXO3B mRNA and protein expression; thus revealing that the human FOXO3B locus encodes a bona fide human gene. Unlike FOXO3A, we find the FOXO3B protein to be cytosolically localized in both the presence and absence of active Akt. The ability to selectively target and efficiently disrupt the expression of the closely-related FOXO3A and FOXO3B genes demonstrates the efficacy of the spJCRISPR approach. Copyright © 2018. Published by Elsevier B.V.

Perispeckles are major assembly sites for the exon junction core complex

PubMed Central

Daguenet, Elisabeth; Baguet, Aurélie; Degot, Sébastien; Schmidt, Ute; Alpy, Fabien; Wendling, Corinne; Spiegelhalter, Coralie; Kessler, Pascal; Rio, Marie-Christine; Le Hir, Hervé; Bertrand, Edouard; Tomasetto, Catherine

2012-01-01

The exon junction complex (EJC) is loaded onto mRNAs as a consequence of splicing and regulates multiple posttranscriptional events. MLN51, Magoh, Y14, and eIF4A3 form a highly stable EJC core, but where this tetrameric complex is assembled in the cell remains unclear. Here we show that EJC factors are enriched in domains that we term perispeckles and are visible as doughnuts around nuclear speckles. Fluorescence resonance energy transfer analyses and EJC assembly mutants show that perispeckles do not store free subunits, but instead are enriched for assembled cores. At the ultrastructural level, perispeckles are distinct from interchromatin granule clusters that may function as storage sites for splicing factors and intermingle with perichromatin fibrils, where nascent RNAs and active RNA Pol II are present. These results support a model in which perispeckles are major assembly sites for the tetrameric EJC core. This subnuclear territory thus represents an intermediate region important for mRNA maturation, between transcription sites and splicing factor reservoirs and assembly sites. PMID:22419818

MeCP2 regulates Tet1-catalyzed demethylation, CTCF binding, and learning-dependent alternative splicing of the BDNF gene in Turtle

PubMed Central

Zheng, Zhaoqing; Ambigapathy, Ganesh; Keifer, Joyce

2017-01-01

MECP2 mutations underlying Rett syndrome cause widespread misregulation of gene expression. Functions for MeCP2 other than transcriptional are not well understood. In an ex vivo brain preparation from the pond turtle Trachemys scripta elegans, an intraexonic splicing event in the brain-derived neurotrophic factor (BDNF) gene generates a truncated mRNA transcript in naïve brain that is suppressed upon classical conditioning. MeCP2 and its partners, splicing factor Y-box binding protein 1 (YB-1) and methylcytosine dioxygenase 1 (Tet1), bind to BDNF chromatin in naïve but dissociate during conditioning; the dissociation correlating with decreased DNA methylation. Surprisingly, conditioning results in new occupancy of BDNF chromatin by DNA insulator protein CCCTC-binding factor (CTCF), which is associated with suppression of splicing in conditioning. Knockdown of MeCP2 shows it is instrumental for splicing and inhibits Tet1 and CTCF binding thereby negatively impacting DNA methylation and conditioning-dependent splicing regulation. Thus, mutations in MECP2 can have secondary effects on DNA methylation and alternative splicing. DOI: http://dx.doi.org/10.7554/eLife.25384.001 PMID:28594324

New Phosphospecific Antibody Reveals Isoform-Specific Phosphorylation of CPEB3 Protein

PubMed Central

Sehgal, Kapil; Sylvester, Marc; Skubal, Magdalena; Josten, Michele; Steinhäuser, Christian; De Koninck, Paul; Theis, Martin

2016-01-01

Cytoplasmic Polyadenylation Element Binding proteins (CPEBs) are a family of polyadenylation factors interacting with 3’UTRs of mRNA and thereby regulating gene expression. Various functions of CPEBs in development, synaptic plasticity, and cellular senescence have been reported. Four CPEB family members of partially overlapping functions have been described to date, each containing a distinct alternatively spliced region. This region is highly conserved between CPEBs-2-4 and contains a putative phosphorylation consensus, overlapping with the exon seven of CPEB3. We previously found CPEBs-2-4 splice isoforms containing exon seven to be predominantly present in neurons, and the isoform expression pattern to be cell type-specific. Here, focusing on the alternatively spliced region of CPEB3, we determined that putative neuronal isoforms of CPEB3 are phosphorylated. Using a new phosphospecific antibody directed to the phosphorylation consensus we found Protein Kinase A and Calcium/Calmodulin-dependent Protein Kinase II to robustly phosphorylate CPEB3 in vitro and in primary hippocampal neurons. Interestingly, status epilepticus induced by systemic kainate injection in mice led to specific upregulation of the CPEB3 isoforms containing exon seven. Extensive analysis of CPEB3 phosphorylation in vitro revealed two other phosphorylation sites. In addition, we found plethora of potential kinases that might be targeting the alternatively spliced kinase consensus site of CPEB3. As this site is highly conserved between the CPEB family members, we suggest the existence of a splicing-based regulatory mechanism of CPEB function, and describe a robust phosphospecific antibody to study it in future. PMID:26915047

A mutational analysis of U12-dependent splice site dinucleotides

PubMed Central

DIETRICH, ROSEMARY C.; FULLER, JOHN D.; PADGETT, RICHARD A.

2005-01-01

Introns spliced by the U12-dependent minor spliceosome are divided into two classes based on their splice site dinucleotides. The /AU-AC/ class accounts for about one-third of U12-dependent introns in humans, while the /GU-AG/ class accounts for the other two-thirds. We have investigated the in vivo and in vitro splicing phenotypes of mutations in these dinucleotide sequences. A 5′ A residue can splice to any 3′ residue, although C is preferred. A 5′ G residue can splice to 3′ G or U residues with a preference for G. Little or no splicing was observed to 3′ A or C residues. A 5′ U or C residue is highly deleterious for U12-dependent splicing, although some combinations, notably 5′ U to 3′ U produced detectable spliced products. The dependence of 3′ splice site activity on the identity of the 5′ residue provides evidence for communication between the first and last nucleotides of the intron. Most mutants in the second position of the 5′ splice site and the next to last position of the 3′ splice site were defective for splicing. Double mutants of these residues showed no evidence of communication between these nucleotides. Varying the distance between the branch site and the 3′ splice site dinucleotide in the /GU-AG/ class showed that a somewhat larger range of distances was functional than for the /AU-AC/ class. The optimum branch site to 3′ splice site distance of 11–12 nucleotides appears to be the same for both classes. PMID:16043500

Quantitation of normal CFTR mRNA in CF patients with splice-site mutations

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

Zhou, Z.; Olsen, J.C.; Silverman, L.M.

Previously we identified two mutations in introns of the CFTR gene associated with partially active splice sites and unusual clinical phenotypes. One mutation in intron 19 (3849+10 kb C to T) is common in CF patients with normal sweat chloride values; an 84 bp sequence from intron 19, which contains a stop codon, is inserted between exon 19 and exon 20 in most nasal CFTR transcripts. The other mutation in intron 14B (2789+5 G to A) is associated with elevated sweat chloride levels, but mild pulmonary disease; exon 14B (38 bp) is spliced out of most nasal CFTR transcipts. Themore » remaining CFTR cDNA sequences, other than the 84 bp insertion of exon 14B deletion, are identical to the published sequence. To correlate genotype and phenotype, we used quantitative RT-PCR to determine the levels of normally-spliced CFTR mRNA in nasal epithelia from these patients. CFTR cDNA was amplified (25 cycles) by using primers specific for normally-spliced species, {gamma}-actin cDNA was amplified as a standard.« less

Enhancer of rudimentary homologue interacts with scaffold attachment factor B at the nuclear matrix to regulate SR protein phosphorylation.

PubMed

Drakouli, Sotiria; Lyberopoulou, Aggeliki; Papathanassiou, Maria; Mylonis, Ilias; Georgatsou, Eleni

2017-08-01

Scaffold attachment factor B1 (SAFB1) is an integral component of the nuclear matrix of vertebrate cells. It binds to DNA on scaffold/matrix attachment region elements, as well as to RNA and a multitude of different proteins, affecting basic cellular activities such as transcription, splicing and DNA damage repair. In the present study, we show that enhancer of rudimentary homologue (ERH) is a new molecular partner of SAFB1 and its 70% homologous paralogue, scaffold attachment factor B2 (SAFB2). ERH interacts directly in the nucleus with the C-terminal Arg-Gly-rich region of SAFB1/2 and co-localizes with it in the insoluble nuclear fraction. ERH, a small ubiquitous protein with striking homology among species and a unique structure, has also been implicated in fundamental cellular mechanisms. Our functional analyses suggest that the SAFB/ERH interaction does not affect SAFB1/2 function in transcription (e.g. as oestrogen receptor α co-repressors), although it reverses the inhibition exerted by SAFB1/2 on the splicing kinase SR protein kinase 1 (SRPK1), which also binds on the C-terminus of SAFB1/2. Accordingly, ERH silencing decreases lamin B receptor and SR protein phosphorylation, which are major SRPK1 substrates, further substantiating the role of SAFB1 and SAFB2 in the co-ordination of nuclear function. © 2017 Federation of European Biochemical Societies.

Functioning of the Drosophila Wilms'-Tumor-1-Associated Protein Homolog, Fl(2)d, in Sex-Lethal-Dependent Alternative Splicing

PubMed Central

Penn, Jill K. M.; Graham, Patricia; Deshpande, Girish; Calhoun, Gretchen; Chaouki, Ahmad Sami; Salz, Helen K.; Schedl, Paul

2008-01-01

fl(2)d, the Drosophila homolog of Wilms'-tumor-1-associated protein (WTAP), regulates the alternative splicing of Sex-lethal (Sxl), transformer (tra), and Ultrabithorax (Ubx). Although WTAP has been found in functional human spliceosomes, exactly how it contributes to the splicing process remains unknown. Here we attempt to identify factors that interact genetically and physically with fl(2)d. We begin by analyzing the Sxl-Fl(2)d protein–protein interaction in detail and present evidence suggesting that the female-specific fl(2)d1 allele is antimorphic with respect to the process of sex determination. Next we show that fl(2)d interacts genetically with early acting general splicing regulators and that Fl(2)d is present in immunoprecipitable complexes with Snf, U2AF50, U2AF38, and U1-70K. By contrast, we could not detect Fl(2)d complexes containing the U5 snRNP protein U5-40K or with a protein that associates with the activated B spliceosomal complex SKIP. Significantly, the genetic and molecular interactions observed for Sxl are quite similar to those detected for fl(2)d. Taken together, our findings suggest that Sxl and fl(2)d function to alter splice-site selection at an early step in spliceosome assembly. PMID:18245840

A short conserved motif in ALYREF directs cap- and EJC-dependent assembly of export complexes on spliced mRNAs.

PubMed

Gromadzka, Agnieszka M; Steckelberg, Anna-Lena; Singh, Kusum K; Hofmann, Kay; Gehring, Niels H

2016-03-18

The export of messenger RNAs (mRNAs) is the final of several nuclear posttranscriptional steps of gene expression. The formation of export-competent mRNPs involves the recruitment of export factors that are assumed to facilitate transport of the mature mRNAs. Using in vitro splicing assays, we show that a core set of export factors, including ALYREF, UAP56 and DDX39, readily associate with the spliced RNAs in an EJC (exon junction complex)- and cap-dependent manner. In order to elucidate how ALYREF and other export adaptors mediate mRNA export, we conducted a computational analysis and discovered four short, conserved, linear motifs present in RNA-binding proteins. We show that mutation in one of the new motifs (WxHD) in an unstructured region of ALYREF reduced RNA binding and abolished the interaction with eIF4A3 and CBP80. Additionally, the mutation impaired proper localization to nuclear speckles and export of a spliced reporter mRNA. Our results reveal important details of the orchestrated recruitment of export factors during the formation of export competent mRNPs. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

A splice-site mutation affecting the paired box of PAX3 in a three generation family with Waardenburg syndrome type I (WS1).

PubMed

Attaie, A; Kim, E; Wilcox, E R; Lalwani, A K

1997-06-01

Waardenburg syndrome, an autosomal dominant disorder characterized by sensorineural hearing loss, pigmentary disturbances and other developmental defects, is the most frequent form of congenital deafness in humans. Mutations in the PAX3 gene, a transcription factor expressed during embryonic development, is associated with WS types I and III. Here we report the identification of a novel acceptor splice site mutation (86-2 A-->G) in the paired domain of the human PAX3 gene causing WS type I in a three generation family.

A cross-sectional study examining the expression of splice variants K-RAS4A and K-RAS4B in advanced non-small-cell lung cancer patients.

PubMed

Aran, Veronica; Masson Domingues, Pedro; Carvalho de Macedo, Fabiane; Moreira de Sousa, Carlos Augusto; Caldas Montella, Tatiane; de Souza Accioly, Maria Theresa; Ferreira, Carlos Gil

2018-02-01

Mammalian cells differently express 4 RAS isoforms: H-RAS, N-RAS, K-RAS4A and K-RAS4B, which are important in promoting oncogenic processes when mutated. In lung cancer, the K-RAS isoform is the most frequently altered RAS protein, being also a difficult therapeutic target. Interestingly, there are two K-RAS splice variants (K-RAS4A and K-RAS4B) and little is known about the role of K-RAS4A. Most studies targeting K-RAS, or analysing it as a prognostic factor, have not taken into account the two isoforms. Consequently, the in-depth investigation of them is needed. The present study analysed 98 specimens from advanced non-small cell lung cancer (NSCLC) adenocarcinoma patients originated from Brazil. The alterations present in K-RAS at the DNA level (Sanger sequencing) as well as the expression of the splicing isoforms at the RNA (qRT-PCR) and protein levels (immunohistochemistry analysis), were evaluated. Possible associations between clinicopathological features and the molecular findings were also investigated. Our results showed that in the non-smoking population, the cancer incidence was higher among women. In contrast, in smokers and former smokers, the incidence was higher among men. Regarding sequencing results, 10.5% of valid samples presented mutations in exon 2, being all wild-type for exon 3, and the most frequently occurring base change was the transversion G → T. Our qRT-PCR and immunohistochemical analysis showed that both, K-RAS4A and K-RAS4B, were differently expressed in NSCLC tumour samples. For example, tumour specimens showed higher K-RAS4A mRNA expression in relation to commercial normal lung control than did K-RAS4B. In addition, K-RAS4B protein expression was frequently stronger than K-RAS4A in the patients analysed. Our results highlight the differential expression of K-RAS4A and K-RAS4B in advanced adenocarcinoma NSCLC patients and underline the need to further clarify the enigma behind their biological significance in various cancer types, including NSCLC. Copyright © 2017 Elsevier B.V. All rights reserved.

Lnx2 ubiquitin ligase is essential for exocrine cell differentiation in the early zebrafish pancreas

PubMed Central

Won, Minho; Ro, Hyunju; Dawid, Igor B.

2015-01-01

The gene encoding the E3 ubiquitin ligase Ligand of Numb protein-X (Lnx)2a is expressed in the ventral-anterior pancreatic bud of zebrafish embryos in addition to its expression in the brain. Knockdown of Lnx2a by using an exon 2/intron 2 splice morpholino resulted in specific inhibition of the differentiation of ventral bud derived exocrine cell types, with little effect on endocrine cell types. A frame shifting null mutation in lnx2a did not mimic this phenotype, but a mutation that removed the exon 2 splice donor site did. We found that Lnx2b functions in a redundant manner with its paralog Lnx2a. Inhibition of lnx2a exon 2/3 splicing causes exon 2 skipping and leads to the production of an N-truncated protein that acts as an interfering molecule. Thus, the phenotype characterized by inhibition of exocrine cell differentiation requires inactivation of both Lnx2a and Lnx2b. Human LNX1 is known to destabilize Numb, and we show that inhibition of Numb expression rescues the Lnx2a/b-deficient phenotype. Further, Lnx2a/b inhibition leads to a reduction in the number of Notch active cells in the pancreas. We suggest that Lnx2a/b function to fine tune the regulation of Notch through Numb in the differentiation of cell types in the early zebrafish pancreas. Further, the complex relationships among genotype, phenotype, and morpholino effect in this case may be instructive in the ongoing consideration of morpholino use. PMID:26392552

Lnx2 ubiquitin ligase is essential for exocrine cell differentiation in the early zebrafish pancreas.

PubMed

Won, Minho; Ro, Hyunju; Dawid, Igor B

2015-10-06

The gene encoding the E3 ubiquitin ligase Ligand of Numb protein-X (Lnx)2a is expressed in the ventral-anterior pancreatic bud of zebrafish embryos in addition to its expression in the brain. Knockdown of Lnx2a by using an exon 2/intron 2 splice morpholino resulted in specific inhibition of the differentiation of ventral bud derived exocrine cell types, with little effect on endocrine cell types. A frame shifting null mutation in lnx2a did not mimic this phenotype, but a mutation that removed the exon 2 splice donor site did. We found that Lnx2b functions in a redundant manner with its paralog Lnx2a. Inhibition of lnx2a exon 2/3 splicing causes exon 2 skipping and leads to the production of an N-truncated protein that acts as an interfering molecule. Thus, the phenotype characterized by inhibition of exocrine cell differentiation requires inactivation of both Lnx2a and Lnx2b. Human LNX1 is known to destabilize Numb, and we show that inhibition of Numb expression rescues the Lnx2a/b-deficient phenotype. Further, Lnx2a/b inhibition leads to a reduction in the number of Notch active cells in the pancreas. We suggest that Lnx2a/b function to fine tune the regulation of Notch through Numb in the differentiation of cell types in the early zebrafish pancreas. Further, the complex relationships among genotype, phenotype, and morpholino effect in this case may be instructive in the ongoing consideration of morpholino use.

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.

Characterization of Pax3 and Pax7 genes and their expression patterns during different development and growth stages of Japanese pufferfish Takifugu rubripes.

PubMed

Akolkar, Dadasaheb B; Asaduzzaman, Md; Kinoshita, Shigeharu; Asakawa, Shuichi; Watabe, Shugo

2016-01-01

Pax3 and Pax7 are the regulators and markers of muscle progenitors and satellite cells that contribute to the embryonic development and postembryonic growth of skeletal muscle in vertebrates, as well as to its repair and regeneration. However, information regarding them in vertebrate genome model, torafugu Takifugu rubripes, has remained unknown. Therefore, as an initial step, here we characterized Pax3 and Pax7 from torafugu and investigated their expression patterns during different developmental stages by RT-PCR. In silico analysis with the Fugu genome database (ver. 4.0) yielded two distinct genes each for Pax3 (Pax3a and Pax3b) and Pax7 (Pax7a and Pax7b). The 75th amino acid, glutamine (Gln75), from the N-terminus was replaced by proline in the paired box domain (PD) of Pax3a. One single cDNA clone encoding Pax3a had deletion of Gln75 in PD, suggesting the presence of alternatively spliced variants (Q+/Q-). This was further supported by identification of two adjacent alternative 3' splice acceptor sites which produce Pax3b Q+ (aagCAGGGA) and Q- (aagcagGGA) variants. Interestingly, torafugu Pax7a, but not Pax7b, had an insert encoding five amino acid residues (SGEAS) in a C-terminal region of PD in two out of three cDNA clones. Genomic analysis showed two alternate splice donor sites at exon 4 of Pax7a. In synteny analysis, torafugu Pax3a showed syntenic relationship with the corresponding regions in other teleosts only, whereas Pax3b and Pax7b showed high syntenic relationship with the corresponding regions of both mammals and other teleosts. RT-PCR revealed that expression of Pax3a and Pax3b transcripts was restricted to embryonic stages only, whereas those of Pax7a and Pax7b was continued to be expressed in larvae and importantly those of Pax7a were found in adult skeletal muscles. Therefore, Pax3 appears to be most important for primary myogenesis and Pax7 for secondary myogenesis and growth by hyperplasia in fish. In this regard, the transcripts of torafugu Pax3 and Pax7 genes might be used for further investigation as a marker for identification of muscle precursor cells during different phases of growth, and this ambiguity is the next target of our research. Copyright © 2015 Elsevier B.V. All rights reserved.

Resveratrol, by modulating RNA processing factor levels, can influence the alternative splicing of pre-mRNAs.

PubMed

Markus, M Andrea; Marques, Francine Z; Morris, Brian J

2011-01-01

Alternative pre-mRNA splicing defects can contribute to, or result from, various diseases, including cancer. Aberrant mRNAs, splicing factors and other RNA processing factors have therefore become targets for new therapeutic interventions. Here we report that the natural polyphenol resveratrol can modulate alternative splicing in a target-specific manner. We transfected minigenes of several alternatively spliceable primary mRNAs into HEK293 cells in the presence or absence of 1, 5, 20 and 50 µM resveratrol and measured exon levels by semi-quantitative PCR after separation by agarose gel electrophoresis. We found that 20 µg/ml and 50 µg/ml of resveratrol affected exon inclusion of SRp20 and SMN2 pre-mRNAs, but not CD44v5 or tau pre-mRNAs. By Western blotting and immunofluorescence we showed that this effect may be due to the ability of resveratrol to change the protein level but not the localization of several RNA processing factors. The processing factors that increased significantly were ASF/SF2, hnRNPA1 and HuR, but resveratrol did not change the levels of RBM4, PTBP1 and U2AF35. By means of siRNA-mediated knockdown we depleted cells of SIRT1, regarded as a major target of resveratrol, and showed that the effect on splicing was not dependent on SIRT1. Our results suggest that resveratrol might be an attractive small molecule to treat diseases in which aberrant splicing has been implicated, and justify more extensive research on the effects of resveratrol on the splicing machinery.

Multivalent binding of formin-binding protein 21 (FBP21)-tandem-WW domains fosters protein recognition in the pre-spliceosome.

PubMed

Klippel, Stefan; Wieczorek, Marek; Schümann, Michael; Krause, Eberhard; Marg, Berenice; Seidel, Thorsten; Meyer, Tim; Knapp, Ernst-Walter; Freund, Christian

2011-11-04

The high abundance of repetitive but nonidentical proline-rich sequences in spliceosomal proteins raises the question of how these known interaction motifs recruit their interacting protein domains. Whereas complex formation of these adaptors with individual motifs has been studied in great detail, little is known about the binding mode of domains arranged in tandem repeats and long proline-rich sequences including multiple motifs. Here we studied the interaction of the two adjacent WW domains of spliceosomal protein FBP21 with several ligands of different lengths and composition to elucidate the hallmarks of multivalent binding for this class of recognition domains. First, we show that many of the proteins that define the cellular proteome interacting with FBP21-WW1-WW2 contain multiple proline-rich motifs. Among these is the newly identified binding partner SF3B4. Fluorescence resonance energy transfer (FRET) analysis reveals the tandem-WW domains of FBP21 to interact with splicing factor 3B4 (SF3B4) in nuclear speckles where splicing takes place. Isothermal titration calorimetry and NMR shows that the tandem arrangement of WW domains and the multivalency of the proline-rich ligands both contribute to affinity enhancement. However, ligand exchange remains fast compared with the NMR time scale. Surprisingly, a N-terminal spin label attached to a bivalent ligand induces NMR line broadening of signals corresponding to both WW domains of the FBP21-WW1-WW2 protein. This suggests that distinct orientations of the ligand contribute to a delocalized and semispecific binding mode that should facilitate search processes within the spliceosome.

Multivalent Binding of Formin-binding Protein 21 (FBP21)-Tandem-WW Domains Fosters Protein Recognition in the Pre-spliceosome*

PubMed Central

Klippel, Stefan; Wieczorek, Marek; Schümann, Michael; Krause, Eberhard; Marg, Berenice; Seidel, Thorsten; Meyer, Tim; Knapp, Ernst-Walter; Freund, Christian

2011-01-01

The high abundance of repetitive but nonidentical proline-rich sequences in spliceosomal proteins raises the question of how these known interaction motifs recruit their interacting protein domains. Whereas complex formation of these adaptors with individual motifs has been studied in great detail, little is known about the binding mode of domains arranged in tandem repeats and long proline-rich sequences including multiple motifs. Here we studied the interaction of the two adjacent WW domains of spliceosomal protein FBP21 with several ligands of different lengths and composition to elucidate the hallmarks of multivalent binding for this class of recognition domains. First, we show that many of the proteins that define the cellular proteome interacting with FBP21-WW1-WW2 contain multiple proline-rich motifs. Among these is the newly identified binding partner SF3B4. Fluorescence resonance energy transfer (FRET) analysis reveals the tandem-WW domains of FBP21 to interact with splicing factor 3B4 (SF3B4) in nuclear speckles where splicing takes place. Isothermal titration calorimetry and NMR shows that the tandem arrangement of WW domains and the multivalency of the proline-rich ligands both contribute to affinity enhancement. However, ligand exchange remains fast compared with the NMR time scale. Surprisingly, a N-terminal spin label attached to a bivalent ligand induces NMR line broadening of signals corresponding to both WW domains of the FBP21-WW1-WW2 protein. This suggests that distinct orientations of the ligand contribute to a delocalized and semispecific binding mode that should facilitate search processes within the spliceosome. PMID:21917930

Transcriptome-wide analysis of alternative RNA splicing events in Epstein-Barr virus-associated gastric carcinomas

PubMed Central

Armero, Victoria E. S.; Tremblay, Marie-Pier; Allaire, Andréa; Boudreault, Simon; Martenon-Brodeur, Camille; Duval, Cyntia; Durand, Mathieu; Lapointe, Elvy; Thibault, Philippe; Tremblay-Létourneau, Maude; Perreault, Jean-Pierre; Scott, Michelle S.

2017-01-01

Multiple human diseases including cancer have been associated with a dysregulation in RNA splicing patterns. In the current study, modifications to the global RNA splicing landscape of cellular genes were investigated in the context of Epstein-Barr virus-associated gastric cancer. Global alterations to the RNA splicing landscape of cellular genes was examined in a large-scale screen from 295 primary gastric adenocarcinomas using high-throughput RNA sequencing data. RT-PCR analysis, mass spectrometry, and co-immunoprecipitation studies were also used to experimentally validate and investigate the differential alternative splicing (AS) events that were observed through RNA-seq studies. Our study identifies alterations in the AS patterns of approximately 900 genes such as tumor suppressor genes, transcription factors, splicing factors, and kinases. These findings allowed the identification of unique gene signatures for which AS is misregulated in both Epstein-Barr virus-associated gastric cancer and EBV-negative gastric cancer. Moreover, we show that the expression of Epstein–Barr nuclear antigen 1 (EBNA1) leads to modifications in the AS profile of cellular genes and that the EBNA1 protein interacts with cellular splicing factors. These findings provide insights into the molecular differences between various types of gastric cancer and suggest a role for the EBNA1 protein in the dysregulation of cellular AS. PMID:28493890

Cytoplasmic mislocalization of RNA splicing factors and aberrant neuronal gene splicing in TDP-43 transgenic pig brain.

PubMed

Wang, Guohao; Yang, Huaqiang; Yan, Sen; Wang, Chuan-En; Liu, Xudong; Zhao, Bentian; Ouyang, Zhen; Yin, Peng; Liu, Zhaoming; Zhao, Yu; Liu, Tao; Fan, Nana; Guo, Lin; Li, Shihua; Li, Xiao-Jiang; Lai, Liangxue

2015-09-03

TAR DNA-binding protein 43 (TDP-43) is a nuclear protein, but it is redistributed in the neuronal cytoplasm in both amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Because small transgenic animal models often lack cytoplasmic TDP-43, how the cytoplasmic accumulation of TDP-43 contributes to these diseases remains unclear. The current study is aimed at studying the mechanism of cytoplasmic pathology of TDP-43. We established transgenic pigs expressing mutant TDP-43 (M337V). This pig model shows severe phenotypes and early death. We found that transgenic TDP-43 is also distributed in the cytoplasm of neuronal cells in the spinal cord and brain. Transgenic TDP-43 interacts with PSF, an RNA splicing factor that associates with NeuN to regulate neuronal RNA splicing. The interaction of TDP-43, PSF and NeuN causes PSF and NeuN mislocalize into the neuronal cytoplasm in transgenic pigs. Consistently, abnormal PSF-related neuronal RNA splicing is seen in TDP-43 transgenic pigs. The cytoplasmic localization of PSF and NeuN as well as abnormal PSF-related neuronal RNA splicing was also found in ALS patient brains. Our findings from a large mammalian model suggest that cytoplasmic mutant TDP-43 could reduce the nuclear function of RNA splicing factors, contributing to neuropathology.

Transcriptome-wide analysis of alternative RNA splicing events in Epstein-Barr virus-associated gastric carcinomas.

PubMed

Armero, Victoria E S; Tremblay, Marie-Pier; Allaire, Andréa; Boudreault, Simon; Martenon-Brodeur, Camille; Duval, Cyntia; Durand, Mathieu; Lapointe, Elvy; Thibault, Philippe; Tremblay-Létourneau, Maude; Perreault, Jean-Pierre; Scott, Michelle S; Bisaillon, Martin

2017-01-01

Multiple human diseases including cancer have been associated with a dysregulation in RNA splicing patterns. In the current study, modifications to the global RNA splicing landscape of cellular genes were investigated in the context of Epstein-Barr virus-associated gastric cancer. Global alterations to the RNA splicing landscape of cellular genes was examined in a large-scale screen from 295 primary gastric adenocarcinomas using high-throughput RNA sequencing data. RT-PCR analysis, mass spectrometry, and co-immunoprecipitation studies were also used to experimentally validate and investigate the differential alternative splicing (AS) events that were observed through RNA-seq studies. Our study identifies alterations in the AS patterns of approximately 900 genes such as tumor suppressor genes, transcription factors, splicing factors, and kinases. These findings allowed the identification of unique gene signatures for which AS is misregulated in both Epstein-Barr virus-associated gastric cancer and EBV-negative gastric cancer. Moreover, we show that the expression of Epstein-Barr nuclear antigen 1 (EBNA1) leads to modifications in the AS profile of cellular genes and that the EBNA1 protein interacts with cellular splicing factors. These findings provide insights into the molecular differences between various types of gastric cancer and suggest a role for the EBNA1 protein in the dysregulation of cellular AS.

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

Conserved Domains in the Transformer Protein Act Complementary to Regulate Sex-Specific Splicing of Its Own Pre-mRNA.

PubMed

Tanaka, Arisa; Aoki, Fugaku; Suzuki, Masataka G

2018-05-26

The transformer (tra) gene, which is a female-determining master gene in the housefly Musca domestica, acts as a memory device for sex determination via its auto-regulatory function, i.e., through the contribution of the TRA protein to female-specific splicing of its own pre-mRNA. The TRA protein contains 4 small domains that are specifically conserved among TRA proteins (domains 1-4). Domain 2, also named TRA-CAM domain, is the most conserved, but its function remains unknown. To examine whether these domains are involved in the auto-regulatory function, we performed in vitro splicing assays using a tra minigene containing a partial genomic sequence of the M. domestica tra (Mdtra) gene. Co-transfection of the Mdtra minigene and an MdTRA protein expression vector into cultured insect cells strongly induced female-specific splicing of the minigene. A series of deletion mutation analyses demonstrated that these domains act complementarily to induce female-specific splicing. Domain 1 and the TRA-CAM domain were necessary for the female-specific splicing when the MdTRA protein lacked both domains 3 and 4. In this situation, mutation of the well-conserved 3 amino acids (GEG) in the TRA-CAM domain significantly reduced the female-specific splicing activity of MdTRA. GST-pull down analyses demonstrated that the MdTRA protein specifically enriched on the male-specific exonic region (exon 2b), which contains the putative TRA/TRA-2 binding sites, and that the GEG mutation disrupts this enrichment. Since the MdTRA protein interacts with its own pre-mRNA through TRA-2, our findings suggest that the conserved amino acid residues in the TRA-CAM domain may be crucial for the interaction between MdTRA and TRA-2, enhancing MdTRA recruitment on its pre-mRNA to induce female-specific splicing of tra in the housefly. © 2018 S. Karger AG, Basel.

Melatonin Inhibits Androgen Receptor Splice Variant-7 (AR-V7)-Induced Nuclear Factor-Kappa B (NF-κB) Activation and NF-κB Activator-Induced AR-V7 Expression in Prostate Cancer Cells: Potential Implications for the Use of Melatonin in Castration-Resistant Prostate Cancer (CRPC) Therapy.

PubMed

Liu, Vincent Wing Sun; Yau, Wing Lung; Tam, Chun Wai; Yao, Kwok-Ming; Shiu, Stephen Yuen Wing

2017-05-31

A major current challenge in the treatment of advanced prostate cancer, which can be initially controlled by medical or surgical castration, is the development of effective, safe, and affordable therapies against progression of the disease to the stage of castration resistance. Here, we showed that in LNCaP and 22Rv1 prostate cancer cells transiently overexpressing androgen receptor splice variant-7 (AR-V7), nuclear factor-kappa B (NF-κB) was activated and could result in up-regulated interleukin ( IL ) -6 gene expression, indicating a positive interaction between AR-V7 expression and activated NF-κB/IL-6 signaling in castration-resistant prostate cancer (CRPC) pathogenesis. Importantly, both AR-V7-induced NF-κB activation and IL-6 gene transcription in LNCaP and 22Rv1 cells could be inhibited by melatonin. Furthermore, stimulation of AR-V7 mRNA expression in LNCaP cells by betulinic acid, a pharmacological NF-κB activator, was reduced by melatonin treatment. Our data support the presence of bi-directional positive interactions between AR-V7 expression and NF-κB activation in CRPC pathogenesis. Of note, melatonin, by inhibiting NF-κB activation via the previously-reported MT₁ receptor-mediated antiproliferative pathway, can disrupt these bi-directional positive interactions between AR-V7 and NF-κB and thereby delay the development of castration resistance in advanced prostate cancer. Apparently, this therapeutic potential of melatonin in advanced prostate cancer/CRPC management is worth translation in the clinic via combined androgen depletion and melatonin repletion.

Melatonin Inhibits Androgen Receptor Splice Variant-7 (AR-V7)-Induced Nuclear Factor-Kappa B (NF-κB) Activation and NF-κB Activator-Induced AR-V7 Expression in Prostate Cancer Cells: Potential Implications for the Use of Melatonin in Castration-Resistant Prostate Cancer (CRPC) Therapy

PubMed Central

Liu, Vincent Wing Sun; Yau, Wing Lung; Tam, Chun Wai; Yao, Kwok-Ming; Shiu, Stephen Yuen Wing

2017-01-01

A major current challenge in the treatment of advanced prostate cancer, which can be initially controlled by medical or surgical castration, is the development of effective, safe, and affordable therapies against progression of the disease to the stage of castration resistance. Here, we showed that in LNCaP and 22Rv1 prostate cancer cells transiently overexpressing androgen receptor splice variant-7 (AR-V7), nuclear factor-kappa B (NF-κB) was activated and could result in up-regulated interleukin (IL)-6 gene expression, indicating a positive interaction between AR-V7 expression and activated NF-κB/IL-6 signaling in castration-resistant prostate cancer (CRPC) pathogenesis. Importantly, both AR-V7-induced NF-κB activation and IL-6 gene transcription in LNCaP and 22Rv1 cells could be inhibited by melatonin. Furthermore, stimulation of AR-V7 mRNA expression in LNCaP cells by betulinic acid, a pharmacological NF-κB activator, was reduced by melatonin treatment. Our data support the presence of bi-directional positive interactions between AR-V7 expression and NF-κB activation in CRPC pathogenesis. Of note, melatonin, by inhibiting NF-κB activation via the previously-reported MT1 receptor-mediated antiproliferative pathway, can disrupt these bi-directional positive interactions between AR-V7 and NF-κB and thereby delay the development of castration resistance in advanced prostate cancer. Apparently, this therapeutic potential of melatonin in advanced prostate cancer/CRPC management is worth translation in the clinic via combined androgen depletion and melatonin repletion. PMID:28561752

Coexpression of the KCNA3B gene product with Kv1.5 leads to a novel A-type potassium channel.

PubMed

Leicher, T; Bähring, R; Isbrandt, D; Pongs, O

1998-12-25

Shaker-related voltage-gated potassium (Kv) channels may be heterooligomers consisting of membrane-integral alpha-subunits associated with auxiliary cytoplasmic beta-subunits. In this study we have cloned the human Kvbeta3.1 subunit and the corresponding KCNA3B gene. Identification of sequence-tagged sites in the gene mapped KCNA3B to band p13.1 of human chromosome 17. Comparison of the KCNA1B, KCNA2B, and KCNA3B gene structures showed that the three Kvbeta genes have very disparate lengths varying from >/=350 kb (KCNA1B) to approximately 7 kb (KCNA3B). Yet, the exon patterns of the three genes, which code for the seven known mammalian Kvbeta subunits, are very similar. The Kvbeta1 and Kvbeta2 splice variants are generated by alternative use of 5'-exons. Mouse Kvbeta4, a potential splice variant of Kvbeta3, is a read-through product where the open reading frame starts within the sequence intervening between Kvbeta3 exons 7 and 8. The human KCNA3B sequence does not contain a mouse Kvbeta4-like open reading frame. Human Kvbeta3 mRNA is specifically expressed in the brain, where it is predominantly detected in the cerebellum. The heterologous coexpression of human Kv1.5 and Kvbeta3.1 subunits in Chinese hamster ovary cells yielded a novel Kv channel mediating very fast inactivating (A-type) outward currents upon depolarization. Thus, the expression of Kvbeta3.1 subunits potentially extends the possibilities to express diverse A-type Kv channels in the human brain.

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

Variants of the Xenopus laevis ribosomal transcription factor xUBF are developmentally regulated by differential splicing.

PubMed

Guimond, A; Moss, T

1992-07-11

XUBF is a Xenopus ribosomal transcription factor of the HMG-box family which contains five tandemly disposed homologies to the HMG1 & 2 DNA binding domains. XUBF has been isolated as a protein doublet and two cDNAs encoding the two molecular weight variants have been characterised. The major two forms of xUBF identified differ by the presence or absence of a 22 amino acid segment lying between HMG-boxes 3 and 4. Here we show that the mRNAs for these two forms of xUBF are regulated during development and differentiation over a range of nearly 20 fold. By isolating two of the xUBF genes, it was possible to show that both encoded the variable 22 amino acid segment in exon 12. Oocyte splicing assays and the sequencing of PCR-generated cDNA fragments, demonstrated that the transcripts from one of these genes were differentially spliced in a developmentally regulated manner. Transcripts from the second gene were found to be predominantly or exclusively spliced to produce the lower molecular weight form of xUBF. Expression of a high molecular weight form from yet a third gene was also detected. Although the intron-exon structures of the Xenopus and mouse UBF genes were found to be essentially identical, the differential splicing of exon 8 found in mammals, was not detected in Xenopus.

LEDGF/p75 interacts with mRNA splicing factors and targets HIV-1 integration to highly spliced genes

PubMed Central

Singh, Parmit Kumar; Plumb, Matthew R.; Ferris, Andrea L.; Iben, James R.; Wu, Xiaolin; Fadel, Hind J.; Luke, Brian T.; Esnault, Caroline; Poeschla, Eric M.; Hughes, Stephen H.; Kvaratskhelia, Mamuka; Levin, Henry L.

2015-01-01

The host chromatin-binding factor LEDGF/p75 interacts with HIV-1 integrase and directs integration to active transcription units. To understand how LEDGF/p75 recognizes transcription units, we sequenced 1 million HIV-1 integration sites isolated from cultured HEK293T cells. Analysis of integration sites showed that cancer genes were preferentially targeted, raising concerns about using lentivirus vectors for gene therapy. Additional analysis led to the discovery that introns and alternative splicing contributed significantly to integration site selection. These correlations were independent of transcription levels, size of transcription units, and length of the introns. Multivariate analysis with five parameters previously found to predict integration sites showed that intron density is the strongest predictor of integration density in transcription units. Analysis of previously published HIV-1 integration site data showed that integration density in transcription units in mouse embryonic fibroblasts also correlated strongly with intron number, and this correlation was absent in cells lacking LEDGF. Affinity purification showed that LEDGF/p75 is associated with a number of splicing factors, and RNA sequencing (RNA-seq) analysis of HEK293T cells lacking LEDGF/p75 or the LEDGF/p75 integrase-binding domain (IBD) showed that LEDGF/p75 contributes to splicing patterns in half of the transcription units that have alternative isoforms. Thus, LEDGF/p75 interacts with splicing factors, contributes to exon choice, and directs HIV-1 integration to transcription units that are highly spliced. PMID:26545813

Rhythmic Behavior Is Controlled by the SRm160 Splicing Factor in Drosophila melanogaster.

PubMed

Beckwith, Esteban J; Hernando, Carlos E; Polcowñuk, Sofía; Bertolin, Agustina P; Mancini, Estefania; Ceriani, M Fernanda; Yanovsky, Marcelo J

2017-10-01

Circadian clocks organize the metabolism, physiology, and behavior of organisms throughout the day-night cycle by controlling daily rhythms in gene expression at the transcriptional and post-transcriptional levels. While many transcription factors underlying circadian oscillations are known, the splicing factors that modulate these rhythms remain largely unexplored. A genome-wide assessment of the alterations of gene expression in a null mutant of the alternative splicing regulator SR-related matrix protein of 160 kDa (SRm160) revealed the extent to which alternative splicing impacts on behavior-related genes. We show that SRm160 affects gene expression in pacemaker neurons of the Drosophila brain to ensure proper oscillations of the molecular clock. A reduced level of SRm160 in adult pacemaker neurons impairs circadian rhythms in locomotor behavior, and this phenotype is caused, at least in part, by a marked reduction in period ( per ) levels. Moreover, rhythmic accumulation of the neuropeptide PIGMENT DISPERSING FACTOR in the dorsal projections of these neurons is abolished after SRm160 depletion. The lack of rhythmicity in SRm160-downregulated flies is reversed by a fully spliced per construct, but not by an extra copy of the endogenous locus, showing that SRm160 positively regulates per levels in a splicing-dependent manner. Our findings highlight the significant effect of alternative splicing on the nervous system and particularly on brain function in an in vivo model. Copyright © 2017 by the Genetics Society of America.

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

PubMed

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

2016-12-27

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

Low loss fusion splicing polarization-maintaining photonic crystal fiber and conventional polarization-maintaining fiber

NASA Astrophysics Data System (ADS)

Zuoming, Sun; Ningfang, Song; Jing, Jin; Jingming, Song; Pan, Ma

2012-12-01

An efficient and simple method of fusion splicing of a Polarization-Maintaining Photonic Crystal Fiber (PM-PCF) and a conventional Polarization-Maintaining Fiber (PMF) with a low loss of 0.65 dB in experiment is reported. The minimum bending diameter of the joint can reach 2 cm. Theoretical calculation of the splicing loss based on mode field diameters (MFDs) mismatch of the two kinds of fibers is given. All parameters affected the splicing loss were studied.

Function of Apollo (SNM1B) at telomere highlighted by a splice variant identified in a patient with Hoyeraal–Hreidarsson syndrome

PubMed Central

Touzot, Fabien; Callebaut, Isabelle; Soulier, Jean; Gaillard, Laetitia; Azerrad, Chantal; Durandy, Anne; Fischer, Alain; de Villartay, Jean-Pierre; Revy, Patrick

2010-01-01

Telomeres, the protein–DNA complexes at the ends of linear chromosomes, are protected and regulated by the shelterin molecules, the telomerase complex, and other accessory factors, among which is Apollo, a DNA repair factor of the β-lactamase/β-CASP family. Impaired telomere protection in humans causes dyskeratosis congenita and Hoyeraal–Hreidarsson (HH) syndrome, characterized by premature aging, bone marrow failure, and immunodeficiency. We identified a unique Apollo splice variant (designated Apollo-Δ) in fibroblasts from a patient with HH syndrome. Apollo-Δ generates a dominant negative form of Apollo lacking the telomeric repeat-binding factor homology (TRFH)-binding motif (TBM) required for interaction with the shelterin TRF2 at telomeres. Apollo-Δ hampers the proper replication of telomeres, leading to major telomeric dysfunction and cellular senescence, but maintains its DNA interstrand cross-link repair function in the whole genome. These results identify Apollo as a crucial actor in telomere maintenance in vivo, independent of its function as a general DNA repair factor. PMID:20479256

Function of Apollo (SNM1B) at telomere highlighted by a splice variant identified in a patient with Hoyeraal-Hreidarsson syndrome.

PubMed

Touzot, Fabien; Callebaut, Isabelle; Soulier, Jean; Gaillard, Laetitia; Azerrad, Chantal; Durandy, Anne; Fischer, Alain; de Villartay, Jean-Pierre; Revy, Patrick

2010-06-01

Telomeres, the protein-DNA complexes at the ends of linear chromosomes, are protected and regulated by the shelterin molecules, the telomerase complex, and other accessory factors, among which is Apollo, a DNA repair factor of the beta-lactamase/beta-CASP family. Impaired telomere protection in humans causes dyskeratosis congenita and Hoyeraal-Hreidarsson (HH) syndrome, characterized by premature aging, bone marrow failure, and immunodeficiency. We identified a unique Apollo splice variant (designated Apollo-Delta) in fibroblasts from a patient with HH syndrome. Apollo-Delta generates a dominant negative form of Apollo lacking the telomeric repeat-binding factor homology (TRFH)-binding motif (TBM) required for interaction with the shelterin TRF2 at telomeres. Apollo-Delta hampers the proper replication of telomeres, leading to major telomeric dysfunction and cellular senescence, but maintains its DNA interstrand cross-link repair function in the whole genome. These results identify Apollo as a crucial actor in telomere maintenance in vivo, independent of its function as a general DNA repair factor.

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.

Sensing and splicing applications of small core Ge-doped photonic crystal fibers

NASA Astrophysics Data System (ADS)

Wang, Yiping; Brueckner, Sven; Kobelke, Jens; Rothhardt, Manfred; Ecke, Wolfgang; Willsch, Reinhardt; Bartelt, Hartmut

2008-04-01

Sensor related properties of a small core (4.1μm) Ge-doped photonic crystal fiber (PCF) are being reported. Fiber Bragg gratings with 35% and almost 100 % reflectivity were written in the Ge-doped PCF before and after hydrogen loading, respectively, by use of a UV laser. A 5.6pm/°C temperature sensitivity of the FBG was observed. Additionally, a novel method is demonstrated to splice such PCF by use of a commercial fusion splicer with default splice parameters for standard single mode fibers (SMF). No parameter adjustments are required to splice the PCF to various SMFs and a low splice loss of 1.0 ~ 1.4dB can be achieved. No splice interface emerges at the splice joint, which is of advantage for the sensing applications of such a PCF.

Functional analysis of sex-determination genes by gene silencing with LNA-DNA gapmers in the silkworm, Bombyx mori.

PubMed

Sakai, Hiroki; Sakaguchi, Honami; Aoki, Fugaku; Suzuki, Masataka G

2015-08-01

The sexual fate of B. mori is determined genetically; ZW, female and ZZ, male. Recently, we successfully identified a strong candidate gene at the top of the sex determination cascade in B. mori. This gene was termed Feminizer (Fem) and revealed to be a source of Fem-piRNA. Further, we found that B. mori doublesex (Bmdsx) splicing was markedly altered to produce the male-type isoform when a Fem-piRNA inhibitor was injected into ZW embryos. Moreover, knockdown of Masculinizer (Masc), a Fem-piRNA target gene, altered to produce the female-type isoform of Bmdsx in male embryos. However, it remains unclear as to whether Masc directly regulates the sex-specific expression of Bmdsx. In previous studies, we determined that the male-specific isoform of the Bombyx homolog of IGF-II mRNA-binding protein (Imp(M)) was involved in the male-specific splicing of Bmdsx. In an attempt to clarify the genetic relationship between Fem, Masc, Imp(M), and Bmdsx, knockdown experiments were performed. Knockdown of Fem shifted into male-type Bmdsx, Imp(M) and Masc in female embryos. Knockdown of Masc led to the production of the female-type Bmdsx and a dramatic reduction in Imp(M) expression in male embryos. Knockdown of Imp(M) shifted Bmdsx splice mode from the male-type into the female-type. Our results suggest that: (1) Fem reduces Masc expression, (2) Masc dramatically induces Imp(M) expression, and (3) Imp(M) shifting Bmdsx splice mode from the female-type into the male-type. Based on these findings, we propose a possible genetic cascade regulating sex determination in B. mori. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

A mammalian germ cell-specific RNA-binding protein interacts with ubiquitously expressed proteins involved in splice site selection

NASA Astrophysics Data System (ADS)

Elliott, David J.; Bourgeois, Cyril F.; Klink, Albrecht; Stévenin, James; Cooke, Howard J.

2000-05-01

RNA-binding motif (RBM) genes are found on all mammalian Y chromosomes and are implicated in spermatogenesis. Within human germ cells, RBM protein shows a similar nuclear distribution to components of the pre-mRNA splicing machinery. To address the function of RBM, we have used protein-protein interaction assays to test for possible physical interactions between these proteins. We find that RBM protein directly interacts with members of the SR family of splicing factors and, in addition, strongly interacts with itself. We have mapped the protein domains responsible for mediating these interactions and expressed the mouse RBM interaction region as a bacterial fusion protein. This fusion protein can pull-down several functionally active SR protein species from cell extracts. Depletion and add-back experiments indicate that these SR proteins are the only splicing factors bound by RBM which are required for the splicing of a panel of pre-mRNAs. Our results suggest that RBM protein is an evolutionarily conserved mammalian splicing regulator which operates as a germ cell-specific cofactor for more ubiquitously expressed pre-mRNA splicing activators.

On splice site prediction using weight array models: a comparison of smoothing techniques

NASA Astrophysics Data System (ADS)

Taher, Leila; Meinicke, Peter; Morgenstern, Burkhard

2007-11-01

In most eukaryotic genes, protein-coding exons are separated by non-coding introns which are removed from the primary transcript by a process called "splicing". The positions where introns are cut and exons are spliced together are called "splice sites". Thus, computational prediction of splice sites is crucial for gene finding in eukaryotes. Weight array models are a powerful probabilistic approach to splice site detection. Parameters for these models are usually derived from m-tuple frequencies in trusted training data and subsequently smoothed to avoid zero probabilities. In this study we compare three different ways of parameter estimation for m-tuple frequencies, namely (a) non-smoothed probability estimation, (b) standard pseudo counts and (c) a Gaussian smoothing procedure that we recently developed.

Isolation of the sex-determining gene Sex-lethal (Sxl) in Penaeus (Litopenaeus) vannamei (Boone, 1931) and characterization of its embryogenic, gametogenic, and tissue-specific expression.

PubMed

López-Cuadros, Itzia; García-Gasca, Alejandra; Gomez-Anduro, Gracia; Escobedo-Fregoso, Cristina; Llera-Herrera, Raúl A; Ibarra, Ana M

2018-08-20

The Pacific white shrimp Penaeus vannamei is the most cultured shrimp species around the world. Because females grow larger than males, the culture of 'only females' is of great interest, but knowledge on sex determination and differentiation is required for producing only females. In an effort to obtain information associated with reproduction in P. vannamei, transcriptomic data from female gonads was generated, and partial sequences of a transcript were identified as Sex-lethal (Sxl). Its characterization indicated that, differently from other penaeids in which this gene has been isolated, there are six isoforms of the Sxl transcript in P. vannamei (PvanSxl 1-6). These isoforms result from alternative splicing at three splice sites (SS1, SS2, SS3). The first splice-site is unique to P. vannamei, as it has not been reported for other Arthropod species; the second splice-site (SS2) is common among crustaceans, and the third splice-site (SS3) is also unique to P. vannamei and when spliced-out, it is always together with SS2. All isoforms are expressed during embryogenesis as well as gametogenesis of both genders. The two shorter isoforms, PvanSxl-5 and PvanSxl-6, which result from the splicing of SS2 and SS3, were found mostly expressed in adult testis, but PvanSxl-6 was also expressed in oocytes during gametogenesis. During oogenesis, the second largest isoform, PvanSxl-2, which splices-out only SS1, and PvanSxl-4 that splices-out SS1 and SS2 were highly expressed. These two isoforms were also highly expressed during embryonic development. In situ hybridization allowed pinpointing more specifically the cells where the PvanSxl transcripts were expressed. During embryogenesis, hybridization was observed from the one-cell stage embryo to late gastrula. In the female gonad in previtellogenesis, hybridization occurred in the nucleus of oocytes, whereas in secondary vitellogenesis the transcript also hybridized cytoplasmic granules and cortical crypts. Finally, in situ hybridization corroborated the expression of PvanSxl also in the male gonad during spermatogenesis, mostly occurring in the cytoplasm from spermatogonia and spermatocytes. Copyright © 2018 Elsevier B.V. All rights reserved.

Gene expression and splicing alterations analyzed by high throughput RNA sequencing of chronic lymphocytic leukemia specimens.

PubMed

Liao, Wei; Jordaan, Gwen; Nham, Phillipp; Phan, Ryan T; Pelegrini, Matteo; Sharma, Sanjai

2015-10-16

To determine differentially expressed and spliced RNA transcripts in chronic lymphocytic leukemia specimens a high throughput RNA-sequencing (HTS RNA-seq) analysis was performed. Ten CLL specimens and five normal peripheral blood CD19+ B cells were analyzed by HTS RNA-seq. The library preparation was performed with Illumina TrueSeq RNA kit and analyzed by Illumina HiSeq 2000 sequencing system. An average of 48.5 million reads for B cells, and 50.6 million reads for CLL specimens were obtained with 10396 and 10448 assembled transcripts for normal B cells and primary CLL specimens respectively. With the Cuffdiff analysis, 2091 differentially expressed genes (DEG) between B cells and CLL specimens based on FPKM (fragments per kilobase of transcript per million reads and false discovery rate, FDR q < 0.05, fold change >2) were identified. Expression of selected DEGs (n = 32) with up regulated and down regulated expression in CLL from RNA-seq data were also analyzed by qRT-PCR in a test cohort of CLL specimens. Even though there was a variation in fold expression of DEG genes between RNA-seq and qRT-PCR; more than 90 % of analyzed genes were validated by qRT-PCR analysis. Analysis of RNA-seq data for splicing alterations in CLL and B cells was performed by Multivariate Analysis of Transcript Splicing (MATS analysis). Skipped exon was the most frequent splicing alteration in CLL specimens with 128 significant events (P-value <0.05, minimum inclusion level difference >0.1). The RNA-seq analysis of CLL specimens identifies novel DEG and alternatively spliced genes that are potential prognostic markers and therapeutic targets. High level of validation by qRT-PCR for a number of DEG genes supports the accuracy of this analysis. Global comparison of transcriptomes of B cells, IGVH non-mutated CLL (U-CLL) and mutated CLL specimens (M-CLL) with multidimensional scaling analysis was able to segregate CLL and B cell transcriptomes but the M-CLL and U-CLL transcriptomes were indistinguishable. The analysis of HTS RNA-seq data to identify alternative splicing events and other genetic abnormalities specific to CLL is an added advantage of RNA-seq that is not feasible with other genome wide analysis.

The B2 Alternatively Spliced Isoform of Nonmuscle Myosin II-B Lacks Actin-activated MgATPase Activity and In Vitro Motility

PubMed Central

Kim, Kye-Young; Kawamoto, Sachiyo; Bao, Jianjun; Sellers, James R.; Adelstein, Robert S.

2008-01-01

We report the initial biochemical characterization of an alternatively spliced isoform of nonmuscle heavy meromyosin (HMM) II-B2 and compare it with HMM II-B0, the non-spliced isoform. HMM II-B2 is the HMM derivative of an alternatively spliced isoform of endogenous nonmuscle myosin (NM) II-B, which has 21-amino acids inserted into loop 2, near the actin-binding region. NM II-B2 is expressed in the Purkinje cells of the cerebellum as well as in other neuronal cells (Ma et al., Mol. Biol. Cell 15 (2006) 2138-2149). In contrast to any of the previously described isoforms of NM II (II-A, II-B0, II-B1, II-C0 and II-C1) or to smooth muscle myosin, the actin-activated MgATPase activity of HMM II-B2 is not significantly increased from a low, basal level by phosphorylation of the 20 kDa myosin light chain (MLC-20). Moreover, although HMM II-B2 can bind to actin in the absence of ATP and is released in its presence, it cannot propel actin in the sliding actin filament assay following MLC-20 phosphorylation. Unlike HMM II-B2, the actin-activated MgATPase activity of a chimeric HMM with the 21-amino acids II-B2 sequence inserted into the homologous location in the heavy chain of HMM II-C is increased following MLC-20 phosphorylation. This indicates that the effect of the II-B2 insert is myosin heavy chain specific. PMID:18060863

The UDP-Glucuronosyltransferase (UGT) 1A Polymorphism c.2042C>G (rs8330) Is Associated with Increased Human Liver Acetaminophen Glucuronidation, Increased UGT1A Exon 5a/5b Splice Variant mRNA Ratio, and Decreased Risk of Unintentional Acetaminophen-Induced Acute Liver FailureS⃞

PubMed Central

Freytsis, Marina; Wang, Xueding; Peter, Inga; Guillemette, Chantal; Hazarika, Suwagmani; Duan, Su X.; Greenblatt, David J.; Lee, William M.

2013-01-01

Acetaminophen is cleared primarily by hepatic glucuronidation. Polymorphisms in genes encoding the acetaminophen UDP-glucuronosyltransferase (UGT) enzymes could explain interindividual variability in acetaminophen glucuronidation and variable risk for liver injury after acetaminophen overdose. In this study, human liver bank samples were phenotyped for acetaminophen glucuronidation activity and genotyped for the major acetaminophen-glucuronidating enzymes (UGTs 1A1, 1A6, 1A9, and 2B15). Of these, only three linked single nucleotide polymorphisms (SNPs) located in the shared UGT1A-3′UTR region (rs10929303, rs1042640, rs8330) were associated with acetaminophen glucuronidation activity, with rs8330 consistently showing higher acetaminophen glucuronidation at all the tested concentrations of acetaminophen. Mechanistic studies using luciferase-UGT1A-3′UTR reporters indicated that these SNPs do not alter mRNA stability or translation efficiency. However, there was evidence for allelic imbalance and a gene-dose proportional increase in the amount of exon 5a versus exon 5b containing UGT1A mRNA spliced transcripts in livers with the rs8330 variant allele. Cotransfection studies demonstrated an inhibitory effect of exon 5b containing cDNAs on acetaminophen glucuronidation by UGT1A1 and UGT1A6 cDNAs containing exon 5a. In silico analysis predicted that rs8330 creates an exon splice enhancer site that could favor exon 5a (over exon 5b) utilization during splicing. Finally, the prevalence of rs8330 was significantly lower (P = 0.027, χ2 test) in patients who had acute liver failure from unintentional acetaminophen overdose compared with patients with acute liver failure from other causes or a race- or ethnicity-matched population. Together, these findings suggest that rs8330 is an important determinant of acetaminophen glucuronidation and could affect an individual’s risk for acetaminophen-induced liver injury. PMID:23408116

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.

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

17A, a novel non-coding RNA, regulates GABA B alternative splicing and signaling in response to inflammatory stimuli and in Alzheimer disease.

PubMed

Massone, Sara; Vassallo, Irene; Fiorino, Gloria; Castelnuovo, Manuele; Barbieri, Federica; Borghi, Roberta; Tabaton, Massimo; Robello, Mauro; Gatta, Elena; Russo, Claudio; Florio, Tullio; Dieci, Giorgio; Cancedda, Ranieri; Pagano, Aldo

2011-02-01

Alternative splicing is a central component of human brain complexity; nonetheless, its regulatory mechanisms are still largely unclear. In this work, we describe a novel non-coding (nc) RNA (named 17A) RNA polymerase (pol) III-dependent embedded in the human G-protein-coupled receptor 51 gene (GPR51, GABA B2 receptor). The stable expression of 17A in SHSY5Y neuroblastoma cells induces the synthesis of an alternative splicing isoform that abolish GABA B2 intracellular signaling (i.e., inhibition of cAMP accumulation and activation of K(+) channels). Indeed, 17A is expressed in human brain, and we report that it is upregulated in cerebral tissues derived from Alzheimer disease patients. We demonstrate that 17A expression in neuroblastoma cells enhances the secretion of amyloid β peptide (Aβ) and the Aβ x-42/Αβ x-40 peptide ratio and that its synthesis is induced in response to inflammatory stimuli. These data correlate, for the first time, the activity of a novel pol III-dependent ncRNA to alternative splicing events and, possibly, to neurodegeneration induced by abnormal GABA B function. We anticipate that further analysis of pol III-dependent regulation of alternative splicing will disclose novel regulatory pathways associated to brain physiology and/or pathology. Copyright © 2010 Elsevier Inc. All rights reserved.

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.

Activation of Antitumorigenic Stat3beta in Breast Cancer by Splicing Redirection

DTIC Science & Technology

2013-07-01

putative mapped ESEs (shown in green). (B) (Top) RT-PCR and (Bottom) Western Blot analysis of STAT3 a/b levels in MDA-MB-435s cells treated with...codon (PTC), ultimately causing RNA degradation following nonsense mediated decay (NMD). (B) RT-PCR and Western Blot analysis of STAT3 α/β levels in MDA...MB-435s cells treated with 16µM of ST6, ST7 or INV for 4 days. α-tubulin was used as loading control. (C) RT-PCR and Western Blot analysis of STAT3

Mutations in POLR3A and POLR3B Encoding RNA Polymerase III Subunits Cause an Autosomal-Recessive Hypomyelinating Leukoencephalopathy

PubMed Central

Saitsu, Hirotomo; Osaka, Hitoshi; Sasaki, Masayuki; Takanashi, Jun-ichi; Hamada, Keisuke; Yamashita, Akio; Shibayama, Hidehiro; Shiina, Masaaki; Kondo, Yukiko; Nishiyama, Kiyomi; Tsurusaki, Yoshinori; Miyake, Noriko; Doi, Hiroshi; Ogata, Kazuhiro; Inoue, Ken; Matsumoto, Naomichi

2011-01-01

Congenital hypomyelinating disorders are a heterogeneous group of inherited leukoencephalopathies characterized by abnormal myelin formation. We have recently reported a hypomyelinating syndrome characterized by diffuse cerebral hypomyelination with cerebellar atrophy and hypoplasia of the corpus callosum (HCAHC). We performed whole-exome sequencing of three unrelated individuals with HCAHC and identified compound heterozygous mutations in POLR3B in two individuals. The mutations include a nonsense mutation, a splice-site mutation, and two missense mutations at evolutionally conserved amino acids. Using reverse transcription-PCR and sequencing, we demonstrated that the splice-site mutation caused deletion of exon 18 from POLR3B mRNA and that the transcript harboring the nonsense mutation underwent nonsense-mediated mRNA decay. We also identified compound heterozygous missense mutations in POLR3A in the remaining individual. POLR3A and POLR3B encode the largest and second largest subunits of RNA Polymerase III (Pol III), RPC1 and RPC2, respectively. RPC1 and RPC2 together form the active center of the polymerase and contribute to the catalytic activity of the polymerase. Pol III is involved in the transcription of small noncoding RNAs, such as 5S ribosomal RNA and all transfer RNAs (tRNA). We hypothesize that perturbation of Pol III target transcription, especially of tRNAs, could be a common pathological mechanism underlying POLR3A and POLR3B mutations. PMID:22036171

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.

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

Intellectual disabilities, neuronal posttranscriptional RNA metabolism, and RNA-binding proteins: three actors for a complex scenario.

PubMed

Bardoni, Barbara; Abekhoukh, Sabiha; Zongaro, Samantha; Melko, Mireille

2012-01-01

Intellectual disability (ID) is the most frequent cause of serious handicap in children and young adults and interests 2-3% of worldwide population, representing a serious problem from the medical, social, and economic points of view. The causes are very heterogeneous. Genes involved in ID have various functions altering different pathways important in neuronal function. Regulation of mRNA metabolism is particularly important in neurons for synaptic structure and function. Here, we review ID due to alteration of mRNA metabolism. Functional absence of some RNA-binding proteins--namely, FMRP, FMR2P, PQBP1, UFP3B, VCX-A--causes different forms of ID. These proteins are involved in different steps of RNA metabolism and, even if a detailed analysis of their RNA targets has been performed so far only for FMRP, it appears clear that they modulate some aspects (translation, stability, transport, and sublocalization) of a subset of RNAs coding for proteins, whose function must be relevant for neurons. Two other proteins, DYRK1A and CDKL5, involved in Down syndrome and Rett syndrome, respectively, have been shown to have an impact on splicing efficiency of specific mRNAs. Both proteins are kinases and their effect is indirect. Interestingly, both are localized in nuclear speckles, the nuclear domains where splicing factors are assembled, stocked, and recycled and influence their biogenesis and/or their organization. Copyright © 2012 Elsevier B.V. All rights reserved.

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

DTIC Science & Technology

2015-10-01

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

A new role for FBP21 as regulator of Brr2 helicase activity.

PubMed

Henning, Lisa M; Santos, Karine F; Sticht, Jana; Jehle, Stefanie; Lee, Chung-Tien; Wittwer, Malte; Urlaub, Henning; Stelzl, Ulrich; Wahl, Markus C; Freund, Christian

2017-07-27

Splicing of eukaryotic pre-mRNA is carried out by the spliceosome, which assembles stepwise on each splicing substrate. This requires the concerted action of snRNPs and non-snRNP accessory proteins, the functions of which are often not well understood. Of special interest are B complex factors that enter the spliceosome prior to catalytic activation and may alter splicing kinetics and splice site selection. One of these proteins is FBP21, for which we identified several spliceosomal binding partners in a yeast-two-hybrid screen, among them the RNA helicase Brr2. Biochemical and biophysical analyses revealed that an intrinsically disordered region of FBP21 binds to an extended surface of the C-terminal Sec63 unit of Brr2. Additional contacts in the C-terminal helicase cassette are required for allosteric inhibition of Brr2 helicase activity. Furthermore, the direct interaction between FBP21 and the U4/U6 di-snRNA was found to reduce the pool of unwound U4/U6 di-snRNA. Our results suggest FBP21 as a novel key player in the regulation of Brr2. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

VEGF(121)b, a new member of the VEGF(xxx)b family of VEGF-A splice isoforms, inhibits neovascularisation and tumour growth in vivo.

PubMed

Rennel, E S; Varey, A H R; Churchill, A J; Wheatley, E R; Stewart, L; Mather, S; Bates, D O; Harper, S J

2009-10-06

The key mediator of new vessel formation in cancer and other diseases is VEGF-A. VEGF-A exists as alternatively spliced isoforms - the pro-angiogenic VEGF(xxx) family generated by exon 8 proximal splicing, and a sister family, termed VEGF(xxx)b, exemplified by VEGF(165)b, generated by distal splicing of exon 8. However, it is unknown whether this anti-angiogenic property of VEGF(165)b is a general property of the VEGF(xxx)b family of isoforms. The mRNA and protein expression of VEGF(121)b was studied in human tissue. The effect of VEGF(121)b was analysed by saturation binding to VEGF receptors, endothelial migration, apoptosis, xenograft tumour growth, pre-retinal neovascularisation and imaging of biodistribution in tumour-bearing mice with radioactive VEGF(121)b. The existence of VEGF(121)b was confirmed in normal human tissues. VEGF(121)b binds both VEGF receptors with similar affinity as other VEGF isoforms, but inhibits endothelial cell migration and is cytoprotective to endothelial cells through VEGFR-2 activation. Administration of VEGF(121)b normalised retinal vasculature by reducing both angiogenesis and ischaemia. VEGF(121)b reduced the growth of xenografted human colon tumours in association with reduced microvascular density, and an intravenous bolus of VEGF(121)b is taken up into colon tumour xenografts. Here we identify a second member of the family, VEGF(121)b, with similar properties to those of VEGF(165)b, and underline the importance of the six amino acids of exon 8b in the anti-angiogenic activity of the VEGF(xxx)b isoforms.

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.

Structure and Function of the Splice Variants of TMPRSS2-ERG, a Prevalent Genomic Alteration in Prostate Cancer

DTIC Science & Technology

2012-09-01

apoptosis through VE-cadherin. Blood 2008; 111: 3498–3506. 20 Ellett F, Kile BT, Lieschke GJ. The role of the ETS factor erg in zebrafish...angiogenesis and endothelial apoptosis through VE-cadherin. Blood 111, 3498–3506 (2008). 46. Ellett, F., Kile , B. T. & Lieschke, G. J. The role of

Genome-wide Association Study Implicates PARD3B-based AIDS Restriction

PubMed Central

Nelson, George W.; Lautenberger, James A.; Chinn, Leslie; McIntosh, Carl; Johnson, Randall C.; Sezgin, Efe; Kessing, Bailey; Malasky, Michael; Hendrickson, Sher L.; Pontius, Joan; Tang, Minzhong; An, Ping; Winkler, Cheryl A.; Limou, Sophie; Le Clerc, Sigrid; Delaneau, Olivier; Zagury, Jean-François; Schuitemaker, Hanneke; van Manen, Daniëlle; Bream, Jay H.; Gomperts, Edward D.; Buchbinder, Susan; Goedert, James J.; Kirk, Gregory D.; O'Brien, Stephen J.

2011-01-01

Background. Host genetic variation influences human immunodeficiency virus (HIV) infection and progression to AIDS. Here we used clinically well-characterized subjects from 5 pretreatment HIV/AIDS cohorts for a genome-wide association study to identify gene associations with rate of AIDS progression. Methods.  European American HIV seroconverters (n = 755) were interrogated for single-nucleotide polymorphisms (SNPs) (n = 700,022) associated with progression to AIDS 1987 (Cox proportional hazards regression analysis, co-dominant model). Results.  Association with slower progression was observed for SNPs in the gene PARD3B. One of these, rs11884476, reached genome-wide significance (relative hazard = 0.3; P =3. 370 × 10−9) after statistical correction for 700,022 SNPs and contributes 4.52% of the overall variance in AIDS progression in this study. Nine of the top-ranked SNPs define a PARD3B haplotype that also displays significant association with progression to AIDS (hazard ratio, 0.3; P = 3.220 × 10−8). One of these SNPs, rs10185378, is a predicted exonic splicing enhancer; significant alteration in the expression profile of PARD3B splicing transcripts was observed in B cell lines with alternate rs10185378 genotypes. This SNP was typed in European cohorts of rapid progressors and was found to be protective for AIDS 1993 definition (odds ratio, 0.43, P = .025). Conclusions. These observations suggest a potential unsuspected pathway of host genetic influence on the dynamics of AIDS progression. PMID:21502085

Structure and Function of the Two Tandem WW Domains of the Pre-mRNA Splicing Factor FBP21 (Formin-binding Protein 21)*

PubMed Central

Huang, Xiaojuan; Beullens, Monique; Zhang, Jiahai; Zhou, Yi; Nicolaescu, Emilia; Lesage, Bart; Hu, Qi; Wu, Jihui; Bollen, Mathieu; Shi, Yunyu

2009-01-01

Human FBP21 (formin-binding protein 21) contains a matrin-type zinc finger and two tandem WW domains. It is a component of the spliceosomes and interacts with several established splicing factors. Here we demonstrate for the first time that FBP21 is an activator of pre-mRNA splicing in vivo and that its splicing activation function and interaction with the splicing factor SIPP1 (splicing factor that interacts with PQBP1 and PP1) are both mediated by the two tandem WW domains of group III. We determined the solution structure of the tandem WW domains of FBP21 and found that the WW domains recognize peptide ligands containing either group II (PPLP) or group III (PPR) motifs. The binding interfaces involve both the XP and XP2 grooves of the two WW domains. Significantly, the tandem WW domains of FBP21 are connected by a highly flexible region, enabling their simultaneous interaction with two proline-rich motifs of SIPP1. The strong interaction between SIPP1 and FBP21 can be explained by the conjugation of two low affinity interactions with the tandem WW domains. Our study provides a structural basis for understanding the molecular mechanism underlying the functional implication of FBP21 and the biological specificity of tandem WW domains. PMID:19592703

Dynamic integration of splicing within gene regulatory pathways

PubMed Central

Braunschweig, Ulrich; Gueroussov, Serge; Plocik, Alex; Graveley, Brenton R.; Blencowe, Benjamin J.

2013-01-01

Precursor mRNA splicing is one of the most highly regulated processes in metazoan species. In addition to generating vast repertoires of RNAs and proteins, splicing has a profound impact on other gene regulatory layers, including mRNA transcription, turnover, transport and translation. Conversely, factors regulating chromatin and transcription complexes impact the splicing process. This extensive cross-talk between gene regulatory layers takes advantage of dynamic spatial, physical and temporal organizational properties of the cell nucleus, and further emphasizes the importance of developing a multidimensional understanding of splicing control. PMID:23498935

Solution structure of the first RNA recognition motif domain of human spliceosomal protein SF3b49 and its mode of interaction with a SF3b145 fragment

PubMed Central

Nameki, Nobukazu; Tsuda, Kengo; Takahashi, Mari; Sato, Atsuko; Tochio, Naoya; Inoue, Makoto; Terada, Takaho; Kigawa, Takanori; Kobayashi, Naohiro; Shirouzu, Mikako; Ito, Takuhiro; Sakamoto, Taiichi; Wakamatsu, Kaori; Güntert, Peter; Takahashi, Seizo; Yokoyama, Shigeyuki

2016-01-01

Abstract The spliceosomal protein SF3b49, a component of the splicing factor 3b (SF3b) protein complex in the U2 small nuclear ribonucleoprotein, contains two RNA recognition motif (RRM) domains. In yeast, the first RRM domain (RRM1) of Hsh49 protein (yeast orthologue of human SF3b49) reportedly interacts with another component, Cus1 protein (orthologue of human SF3b145). Here, we solved the solution structure of the RRM1 of human SF3b49 and examined its mode of interaction with a fragment of human SF3b145 using NMR methods. Chemical shift mapping showed that the SF3b145 fragment spanning residues 598–631 interacts with SF3b49 RRM1, which adopts a canonical RRM fold with a topology of β1‐α1‐β2‐β3‐α2‐β4. Furthermore, a docking model based on NOESY measurements suggests that residues 607–616 of the SF3b145 fragment adopt a helical structure that binds to RRM1 predominantly via α1, consequently exhibiting a helix–helix interaction in almost antiparallel. This mode of interaction was confirmed by a mutational analysis using GST pull‐down assays. Comparison with structures of all RRM domains when complexed with a peptide found that this helix–helix interaction is unique to SF3b49 RRM1. Additionally, all amino acid residues involved in the interaction are well conserved among eukaryotes, suggesting evolutionary conservation of this interaction mode between SF3b49 RRM1 and SF3b145. PMID:27862552

Splicing Efficiently Couples Optical Fibers

NASA Technical Reports Server (NTRS)

Lutes, G. F.

1985-01-01

Method of splicing single-mode optical fibers results in very low transmission losses through joined fiber ends. Coupling losses between joined optical-fiber ends only 0.1 dB. Method needs no special operator training.

Genome-based identification of spliceosomal proteins in the silk moth Bombyx mori.

PubMed

Somarelli, Jason A; Mesa, Annia; Fuller, Myron E; Torres, Jacqueline O; Rodriguez, Carol E; Ferrer, Christina M; Herrera, Rene J

2010-12-01

Pre-messenger RNA splicing is a highly conserved eukaryotic cellular function that takes place by way of a large, RNA-protein assembly known as the spliceosome. In the mammalian system, nearly 300 proteins associate with uridine-rich small nuclear (sn)RNAs to form this complex. Some of these splicing factors are ubiquitously present in the spliceosome, whereas others are involved only in the processing of specific transcripts. Several proteomics analyses have delineated the proteins of the spliceosome in several species. In this study, we mine multiple sequence data sets of the silk moth Bombyx mori in an attempt to identify the entire set of known spliceosomal proteins. Five data sets were utilized, including the 3X, 6X, and Build 2.0 genomic contigs as well as the expressed sequence tag and protein libraries. While homologs for 88% of vertebrate splicing factors were delineated in the Bombyx mori genome, there appear to be several spliceosomal polypeptides absent in Bombyx mori and seven additional insect species. This apparent increase in spliceosomal complexity in vertebrates may reflect the tissue-specific and developmental stage-specific alternative pre-mRNA splicing requirements in vertebrates. Phylogenetic analyses of 15 eukaryotic taxa using the core splicing factors suggest that the essential functional units of the pre-mRNA processing machinery have remained highly conserved from yeast to humans. The Sm and LSm proteins are the most conserved, whereas proteins of the U1 small nuclear ribonucleoprotein particle are the most divergent. These data highlight both the differential conservation and relative phylogenetic signals of the essential spliceosomal components throughout evolution. © 2010 Wiley Periodicals, Inc.

Par-4/THAP1 complex and Notch3 competitively regulated pre-mRNA splicing of CCAR1 and affected inversely the survival of T-cell acute lymphoblastic leukemia cells

PubMed Central

Lu, C; Li, J-Y; Ge, Z; Zhang, L; Zhou, G-P

2013-01-01

Although the intensification of therapy for children with T-cell acute lymphoblastic leukemia (T-ALL) has substantially improved clinical outcomes, T-ALL remains an important challenge in pediatric oncology. Here, we report that the cooperative synergy between prostate apoptosis response factor-4 (Par-4) and THAP1 induces cell cycle and apoptosis regulator 1 (CCAR1) gene expression and cellular apoptosis in human T-ALL cell line Jurkat cells, CEM cells and primary cultured neoplastic T lymphocytes from children with T-ALL. Par-4 and THAP1 collaborated to activate the promoter of CCAR1 gene. Mechanistic investigations revealed that Par-4 and THAP1 formed a protein complex by the interaction of their carboxyl termini, and THAP1 bound to CCAR1 promoter though its zinc-dependent DNA-binding domain at amino terminus. Par-4/THAP1 complex and Notch3 competitively bound to CCAR1 promoter and competitively modulated alternative pre-mRNA splicing of CCAR1, which resulted in two different transcripts and played an opposite role in T-ALL cell survival. Despite Notch3 induced a shift splicing from the full-length isoform toward a shorter form of CCAR1 mRNA by splicing factor SRp40 and SRp55, Par-4/THAP1 complex strongly antagonized this inductive effect. Our finding revealed a mechanistic rationale for Par-4/THAP1-induced apoptosis in T-ALL cells that would be of benefit to develop a new therapy strategy for T-ALL. PMID:23975424

Tissue-specific expression and regulation of the alternatively-spliced forms of lysyl hydroxylase 2 (LH2) in human kidney cells and skin fibroblasts.

PubMed

Walker, Linda C; Overstreet, Mayra A; Yeowell, Heather N

2005-01-01

Lysyl hydroxylases 1, 2, and 3 catalyse the hydroxylation of specific lysines in collagen. A small percentage of these hydroxylysine residues are precursors for the cross-link formation essential for the tensile strength of collagen. Lysyl hydroxylase 2 (LH2) exists as two alternatively-spliced forms; the long transcript (the major ubiquitously-expressed form) includes a 63 bp exon (13A) that is spliced out in the short form (expressed, together with the long form, in human kidney, spleen, liver, and placenta). This study shows that this alternative splicing event can be regulated by both cell density and cycloheximide (CHX). Although only the long form of LH2 is detected in untreated confluent human skin fibroblasts, after 24 h treatment with CHX the short LH2 transcript is also expressed. In kidney cells, in which both LH2 transcripts are equally expressed, the long LH2 transcript is significantly decreased after 24 h CHX treatment, whereas expression of the short transcript is slightly increased. This suggests that, in kidney cells, the splicing mechanism for the inclusion of exon 13A in LH2 requires a newly-synthesized protein factor that is suppressed by CHX, whereas, in skin fibroblasts in which levels of LH2 (long) are unaffected, CHX appears to suppress a factor that inhibits exclusion of exon 13A, thereby promoting expression of LH2 (short). As these alternate transcripts of LH2 may have specificity for hydroxylation of lysines in either telopeptide or helical collagen domains, their relative expression determines the type of cross-links formed, thereby affecting collagen strength. Therefore, any perturbation of the regulation of LH2 splicing could influence the stability of the extracellular matrix and contribute to specific connective tissue disorders.

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

A single splice site mutation in human-specific ARHGAP11B causes basal progenitor amplification

PubMed Central

Florio, Marta; Namba, Takashi; Pääbo, Svante; Hiller, Michael; Huttner, Wieland B.

2016-01-01

The gene ARHGAP11B promotes basal progenitor amplification and is implicated in neocortex expansion. It arose on the human evolutionary lineage by partial duplication of ARHGAP11A, which encodes a Rho guanosine triphosphatase–activating protein (RhoGAP). However, a lack of 55 nucleotides in ARHGAP11B mRNA leads to loss of RhoGAP activity by GAP domain truncation and addition of a human-specific carboxy-terminal amino acid sequence. We show that these 55 nucleotides are deleted by mRNA splicing due to a single C→G substitution that creates a novel splice donor site. We reconstructed an ancestral ARHGAP11B complementary DNA without this substitution. Ancestral ARHGAP11B exhibits RhoGAP activity but has no ability to increase basal progenitors during neocortex development. Hence, a single nucleotide substitution underlies the specific properties of ARHGAP11B that likely contributed to the evolutionary expansion of the human neocortex. PMID:27957544

Development of Potent, Selective SRPK1 Inhibitors as Potential Topical Therapeutics for Neovascular Eye Disease.

PubMed

Batson, Jennifer; Toop, Hamish D; Redondo, Clara; Babaei-Jadidi, Roya; Chaikuad, Apirat; Wearmouth, Stephen F; Gibbons, Brian; Allen, Claire; Tallant, Cynthia; Zhang, Jingxue; Du, Chunyun; Hancox, Jules C; Hawtrey, Tom; Da Rocha, Joana; Griffith, Renate; Knapp, Stefan; Bates, David O; Morris, Jonathan C

2017-03-17

Serine/arginine-protein kinase 1 (SRPK1) regulates alternative splicing of VEGF-A to pro-angiogenic isoforms and SRPK1 inhibition can restore the balance of pro/antiangiogenic isoforms to normal physiological levels. The lack of potency and selectivity of available compounds has limited development of SRPK1 inhibitors, with the control of alternative splicing by splicing factor-specific kinases yet to be translated. We present here compounds that occupy a binding pocket created by the unique helical insert of SRPK1, and trigger a backbone flip in the hinge region, that results in potent (<10 nM) and selective inhibition of SRPK1 kinase activity. Treatment with these inhibitors inhibited SRPK1 activity and phosphorylation of serine/arginine splicing factor 1 (SRSF1), resulting in alternative splicing of VEGF-A from pro-angiogenic to antiangiogenic isoforms. This property resulted in potent inhibition of blood vessel growth in models of choroidal angiogenesis in vivo. This work identifies tool compounds for splice isoform selective targeting of pro-angiogenic VEGF, which may lead to new therapeutic strategies for a diversity of diseases where dysfunctional splicing drives disease development.

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

DTIC Science & Technology

2015-10-01

Populations: Contributing Factor in Prostate Cancer Disparities? PRINCIPAL INVESTIGATOR: Norman H Lee, Ph.D. CONTRACTING ORGANIZATION: George Washington...Prostate Cancer Disparities? 5b. GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) Norman H Lee, PhD; Bi-Dar Wang, PhD; Jacqueline Olender (PhD graduate...suppressor genes in prostate cancer disparities between African American (AA) and Caucasian American (CA) prostate cancer (PCa). In year 1 of this award

Tannic acid facilitates expression of the polypyrimidine tract binding protein and alleviates deleterious inclusion of CHRNA1 exon P3A due to an hnRNP H-disrupting mutation in congenital myasthenic syndrome

PubMed Central

Bian, Yang; Masuda, Akio; Matsuura, Tohru; Ito, Mikako; Okushin, Kazuya; Engel, Andrew G.; Ohno, Kinji

2009-01-01

We recently reported that the intronic splice-site mutation IVS3-8G>A of CHRNA1 that encodes the muscle nicotinic acetylcholine receptor α subunit disrupts binding of a splicing repressor, hnRNP H. This, in turn, results in exclusive inclusion of the downstream exon P3A. The P3A(+) transcript encodes a non-functional α subunit that comprises 50% of the transcripts in normal human skeletal muscle, but its functional significance remains undetermined. In an effort to search for a potential therapy, we screened off-label effects of 960 bioactive chemical compounds and found that tannic acid ameliorates the aberrant splicing due to IVS3-8G>A but without altering the expression of hnRNP H. Therefore, we searched for another splicing trans-factor. We found that the polypyrimidine tract binding protein (PTB) binds close to the 3′ end of CHRNA1 intron 3, that PTB induces skipping of exon P3A and that tannic acid increases the expression of PTB in a dose-dependent manner. Deletion assays of the PTB promoter region revealed that the tannic acid-responsive element is between positions −232 and −74 from the translation initiation site. These observations open the door to the discovery of novel therapies based on PTB overexpression and to detecting possible untoward effects of the overexpression. PMID:19147685

CELF1 preferentially binds to exon-intron boundary and regulates alternative splicing in HeLa cells.

PubMed

Xia, Heng; Chen, Dong; Wu, Qijia; Wu, Gang; Zhou, Yanhong; Zhang, Yi; Zhang, Libin

2017-09-01

The current RIP-seq approach has been developed for the identification of genome-wide interaction between RNA binding protein (RBP) and the bound RNA transcripts, but still rarely for identifying its binding sites. In this study, we performed RIP-seq experiments in HeLa cells using a monoclonal antibody against CELF1. Mapping of the RIP-seq reads showed a biased distribution at the 3'UTR and intronic regions. A total of 15,285 and 1384 CELF1-specific sense and antisense peaks were identified using the ABLIRC software tool. Our bioinformatics analyses revealed that 5' and 3' splice site motifs and GU-rich motifs were highly enriched in the CELF1-bound peaks. Furthermore, transcriptome analyses revealed that alternative splicing was globally regulated by CELF1 in HeLa cells. For example, the inclusion of exon 16 of LMO7 gene, a marker gene of breast cancer, is positively regulated by CELF1. Taken together, we have shown that RIP-seq data can be used to decipher RBP binding sites and reveal an unexpected landscape of the genome-wide CELF1-RNA interactions in HeLa cells. In addition, we found that CELF1 globally regulates the alternative splicing by binding the exon-intron boundary in HeLa cells, which will deepen our understanding of the regulatory roles of CELF1 in the pre-mRNA splicing process. Copyright © 2017 Elsevier B.V. All rights reserved.

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.

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

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

Analyses of publicly available genomics resources define FGF-2-expressing bladder carcinomas as EMT-prone, proliferative tumors with low mutation rates and high expression of CTLA-4, PD-1 and PD-L1

PubMed Central

McNiel, Elizabeth A; Tsichlis, Philip N

2017-01-01

Fibroblast growth factor 2 (FGF-2) is overexpressed in a subset of invasive bladder carcinomas and its overexpression correlates with poor prognosis. Analyses of publicly available databases addressing the molecular mechanisms that may be responsible for the poor prognosis of these tumors, revealed that FGF-2 expression correlates positively with the expression of epithelial to mesenchymal transition (EMT)-promoting transcription factors and with changes in gene expression that are characteristic of EMT. The same analyses also revealed that FGF-2 correlates negatively with the expression, mutation and copy number variations of FGFR-3, all of which are associated with noninvasive bladder carcinomas. Finally, they showed that FGF-2 expression correlates with the expression of FGFR-1, the expression of the IIIc variant of FGFR-2 and with the expression of Akt3. The latter observation is significant because our earlier studies had shown that Akt3 regulates FGFR-2 alternative splicing, shifting the balance toward the IIIc relative to the IIIb FGFR-2 splice variant. As the IIIc variant is recognized by FGF-2, while the IIIb variant is not, we conclude that Akt3 may facilitate the FGF-2 response. FGF-2 is known to promote the expression of KDM2B, which functions in concert with EZH2 to repress the EZH2-targeting microRNA miR-101, activating a switch, which stably upregulates EZH2. The cancer genome atlas (TCGA) data showing a correlation between KDM2B and EZH2 expression and Oncomine data, showing a correlation between KDM2B and tumor progression, strongly support the role of the FGF-2/KDM2B/miR-101/EZH2 pathway in bladder cancer. These observations combined, suggest a model according to which FGF-2 induces EMT, cell proliferation and cancer stem cell self-renewal by coupling the Akt3 and KDM2B-controlled pathways outlined above, in bladder carcinomas. Further analyses of publicly available databases, revealed that FGF-2-expressing bladder carcinomas carry fewer genetic alterations and they tend to express high levels of CTLA-4, PD-1 and PD-L1, which suggests immune blockade by checkpoint activation. EMT, enhanced proliferation and immune checkpoint activation combined, may be responsible for the poor prognosis of FGF-2-expressing bladder carcinomas. PMID:28515962

Using a minigene approach to characterize a novel splice site mutation in human F7 gene causing inherited factor VII deficiency in a Chinese pedigree.

PubMed

Yu, T; Wang, X; Ding, Q; Fu, Q; Dai, J; Lu, Y; Xi, X; Wang, H

2009-11-01

Factor VII deficiency which transmitted as an autosomal recessive disorder is a rare haemorrhagic condition. The aim of this study was to identify the molecular genetic defect and determine its functional consequences in a Chinese pedigree with FVII deficiency. The proband was diagnosed as inherited coagulation FVII deficiency by reduced plasma levels of FVII activity (4.4%) and antigen (38.5%). All nine exons and their flanking sequence of F7 gene were amplified by polymerase chain reaction (PCR) for the proband and the PCR products were directly sequenced. The compound heterozygous mutations of F7 (NM_000131.3) c.572-1G>A and F7 (NM_000131.3) c.1165T>G; p.Cys389Gly were identified in the proband's F7 gene. To investigate the splicing patterns associated with F7 c.572-1G>A, ectopic transcripts in leucocytes of the proband were analyzed. F7 minigenes, spanning from intron 4 to intron 7 and carrying either an A or a G at position -1 of intron 5, were constructed and transiently transfected into human embryonic kidney (HEK) 293T cells, followed by RT-PCR analysis. The aberrant transcripts from the F7 c.572-1G>A mutant allele were not detected by ectopic transcription study. Sequencing of the RT-PCR products from the mutant transfectant demonstrated the production of an erroneously spliced mRNA with exon 6 skipping, whereas a normal splicing occurred in the wide type transfectant. The aberrant mRNA produced from the F7 c.572-1G>A mutant allele is responsible for the factor VII deficiency in this pedigree.

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.

The group II intron maturase: a reverse transcriptase and splicing factor go hand in hand.

PubMed

Zhao, Chen; Pyle, Anna Marie

2017-12-01

The splicing of group II introns in vivo requires the assistance of a multifunctional intron encoded protein (IEP, or maturase). Each IEP is also a reverse-transcriptase enzyme that enables group II introns to behave as mobile genetic elements. During splicing or retro-transposition, each group II intron forms a tight, specific complex with its own encoded IEP, resulting in a highly reactive holoenzyme. This review focuses on the structural basis for IEP function, as revealed by recent crystal structures of an IEP reverse transcriptase domain and cryo-EM structures of an IEP-intron complex. These structures explain how the same IEP scaffold is utilized for intron recognition, splicing and reverse transcription, while providing a physical basis for understanding the evolutionary transformation of the IEP into the eukaryotic splicing factor Prp8. Copyright © 2017 Elsevier Ltd. All rights reserved.

Antisense oligonucleotide–mediated MDM4 exon 6 skipping impairs tumor growth

PubMed Central

Dewaele, Michael; Tabaglio, Tommaso; Willekens, Karen; Bezzi, Marco; Teo, Shun Xie; Low, Diana H.P.; Koh, Cheryl M.; Rambow, Florian; Fiers, Mark; Rogiers, Aljosja; Radaelli, Enrico; Al-Haddawi, Muthafar; Tan, Soo Yong; Hermans, Els; Amant, Frederic; Yan, Hualong; Lakshmanan, Manikandan; Koumar, Ratnacaram Chandrahas; Lim, Soon Thye; Derheimer, Frederick A.; Campbell, Robert M.; Bonday, Zahid; Tergaonkar, Vinay; Shackleton, Mark; Blattner, Christine; Marine, Jean-Christophe; Guccione, Ernesto

2015-01-01

MDM4 is a promising target for cancer therapy, as it is undetectable in most normal adult tissues but often upregulated in cancer cells to dampen p53 tumor-suppressor function. The mechanisms that underlie MDM4 upregulation in cancer cells are largely unknown. Here, we have shown that this key oncogenic event mainly depends on a specific alternative splicing switch. We determined that while a nonsense-mediated, decay-targeted isoform of MDM4 (MDM4-S) is produced in normal adult tissues as a result of exon 6 skipping, enhanced exon 6 inclusion leads to expression of full-length MDM4 in a large number of human cancers. Although this alternative splicing event is likely regulated by multiple splicing factors, we identified the SRSF3 oncoprotein as a key enhancer of exon 6 inclusion. In multiple human melanoma cell lines and in melanoma patient–derived xenograft (PDX) mouse models, antisense oligonucleotide–mediated (ASO-mediated) skipping of exon 6 decreased MDM4 abundance, inhibited melanoma growth, and enhanced sensitivity to MAPK-targeting therapeutics. Additionally, ASO-based MDM4 targeting reduced diffuse large B cell lymphoma PDX growth. As full-length MDM4 is enhanced in multiple human tumors, our data indicate that this strategy is applicable to a wide range of tumor types. We conclude that enhanced MDM4 exon 6 inclusion is a common oncogenic event and has potential as a clinically compatible therapeutic target. PMID:26595814

Identification of a novel splicing mutation within SLC17A8 in a Korean family with hearing loss by whole-exome sequencing.

PubMed

Ryu, Nari; Lee, Seokwon; Park, Hong-Joon; Lee, Byeonghyeon; Kwon, Tae-Jun; Bok, Jinwoong; Park, Chan Ik; Lee, Kyu-Yup; Baek, Jeong-In; Kim, Un-Kyung

2017-09-05

Hereditary hearing loss (HHL) is a common genetically heterogeneous disorder, which follows Mendelian inheritance in humans. Because of this heterogeneity, the identification of the causative gene of HHL by linkage analysis or Sanger sequencing have shown economic and temporal limitations. With recent advances in next-generation sequencing (NGS) techniques, rapid identification of a causative gene via massively parallel sequencing is now possible. We recruited a Korean family with three generations exhibiting autosomal dominant inheritance of hearing loss (HL), and the clinical information about this family revealed that there are no other symptoms accompanied with HL. To identify a causative mutation of HL in this family, we performed whole-exome sequencing of 4 family members, 3 affected and an unaffected. As the result, A novel splicing mutation, c.763+1G>T, in the solute carrier family 17, member 8 (SLC17A8) gene was identified in the patients, and the genotypes of the mutation were co-segregated with the phenotype of HL. Additionally, this mutation was not detected in 100 Koreans with normal hearing. Via NGS, we detected a novel splicing mutation that might influence the hearing ability within the patients with autosomal dominant non-syndromic HL. Our data suggests that this technique is a powerful tool to discover causative genetic factors of HL and facilitate diagnoses of the primary cause of HHL. Copyright © 2017 Elsevier B.V. All rights reserved.

Functional Cus1p Is Found with Hsh155p in a Multiprotein Splicing Factor Associated with U2 snRNA

PubMed Central

Pauling, Michelle Haynes; McPheeters, David S.; Ares, Manuel

2000-01-01

To explore the dynamics of snRNP structure and function, we have studied Cus1p, identified as a suppressor of U2 snRNA mutations in budding yeast. Cus1p is homologous to human SAP145, a protein present in the 17S form of the human U2 snRNP. Here, we define the Cus1p amino acids required for function in yeast. The segment of Cus1p required for binding to Hsh49p, a homolog of human SAP49, is contained within an essential region of Cus1p. Antibodies against Cus1p coimmunoprecipitate U2 snRNA, as well as Hsh155p, a protein homologous to human SAP155. Biochemical fractionation of splicing extracts and reconstitution of heat-inactivated splicing extracts from strains carrying a temperature-sensitive allele of CUS1 indicate that Cus1p and Hsh155p reside in a functional, high-salt-stable complex that is salt-dissociable from U2 snRNA. We propose that Cus1p, Hsh49p, and Hsh155p exist in a stable protein complex which can exchange with a core U2 snRNP and which is necessary for U2 snRNP function in prespliceosome assembly. The Cus1p complex shares functional as well as structural similarities with human SF3b. PMID:10688664

Alternative splicing at the intersection of biological timing, development, and stress responses.

PubMed

Staiger, Dorothee; Brown, John W S

2013-10-01

High-throughput sequencing for transcript profiling in plants has revealed that alternative splicing (AS) affects a much higher proportion of the transcriptome than was previously assumed. AS is involved in most plant processes and is particularly prevalent in plants exposed to environmental stress. The identification of mutations in predicted splicing factors and spliceosomal proteins that affect cell fate, the circadian clock, plant defense, and tolerance/sensitivity to abiotic stress all point to a fundamental role of splicing/AS in plant growth, development, and responses to external cues. Splicing factors affect the AS of multiple downstream target genes, thereby transferring signals to alter gene expression via splicing factor/AS networks. The last two to three years have seen an ever-increasing number of examples of functional AS. At a time when the identification of AS in individual genes and at a global level is exploding, this review aims to bring together such examples to illustrate the extent and importance of AS, which are not always obvious from individual publications. It also aims to ensure that plant scientists are aware that AS is likely to occur in the genes that they study and that dynamic changes in AS and its consequences need to be considered routinely.

Alternative Splicing at the Intersection of Biological Timing, Development, and Stress Responses[OPEN

PubMed Central

Staiger, Dorothee; Brown, John W.S.

2013-01-01

High-throughput sequencing for transcript profiling in plants has revealed that alternative splicing (AS) affects a much higher proportion of the transcriptome than was previously assumed. AS is involved in most plant processes and is particularly prevalent in plants exposed to environmental stress. The identification of mutations in predicted splicing factors and spliceosomal proteins that affect cell fate, the circadian clock, plant defense, and tolerance/sensitivity to abiotic stress all point to a fundamental role of splicing/AS in plant growth, development, and responses to external cues. Splicing factors affect the AS of multiple downstream target genes, thereby transferring signals to alter gene expression via splicing factor/AS networks. The last two to three years have seen an ever-increasing number of examples of functional AS. At a time when the identification of AS in individual genes and at a global level is exploding, this review aims to bring together such examples to illustrate the extent and importance of AS, which are not always obvious from individual publications. It also aims to ensure that plant scientists are aware that AS is likely to occur in the genes that they study and that dynamic changes in AS and its consequences need to be considered routinely. PMID:24179132

Hypoxia regulates alternative splicing of HIF and non-HIF target genes.

PubMed

Sena, Johnny A; Wang, Liyi; Heasley, Lynn E; Hu, Cheng-Jun

2014-09-01

Hypoxia is a common characteristic of many solid tumors. The hypoxic microenvironment stabilizes hypoxia-inducible transcription factor 1α (HIF1α) and 2α (HIF2α/EPAS1) to activate gene transcription, which promotes tumor cell survival. The majority of human genes are alternatively spliced, producing RNA isoforms that code for functionally distinct proteins. Thus, an effective hypoxia response requires increased HIF target gene expression as well as proper RNA splicing of these HIF-dependent transcripts. However, it is unclear if and how hypoxia regulates RNA splicing of HIF targets. This study determined the effects of hypoxia on alternative splicing (AS) of HIF and non-HIF target genes in hepatocellular carcinoma cells and characterized the role of HIF in regulating AS of HIF-induced genes. The results indicate that hypoxia generally promotes exon inclusion for hypoxia-induced, but reduces exon inclusion for hypoxia-reduced genes. Mechanistically, HIF activity, but not hypoxia per se is found to be necessary and sufficient to increase exon inclusion of several HIF targets, including pyruvate dehydrogenase kinase 1 (PDK1). PDK1 splicing reporters confirm that transcriptional activation by HIF is sufficient to increase exon inclusion of PDK1 splicing reporter. In contrast, transcriptional activation of a PDK1 minigene by other transcription factors in the absence of endogenous HIF target gene activation fails to alter PDK1 RNA splicing. This study demonstrates a novel function of HIF in regulating RNA splicing of HIF target genes. ©2014 American Association for Cancer Research.

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

A CRM domain protein functions dually in group I and group II intron splicing in land plant chloroplasts.

PubMed

Asakura, Yukari; Barkan, Alice

2007-12-01

The CRM domain is a recently recognized RNA binding domain found in three group II intron splicing factors in chloroplasts, in a bacterial protein that associates with ribosome precursors, and in a family of uncharacterized proteins in plants. To elucidate the functional repertoire of proteins with CRM domains, we studied CFM2 (for CRM Family Member 2), which harbors four CRM domains. RNA coimmunoprecipitation assays showed that CFM2 in maize (Zea mays) chloroplasts is associated with the group I intron in pre-trnL-UAA and group II introns in the ndhA and ycf3 pre-mRNAs. T-DNA insertions in the Arabidopsis thaliana ortholog condition a defective-seed phenotype (strong allele) or chlorophyll-deficient seedlings with impaired splicing of the trnL group I intron and the ndhA, ycf3-int1, and clpP-int2 group II introns (weak alleles). CFM2 and two previously described CRM proteins are bound simultaneously to the ndhA and ycf3-int1 introns and act in a nonredundant fashion to promote their splicing. With these findings, CRM domain proteins are implicated in the activities of three classes of catalytic RNA: group I introns, group II introns, and 23S rRNA.

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

Microprocessor-dependent processing of Splice site Overlapping microRNA exons does not result in changes in alternative splicing.

PubMed

Pianigiani, Giulia; Licastro, Danilo; Fortugno, Paola; Castiglia, Daniele; Petrovic, Ivana; Pagani, Franco

2018-06-12

MicroRNAs are found throughout the genome and are processed by the microprocessor complex (MPC) from longer precursors. Some precursor miRNAs overlap intron:exon junctions. These Splice site Overlapping microRNAs (SO-miRNAs) are mostly located in coding genes. It has been intimated, in the rarer examples of SO-miRNAs in non-coding RNAs, that the competition between the spliceosome and the MPC modulates alternative splicing. However, the effect of this overlap on coding transcripts is unknown. Unexpectedly, we show that neither Drosha silencing nor SF3b1 silencing changed the inclusion ratio of SO-miRNA exons. Two SO-miRNAs, located in genes that code for basal membrane proteins, are known to inhibit proliferation in primary keratinocytes. These SO-miRNAs were upregulated during differentiation and the host mRNAs were downregulated, but again there was no change in inclusion ratio of the SO-miRNA exons. Interestingly, Drosha silencing increased nascent RNA density, on chromatin, downstream of SO-miRNA exons. Overall our data suggest a novel mechanism for regulating gene expression in which MPC-dependent cleavage of SO-miRNA exons could cause premature transcriptional termination of coding genes rather than affecting alternative splicing. Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Distinctive expression pattern of OCT4 variants in different types of breast cancer.

PubMed

Soheili, Saamaaneh; Asadi, Malek Hossein; Farsinejad, Alireza

2017-01-01

OCT4 is a key regulator of self-renewal and pluripotency in embryonic stem cells which can potentially encode three spliced variants designated OCT4A, OCT4B and OCT4B1. Based on cancer stem cell concept, it is suggested that the stemness factors misexpressed in cancer cells and potentially is involved in tumorigenesis. Accordingly, in this study, we investigated the potential expression of OCT4 variants in breast cancer tissues. A total of 94 tumoral and peritumoral breast specimens were evaluated with respect to the expression of OCT4 variants using quantitative RT-PCR and immunohistochemical (IHC) analysis. We detected the expression of OCT4 variants in breast tumor tissues with no or very low levels of expression in peritumoral samples of the same patients. While OCT4B was highly expressed in lobular type of breast cancer, OCT4A and OCTB1 variants are highly expressed in low grade (I and II) ductal tumors. Furthermore, the results of this study revealed a considerable association between the expression level of OCT4 variants and the expression of ER, PR, Her2 and P53 factors. All data demonstrated a distinctive expression pattern of OCT4 spliced variants in different types of breast cancer and provide further evidence for the involvement of embryonic genes in carcinogenesis.

Mechanistic Evaluation for Mixed-field Agglutination in the K562 Cell Study Model with Exon 3 Deletion of A1 Gene.

PubMed

Chen, Ding-Ping; Tseng, Ching-Ping; Lin, Chi-Jui; Wang, Wei-Ting; Sun, Chien-Feng

2015-01-01

In the case of blood type B3 with typical mixed-field agglutination of RBCs in the presence of anti-B or anti-AB antibody, a number of genetic alternations have been reported. It is well known that the IVS3+5G→A mutation in the B gene destroys the consensus of the splice donor site leading to exon 3 skipping during mRNA splicing. The lack of exon 3 likely causes a short stem region, producing an unstable B3 protein, and is concomitant with a decrease in B3 protein expression. Whether the phenomenon also appears in the type A blood group is of question. In this study, we evaluate whether exon 3 deletion in the blood type A gene also results in mixed-field phenotype. Site-directed mutagenesis was used to generate cDNA encoding A1 gene with exon 3 deletion. The cDNA was stably expressed in K562 cells. The expression of A antigen was compared with expression in parental K562 cells that did not express A antigen and in the stable K562 cell line expressing A(1) cDNA by flow cytometry analyses. The expression of A antigen in A1 stable cells and parental K562 cells was set as 100% and 0%, respectively. The mean relative percentage of A antigen expression for the cells of A1 with exon 3 deletion was 59.9% of A1 stable cells. Consistent with the observations of B3, which is B gene with exon 3 deletion, mixed field agglutination was observed for the cells expressing A1 with exon 3 deletion. Exon 3 deletion results in mixed field phenotype in both type A and B RBCs. However, the degree of antigen expression change for exon 3 deletion in A gene was less severe when compared with the deletion occurred in B gene. © 2015 by the Association of Clinical Scientists, Inc.

Impaired Spermatogenesis, Muscle, and Erythrocyte Function in U12 Intron Splicing-Defective Zrsr1 Mutant Mice.

PubMed

Horiuchi, Keiko; Perez-Cerezales, Serafín; Papasaikas, Panagiotis; Ramos-Ibeas, Priscila; López-Cardona, Angela Patricia; Laguna-Barraza, Ricardo; Fonseca Balvís, Noelia; Pericuesta, Eva; Fernández-González, Raul; Planells, Benjamín; Viera, Alberto; Suja, Jose Angel; Ross, Pablo Juan; Alén, Francisco; Orio, Laura; Rodriguez de Fonseca, Fernando; Pintado, Belén; Valcárcel, Juan; Gutiérrez-Adán, Alfonso

2018-04-03

The U2AF35-like ZRSR1 has been implicated in the recognition of 3' splice site during spliceosome assembly, but ZRSR1 knockout mice do not show abnormal phenotypes. To analyze ZRSR1 function and its precise role in RNA splicing, we generated ZRSR1 mutant mice containing truncating mutations within its RNA-recognition motif. Homozygous mutant mice exhibited severe defects in erythrocytes, muscle stretch, and spermatogenesis, along with germ cell sloughing and apoptosis, ultimately leading to azoospermia and male sterility. Testis RNA sequencing (RNA-seq) analyses revealed increased intron retention of both U2- and U12-type introns, including U12-type intron events in genes with key functions in spermatogenesis and spermatid development. Affected U2 introns were commonly found flanking U12 introns, suggesting functional cross-talk between the two spliceosomes. The splicing and tissue defects observed in mutant mice attributed to ZRSR1 loss of function suggest a physiological role for this factor in U12 intron splicing. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

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 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.

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.

CYCLIN-DEPENDENT KINASE G1 Is Associated with the Spliceosome to Regulate CALLOSE SYNTHASE5 Splicing and Pollen Wall Formation in Arabidopsis[C][W][OA

PubMed Central

Huang, Xue-Yong; Niu, Jin; Sun, Ming-Xi; Zhu, Jun; Gao, Ju-Fang; Yang, Jun; Zhou, Que; Yang, Zhong-Nan

2013-01-01

Arabidopsis thaliana CYCLIN-DEPEDENT KINASE G1 (CDKG1) belongs to the family of cyclin-dependent protein kinases that were originally characterized as cell cycle regulators in eukaryotes. Here, we report that CDKG1 regulates pre-mRNA splicing of CALLOSE SYNTHASE5 (CalS5) and, therefore, pollen wall formation. The knockout mutant cdkg1 exhibits reduced male fertility with impaired callose synthesis and abnormal pollen wall formation. The sixth intron in CalS5 pre-mRNA, a rare type of intron with a GC 5′ splice site, is abnormally spliced in cdkg1. RNA immunoprecipitation analysis suggests that CDKG1 is associated with this intron. CDKG1 contains N-terminal Ser/Arg (RS) motifs and interacts with splicing factor Arginine/Serine-Rich Zinc Knuckle-Containing Protein33 (RSZ33) through its RS region to regulate proper splicing. CDKG1 and RS-containing Zinc Finger Protein22 (SRZ22), a splicing factor interacting with RSZ33 and U1 small nuclear ribonucleoprotein particle (snRNP) component U1-70k, colocalize in nuclear speckles and reside in the same complex. We propose that CDKG1 is recruited to U1 snRNP through RSZ33 to facilitate the splicing of the sixth intron of CalS5. PMID:23404887

Arrest is a regulator of fiber-specific alternative splicing in the indirect flight muscles of Drosophila

PubMed Central

Oas, Sandy T.

2014-01-01

Drosophila melanogaster flight muscles are distinct from other skeletal muscles, such as jump muscles, and express several uniquely spliced muscle-associated transcripts. We sought to identify factors mediating splicing differences between the flight and jump muscle fiber types. We found that the ribonucleic acid–binding protein Arrest (Aret) is expressed in flight muscles: in founder cells, Aret accumulates in a novel intranuclear compartment that we termed the Bruno body, and after the onset of muscle differentiation, Aret disperses in the nucleus. Down-regulation of the aret gene led to ultrastructural changes and functional impairment of flight muscles, and transcripts of structural genes expressed in the flight muscles became spliced in a manner characteristic of jump muscles. Aret also potently promoted flight muscle splicing patterns when ectopically expressed in jump muscles or tissue culture cells. Genetically, aret is located downstream of exd (extradenticle), hth (homothorax), and salm (spalt major), transcription factors that control fiber identity. Our observations provide insight into a transcriptional and splicing regulatory network for muscle fiber specification. PMID:25246617

Implementing a rational and consistent nomenclature for serine/arginine-rich protein splicing factors (SR proteins) in plants.

PubMed

Barta, Andrea; Kalyna, Maria; Reddy, Anireddy S N

2010-09-01

Growing interest in alternative splicing in plants and the extensive sequencing of new plant genomes necessitate more precise definition and classification of genes coding for splicing factors. SR proteins are a family of RNA binding proteins, which function as essential factors for constitutive and alternative splicing. We propose a unified nomenclature for plant SR proteins, taking into account the newly revised nomenclature of the mammalian SR proteins and a number of plant-specific properties of the plant proteins. We identify six subfamilies of SR proteins in Arabidopsis thaliana and rice (Oryza sativa), three of which are plant specific. The proposed subdivision of plant SR proteins into different subfamilies will allow grouping of paralogous proteins and simple assignment of newly discovered SR orthologs from other plant species and will promote functional comparisons in diverse plant species.

Nuclear m6A reader YTHDC1 regulates alternative polyadenylation and splicing during mouse oocyte development.

PubMed

Kasowitz, Seth D; Ma, Jun; Anderson, Stephen J; Leu, N Adrian; Xu, Yang; Gregory, Brian D; Schultz, Richard M; Wang, P Jeremy

2018-05-25

The N6-methyladenosine (m6A) modification is the most prevalent internal RNA modification in eukaryotes. The majority of m6A sites are found in the last exon and 3' UTRs. Here we show that the nuclear m6A reader YTHDC1 is essential for embryo viability and germline development in mouse. Specifically, YTHDC1 is required for spermatogonial development in males and for oocyte growth and maturation in females; Ythdc1-deficient oocytes are blocked at the primary follicle stage. Strikingly, loss of YTHDC1 leads to extensive alternative polyadenylation in oocytes, altering 3' UTR length. Furthermore, YTHDC1 deficiency causes massive alternative splicing defects in oocytes. The majority of splicing defects in mutant oocytes are rescued by introducing wild-type, but not m6A-binding-deficient, YTHDC1. YTHDC1 is associated with the pre-mRNA 3' end processing factors CPSF6, SRSF3, and SRSF7. Thus, YTHDC1 plays a critical role in processing of pre-mRNA transcripts in the oocyte nucleus and may have similar non-redundant roles throughout fetal development.

The low information content of Neurospora splicing signals: implications for RNA splicing and intron origin.

PubMed

Collins, Richard A; Stajich, Jason E; Field, Deborah J; Olive, Joan E; DeAbreu, Diane M

2015-05-01

When we expressed a small (0.9 kb) nonprotein-coding transcript derived from the mitochondrial VS plasmid in the nucleus of Neurospora we found that it was efficiently spliced at one or more of eight 5' splice sites and ten 3' splice sites, which are present apparently by chance in the sequence. Further experimental and bioinformatic analyses of other mitochondrial plasmids, random sequences, and natural nuclear genes in Neurospora and other fungi indicate that fungal spliceosomes recognize a wide range of 5' splice site and branchpoint sequences and predict introns to be present at high frequency in random sequence. In contrast, analysis of intronless fungal nuclear genes indicates that branchpoint, 5' splice site and 3' splice site consensus sequences are underrepresented compared with random sequences. This underrepresentation of splicing signals is sufficient to deplete the nuclear genome of splice sites at locations that do not comprise biologically relevant introns. Thus, the splicing machinery can recognize a wide range of splicing signal sequences, but splicing still occurs with great accuracy, not because the splicing machinery distinguishes correct from incorrect introns, but because incorrect introns are substantially depleted from the genome. © 2015 Collins et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

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.

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.

Cellular organization of pre-mRNA splicing factors in several tissues. Changes in the uterus by hormone action.

PubMed

George-Téllez, R; Segura-Valdez, M L; González-Santos, L; Jiménez-García, L F

2002-05-01

In the mammalian cell nucleus, splicing factors are distributed in nuclear domains known as speckles or splicing factor compartments (SFCs). In cultured cells, these domains are dynamic and reflect transcriptional and splicing activities. We used immunofluorescence and confocal microscopy to monitor whether splicing factors in differentiated cells display similar features. Speckled patterns are observed in rat hepatocytes, beta-cells, bronchial and intestine epithelia and also in three cell types of the uterus. Moreover, the number, distribution and sizes of the speckles vary among them. In addition, we studied variations in the circular form (shape) of speckles in uterine cells that are transcriptionally modified by a hormone action. During proestrus of the estral cycle, speckles are irregular in shape while in diestrus I they are circular. Experimentally, in castrated rats luminal epithelial cells show a pattern where speckles are dramatically rounded, but they recover their irregular shape rapidly after an injection of estradiol. The same results were observed in muscle and gland epithelial cells of the uterus. We concluded that different speckled patterns are present in various cells types in differentiated tissues and that these patterns change in the uterus depending upon the presence or absence of hormones such as estradiol.

Suppression of HPV-16 late L1 5′-splice site SD3632 by binding of hnRNP D proteins and hnRNP A2/B1 to upstream AUAGUA RNA motifs

PubMed Central

Li, Xiaoze; Johansson, Cecilia; Glahder, Jacob; Mossberg, Ann-Kristin; Schwartz, Stefan

2013-01-01

Human papillomavirus type 16 (HPV-16) 5′-splice site SD3632 is used exclusively to produce late L1 mRNAs. We identified a 34-nt splicing inhibitory element located immediately upstream of HPV-16 late 5′-splice site SD3632. Two AUAGUA motifs located in these 34 nt inhibited SD3632. Two nucleotide substitutions in each of the HPV-16 specific AUAGUA motifs alleviated splicing inhibition and induced late L1 mRNA production from episomal forms of the HPV-16 genome in primary human keratinocytes. The AUAGUA motifs bind specifically not only to the heterogeneous nuclear RNP (hnRNP) D family of RNA-binding proteins including hnRNP D/AUF, hnRNP DL and hnRNP AB but also to hnRNP A2/B1. Knock-down of these proteins induced HPV-16 late L1 mRNA expression, and overexpression of hnRNP A2/B1, hnRNP AB, hnRNP DL and the two hnRNP D isoforms hnRNP D37 and hnRNP D40 further suppressed L1 mRNA expression. This inhibition may allow HPV-16 to hide from the immune system and establish long-term persistent infections with enhanced risk at progressing to cancer. There is an inverse correlation between expression of hnRNP D proteins and hnRNP A2/B1 and HPV-16 L1 production in the cervical epithelium, as well as in cervical cancer, supporting the conclusion that hnRNP D proteins and A2/B1 inhibit HPV-16 L1 mRNA production. PMID:24013563

A novel polymorphic cytochrome P450 formed by splicing of CYP3A7 and the pseudogene CYP3AP1.

PubMed

Rodriguez-Antona, Cristina; Axelson, Magnus; Otter, Charlotta; Rane, Anders; Ingelman-Sundberg, Magnus

2005-08-05

The cytochrome P450 3A7 (CYP3A7) is the most abundant CYP in human liver during fetal development and first months of postnatal age, playing an important role in the metabolism of endogenous hormones, drugs, differentiation factors, and potentially toxic and teratogenic substrates. Here we describe and characterize a novel enzyme, CYP3A7.1L, encompassing the CYP3A7.1 protein with the last four carboxyl-terminal amino acids replaced by a unique sequence of 36 amino acids, generated by splicing of CYP3A7 with CYP3AP1 RNA. The corresponding CYP3A7-3AP1 mRNA had a significant expression in liver, kidney, and gastrointestinal tract, and its presence was found to be tissue-specific and dependent on the developmental stage. Heterologous expression in yeast revealed that CYP3A7.1L was a functional enzyme with a specific activity similar to that of CYP3A7.1 and, in some conditions, a different hydroxylation specificity than CYP3A7.1 using dehydroepiandrosterone as a substrate. CYP3A7.1L was found to be polymorphic due to a mutation at position -6 of the first splicing site of CYP3AP1 (CYP3A7_39256T-->A), which abrogates the pseudogene splicing. This polymorphism had pronounced interethnic differences and was in linkage disequilibrium with other functional polymorphisms described in the CYP3A locus: CYP3A7*2 and CYP3A5*1. Therefore, the resulting CYP3A haplotypes express different sets of enzymes within the population. In conclusion, a novel mechanism, consisting of the splicing of the pseudogene CYP3AP1 to CYP3A7, causes the formation of the novel CYP3A7.1L having a different tissue distribution and functional properties than the parent CYP3A7 enzyme, with possible developmental, physiological, and toxicological consequences.

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.

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

PubMed Central

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

2016-01-01

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

Identification of novel 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

Brain, blood, cerebrospinal fluid, and serum biomarkers in schizophrenia.

PubMed

Mohammadi, Alireza; Rashidi, Ehsan; Amooeian, Vahid Ghasem

2018-04-13

Over the last decade, finding a reliable biomarker for the early detection of schizophrenia (Scz) has been a topic of interest. The main goal of the current review is to provide a comprehensive view of the brain, blood, cerebrospinal fluid (CSF), and serum biomarkers of Scz disease. Imaging studies have demonstrated that the volumes of the corpus callosum, thalamus, hippocampal formation, subiculum, parahippocampal gyrus, superior temporal gyrus, prefrontal and orbitofrontal cortices, and amygdala-hippocampal complex were reduced in patients diagnosed with Scz. It has been revealed that the levels of interleukin 1β (IL-1β), IL-6, IL-8, and TNF-α were increased in patients with Scz. Decreased mRNA levels of brain-derived neurotrophic factor (BDNF), tropomyosin receptor kinase B (TrkB), neurotrophin-3 (NT-3), nerve growth factor (NGF), and vascular endothelial growth factor (VEGF) genes have also been reported in Scz patients. Genes with known strong relationships with this disease include BDNF, catechol-O-methyltransferase (COMT), regulator of G-protein signaling 4 (RGS4), dystrobrevin-binding protein 1 (DTNBP1), neuregulin 1 (NRG1), Reelin (RELN), Selenium-binding protein 1 (SELENBP1), glutamic acid decarboxylase 67 (GAD 67), and disrupted in schizophrenia 1 (DISC1). The levels of dopamine, tyrosine hydroxylase (TH), serotonin or 5-hydroxytryptamine (5-HT) receptor 1A and B (5-HTR1A and 5-HTR1B), and 5-HT1B were significantly increased in Scz patients, while the levels of gamma-aminobutyric acid (GABA), 5-HT transporter (5-HTT), and 5-HT receptor 2A (5-HTR2A) were decreased. The increased levels of SELENBP1 and Glycogen synthase kinase 3 subunit α (GSK3α) genes in contrast with reduced levels of B-cell translocation gene 1 (BTG1), human leukocyte antigen DRB1 (HLA-DRB1), heterogeneous nuclear ribonucleoprotein A3 (HNRPA3), and serine/arginine-rich splicing factor 1 (SFRS1) genes have also been reported. This review covers various dysregulation of neurotransmitters and also highlights the strengths and weaknesses of studies attempting to identify candidate biomarkers. Copyright © 2018 Elsevier B.V. All rights reserved.

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.

Splicing factor SFRS1 recognizes a functionally diverse landscape of RNA transcripts.

PubMed

Sanford, Jeremy R; Wang, Xin; Mort, Matthew; Vanduyn, Natalia; Cooper, David N; Mooney, Sean D; Edenberg, Howard J; Liu, Yunlong

2009-03-01

Metazoan genes are encrypted with at least two superimposed codes: the genetic code to specify the primary structure of proteins and the splicing code to expand their proteomic output via alternative splicing. Here, we define the specificity of a central regulator of pre-mRNA splicing, the conserved, essential splicing factor SFRS1. Cross-linking immunoprecipitation and high-throughput sequencing (CLIP-seq) identified 23,632 binding sites for SFRS1 in the transcriptome of cultured human embryonic kidney cells. SFRS1 was found to engage many different classes of functionally distinct transcripts including mRNA, miRNA, snoRNAs, ncRNAs, and conserved intergenic transcripts of unknown function. The majority of these diverse transcripts share a purine-rich consensus motif corresponding to the canonical SFRS1 binding site. The consensus site was not only enriched in exons cross-linked to SFRS1 in vivo, but was also enriched in close proximity to splice sites. mRNAs encoding RNA processing factors were significantly overrepresented, suggesting that SFRS1 may broadly influence the post-transcriptional control of gene expression in vivo. Finally, a search for the SFRS1 consensus motif within the Human Gene Mutation Database identified 181 mutations in 82 different genes that disrupt predicted SFRS1 binding sites. This comprehensive analysis substantially expands the known roles of human SR proteins in the regulation of a diverse array of RNA transcripts.

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.

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

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

Insertion of part of an intron into the 5[prime] untranslated region of a Caenorhabditis elegans gene converts it into a trans-spliced gene

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

Conrad, R.; Thomas, J.; Spieth, J.

In nematodes, the RNA products of some genes are trans-spliced to a 22-nucleotide spliced leader (SL), while the RNA products of other genes are not. In Caenorhabditis elegans, there are two SLs, Sl1 and SL2, donated by two distinct small nuclear ribonucleoprotein particles in a process functionally quite similar to nuclear intron removal. The authors demonstrate here that it is possible to convert a non-trans-spliced gene into a trans-spliced gene by placement of an intron missing only the 5[prime] splice site into the 5[prime] untranslated region. Stable transgenic strains were isolated expressing a gene in which 69 nucleotides of amore » vit-5 intron, including the 3[prime] splice site, were inserted into the 5[prime] untranslated region of a vit-2/vit-6 fusion gene. The RNA product of this gene was examined by primer extension and PCR amplification. Although the vit-2/vit-6 transgene product is not normally trans-spliced, the majority of transcripts from this altered gene were trans-spliced to SL1. They termed the region of a trans-spliced mRNA precursor between the 5[prime] end and the first 3[prime] splice site an 'outrun'. The results suggest that if a transcript begins with intronlike sequence followed by a 3[prime] splice site, this alone may constitute an outrun and be sufficient to demarcate a transcript as a trans-splice acceptor. These findings leave open the possibility that specific sequences are required to increase the efficiency of trans-splicing.« less

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.

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

Caenorhabditis elegans PRMT-7 and PRMT-9 Are Evolutionarily Conserved Protein Arginine Methyltransferases with Distinct Substrate Specificities.

PubMed

Hadjikyriacou, Andrea; Clarke, Steven G

2017-05-23

Caenorhabditis elegans protein arginine methyltransferases PRMT-7 and PRMT-9 are two evolutionarily conserved enzymes, with distinct orthologs in plants, invertebrates, and vertebrates. Biochemical characterization of these two enzymes reveals that they share much in common with their mammalian orthologs. C. elegans PRMT-7 produces only monomethylarginine (MMA) and preferentially methylates R-X-R motifs in a broad collection of substrates, including human histone peptides and RG-rich peptides. In addition, the activity of the PRMT-7 enzyme is dependent on temperature, the presence of metal ions, and the reducing agent dithiothreitol. C. elegans PRMT-7 has a substrate specificity and a substrate preference different from those of mammalian PRMT7, and the available X-ray crystal structures of the PRMT7 orthologs show differences in active site architecture. C. elegans PRMT-9, on the other hand, produces symmetric dimethylarginine and MMA on SFTB-2, the conserved C. elegans ortholog of human RNA splicing factor SF3B2, indicating a possible role in the regulation of nematode splicing. In contrast to PRMT-7, C. elegans PRMT-9 appears to be biochemically indistinguishable from its human ortholog.

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

Lindner, I.; Ehlers, B.; Noack, S.

The porcine lymphotropic herpesviruses (PLHV) are discussed as possible risk factors in xenotransplantation because of the high prevalence of PLHV-1, PLHV-2 and PLHV-3 in pig populations world-wide and the fact that PLHV-1 has been found to be associated with porcine post-transplant lymphoproliferative disease. To provide structural and functional knowledge on the PLHV immediate-early (IE) transactivator genes, the central regions of the PLHV genomes were characterized by genome walking, sequence and splicing analysis. Three spliced genes were identified (ORF50, ORFA6/BZLF1{sub h}, ORF57) encoding putative IE transactivators, homologous to (i) ORF50 and BRLF1/Rta (ii) K8/K-bZIP and BZLF1/Zta and (iii) ORF57 and BMLF1more » of HHV-8 and EBV, respectively. Expressed as myc-tag or HA-tag fusion proteins, they were located to the cellular nucleus. In reporter gene assays, several PLHV-promoters were mainly activated by PLHV-1 ORF50, to a lower level by PLHV-1 ORFA6/BZLF1{sub h} and not by PLHV-1 ORF57. However, the ORF57-encoded protein acted synergistically on ORF50-mediated activation.« less

The Novel α4B Murine α4 Integrin Protein Splicing Variant Inhibits α4 Protein-dependent Cell Adhesion*

PubMed Central

Kouro, Hitomi; Kon, Shigeyuki; Matsumoto, Naoki; Miyashita, Tomoe; Kakuchi, Ayaka; Ashitomi, Dai; Saitoh, Kodai; Nakatsuru, Takuya; Togi, Sumihito; Muromoto, Ryuta; Matsuda, Tadashi

2014-01-01

Integrins affect the motility of multiple cell types to control cell survival, growth, or differentiation, which are mediated by cell-cell and cell-extracellular matrix interactions. We reported previously that the α9 integrin splicing variant, SFα9, promotes WT α9 integrin-dependent adhesion. In this study, we introduced a new murine α4 integrin splicing variant, α4B, which has a novel short cytoplasmic tail. In inflamed tissues, the expression of α4B, as well as WT α4 integrin, was up-regulated. Cells expressing α4B specifically bound to VCAM-1 but not other α4 integrin ligands, such as fibronectin CS1 or osteopontin. The binding of cells expressing WT α4 integrin to α4 integrin ligands is inhibited by coexpression of α4B. Knockdown of α4B in metastatic melanoma cell lines results in a significant increase in lung metastasis. Expression levels of WT α4 integrin are unaltered by α4B, with α4B acting as a regulatory subunit for WT α4 integrin by a dominant-negative effect or inhibiting α4 integrin activation. PMID:24755217

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

The Tissue-Specific RNA Binding Protein T-STAR Controls Regional Splicing Patterns of Neurexin Pre-mRNAs in the Brain

PubMed Central

Ehrmann, Ingrid; Dalgliesh, Caroline; Liu, Yilei; Danilenko, Marina; Crosier, Moira; Overman, Lynn; Arthur, Helen M.; Lindsay, Susan; Clowry, Gavin J.; Venables, Julian P.; Fort, Philippe; Elliott, David J.

2013-01-01

The RNA binding protein T-STAR was created following a gene triplication 520–610 million years ago, which also produced its two parologs Sam68 and SLM-1. Here we have created a T-STAR null mouse to identify the endogenous functions of this RNA binding protein. Mice null for T-STAR developed normally and were fertile, surprisingly, given the high expression of T-STAR in the testis and the brain, and the known infertility and pleiotropic defects of Sam68 null mice. Using a transcriptome-wide search for splicing targets in the adult brain, we identified T-STAR protein as a potent splicing repressor of the alternatively spliced segment 4 (AS4) exons from each of the Neurexin1-3 genes, and exon 23 of the Stxbp5l gene. T-STAR protein was most highly concentrated in forebrain-derived structures like the hippocampus, which also showed maximal Neurexin1-3 AS4 splicing repression. In the absence of endogenous T-STAR protein, Nrxn1-3 AS4 splicing repression dramatically decreased, despite physiological co-expression of Sam68. In transfected cells Neurexin3 AS4 alternative splicing was regulated by either T-STAR or Sam68 proteins. In contrast, Neurexin2 AS4 splicing was only regulated by T-STAR, through a UWAA-rich response element immediately downstream of the regulated exon conserved since the radiation of bony vertebrates. The AS4 exons in the Nrxn1 and Nrxn3 genes were also associated with distinct patterns of conserved UWAA repeats. Consistent with an ancient mechanism of splicing control, human T-STAR protein was able to repress splicing inclusion of the zebrafish Nrxn3 AS4 exon. Although Neurexin1-3 and Stxbp5l encode critical synaptic proteins, T-STAR null mice had no detectable spatial memory deficits, despite an almost complete absence of AS4 splicing repression in the hippocampus. Our work identifies T-STAR as an ancient and potent tissue-specific splicing regulator that uses a concentration-dependent mechanism to co-ordinately regulate regional splicing patterns of the Neurexin1-3 AS4 exons in the mouse brain. PMID:23637638

RNA-Seq analysis reveals new evidence for inflammation-related changes in aged kidney

PubMed Central

Park, Daeui; Kim, Byoung-Chul; Kim, Chul-Hong; Choi, Yeon Ja; Jeong, Hyoung Oh; Kim, Mi Eun; Lee, Jun Sik; Park, Min Hi; Chung, Ki Wung; Kim, Dae Hyun; Lee, Jaewon; Im, Dong-Soon; Yoon, Seokjoo; Lee, Sunghoon; Yu, Byung Pal; Bhak, Jong; Chung, Hae Young

2016-01-01

Age-related dysregulated inflammation plays an essential role as a major risk factor underlying the pathophysiological aging process. To better understand how inflammatory processes are related to aging at the molecular level, we sequenced the transcriptome of young and aged rat kidney using RNA-Seq to detect known genes, novel genes, and alternative splicing events that are differentially expressed. By comparing young (6 months of age) and old (25 months of age) rats, we detected 722 up-regulated genes and 111 down-regulated genes. In the aged rats, we found 32 novel genes and 107 alternatively spliced genes. Notably, 6.6% of the up-regulated genes were related to inflammation (P < 2.2 × 10−16, Fisher exact t-test); 15.6% were novel genes with functional protein domains (P = 1.4 × 10−5); and 6.5% were genes showing alternative splicing events (P = 3.3 × 10−4). Based on the results of pathway analysis, we detected the involvement of inflammation-related pathways such as cytokines (P = 4.4 × 10−16), which were found up-regulated in the aged rats. Furthermore, an up-regulated inflammatory gene analysis identified the involvement of transcription factors, such as STAT4, EGR1, and FOSL1, which regulate cancer as well as inflammation in aging processes. Thus, RNA changes in these pathways support their involvement in the pro-inflammatory status during aging. We propose that whole RNA-Seq is a useful tool to identify novel genes and alternative splicing events by documenting broadly implicated inflammation-related genes involved in aging processes. PMID:27153548

Lentiviral Vector Induced Insertional Haploinsufficiency of Ebf1 Causes Murine Leukemia

PubMed Central

Heckl, Dirk; Schwarzer, Adrian; Haemmerle, Reinhard; Steinemann, Doris; Rudolph, Cornelia; Skawran, Britta; Knoess, Sabine; Krause, Johanna; Li, Zhixiong; Schlegelberger, Brigitte; Baum, Christopher; Modlich, Ute

2012-01-01

Integrating vectors developed on the basis of various retroviruses have demonstrated therapeutic potential following genetic modification of long-lived hematopoietic stem and progenitor cells. Lentiviral vectors (LV) are assumed to circumvent genotoxic events previously observed with γ-retroviral vectors, due to their integration bias to transcription units in comparison to the γ-retroviral preference for promoter regions and CpG islands. However, recently several studies have revealed the potential for gene activation by LV insertions. Here, we report a murine acute B-lymphoblastic leukemia (B-ALL) triggered by insertional gene inactivation. LV integration occurred into the 8th intron of Ebf1, a major regulator of B-lymphopoiesis. Various aberrant splice variants could be detected that involved splice donor and acceptor sites of the lentiviral construct, inducing downregulation of Ebf1 full-length message. The transcriptome signature was compatible with loss of this major determinant of B-cell differentiation, with partial acquisition of myeloid markers, including Csf1r (macrophage colony-stimulating factor (M-CSF) receptor). This was accompanied by receptor phosphorylation and STAT5 activation, both most likely contributing to leukemic progression. Our results highlight the risk of intragenic vector integration to initiate leukemia by inducing haploinsufficiency of a tumor suppressor gene. We propose to address this risk in future vector design. PMID:22472950

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

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.

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.

The Clk/Sty protein kinase phosphorylates SR splicing factors and regulates their intranuclear distribution.

PubMed Central

Colwill, K; Pawson, T; Andrews, B; Prasad, J; Manley, J L; Bell, J C; Duncan, P I

1996-01-01

Mammalian Clk/Sty is the prototype for a family of dual specificity kinases (termed LAMMER kinases) that have been conserved in evolution, but whose physiological substrates are unknown. In a yeast two-hybrid screen, the Clk/Sty kinase specifically interacted with RNA binding proteins, particularly members of the serine/arginine-rich (SR) family of splicing factors. Clk/Sty itself has an serine/arginine-rich non-catalytic N-terminal region which is important for its association with SR splicing factors. In vitro, Clk/Sty efficiently phosphorylated the SR family member ASF/SF2 on serine residues located within its serine/arginine-rich region (the RS domain). Tryptic phosphopeptide mapping demonstrated that the sites on ASF/SF2 phosphorylated in vitro overlap with those phosphorylated in vivo. Immunofluorescence studies showed that a catalytically inactive form of Clk/Sty co-localized with SR proteins in nuclear speckles. Overexpression of the active Clk/Sty kinase caused a redistribution of SR proteins within the nucleus. These results suggest that Clk/Sty kinase directly regulates the activity and compartmentalization of SR splicing factors. Images PMID:8617202

HnRNP A3 genes and pseudogenes in the vertebrate genomes.

PubMed

Makeyev, Aleksandr V; Kim, Chang Bae; Ruddle, Frank H; Enkhmandakh, Badam; Erdenechimeg, Lkhamsuren; Bayarsaihan, Dashzeveg

2005-04-01

The hnRNP A/B type proteins are abundant nuclear factors that bind to Pol II transcripts and are involved in numerous RNA-related activities. To date most data on the hnRNP A/B family have been obtained with recombinant proteins and cell cultures. Further characterization can result from an examination of the impact of various modifications in intact functional loci; however, such characterization is hampered by the presence of numerous and widely dispersed hnRNP A/B-related sequences in the mammalian genome. We have found hnRNP A3, a poorly recognized member of the hnRNP A/B family, among candidate transcription factors that interact with the regulatory region of the Hoxc8 gene and screened the human and mouse genomes for genes that encode hnRNP A3. We demonstrate that the sequence reported previously as the human hnRNP A3 gene (Accession number S63912) and located on 10p11.1 belongs to a processed pseudogene of the functional intron-containing locus HNRPA3, which we have identified on 2q31.2. We have also identified its murine orthologs on mouse chromosome 2D and rat chromosome 3q23. Alternative splices were revealed at the N-terminus and in the middle of hnRNP A3. 14 and 28 additional loci in the human and mouse genome, respectively, were mapped and identified as hnRNP A3 processed pseudogenes. In addition, we have found and compared hnRNP A3 orthologous genes in Gallus gallus, Xenopus tropicalis, and Danio rerio. The present in silico analysis serves as a necessary step toward a further functional characterization of hnRNP A3. (c) 2005 Wiley-Liss, Inc.

The Choice of Alternative 5' Splice Sites in Influenza Virus M1 mRNA is Regulated by the Viral Polymerase Complex

NASA Astrophysics Data System (ADS)

Shih, Shin-Ru; Nemeroff, Martin E.; Krug, Robert M.

1995-07-01

The influenza virus M1 mRNA has two alternative 5' splice sites: a distal 5' splice site producing mRNA_3 that has the coding potential for 9 amino acids and a proximal 5' splice site producing M2 mRNA encoding the essential M2 ion-channel protein. Only mRNA_3 was made in uninfected cells transfected with DNA expressing M1 mRNA. Similarly, using nuclear extracts from uninfected cells, in vitro splicing of M1 mRNA yielded only mRNA_3. Only when the mRNA_3 5' splice site was inactivated by mutation was M2 mRNA made in uninfected cells and in uninfected cell extracts. In influenza virus-infected cells, M2 mRNA was made, but only after a delay, suggesting that newly synthesized viral gene product(s) were needed to activate the M2 5' splice site. We present strong evidence that these gene products are the complex of the three polymerase proteins, the same complex that functions in the transcription and replication of the viral genome. Gel shift experiments showed that the viral polymerase complex bound to the 5' end of the viral M1 mRNA in a sequence-specific and cap-dependent manner. During in vitro splicing catalyzed by uninfected cell extracts, the binding of the viral polymerase complex blocked the mRNA_3 5' splice site, resulting in the switch to the M2 mRNA 5' splice site and the production of M2 mRNA.

Characterization of Ether-à-go-go Channels Present in Photoreceptors Reveals Similarity to IKx, a K+ Current in Rod Inner Segments

PubMed Central

Frings, Stephan; Brüll, Nicole; Dzeja, Claudia; Angele, Albert; Hagen, Volker; Kaupp, U. Benjamin; Baumann, Arnd

1998-01-01

In this study, we describe two splice variants of an ether-à-go-go (EAG) K+ channel cloned from bovine retina: bEAG1 and bEAG2. The bEAG2 polypeptide contains an additional insertion of 27 amino acids in the extracellular linker between transmembrane segments S3 and S4. The heterologously expressed splice variants differ in their activation kinetics and are differently modulated by extracellular Mg2+. Cooperativity of modulation by Mg2+ suggests that each subunit of the putative tetrameric channel binds a Mg2+ ion. The channels are neither permeable to Ca2+ ions nor modulated by cyclic nucleotides. In situ hybridization localizes channel transcripts to photoreceptors and retinal ganglion cells. Comparison of EAG currents with IKx, a noninactivating K+ current in the inner segment of rod photoreceptors, reveals an intriguing similarity, suggesting that EAG polypeptides are involved in the formation of Kx channels. PMID:9524140

Transcriptional and functional studies of Human Endogenous Retrovirus envelope EnvP(b) and EnvV genes in human trophoblasts

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

Vargas, Amandine, E-mail: amandine.vargas@voila.fr; Thiery, Maxime, E-mail: thiery.maxime@courrier.uqam.ca; Lafond, Julie, E-mail: lafond.julie@uqam.ca

2012-03-30

HERV (Human Endogenous Retrovirus)-encoded envelope proteins are implicated in the development of the placenta. Indeed, Syncytin-1 and -2 play a crucial role in the fusion of human trophoblasts, a key step in placentation. Other studies have identified two other HERV env proteins, namely EnvP(b) and EnvV, both expressed in the placenta. In this study, we have fully characterized both env transcripts and their expression pattern and have assessed their implication in trophoblast fusion. Through RACE analyses, standard spliced transcripts were detected, while EnvV transcripts demonstrated alternative splicing at its 3 Prime end. Promoter activity and expression of both genes weremore » induced in forskolin-stimulated BeWo cells and in primary trophoblasts. Although we have confirmed the fusogenic activity of EnvP(b), overexpression or silencing experiments revealed no impact of this protein on trophoblast fusion. Our results demonstrate that both env genes are expressed in human trophoblasts but are not required for syncytialization.« less

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.

An in vivo genetic screen for genes involved in spliced leader trans-splicing indicates a crucial role for continuous de novo spliced leader RNP assembly.

PubMed

Philippe, Lucas; Pandarakalam, George C; Fasimoye, Rotimi; Harrison, Neale; Connolly, Bernadette; Pettitt, Jonathan; Müller, Berndt

2017-08-21

Spliced leader (SL) trans-splicing is a critical element of gene expression in a number of eukaryotic groups. This process is arguably best understood in nematodes, where biochemical and molecular studies in Caenorhabditis elegans and Ascaris suum have identified key steps and factors involved. Despite this, the precise details of SL trans-splicing have yet to be elucidated. In part, this is because the systematic identification of the molecules involved has not previously been possible due to the lack of a specific phenotype associated with defects in this process. We present here a novel GFP-based reporter assay that can monitor SL1 trans-splicing in living C. elegans. Using this assay, we have identified mutants in sna-1 that are defective in SL trans-splicing, and demonstrate that reducing function of SNA-1, SNA-2 and SUT-1, proteins that associate with SL1 RNA and related SmY RNAs, impairs SL trans-splicing. We further demonstrate that the Sm proteins and pICln, SMN and Gemin5, which are involved in small nuclear ribonucleoprotein assembly, have an important role in SL trans-splicing. Taken together these results provide the first in vivo evidence for proteins involved in SL trans-splicing, and indicate that continuous replacement of SL ribonucleoproteins consumed during trans-splicing reactions is essential for effective trans-splicing. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Non-coding recurrent mutations in chronic lymphocytic leukaemia.

PubMed

Puente, Xose S; Beà, Silvia; Valdés-Mas, Rafael; Villamor, Neus; Gutiérrez-Abril, Jesús; Martín-Subero, José I; Munar, Marta; Rubio-Pérez, Carlota; Jares, Pedro; Aymerich, Marta; Baumann, Tycho; Beekman, Renée; Belver, Laura; Carrio, Anna; Castellano, Giancarlo; Clot, Guillem; Colado, Enrique; Colomer, Dolors; Costa, Dolors; Delgado, Julio; Enjuanes, Anna; Estivill, Xavier; Ferrando, Adolfo A; Gelpí, Josep L; González, Blanca; González, Santiago; González, Marcos; Gut, Marta; Hernández-Rivas, Jesús M; López-Guerra, Mónica; Martín-García, David; Navarro, Alba; Nicolás, Pilar; Orozco, Modesto; Payer, Ángel R; Pinyol, Magda; Pisano, David G; Puente, Diana A; Queirós, Ana C; Quesada, Víctor; Romeo-Casabona, Carlos M; Royo, Cristina; Royo, Romina; Rozman, María; Russiñol, Nuria; Salaverría, Itziar; Stamatopoulos, Kostas; Stunnenberg, Hendrik G; Tamborero, David; Terol, María J; Valencia, Alfonso; López-Bigas, Nuria; Torrents, David; Gut, Ivo; López-Guillermo, Armando; López-Otín, Carlos; Campo, Elías

2015-10-22

Chronic lymphocytic leukaemia (CLL) is a frequent disease in which the genetic alterations determining the clinicobiological behaviour are not fully understood. Here we describe a comprehensive evaluation of the genomic landscape of 452 CLL cases and 54 patients with monoclonal B-lymphocytosis, a precursor disorder. We extend the number of CLL driver alterations, including changes in ZNF292, ZMYM3, ARID1A and PTPN11. We also identify novel recurrent mutations in non-coding regions, including the 3' region of NOTCH1, which cause aberrant splicing events, increase NOTCH1 activity and result in a more aggressive disease. In addition, mutations in an enhancer located on chromosome 9p13 result in reduced expression of the B-cell-specific transcription factor PAX5. The accumulative number of driver alterations (0 to ≥4) discriminated between patients with differences in clinical behaviour. This study provides an integrated portrait of the CLL genomic landscape, identifies new recurrent driver mutations of the disease, and suggests clinical interventions that may improve the management of this neoplasia.

Selective aggregation of the splicing factor Hsh155 suppresses splicing upon genotoxic stress.

PubMed

Mathew, Veena; Tam, Annie S; Milbury, Karissa L; Hofmann, Analise K; Hughes, Christopher S; Morin, Gregg B; Loewen, Christopher J R; Stirling, Peter C

2017-12-04

Upon genotoxic stress, dynamic relocalization events control DNA repair as well as alterations of the transcriptome and proteome, enabling stress recovery. How these events may influence one another is only partly known. Beginning with a cytological screen of genome stability proteins, we find that the splicing factor Hsh155 disassembles from its partners and localizes to both intranuclear and cytoplasmic protein quality control (PQC) aggregates under alkylation stress. Aggregate sequestration of Hsh155 occurs at nuclear and then cytoplasmic sites in a manner that is regulated by molecular chaperones and requires TORC1 activity signaling through the Sfp1 transcription factor. This dynamic behavior is associated with intron retention in ribosomal protein gene transcripts, a decrease in splicing efficiency, and more rapid recovery from stress. Collectively, our analyses suggest a model in which some proteins evicted from chromatin and undergoing transcriptional remodeling during stress are targeted to PQC sites to influence gene expression changes and facilitate stress recovery. © 2017 Mathew et al.

Serine 192 in the tiny RS repeat of the adenoviral L4-33K splicing enhancer protein is essential for function and reorganization of the protein to the periphery of viral replication centers

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

Oestberg, Sara, E-mail: sara.ostberg@imbim.uu.se; Toermaenen Persson, Heidi, E-mail: heidi.tormanen.persson@imbim.uu.se; Akusjaervi, Goeran, E-mail: goran.akusjarvi@imbim.uu.se

2012-11-25

The adenovirus L4-33K protein is a key regulator involved in the temporal shift from early to late pattern of mRNA expression from the adenovirus major late transcription unit. L4-33K is a virus-encoded alternative splicing factor, which enhances processing of 3 Prime splice sites with a weak sequence context. Here we show that L4-33K expressed from a plasmid is localized at the nuclear margin of uninfected cells. During an infection L4-33K is relocalized to the periphery of E2A-72K containing viral replication centers. We also show that serine 192 in the tiny RS repeat of the conserved carboxy-terminus of L4-33K, which ismore » critical for the splicing enhancer function of L4-33K, is necessary for the nuclear localization and redistribution of the protein to viral replication sites. Collectively, our results show a good correlation between the activity of L4-33K as a splicing enhancer protein and its localization to the periphery of viral replication centers.« less

Alternative Splicing of a Novel Inducible Exon Diversifies the CASK Guanylate Kinase Domain

PubMed Central

Dembowski, Jill A.; An, Ping; Scoulos-Hanson, Maritsa; Yeo, Gene; Han, Joonhee; Fu, Xiang-Dong; Grabowski, Paula J.

2012-01-01

Alternative pre-mRNA splicing has a major impact on cellular functions and development with the potential to fine-tune cellular localization, posttranslational modification, interaction properties, and expression levels of cognate proteins. The plasticity of regulation sets the stage for cells to adjust the relative levels of spliced mRNA isoforms in response to stress or stimulation. As part of an exon profiling analysis of mouse cortical neurons stimulated with high KCl to induce membrane depolarization, we detected a previously unrecognized exon (E24a) of the CASK gene, which encodes for a conserved peptide insertion in the guanylate kinase interaction domain. Comparative sequence analysis shows that E24a appeared selectively in mammalian CASK genes as part of a >3,000 base pair intron insertion. We demonstrate that a combination of a naturally defective 5′ splice site and negative regulation by several splicing factors, including SC35 (SRSF2) and ASF/SF2 (SRSF1), drives E24a skipping in most cell types. However, this negative regulation is countered with an observed increase in E24a inclusion after neuronal stimulation and NMDA receptor signaling. Taken together, E24a is typically a skipped exon, which awakens during neuronal stimulation with the potential to diversify the protein interaction properties of the CASK polypeptide. PMID:23008758

CEF1/CDC5 alleles modulate transitions between catalytic conformations of the spliceosome

PubMed Central

Query, Charles C.; Konarska, Maria M.

2012-01-01

Conformational change within the spliceosome is required between the first and second catalytic steps of pre-mRNA splicing. A prior genetic screen for suppressors of an intron mutant that stalls between the two steps yielded both prp8 and non-prp8 alleles that suppressed second-step splicing defects. We have now identified the strongest non-prp8 suppressors as alleles of the NTC (Prp19 complex) component, CEF1. These cef1 alleles generally suppress second-step defects caused by a variety of intron mutations, mutations in U6 snRNA, or deletion of the second-step protein factor Prp17, and they can activate alternative 3′ splice sites. Genetic and functional interactions between cef1 and prp8 alleles suggest that they modulate the same event(s) in the first-to-second-step transition, most likely by stabilization of the second-step spliceosome; in contrast, alleles of U6 snRNA that also alter this transition modulate a distinct event, most likely by stabilization of the first-step spliceosome. These results implicate a myb-like domain of Cef1/CDC5 in interactions that modulate conformational states of the spliceosome and suggest that alteration of these events affects splice site use, resulting in alternative splicing-like patterns in yeast. PMID:22408182

Regulation of human adenovirus alternative RNA splicing by the adenoviral L4-33K and L4-22K proteins.

PubMed

Biasiotto, Roberta; Akusjärvi, Göran

2015-01-28

Adenovirus makes extensive use of alternative RNA splicing to produce a complex set of spliced viral mRNAs. Studies aimed at characterizing the interactions between the virus and the host cell RNA splicing machinery have identified three viral proteins of special significance for the control of late viral gene expression: L4-33K, L4-22K, and E4-ORF4. L4-33K is a viral alternative RNA splicing factor that controls L1 alternative splicing via an interaction with the cellular protein kinases Protein Kinase A (PKA) and DNA-dependent protein kinase (DNA-PK). L4-22K is a viral transcription factor that also has been implicated in the splicing of a subset of late viral mRNAs. E4-ORF4 is a viral protein that binds the cellular protein phosphatase IIA (PP2A) and controls Serine/Arginine (SR)-rich protein activity by inducing SR protein dephosphorylation. The L4-33K, and most likely also the L4-22K protein, are highly phosphorylated in vivo. Here we will review the function of these viral proteins in the post-transcriptional control of adenoviral gene expression and further discuss the significance of potential protein kinases phosphorylating the L4-33K and/or L4-22K proteins.

Triplex DNA-binding proteins are associated with clinical outcomes revealed by proteomic measurements in patients with colorectal cancer

PubMed Central

2012-01-01

Background Tri- and tetra-nucleotide repeats in mammalian genomes can induce formation of alternative non-B DNA structures such as triplexes and guanine (G)-quadruplexes. These structures can induce mutagenesis, chromosomal translocations and genomic instability. We wanted to determine if proteins that bind triplex DNA structures are quantitatively or qualitatively different between colorectal tumor and adjacent normal tissue and if this binding activity correlates with patient clinical characteristics. Methods Extracts from 63 human colorectal tumor and adjacent normal tissues were examined by gel shifts (EMSA) for triplex DNA-binding proteins, which were correlated with clinicopathological tumor characteristics using the Mann-Whitney U, Spearman’s rho, Kaplan-Meier and Mantel-Cox log-rank tests. Biotinylated triplex DNA and streptavidin agarose affinity binding were used to purify triplex-binding proteins in RKO cells. Western blotting and reverse-phase protein array were used to measure protein expression in tissue extracts. Results Increased triplex DNA-binding activity in tumor extracts correlated significantly with lymphatic disease, metastasis, and reduced overall survival. We identified three multifunctional splicing factors with biotinylated triplex DNA affinity: U2AF65 in cytoplasmic extracts, and PSF and p54nrb in nuclear extracts. Super-shift EMSA with anti-U2AF65 antibodies produced a shifted band of the major EMSA H3 complex, identifying U2AF65 as the protein present in the major EMSA band. U2AF65 expression correlated significantly with EMSA H3 values in all extracts and was higher in extracts from Stage III/IV vs. Stage I/II colon tumors (p = 0.024). EMSA H3 values and U2AF65 expression also correlated significantly with GSK3 beta, beta-catenin, and NF- B p65 expression, whereas p54nrb and PSF expression correlated with c-Myc, cyclin D1, and CDK4. EMSA values and expression of all three splicing factors correlated with ErbB1, mTOR, PTEN, and Stat5. Western blots confirmed that full-length and truncated beta-catenin expression correlated with U2AF65 expression in tumor extracts. Conclusions Increased triplex DNA-binding activity in vitro correlates with lymph node disease, metastasis, and reduced overall survival in colorectal cancer, and increased U2AF65 expression is associated with total and truncated beta-catenin expression in high-stage colorectal tumors. PMID:22682314

Advanced cell-based modeling of the royal disease: characterization of the mutated F9 mRNA.

PubMed

Martorell, L; Luce, E; Vazquez, J L; Richaud-Patin, Y; Jimenez-Delgado, S; Corrales, I; Borras, N; Casacuberta-Serra, S; Weber, A; Parra, R; Altisent, C; Follenzi, A; Dubart-Kupperschmitt, A; Raya, A; Vidal, F; Barquinero, J

2017-11-01

Essentials The Royal disease (RD) is a form of hemophilia B predicted to be caused by a splicing mutation. We generated an iPSC-based model of the disease allowing mechanistic studies at the RNA level. F9 mRNA analysis in iPSC-derived hepatocyte-like cells showed the predicted abnormal splicing. Mutated F9 mRNA level was very low but we also found traces of wild type transcripts. Background The royal disease is a form of hemophilia B (HB) that affected many descendants of Queen Victoria in the 19th and 20th centuries. It was found to be caused by the mutation F9 c.278-3A>G. Objective To generate a physiological cell model of the disease and to study F9 expression at the RNA level. Methods Using fibroblasts from skin biopsies of a previously identified hemophilic patient bearing the F9 c.278-3A>G mutation and his mother, we generated induced pluripotent stem cells (iPSCs). Both the patient's and mother's iPSCs were differentiated into hepatocyte-like cells (HLCs) and their F9 mRNA was analyzed using next-generation sequencing (NGS). Results and Conclusion We demonstrated the previously predicted aberrant splicing of the F9 transcript as a result of an intronic nucleotide substitution leading to a frameshift and the generation of a premature termination codon (PTC). The F9 mRNA level in the patient's HLCs was significantly reduced compared with that of his mother, suggesting that mutated transcripts undergo nonsense-mediated decay (NMD), a cellular mechanism that degrades PTC-containing mRNAs. We also detected small proportions of correctly spliced transcripts in the patient's HLCs, which, combined with genetic variability in splicing and NMD machineries, could partially explain some clinical variability among affected members of the European royal families who had lifespans above the average. This work allowed the demonstration of the pathologic consequences of an intronic mutation in the F9 gene and represents the first bona fide cellular model of HB allowing the study of rare mutations at the RNA level. © 2017 International Society on Thrombosis and Haemostasis.

Analyses of publicly available genomics resources define FGF-2-expressing bladder carcinomas as EMT-prone, proliferative tumors with low mutation rates and high expression of CTLA-4, PD-1 and PD-L1.

PubMed

McNiel, Elizabeth A; Tsichlis, Philip N

2017-01-01

FGF-2 is overexpressed in a subset of invasive bladder carcinomas and its overexpression correlates with poor prognosis. Analyses of publicly available databases addressing the molecular mechanisms that may be responsible for the poor prognosis of these tumors, revealed that FGF-2 expression correlates positively with the expression of EMT-promoting transcription factors and with changes in gene expression that are characteristic of EMT. The same analyses also revealed that FGF-2 correlates negatively with the expression, mutation and copy number variations of FGFR-3, all of which are associated with non-invasive bladder carcinomas. Finally, they showed that FGF-2 expression correlates with the expression of FGFR-1, the expression of the IIIc variant of FGFR-2 and with the expression of Akt3. The latter observation is significant because our earlier studies had shown that Akt3 regulates FGFR-2 alternative splicing, shifting the balance toward the IIIc relative to the IIIb FGFR-2 splice variant. Since the IIIc variant is recognized by FGF-2, while the IIIb variant is not, we conclude that Akt3 may facilitate the FGF-2 response. FGF-2 is known to promote the expression of KDM2B, which functions in concert with EZH2 to repress the EZH2-targeting microRNA miR-101, activating a switch, which stably upregulates EZH2. TCGA data showing a correlation between KDM2B and EZH2 expression and Oncomine data, showing a correlation between KDM2B and tumor progression, strongly support the role of the FGF-2/KDM2B/miR-101/EZH2 pathway in bladder cancer. These observations combined, suggest a model according to which FGF-2 induces EMT, cell proliferation and cancer stem cell self-renewal by coupling the Akt3 and KDM2B-controlled pathways outlined above, in bladder carcinomas. Further analyses of publicly-available databases, revealed that FGF-2-expressing bladder carcinomas carry fewer genetic alterations and they tend to express high levels of CTLA-4, PD-1 and PD-L1, which suggests immune blockade by checkpoint activation. EMT, enhanced proliferation and immune checkpoint activation combined, may be responsible for the poor prognosis of FGF-2-expressing bladder carcinomas.

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.

Cold-dependent alternative splicing of a Jumonji C domain-containing gene MtJMJC5 in Medicago truncatula.

PubMed

Shen, Yingfang; Wu, Xiaopei; Liu, Demei; Song, Shengjing; Liu, Dengcai; Wang, Haiqing

2016-05-27

Histone methylation is an epigenetic modification mechanism that regulates gene expression in eukaryotic cells. Jumonji C domain-containing demethylases are involved in removal of methyl groups at lysine or arginine residues. The JmjC domain-only member, JMJ30/JMJD5 of Arabidopsis, is a component of the plant circadian clock. Although some plant circadian clock genes undergo alternative splicing in response to external cues, there is no evidence that JMJ30/JMJD5 is regulated by alternative splicing. In this study, the expression of an Arabidopsis JMJ30/JMJD5 ortholog in Medicago truncatula, MtJMJC5, in response to circadian clock and abiotic stresses were characterized. The results showed that MtJMJC5 oscillates with a circadian rhythm, and undergoes cold specifically induced alternative splicing. The cold-induced alternative splicing could be reversed after ambient temperature returning to the normal. Sequencing results revealed four alternative splicing RNA isoforms including a full-length authentic protein encoding variant, and three premature termination condon-containing variants due to alternative 3' splice sites at the first and second intron. Under cold treatment, the variants that share a common 3' alternative splicing site at the second intron were intensively up-regulated while the authentic protein encoding variant and the premature termination condon-containing variant only undergoing a 3' alternative splicing at the first intron were down regulated. Although all the premature termination condon-harboring alternative splicing variants were sensitive to nonsense-mediated decay, the premature termination codon-harboring alternative splicing variants sharing the 3' alternative splicing site at the second intron showed less sensitivity than the one only containing the 3' alternative slicing site at the first intron under cold treatment. These results suggest that the cold-dependent alternative splicing of MtJMJC5 is likely a species or genus-specific mechanism of gene expression regulation on RNA levels, and might play a role in epigenetic regulation of the link between the circadian clock and ambient temperature fluctuation in Medicago. Copyright © 2016 Elsevier Inc. All rights reserved.

Prolyl hydroxylation regulates protein degradation, synthesis, and splicing in human induced pluripotent stem cell-derived cardiomyocytes

PubMed Central

Stoehr, Andrea; Yang, Yanqin; Patel, Sajni; Evangelista, Alicia M.; Aponte, Angel; Wang, Guanghui; Liu, Poching; Boylston, Jennifer; Kloner, Philip H.; Lin, Yongshun; Gucek, Marjan; Zhu, Jun; Murphy, Elizabeth

2016-01-01

Aims Protein hydroxylases are oxygen- and α-ketoglutarate-dependent enzymes that catalyse hydroxylation of amino acids such as proline, thus linking oxygen and metabolism to enzymatic activity. Prolyl hydroxylation is a dynamic post-translational modification that regulates protein stability and protein–protein interactions; however, the extent of this modification is largely uncharacterized. The goals of this study are to investigate the biological consequences of prolyl hydroxylation and to identify new targets that undergo prolyl hydroxylation in human cardiomyocytes. Methods and results We used human induced pluripotent stem cell-derived cardiomyocytes in combination with pulse-chase amino acid labelling and proteomics to analyse the effects of prolyl hydroxylation on protein degradation and synthesis. We identified 167 proteins that exhibit differences in degradation with inhibition of prolyl hydroxylation by dimethyloxalylglycine (DMOG); 164 were stabilized. Proteins involved in RNA splicing such as serine/arginine-rich splicing factor 2 (SRSF2) and splicing factor and proline- and glutamine-rich (SFPQ) were stabilized with DMOG. DMOG also decreased protein translation of cytoskeletal and sarcomeric proteins such as α-cardiac actin. We searched the mass spectrometry data for proline hydroxylation and identified 134 high confidence peptides mapping to 78 unique proteins. We identified SRSF2, SFPQ, α-cardiac actin, and cardiac titin as prolyl hydroxylated. We identified 29 prolyl hydroxylated proteins that showed a significant difference in either protein degradation or synthesis. Additionally, we performed next-generation RNA sequencing and showed that the observed decrease in protein synthesis was not due to changes in mRNA levels. Because RNA splicing factors were prolyl hydroxylated, we investigated splicing ± inhibition of prolyl hydroxylation and detected 369 alternative splicing events, with a preponderance of exon skipping. Conclusions This study provides the first extensive characterization of the cardiac prolyl hydroxylome and demonstrates that inhibition of α-ketoglutarate hydroxylases alters protein stability, translation, and splicing. PMID:27095734

Whole-exome sequencing identifies recurrent SF3B1 R625 mutation and comutation of NF1 and KIT in mucosal melanoma.

PubMed

Hintzsche, Jennifer D; Gorden, Nicholas T; Amato, Carol M; Kim, Jihye; Wuensch, Kelsey E; Robinson, Steven E; Applegate, Allison J; Couts, Kasey L; Medina, Theresa M; Wells, Keith R; Wisell, Joshua A; McCarter, Martin D; Box, Neil F; Shellman, Yiqun G; Gonzalez, Rene C; Lewis, Karl D; Tentler, John J; Tan, Aik Choon; Robinson, William A

2017-06-01

Mucosal melanomas are a rare subtype of melanoma, arising in mucosal tissues, which have a very poor prognosis due to the lack of effective targeted therapies. This study aimed to better understand the molecular landscape of these cancers and find potential new therapeutic targets. Whole-exome sequencing was performed on mucosal melanomas from 19 patients and 135 sun-exposed cutaneous melanomas, with matched peripheral blood samples when available. Mutational profiles were compared between mucosal subgroups and sun-exposed cutaneous melanomas. Comparisons of molecular profiles identified 161 genes enriched in mucosal melanoma (P<0.05). KIT and NF1 were frequently comutated (32%) in the mucosal subgroup, with a significantly higher incidence than that in cutaneous melanoma (4%). Recurrent SF3B1 R625H/S/C mutations were identified and validated in 7 of 19 (37%) mucosal melanoma patients. Mutations in the spliceosome pathway were found to be enriched in mucosal melanomas when compared with cutaneous melanomas. Alternative splicing in four genes were observed in SF3B1-mutant samples compared with the wild-type samples. This study identified potential new therapeutic targets for mucosal melanoma, including comutation of NF1 and KIT, and recurrent R625 mutations in SF3B1. This is the first report of SF3B1 R625 mutations in vulvovaginal mucosal melanoma, with the largest whole-exome sequencing project of mucosal melanomas to date. The results here also indicated that the mutations in SF3B1 lead to alternative splicing in multiple genes. These findings expand our knowledge of this rare disease.

Whole-exome sequencing identifies recurrent SF3B1 R625 mutation and comutation of NF1 and KIT in mucosal melanoma

PubMed Central

Hintzsche, Jennifer D.; Gorden, Nicholas T.; Amato, Carol M.; Kim, Jihye; Wuensch, Kelsey E.; Robinson, Steven E.; Applegate, Allison J.; Couts, Kasey L.; Medina, Theresa M.; Wells, Keith R.; Wisell, Joshua A.; McCarter, Martin D.; Box, Neil F.; Shellman, Yiqun G.; Gonzalez, Rene C.; Lewis, Karl D.; Tentler, John J.

2017-01-01

Mucosal melanomas are a rare subtype of melanoma, arising in mucosal tissues, which have a very poor prognosis due to the lack of effective targeted therapies. This study aimed to better understand the molecular landscape of these cancers and find potential new therapeutic targets. Whole-exome sequencing was performed on mucosal melanomas from 19 patients and 135 sun-exposed cutaneous melanomas, with matched peripheral blood samples when available. Mutational profiles were compared between mucosal subgroups and sun-exposed cutaneous melanomas. Comparisons of molecular profiles identified 161 genes enriched in mucosal melanoma (P<0.05). KIT and NF1 were frequently comutated (32%) in the mucosal subgroup, with a significantly higher incidence than that in cutaneous melanoma (4%). Recurrent SF3B1 R625H/S/C mutations were identified and validated in 7 of 19 (37%) mucosal melanoma patients. Mutations in the spliceosome pathway were found to be enriched in mucosal melanomas when compared with cutaneous melanomas. Alternative splicing in four genes were observed in SF3B1-mutant samples compared with the wild-type samples. This study identified potential new therapeutic targets for mucosal melanoma, including comutation of NF1 and KIT, and recurrent R625 mutations in SF3B1. This is the first report of SF3B1 R625 mutations in vulvovaginal mucosal melanoma, with the largest whole-exome sequencing project of mucosal melanomas to date. The results here also indicated that the mutations in SF3B1 lead to alternative splicing in multiple genes. These findings expand our knowledge of this rare disease. PMID:28296713

Overcoming imatinib resistance conferred by the BIM deletion polymorphism in chronic myeloid leukemia with splice-switching antisense oligonucleotides

PubMed Central

Liu, Jun; Bhadra, Malini; Sinnakannu, Joanna Rajeswary; Yue, Wan Lin; Tan, Cheryl Weiqi; Rigo, Frank; Ong, S.Tiong; Roca, Xavier

2017-01-01

Many tyrosine kinase-driven cancers, including chronic myeloid leukemia (CML), are characterized by high response rates to specific tyrosine kinase inhibitors (TKIs) like imatinib. In East Asians, primary imatinib resistance is caused by a deletion polymorphism in Intron 2 of the BIM gene, whose product is required for TKI-induced apoptosis. The deletion biases BIM splicing from exon 4 to exon 3, generating splice isoforms lacking the exon 4-encoded pro-apoptotic BH3 domain, which impairs the ability of TKIs to induce apoptosis. We sought to identify splice-switching antisense oligonucleotides (ASOs) that block exon 3 but enhance exon 4 splicing, and thereby resensitize BIM deletion-containing cancers to imatinib. First, we mapped multiple cis-acting splicing elements around BIM exon 3 by minigene mutations, and found an exonic splicing enhancer acting via SRSF1. Second, by a systematic ASO walk, we isolated ASOs that corrected the aberrant BIM splicing. Eight of 67 ASOs increased exon 4 levels in BIM deletion-containing cells, and restored imatinib-induced apoptosis and TKI sensitivity. This proof-of-principle study proves that resistant CML cells by BIM deletion polymorphism can be resensitized to imatinib via splice-switching BIM ASOs. Future optimizations might yield a therapeutic ASO as precision-medicine adjuvant treatment for BIM-polymorphism-associated TKI-resistant CML and other cancers. PMID:29100409

Overcoming imatinib resistance conferred by the BIM deletion polymorphism in chronic myeloid leukemia with splice-switching antisense oligonucleotides.

PubMed

Liu, Jun; Bhadra, Malini; Sinnakannu, Joanna Rajeswary; Yue, Wan Lin; Tan, Cheryl Weiqi; Rigo, Frank; Ong, S Tiong; Roca, Xavier

2017-09-29

Many tyrosine kinase-driven cancers, including chronic myeloid leukemia (CML), are characterized by high response rates to specific tyrosine kinase inhibitors (TKIs) like imatinib. In East Asians, primary imatinib resistance is caused by a deletion polymorphism in Intron 2 of the BIM gene, whose product is required for TKI-induced apoptosis. The deletion biases BIM splicing from exon 4 to exon 3, generating splice isoforms lacking the exon 4-encoded pro-apoptotic BH3 domain, which impairs the ability of TKIs to induce apoptosis. We sought to identify splice-switching antisense oligonucleotides (ASOs) that block exon 3 but enhance exon 4 splicing, and thereby resensitize BIM deletion-containing cancers to imatinib. First, we mapped multiple cis -acting splicing elements around BIM exon 3 by minigene mutations, and found an exonic splicing enhancer acting via SRSF1. Second, by a systematic ASO walk, we isolated ASOs that corrected the aberrant BIM splicing. Eight of 67 ASOs increased exon 4 levels in BIM deletion-containing cells, and restored imatinib-induced apoptosis and TKI sensitivity. This proof-of-principle study proves that resistant CML cells by BIM deletion polymorphism can be resensitized to imatinib via splice-switching BIM ASOs. Future optimizations might yield a therapeutic ASO as precision-medicine adjuvant treatment for BIM -polymorphism-associated TKI-resistant CML and other cancers.

Mutations in the Promoter Region of the Aldolase B Gene that cause Hereditary Fructose Intolerance

PubMed Central

Coffee, Erin M.; Tolan, Dean R.

2010-01-01

SUMMARY Hereditary fructose intolerance (HFI) is a potentially fatal inherited metabolic disease caused by a deficiency of aldolase B activity in the liver and kidney. Over 40 disease-causing mutations are known in the protein-coding region of ALDOB. Mutations upstream of the protein-coding portion of ALDOB are reported here for the first time. DNA sequence analysis of 61 HFI patients revealed single base mutations in the promoter, intronic enhancer, and the first exon, which is entirely untranslated. One mutation, g.–132G>A, is located within the promoter at an evolutionarily conserved nucleotide within a transcription factor-binding site. A second mutation, IVS1+1G>C, is at the donor splice site of the first exon. In vitro electrophoretic mobility shift assays show a decrease in nuclear extract-protein binding at the g.–132G>A mutant site. The promoter mutation results in decreased transcription using luciferase reporter plasmids. Analysis of cDNA from cells transfected with plasmids harboring the IVS1+1G>C mutation results in aberrant splicing leading to complete retention of the first intron (~ 5 kb). The IVS1+1G>C splicing mutation results in loss of luciferase activity from a reporter plasmid. These novel mutations in ALDOB represent 2% of alleles in American HFI patients, with IVS1+1G>C representing a significantly higher allele frequency (6%) among HFI patients of Hispanic and African-American ethnicity. PMID:20882353

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

DTIC Science & Technology

2017-10-01

aggressive disease. 15. SUBJECT TERMS Prostate cancer, health disparities among racial groups, molecular mechanisms, differential gene expression...identify molecular mechanisms of tumor aggressiveness. The studies proposed here address the urgent need to elucidate the molecular mechanisms underlying... genetic /epigenetic/post-transcriptional factors in AA prostate cancer and Gleason grade and 2) manipulate splicing using novel splice-switching

Functional impact of splice isoform diversity in individual cells

PubMed Central

Yap, Karen; Makeyev, Eugene V.

2016-01-01

Alternative pre-mRNA splicing provides an effective means for expanding coding capacity of eukaryotic genomes. Recent studies suggest that co-expression of different splice isoforms may increase diversity of RNAs and proteins at a single-cell level. A pertinent question in the field is whether such co-expression is biologically meaningful or, rather, represents insufficiently stringent splicing regulation. Here we argue that isoform co-expression may produce functional outcomes that are difficult and sometimes impossible to achieve using other regulation strategies. Far from being a ‘splicing noise’, co-expression is often established through co-ordinated activity of specific cis-elements and trans-acting factors. Further work in this area may uncover new biological functions of alternative splicing (AS) and generate important insights into mechanisms allowing different cell types to attain their unique molecular identities. PMID:27528755

Functional impact of splice isoform diversity in individual cells.

PubMed

Yap, Karen; Makeyev, Eugene V

2016-08-15

Alternative pre-mRNA splicing provides an effective means for expanding coding capacity of eukaryotic genomes. Recent studies suggest that co-expression of different splice isoforms may increase diversity of RNAs and proteins at a single-cell level. A pertinent question in the field is whether such co-expression is biologically meaningful or, rather, represents insufficiently stringent splicing regulation. Here we argue that isoform co-expression may produce functional outcomes that are difficult and sometimes impossible to achieve using other regulation strategies. Far from being a 'splicing noise', co-expression is often established through co-ordinated activity of specific cis-elements and trans-acting factors. Further work in this area may uncover new biological functions of alternative splicing (AS) and generate important insights into mechanisms allowing different cell types to attain their unique molecular identities. © 2016 The Author(s).

Reinforcement of a minor alternative splicing event in MYO7A due to a missense mutation results in a mild form of retinopathy and deafness.

PubMed

Ben Rebeh, Imen; Morinière, Madeleine; Ayadi, Leila; Benzina, Zeineb; Charfedine, Ilhem; Feki, Jamel; Ayadi, Hammadi; Ghorbel, Abdelmonem; Baklouti, Faouzi; Masmoudi, Saber

2010-09-30

Recessive mutations of the myosin VIIA (MYO7A) gene are reported to be responsible for both a deaf-blindness syndrome (Usher type 1B [USH1B] and atypical Usher syndrome) and nonsyndromic hearing loss (HL; Deafness, Neurosensory, Autosomal Recessive 2 [DFNB2]). The existence of DFNB2 is controversial, and often there is no relationship between the type and location of the MYO7A mutations corresponding to the USH1B and DFNB2 phenotype. We investigated the molecular determinant of a mild form of retinopathy in association with a subtle splicing modulation of MYO7A mRNA. Affected members underwent detailed audiologic and ocular characterization. DNA samples from family members were genotyped with polymorphic microsatellite markers. Sequencing of MYO7A was performed. Endogenous lymphoid RNA analysis and a splicing minigene assay were used to study the effect of the c.1935G>A mutation. Funduscopy showed mild retinitis pigmentosa in adults with HL. Microsatellite analysis showed linkage to markers in the region on chromosome 11q13.5. Sequencing of MYO7A revealed a mutation in the last nucleotide of exon 16 (c.1935G>A), which corresponds to a substitution of a methionine to an isoleucine residue at amino acid 645 of the myosin VIIA. However, structural prediction of the molecular model of myosin VIIA shows that this amino acid replacement induces only minor structural changes in the immediate environment of the mutation and thus does not alter the overall native structure. We found that, although predominantly included in mature mRNA, exon 16 is in fact alternatively spliced in control cells and that the mutation at the very last position is associated with a switch toward a predominant exclusion of that exon. This observation was further supported using a splicing minigene transfection assay; the c.1935G>A mutation was found to trigger a partial impairment of the adjacent donor splice site, suggesting that the unique change at the last position of the exon is responsible for the enhanced exon exclusion in this family. This study shows how an exonic mutation that weakens the 5' splice site enhances a minor alternative splicing without abolishing a complete exclusion of the exon and therefore causes a less severe retinitis pigmentosa than the USH1B-associated alleles. It would be interesting to examine a possible correlation between intrafamilial phenotypic variability and the subtle variation in exon 16 inclusion, probably related to genetic background specificities.

Crosstalk between Wnt Signaling and RNA Processing in Colorectal Cancer.

PubMed

Bordonaro, Michael

2013-01-01

RNA processing involves a variety of processes affecting gene expression, including the removal of introns through RNA splicing, as well as 3' end processing (cleavage and polyadenylation). Alternative RNA processing is fundamentally important for gene regulation, and aberrant processing is associated with the initiation and progression of cancer. Deregulated Wnt signaling, which is the initiating event in the development of most cases of human colorectal cancer (CRC), has been linked to modified RNA processing, which may contribute to Wnt-mediated colonic carcinogenesis. Crosstalk between Wnt signaling and alternative RNA splicing with relevance to CRC includes effects on the expression of Rac1b, an alternatively spliced gene associated with tumorigenesis, which exhibits alternative RNA splicing that is influenced by Wnt activity. In addition, Tcf4, a crucial component of Wnt signaling, also exhibits alternative splicing, which is likely involved in colonic tumorigenesis. Modulation of 3' end formation, including of the Wnt target gene COX-2, also can influence the neoplastic process, with implications for CRC. While many human genes are dependent on introns and splicing for normal levels of gene expression, naturally intronless genes exist with a unique metabolism that allows for intron-independent gene expression. Effects of Wnt activity on the RNA metabolism of the intronless Wnt-target gene c-jun is a likely contributor to cancer development. Further, butyrate, a breakdown product of dietary fiber and a histone deacetylase inhibitor, upregulates Wnt activity in CRC cells, and also modulates RNA processing; therefore, the interplay between Wnt activity, the modulation of this activity by butyrate, and differential RNA metabolism in colonic cells can significantly influence tumorigenesis. Determining the role played by altered RNA processing in Wnt-mediated neoplasia may lead to novel interventions aimed at restoring normal RNA metabolism for therapeutic benefit. Therefore, this minireview presents a brief overview of several aspects of RNA processing of relevance to cancer, which potentially influence, or are influenced by, Wnt signaling activity.

Digital holographic microtomography of fusion spliced optical fibers

NASA Astrophysics Data System (ADS)

Deng, Yating; Xiao, Wen; Ma, Xichao; Pan, Feng

2017-03-01

In this paper, we report three-dimensional(3D) measurement results of structural parameters of fusion spliced optical fibers using digital holographic microtomography. A holographic setup in microscopy configuration with the sample-fixed and setup-rotating scheme is established. A series of holograms is recorded from various incident angles. Then the filtered backprojection algorithm is applied to reconstruct the 3D refractive index (RI) distributions of the fusion spliced optical fibers inserted in the index-matching liquid. Experimental results exhibit the internal and external shapes of three kinds of fusion splices between different fibers, including a single-mode fiber(SMF) and a multimode fiber, an SMF and a panda polarization maintaining fiber (Panda PMF), and an SMF and a bow-tie polarization maintaining fiber (Bow-Tie PMF). With 3D maps of RI, it is intuitive to observe internal structural details of fused fibers and evaluate the splicing quality. This paper describes a powerful method for non-invasive microscopic measurement of fiber splicing. Furthermore, it provides the possibility of detecting fiber splicing loss by 3D structures.

Alternative splicing of TIA-1 in human colon cancer regulates VEGF isoform expression, angiogenesis, tumour growth and bevacizumab resistance.

PubMed

Hamdollah Zadeh, Maryam A; Amin, Elianna M; Hoareau-Aveilla, Coralie; Domingo, Enric; Symonds, Kirsty E; Ye, Xi; Heesom, Katherine J; Salmon, Andrew; D'Silva, Olivia; Betteridge, Kai B; Williams, Ann C; Kerr, David J; Salmon, Andrew H J; Oltean, Sebastian; Midgley, Rachel S; Ladomery, Michael R; Harper, Steven J; Varey, Alexander H R; Bates, David O

2015-01-01

The angiogenic capability of colorectal carcinomas (CRC), and their susceptibility to anti-angiogenic therapy, is determined by expression of vascular endothelial growth factor (VEGF) isoforms. The intracellular protein T-cell Intracellular Antigen (TIA-1) alters post-transcriptional RNA processing and binds VEGF-A mRNA. We therefore tested the hypothesis that TIA-1 could regulate VEGF-A isoform expression in colorectal cancers. TIA-1 and VEGF-A isoform expression was measured in colorectal cancers and cell lines. We discovered that an endogenous splice variant of TIA-1 encoding a truncated protein, short TIA-1 (sTIA-1) was expressed in CRC tissues and invasive K-Ras mutant colon cancer cells and tissues but not in adenoma cell lines. sTIA-1 was more highly expressed in CRC than in normal tissues and increased with tumour stage. Knockdown of sTIA-1 or over-expression of full length TIA-1 (flTIA-1) induced expression of the anti-angiogenic VEGF isoform VEGF-A165b. Whereas flTIA-1 selectively bound VEGF-A165 mRNA and increased translation of VEGF-A165b, sTIA-1 prevented this binding. In nude mice, xenografted colon cancer cells over-expressing flTIA-1 formed smaller, less vascular tumours than those expressing sTIA-1, but flTIA-1 expression inhibited the effect of anti-VEGF antibodies. These results indicate that alternative splicing of an RNA binding protein can regulate isoform specific expression of VEGF providing an added layer of complexity to the angiogenic profile of colorectal cancer and their resistance to anti-angiogenic therapy. Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.

Nuclear m6A reader YTHDC1 regulates alternative polyadenylation and splicing during mouse oocyte development

PubMed Central

Leu, N. Adrian; Xu, Yang; Schultz, Richard M.

2018-01-01

The N6-methyladenosine (m6A) modification is the most prevalent internal RNA modification in eukaryotes. The majority of m6A sites are found in the last exon and 3’ UTRs. Here we show that the nuclear m6A reader YTHDC1 is essential for embryo viability and germline development in mouse. Specifically, YTHDC1 is required for spermatogonial development in males and for oocyte growth and maturation in females; Ythdc1-deficient oocytes are blocked at the primary follicle stage. Strikingly, loss of YTHDC1 leads to extensive alternative polyadenylation in oocytes, altering 3’ UTR length. Furthermore, YTHDC1 deficiency causes massive alternative splicing defects in oocytes. The majority of splicing defects in mutant oocytes are rescued by introducing wild-type, but not m6A-binding-deficient, YTHDC1. YTHDC1 is associated with the pre-mRNA 3’ end processing factors CPSF6, SRSF3, and SRSF7. Thus, YTHDC1 plays a critical role in processing of pre-mRNA transcripts in the oocyte nucleus and may have similar non-redundant roles throughout fetal development. PMID:29799838

Protein Kinase C δ (PKCδ) Splice Variants Modulate Apoptosis Pathway in 3T3L1 Cells during Adipogenesis

PubMed Central

Patel, Rekha; Apostolatos, André; Carter, Gay; Ajmo, Joanne; Gali, Meghanath; Cooper, Denise R.; You, Min; Bisht, Kirpal S.; Patel, Niketa A.

2013-01-01

Increased food intake and lack of physical activity results in excess energy stored in adipocytes, and this imbalance contributes to obesity. New adipocytes are required for storage of energy in the white adipose tissue. This process of adipogenesis is widely studied in differentiating 3T3L1 preadipocytes in vitro. We have identified a key signaling kinase, protein kinase C delta (PKCδ), whose alternative splice variant expression is modulated during adipogenesis. We demonstrate that PKCδII splice variant promotes survival in differentiating 3T3L1 cells through the Bcl2 pathway. Here we demonstrate that resveratrol, a naturally occurring polyphenol, increases apoptosis and inhibits adipogenesis along with disruption of PKCδ alternative splicing during 3T3L1 differentiation. Importantly, we have identified a PKCδII splice variant inhibitor. This inhibitor may be a valuable tool with therapeutic implications in obesity. PMID:23902767

A Pre-mRNA-Splicing Factor Is Required for RNA-Directed DNA Methylation in Arabidopsis

PubMed Central

Huang, Chao-Feng; Miki, Daisuke; Tang, Kai; Zhou, Hao-Ran; Zheng, Zhimin; Chen, Wei; Ma, Ze-Yang; Yang, Lan; Zhang, Heng; Liu, Renyi; He, Xin-Jian; Zhu, Jian-Kang

2013-01-01

Cytosine DNA methylation is a stable epigenetic mark that is frequently associated with the silencing of genes and transposable elements (TEs). In Arabidopsis, the establishment of DNA methylation is through the RNA-directed DNA methylation (RdDM) pathway. Here, we report the identification and characterization of RDM16, a new factor in the RdDM pathway. Mutation of RDM16 reduced the DNA methylation levels and partially released the silencing of a reporter gene as well as some endogenous genomic loci in the DNA demethylase ros1-1 mutant background. The rdm16 mutant had morphological defects and was hypersensitive to salt stress and abscisic acid (ABA). Map-based cloning and complementation test led to the identification of RDM16, which encodes a pre-mRNA-splicing factor 3, a component of the U4/U6 snRNP. RNA-seq analysis showed that 308 intron retention events occurred in rdm16, confirming that RDM16 is involved in pre-mRNA splicing in planta. RNA-seq and mRNA expression analysis also revealed that the RDM16 mutation did not affect the pre-mRNA splicing of known RdDM genes, suggesting that RDM16 might be directly involved in RdDM. Small RNA expression analysis on loci showing RDM16-dependent DNA methylation suggested that unlike the previously reported putative splicing factor mutants, rdm16 did not affect small RNA levels; instead, the rdm16 mutation caused a decrease in the levels of Pol V transcripts. ChIP assays revealed that RDM16 was enriched at some Pol V target loci. Our results suggest that RDM16 regulates DNA methylation through influencing Pol V transcript levels. Finally, our genome-wide DNA methylation analysis indicated that RDM16 regulates the overall methylation of TEs and gene-surrounding regions, and preferentially targets Pol IV-dependent DNA methylation loci and the ROS1 target loci. Our work thus contributes to the understanding of RdDM and its interactions with active DNA demethylation. PMID:24068953

More than threefold expansion of highly nonlinear photonic crystal fiber cores for low-loss fusion splicing.

PubMed

Chen, Z; Xiong, C; Xiao, L M; Wadsworth, W J; Birks, T A

2009-07-15

We have formed low-loss fusion splices from highly nonlinear (HNL) photonic crystal fibers (PCFs) with small cores and high air-filling fractions to fibers with much larger mode field diameters (MFDs). The PCF core was locally enlarged by the controlled collapse of holes around the core while keeping other holes open. The fiber was then cleaved at the enlarged core and spliced to the large MFD fiber with a conventional electric arc fusion splicer. Splice losses as low as 0.36 dB were achieved between a PCF and a standard single-mode fiber (SMF) with MFDs of 1.8 microm and 5.9 microm, respectively.

Tissue-selective restriction of RNA editing of CaV1.3 by splicing factor SRSF9.

PubMed

Huang, Hua; Kapeli, Katannya; Jin, Wenhao; Wong, Yuk Peng; Arumugam, Thiruma Valavan; Koh, Joanne Huifen; Srimasorn, Sumitra; Mallilankaraman, Karthik; Chua, John Jia En; Yeo, Gene W; Soong, Tuck Wah

2018-05-04

Adenosine DeAminases acting on RNA (ADAR) catalyzes adenosine-to-inosine (A-to-I) conversion within RNA duplex structures. While A-to-I editing is often dynamically regulated in a spatial-temporal manner, the mechanisms underlying its tissue-selective restriction remain elusive. We have previously reported that transcripts of voltage-gated calcium channel CaV1.3 are subject to brain-selective A-to-I RNA editing by ADAR2. Here, we show that editing of CaV1.3 mRNA is dependent on a 40 bp RNA duplex formed between exon 41 and an evolutionarily conserved editing site complementary sequence (ECS) located within the preceding intron. Heterologous expression of a mouse minigene that contained the ECS, intermediate intronic sequence and exon 41 with ADAR2 yielded robust editing. Interestingly, editing of CaV1.3 was potently inhibited by serine/arginine-rich splicing factor 9 (SRSF9). Mechanistically, the inhibitory effect of SRSF9 required direct RNA interaction. Selective down-regulation of SRSF9 in neurons provides a basis for the neuron-specific editing of CaV1.3 transcripts.

NF90 isoforms, a new family of cellular proteins involved in viral replication?

PubMed

Patiño, Claudia; Haenni, Anne-Lise; Urcuqui-Inchima, Silvio

2015-01-01

The Nuclear Factor 90 (NF90) and its isoforms constitute a family of proteins that can interact with double-stranded (ds) RNA, through its dsRNA binding motifs. Due to various potential translational events such as alternative splicing, the human Interleukin enhancer binding factor 3 (ilf3) gene codes for multifunctional proteins that are NF90 and its isoforms, involved in transcription, translation, mRNA export and microRNA biogenesis. These proteins can act as cellular partners affecting viral replication and they are also implicated in host defense. As a result of these numerous functions, these protein isoforms have been given various names over the years, leading to confusion in determining their specific functions. In this review we focus on the role of the human NF90 protein isoforms in DNA and RNA virus replication. Copyright © 2014 Elsevier B.V. and Société française de biochimie et biologie Moléculaire (SFBBM). All rights reserved.

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.

Operon Conservation and the Evolution of trans-Splicing in the Phylum Nematoda

PubMed Central

Guiliano, David B; Blaxter, Mark L

2006-01-01

The nematode Caenorhabditis elegans is unique among model animals in that many of its genes are cotranscribed as polycistronic pre-mRNAs from operons. The mechanism by which these operonic transcripts are resolved into mature mRNAs includes trans-splicing to a family of SL2-like spliced leader exons. SL2-like spliced leaders are distinct from SL1, the major spliced leader in C. elegans and other nematode species. We surveyed five additional nematode species, representing three of the five major clades of the phylum Nematoda, for the presence of operons and the use of trans-spliced leaders in resolution of polycistronic pre-mRNAs. Conserved operons were found in Pristionchus pacificus, Nippostrongylus brasiliensis, Strongyloides ratti, Brugia malayi, and Ascaris suum. In nematodes closely related to the rhabditine C. elegans, a related family of SL2-like spliced leaders is used for operonic transcript resolution. However, in the tylenchine S. ratti operonic transcripts are resolved using a family of spliced leaders related to SL1. Non-operonic genes in S. ratti may also receive these SL1 variants. In the spirurine nematodes B. malayi and A. suum operonic transcripts are resolved using SL1. Mapping these phenotypes onto the robust molecular phylogeny for the Nematoda suggests that operons evolved before SL2-like spliced leaders, which are an evolutionary invention of the rhabditine lineage. PMID:17121468

Poly(A) RNA a new component of Cajal bodies.

PubMed

Kołowerzo, Agnieszka; Smoliński, Dariusz Jan; Bednarska, Elzbieta

2009-07-01

In European larch microsporocytes, spherical structures 0.5 to 6 microm in diameter are present in which poly(A) RNA accumulates. There were one to several bodies per cell and they were often present in the vicinity of the nucleolus. No nascent transcripts were observed within them. Splicing factors of the SR family, including protein SC35, which participates in bringing the 3' and 5' sites closer in the splicing reaction, were also not observed. The absence of the above-mentioned elements within bodies containing poly(A) RNA disqualifies them as sites of synthesis and preliminary stages of primary transcript maturation. However, they contained abundant elements of the splicing machinery commonly occurring in Cajal bodies, i.e., Sm proteins or small nuclear RNA (snRNA). The molecular composition as well as the characteristic ultrastructure of bodies containing poly(A) RNA proves that these were Cajal bodies. This is the first report of such poly(A) RNA localization.

Design of Multi-core Fiber Patch Panel for Space Division Multiplexing Implementations

NASA Astrophysics Data System (ADS)

González, Luz E.; Morales, Alvaro; Rommel, Simon; Jørgensen, Bo F.; Porras-Montenegro, N.; Tafur Monroy, Idelfonso

2018-03-01

A multi-core fiber (MCF) patch panel was designed, allowing easy coupling of individual signals to and from a 7-core MCF. The device was characterized, measuring insertion loss and cross talk, finding highest insertion loss and lowest crosstalk at 1300 nm with values of 9.7 dB and -36.5 dB respectively, while at 1600 nm insertion loss drops to 4.8 dB and crosstalk increases to -24.1 dB. Two MCF splices between the fan-in module, the MCF, and the fan-out module are included in the characterization, and splicing parameters are discussed.

Multiple splicing defects in an intronic false exon.

PubMed

Sun, H; Chasin, L A

2000-09-01

Splice site consensus sequences alone are insufficient to dictate the recognition of real constitutive splice sites within the typically large transcripts of higher eukaryotes, and large numbers of pseudoexons flanked by pseudosplice sites with good matches to the consensus sequences can be easily designated. In an attempt to identify elements that prevent pseudoexon splicing, we have systematically altered known splicing signals, as well as immediately adjacent flanking sequences, of an arbitrarily chosen pseudoexon from intron 1 of the human hprt gene. The substitution of a 5' splice site that perfectly matches the 5' consensus combined with mutation to match the CAG/G sequence of the 3' consensus failed to get this model pseudoexon included as the central exon in a dhfr minigene context. Provision of a real 3' splice site and a consensus 5' splice site and removal of an upstream inhibitory sequence were necessary and sufficient to confer splicing on the pseudoexon. This activated context also supported the splicing of a second pseudoexon sequence containing no apparent enhancer. Thus, both the 5' splice site sequence and the polypyrimidine tract of the pseudoexon are defective despite their good agreement with the consensus. On the other hand, the pseudoexon body did not exert a negative influence on splicing. The introduction into the pseudoexon of a sequence selected for binding to ASF/SF2 or its replacement with beta-globin exon 2 only partially reversed the effect of the upstream negative element and the defective polypyrimidine tract. These results support the idea that exon-bridging enhancers are not a prerequisite for constitutive exon definition and suggest that intrinsically defective splice sites and negative elements play important roles in distinguishing the real splicing signal from the vast number of false splicing signals.

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

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.

Transcriptomic insights into the alternative splicing-mediated adaptation of the entomopathogenic fungus Beauveria bassiana to host niches: autophagy-related gene 8 as an example.

PubMed

Dong, Wei-Xia; Ding, Jin-Li; Gao, Yang; Peng, Yue-Jin; Feng, Ming-Guang; Ying, Sheng-Hua

2017-10-01

Alternative splicing (AS) regulates various biological processes in fungi by extending the cellular proteome. However, comprehensive studies investigating AS in entomopathogenic fungi are lacking. Based on transcriptome data obtained via dual RNA-seq, the first overview of AS events was developed for Beauveria bassiana growing in an insect haemocoel. The AS was demonstrated for 556 of 8840 expressed genes, accounting for 5.4% of the total genes in B. bassiana. Intron retention was the most abundant type of AS, accounting for 87.1% of all splicing events and exon skipping events were rare, only accounting for 2.0% of all events. Functional distribution analysis indicated an association between alternatively spliced genes and several physiological processes. Notably, B. bassiana autophagy-related gene 8 (BbATG8), an indispensable gene for autophagy, was spliced at an alternative 5' splice site to generate two transcripts (BbATG8-α and BbATG8-β). The BbATG8-α transcript was necessary for fungal autophagy and oxidation tolerance, while the BbATG8-β transcript was not. These two transcripts differentially contributed to the formation of conidia or blastospores as well as fungal virulence. Thus, AS acts as a powerful post-transcriptional regulatory strategy in insect mycopathogens and significantly mediates fungal transcriptional adaption to host niches. © 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.

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

Identification of an Intronic Splicing Enhancer Essential for the Inclusion of FGFR2 Exon IIIc*S⃞

PubMed Central

Seth, Puneet; Miller, Heather B.; Lasda, Erika L.; Pearson, James L.; Garcia-Blanco, Mariano A.

2008-01-01

The ligand specificity of fibroblast growth factor receptor 2 (FGFR2) is determined by the alternative splicing of exons 8 (IIIb) or 9 (IIIc). Exon IIIb is included in epithelial cells, whereas exon IIIc is included in mesenchymal cells. Although a number of cis elements and trans factors have been identified that play a role in exon IIIb inclusion in epithelium, little is known about the activation of exon IIIc in mesenchyme. We report here the identification of a splicing enhancer required for IIIc inclusion. This 24-nucleotide (nt) downstream intronic splicing enhancer (DISE) is located within intron 9 immediately downstream of exon IIIc. DISE was able to activate the inclusion of heterologous exons rat FGFR2 IIIb and human β-globin exon 2 in cell lines from different tissues and species and also in HeLa cell nuclear extracts in vitro. DISE was capable of replacing the intronic activator sequence 1 (IAS1), a known IIIb splicing enhancer and vice versa. This fact, together with the requirement for DISE to be close to the 5′-splice site and the ability of DISE to promote binding of U1 snRNP, suggested that IAS1 and DISE belong to the same class of cis-acting elements. PMID:18256031

Arc fusion splicing of photonic crystal fibers to standard single mode fibers

NASA Astrophysics Data System (ADS)

Borzycki, Krzysztof; Kobelke, Jens; Schuster, Kay; Wójcik, Jan

2010-04-01

Coupling a photonic crystal fiber (PCF) to measuring instruments or optical subsystems is often done by splicing it to short lengths of single mode fiber (SMF) used for interconnections, as SMF is standardized, widely available and compatible with most fiber optic components and measuring instruments. This paper presents procedures and results of loss measurements during fusion splicing of five PCFs tested at NIT laboratory within activities of COST Action 299 "FIDES". Investigated silica-based fibers had 80-200 μm cladding diameter and were designed as single mode. A standard splicing machine designed for telecom fibers was used, but splicing procedure and arc power were tailored to each PCF. Splice loss varied between 0.7 and 2.8 dB at 1550 nm. Splices protected with heat-shrinkable sleeves served well for gripping fibers during mechanical tests and survived temperature cycling from -30°C to +70°C with stable loss. Collapse of holes in the PCF was limited by reducing fusion time to 0.2-0.5 s; additional measures included reduction of discharge power and shifting SMF-PCF contact point away from the axis of electrodes. Unfortunately, short fusion time sometimes precluded proper smoothing of glass surface, leading to a trade-off between splice loss and strength.

An essential role for trimethylguanosine RNA caps in Saccharomyces cerevisiae meiosis and their requirement for splicing of SAE3 and PCH2 meiotic pre-mRNAs

PubMed Central

Qiu, Zhicheng R.; Shuman, Stewart; Schwer, Beate

2011-01-01

Tgs1 is the enzyme that converts m7G RNA caps to the 2,2,7-trimethylguanosine (TMG) caps characteristic of spliceosomal snRNAs. Fungi grow vegetatively without TMG caps, thereby raising the question of what cellular transactions, if any, are TMG cap-dependent. Here, we report that Saccharomyces cerevisiae Tgs1 methyltransferase activity is essential for meiosis. tgs1Δ cells are specifically defective in splicing PCH2 and SAE3 meiotic pre-mRNAs. The TMG requirement for SAE3 splicing is alleviated by two intron mutations: a UAUUAAC to UACUAAC change that restores a consensus branchpoint and disruption of a stem–loop encompassing the branchpoint. The TMG requirement for PCH2 splicing is alleviated by a CACUAAC to UACUAAC change restoring a consensus branchpoint and by shortening the PCH2 5′ exon. Placing the SAE3 and PCH2 introns within a HIS3 reporter confers Tgs1-dependent histidine prototrophy, signifying that the respective introns are portable determinants of TMG-dependent gene expression. Analysis of in vitro splicing in extracts of TGS1 versus tgs1Δ cells showed that SAE3 intron removal was enfeebled without TMG caps, whereas splicing of ACT1 was unaffected. Our findings illuminate a new mode of tunable splicing, a reliance on TMG caps for an essential developmental RNA transaction, and three genetically distinct meiotic splicing regulons in budding yeast. PMID:21398639

An essential role for trimethylguanosine RNA caps in Saccharomyces cerevisiae meiosis and their requirement for splicing of SAE3 and PCH2 meiotic pre-mRNAs.

PubMed

Qiu, Zhicheng R; Shuman, Stewart; Schwer, Beate

2011-07-01

Tgs1 is the enzyme that converts m(7)G RNA caps to the 2,2,7-trimethylguanosine (TMG) caps characteristic of spliceosomal snRNAs. Fungi grow vegetatively without TMG caps, thereby raising the question of what cellular transactions, if any, are TMG cap-dependent. Here, we report that Saccharomyces cerevisiae Tgs1 methyltransferase activity is essential for meiosis. tgs1Δ cells are specifically defective in splicing PCH2 and SAE3 meiotic pre-mRNAs. The TMG requirement for SAE3 splicing is alleviated by two intron mutations: a UAUUAAC to UACUAAC change that restores a consensus branchpoint and disruption of a stem-loop encompassing the branchpoint. The TMG requirement for PCH2 splicing is alleviated by a CACUAAC to UACUAAC change restoring a consensus branchpoint and by shortening the PCH2 5' exon. Placing the SAE3 and PCH2 introns within a HIS3 reporter confers Tgs1-dependent histidine prototrophy, signifying that the respective introns are portable determinants of TMG-dependent gene expression. Analysis of in vitro splicing in extracts of TGS1 versus tgs1Δ cells showed that SAE3 intron removal was enfeebled without TMG caps, whereas splicing of ACT1 was unaffected. Our findings illuminate a new mode of tunable splicing, a reliance on TMG caps for an essential developmental RNA transaction, and three genetically distinct meiotic splicing regulons in budding yeast.

Kaposi's Sarcoma-Associated Herpesvirus Utilizes and Manipulates RNA N6-Adenosine Methylation To Promote Lytic Replication

PubMed Central

Chen, E. Ricky; Nilsen, Timothy W.

2017-01-01

ABSTRACT N6-adenosine methylation (m6A) is the most common posttranscriptional RNA modification in mammalian cells. We found that most transcripts encoded by the Kaposi's sarcoma-associated herpesvirus (KSHV) genome undergo m6A modification. The levels of m6A-modified mRNAs increased substantially upon stimulation for lytic replication. The blockage of m6A inhibited splicing of the pre-mRNA encoding the replication transcription activator (RTA), a key KSHV lytic switch protein, and halted viral lytic replication. We identified several m6A sites in RTA pre-mRNA crucial for splicing through interactions with YTH domain containing 1 (YTHDC1), an m6A nuclear reader protein, in conjunction with serine/arginine-rich splicing factor 3 (SRSF3) and SRSF10. Interestingly, RTA induced m6A and enhanced its own pre-mRNA splicing. Our results not only demonstrate an essential role of m6A in regulating RTA pre-mRNA splicing but also suggest that KSHV has evolved a mechanism to manipulate the host m6A machinery to its advantage in promoting lytic replication. IMPORTANCE KSHV productive lytic replication plays a pivotal role in the initiation and progression of Kaposi's sarcoma tumors. Previous studies suggested that the KSHV switch from latency to lytic replication is primarily controlled at the chromatin level through histone and DNA modifications. The present work reports for the first time that KSHV genome-encoded mRNAs undergo m6A modification, which represents a new mechanism at the posttranscriptional level in the control of viral replication. PMID:28592530

Transcriptional diversity during lineage commitment of human blood progenitors.

PubMed

Chen, Lu; Kostadima, Myrto; Martens, Joost H A; Canu, Giovanni; Garcia, Sara P; Turro, Ernest; Downes, Kate; Macaulay, Iain C; Bielczyk-Maczynska, Ewa; Coe, Sophia; Farrow, Samantha; Poudel, Pawan; Burden, Frances; Jansen, Sjoert B G; Astle, William J; Attwood, Antony; Bariana, Tadbir; de Bono, Bernard; Breschi, Alessandra; Chambers, John C; Consortium, Bridge; Choudry, Fizzah A; Clarke, Laura; Coupland, Paul; van der Ent, Martijn; Erber, Wendy N; Jansen, Joop H; Favier, Rémi; Fenech, Matthew E; Foad, Nicola; Freson, Kathleen; van Geet, Chris; Gomez, Keith; Guigo, Roderic; Hampshire, Daniel; Kelly, Anne M; Kerstens, Hindrik H D; Kooner, Jaspal S; Laffan, Michael; Lentaigne, Claire; Labalette, Charlotte; Martin, Tiphaine; Meacham, Stuart; Mumford, Andrew; Nürnberg, Sylvia; Palumbo, Emilio; van der Reijden, Bert A; Richardson, David; Sammut, Stephen J; Slodkowicz, Greg; Tamuri, Asif U; Vasquez, Louella; Voss, Katrin; Watt, Stephen; Westbury, Sarah; Flicek, Paul; Loos, Remco; Goldman, Nick; Bertone, Paul; Read, Randy J; Richardson, Sylvia; Cvejic, Ana; Soranzo, Nicole; Ouwehand, Willem H; Stunnenberg, Hendrik G; Frontini, Mattia; Rendon, Augusto

2014-09-26

Blood cells derive from hematopoietic stem cells through stepwise fating events. To characterize gene expression programs driving lineage choice, we sequenced RNA from eight primary human hematopoietic progenitor populations representing the major myeloid commitment stages and the main lymphoid stage. We identified extensive cell type-specific expression changes: 6711 genes and 10,724 transcripts, enriched in non-protein-coding elements at early stages of differentiation. In addition, we found 7881 novel splice junctions and 2301 differentially used alternative splicing events, enriched in genes involved in regulatory processes. We demonstrated experimentally cell-specific isoform usage, identifying nuclear factor I/B (NFIB) as a regulator of megakaryocyte maturation-the platelet precursor. Our data highlight the complexity of fating events in closely related progenitor populations, the understanding of which is essential for the advancement of transplantation and regenerative medicine. Copyright © 2014, American Association for the Advancement of Science.

SMITten by the Speed of Splicing.

PubMed

Johnson, Tracy L; Ares, Manuel

2016-04-07

Splicing occurs co-transcriptionally, but relative rates of splicing and transcription that might reveal mechanisms of their coordinated control have remained mysterious. Now, Carrillo Oesterreich et al. show that the fastest introns are gone nearly as soon as the 3' splice site is transcribed and that introns have distinct splicing kinetics with respect to polymerase progression along the gene. Copyright © 2016 Elsevier Inc. All rights reserved.

RANK-c attenuates aggressive properties of ER-negative breast cancer by inhibiting NF-κB activation and EGFR signaling.

PubMed

Sirinian, Chaido; Papanastasiou, Anastasios D; Schizas, Michail; Spella, Magda; Stathopoulos, Georgios T; Repanti, Maria; Zarkadis, Ioannis K; King, Tari A; Kalofonos, Haralabos P

2018-05-29

The RANK/RANKL axis emerges as a key regulator of breast cancer initiation, progression, and metastasis. RANK-c is a RANK receptor isoform produced through alternative splicing of the TNFRSF11A (RANK) gene and a dominant-negative regulator of RANK-induced nuclear factor-κB (NF-κB) activation. Here we report that RANK-c transcript is expressed in 3.2% of cases in The Cancer Genome Atlas breast cancer cohort evenly between ER-positive and ER-negative cases. Nevertheless, the ratio of RANK to RANK-c (RANK/RANK-c) is increased in ER-negative breast cancer cell lines compared to ER-positive breast cancer cell lines. In addition, forced expression of RANK-c in ER-negative breast cancer cell lines inhibited stimuli-induced NF-κB activation and attenuated migration, invasion, colony formation, and adhesion of cancer cells. Further, RANK-c expression in MDA-MB-231 cells inhibited lung metastasis and colonization in vivo. The RANK-c-mediated inhibition of cancer cell aggressiveness and nuclear factor-κB (NF-κB) activation in breast cancer cells seems to rely on a RANK-c/TNF receptor-associated factor-2 (TRAF2) protein interaction. This was further confirmed by a mutated RANK-c that is unable to interact with TRAF2 and abolishes the ability to attenuate NF-κB activation, migration, and invasion. Additional protein interaction characterization revealed epidermal growth factor receptor (EGFR) as a novel interacting partner for RANK-c in breast cancer cells with a negative effect on EGFR phosphorylation and EGF-dependent downstream signaling pathway activation. Our findings further elucidate the complex molecular biology of the RANKL/RANK system in breast cancer and provide preliminary data for RANK-c as a possible marker for disease progression and aggressiveness.

Identification of protein features encoded by alternative exons using Exon Ontology.

PubMed

Tranchevent, Léon-Charles; Aubé, Fabien; Dulaurier, Louis; Benoit-Pilven, Clara; Rey, Amandine; Poret, Arnaud; Chautard, Emilie; Mortada, Hussein; Desmet, François-Olivier; Chakrama, Fatima Zahra; Moreno-Garcia, Maira Alejandra; Goillot, Evelyne; Janczarski, Stéphane; Mortreux, Franck; Bourgeois, Cyril F; Auboeuf, Didier

2017-06-01

Transcriptomic genome-wide analyses demonstrate massive variation of alternative splicing in many physiological and pathological situations. One major challenge is now to establish the biological contribution of alternative splicing variation in physiological- or pathological-associated cellular phenotypes. Toward this end, we developed a computational approach, named "Exon Ontology," based on terms corresponding to well-characterized protein features organized in an ontology tree. Exon Ontology is conceptually similar to Gene Ontology-based approaches but focuses on exon-encoded protein features instead of gene level functional annotations. Exon Ontology describes the protein features encoded by a selected list of exons and looks for potential Exon Ontology term enrichment. By applying this strategy to exons that are differentially spliced between epithelial and mesenchymal cells and after extensive experimental validation, we demonstrate that Exon Ontology provides support to discover specific protein features regulated by alternative splicing. We also show that Exon Ontology helps to unravel biological processes that depend on suites of coregulated alternative exons, as we uncovered a role of epithelial cell-enriched splicing factors in the AKT signaling pathway and of mesenchymal cell-enriched splicing factors in driving splicing events impacting on autophagy. Freely available on the web, Exon Ontology is the first computational resource that allows getting a quick insight into the protein features encoded by alternative exons and investigating whether coregulated exons contain the same biological information. © 2017 Tranchevent et al.; Published by Cold Spring Harbor Laboratory Press.

[RNA polymerase II and pre-mRNA splicing factors in diplotene oocyte nuclei of the giant African gastropod Achatina fulica].

PubMed

Stepanova, I S; Bogoliubov, D S

2003-01-01

The nuclear distribution of pre-mRNA splicing factors (snRNPs and SR-protein SC35) and unphosphorylated from of RNA polymerase II (Pol II) was studied using fluorescent and immunoelectron cytochemistry in diplotene oocytes of the gastropod Achatina fulica. Association of Pol II and splicing factors with oocyte nuclear structures was analysed. The antibodies against splicing factors and Pol II were shown to label perichromatin fibrils at the periphery of condensed chromatin blocks as well as those in interchromatin regions of nucleoplasm. The revealed character of distribution of snRNPs, SC35 protein, and Pol II, together with the decondensed chromatin and absence of karyosphere, enable us to suggest that oocyte chromosomes maintain their transcriptional activity at the diplotene stage of oogenesis. In A. fulica oocytes, sparse nuclear bodies (NBs) of a complex morphological structure were revealed. These NBs contain snRNPs rather than SC35 protein. NBs are associated with a fibrogranular material (FGM), which contains SC35 protein. No snRNPs were revealed in this material. Homology of A. fulica oocyte nuclear structures to Cajal bodies and interchromatin granule clusters is discussed.

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

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

Salsman, Jayme; Pinder, Jordan; Tse, Brenda

2013-10-15

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

New approach for high reliability, low loss splicing between silica and ZBLAN fibers

NASA Astrophysics Data System (ADS)

Carbonnier, Robin; Zheng, Wenxin

2018-02-01

In the past decade, ZBLAN (ZrF4-BaF2-LaF3-NaF) fibers have drawn increasing interest for laser operations at wavelengths where Fused Silica-based (SiO2) fibers do not perform well. One limitation to the expansion of ZBLAN fiber lasers today is the difficulty to efficiently inject and extract light in/from the guiding medium using SiO2 fibers. Although free space and butt coupling have provided acceptable results, consistent and long lasting physical joints between SiO2 and ZBLAN fibers will allow smaller, cheaper, and more robust component manufacturing. While low loss splices have been reported using a traditional splicing approach, the very low mechanical strength of the joint makes it difficult to scale. Difficulties in achieving a strong bond are mainly due to the large difference of transition temperature between ZBLAN and SiO2 fibers ( 260°C vs 1175°C). This paper presents results obtained by using the high thermal expansion coefficient of the ZBLAN fiber to encapsulate a smaller SiO2 fiber. A CO2 laser glass processing system was used to control the expansion and contraction of the ZBLAN material during the splicing process for optimum reliability. This method produced splices between 125μm ZBLAN to 80μm SiO2 fibers with average transmission loss of 0.225dB (measured at 1550nm) and average ultimate tension strength of 121.4gf. The Resulting splices can be durably packaged without excessive care. Other combinations using 125μm SiO2 fibers tapered to 80μm are also discussed.

Transcription of mouse Sp2 yields alternatively spliced and sub-genomic mRNAs in a tissue- and cell-type-specific fashion.

PubMed

Yin, Haifeng; Nichols, Teresa D; Horowitz, Jonathan M

2010-07-01

The Sp-family of transcription factors is comprised by nine members, Sp1-9, that share a highly conserved DNA-binding domain. Sp2 is a poorly characterized member of this transcription factor family that is widely expressed in murine and human cell lines yet exhibits little DNA-binding or trans-activation activity in these settings. As a prelude to the generation of a "knock-out" mouse strain, we isolated a mouse Sp2 cDNA and performed a detailed analysis of Sp2 transcription in embryonic and adult mouse tissues. We report that (1) the 5' untranslated region of Sp2 is subject to alternative splicing, (2) Sp2 transcription is regulated by at least two promoters that differ in their cell-type specificity, (3) one Sp2 promoter is highly active in nine mammalian cell lines and strains and is regulated by at least five discrete stimulatory and inhibitory elements, (4) a variety of sub-genomic messages are synthesized from the Sp2 locus in a tissue- and cell-type-specific fashion and these transcripts have the capacity to encode a novel partial-Sp2 protein, and (5) RNA in situ hybridization assays indicate that Sp2 is widely expressed during mouse embryogenesis, particularly in the embryonic brain, and robust Sp2 expression occurs in neurogenic regions of the post-natal and adult brain. Copyright (c) 2010 Elsevier B.V. All rights reserved.

Vitamin D and alternative splicing of RNA

PubMed Central

Zhou, Rui; Chun, Rene F.; Lisse, Thomas S.; Garcia, Alejandro J.; Xu, Jianzhong; Adams, John S.; Hewison, Martin

2014-01-01

The active form of vitamin D (1α,25-dihydroxyvitamin D, 1,25(OH)2D) exerts its genomic effects via binding to a nuclear high-affinity vitamin D receptor (VDR). Recent deep sequencing analysis of VDR binding locations across the complete genome has significantly expanded our understanding of the actions of vitamin D and VDR on gene transcription. However, these studies have also promoted appreciation of the extra-transcriptional impact of vitamin D on gene expression. It is now clear that vitamin D interacts with the epigenome via effects on DNA methylation, histone acetylation, and microRNA generation to maintain normal biological functions. There is also increasing evidence that vitamin D can influence pre-mRNA constitutive splicing and alternative splicing, although the mechanism for this remains unclear. Pre-mRNA splicing has long been thought to be a post-transcription RNA processing event, but current data indicate that this occurs co-transcriptionally. Several steroid hormones have been recognized to coordinately control gene transcription and pre-mRNA splicing through the recruitment of nuclear receptor co-regulators that can both control gene transcription and splicing. The current review will discuss this concept with specific reference to vitamin D, and the potential role of heterogeneous nuclear ribonucleoprotein C (hnRNPC), a nuclear factor with an established function in RNA splicing. hnRNPC, has been shown to be involved in the VDR transcriptional complex as a vitamin D-response element-binding protein (VDRE-BP), and may act as a coupling factor linking VDR-directed gene transcription with RNA splicing. In this way hnRNPC may provide an additional mechanism for the fine-tuning of vitamin D-regulated target gene expression. PMID:25447737

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

Galloway, Chad A.; Smith, Harold C., E-mail: harold.smith@rochester.edu

Apolipoprotein B mRNA is edited at cytidine 6666 in the enterocytes lining the small intestine of all mammals; converting a CAA codon to a UAA stop codon. The conversion is {approx}80% efficient in this tissue and leads to the expression of the truncated protein, ApoB48, essential for secretion of dietary lipid as chylomicrons. Caco-2 cell raft cultures have been used as an in vitro model for the induction of editing activity during human small intestinal cell differentiation. This induction of apoB mRNA editing has been ascribed to the expression of APOBEC-1. In agreement our data demonstrated differentiation-dependent induction of expressionmore » of the editing enzyme APOBEC-1 and in addition we show alternative splicing of the essential auxiliary factor ACF. However, transfection of these editing factors in undifferentiated proliferating Caco-2 cells was not sufficient to induce robust apoB mRNA editing activity. Only differentiation of Caco-2 cells could induce more physiological like levels of apoB mRNA editing. The data suggested that additional regulatory mechanism(s) were induced by differentiation that controlled the functional activity of editing factors.« less

FOX-2 Dependent Splicing of Ataxin-2 Transcript Is Affected by Ataxin-1 Overexpression

PubMed Central

Welzel, Franziska; Kaehler, Christian; Isau, Melanie; Hallen, Linda; Lehrach, Hans; Krobitsch, Sylvia

2012-01-01

Alternative splicing is a fundamental posttranscriptional mechanism for controlling gene expression, and splicing defects have been linked to various human disorders. The splicing factor FOX-2 is part of a main protein interaction hub in a network related to human inherited ataxias, however, its impact remains to be elucidated. Here, we focused on the reported interaction between FOX-2 and ataxin-1, the disease-causing protein in spinocerebellar ataxia type 1. In this line, we further evaluated this interaction by yeast-2-hybrid analyses and co-immunoprecipitation experiments in mammalian cells. Interestingly, we discovered that FOX-2 localization and splicing activity is affected in the presence of nuclear ataxin-1 inclusions. Moreover, we observed that FOX-2 directly interacts with ataxin-2, a protein modulating spinocerebellar ataxia type 1 pathogenesis. Finally, we provide evidence that splicing of pre-mRNA of ataxin-2 depends on FOX-2 activity, since reduction of FOX-2 levels led to increased skipping of exon 18 in ataxin-2 transcripts. Most striking, we observed that ataxin-1 overexpression has an effect on this splicing event as well. Thus, our results demonstrate that FOX-2 is involved in splicing of ataxin-2 transcripts and that this splicing event is altered by overexpression of ataxin-1. PMID:22666429

Sf3b4-depleted Xenopus embryos: a model to study the pathogenesis of craniofacial defects in Nager syndrome

PubMed Central

Devotta, Arun; Juraver-Geslin, Hugo; Gonzalez, Jose Antonio; Hong, Chang-Soo; Saint-Jeannet, Jean-Pierre

2016-01-01

Mandibulofacial dysostosis (MFD) is a human developmental disorder characterized by defects of the facial bones. It is the second most frequent craniofacial malformation after cleft lip and palate. Nager syndrome combines many features of MFD with a variety of limb defects. Mutations in SF3B4 (splicing factor 3b, subunit 4) gene, which encodes a component of the pre-mRNA spliceosomal complex, were recently identified as a cause for Nager syndrome, accounting for 60% of affected individuals. Nothing is known about the cellular pathogenesis underlying Nager type MFD. Here we describe the first animal model for Nager syndrome, generated by knocking down Sf3b4 function in Xenopus laevis embryos, using morpholino antisense oligonucleotides. Our results indicate that Sf3b4-depleted embryos show reduced expression of the neural crest genes sox10, snail2 and twist at the neural plate border, associated with a broadening of the neural plate. This phenotype can be rescued by injection of wild-type human SF3B4 mRNA but not by mRNAs carrying mutations that cause Nager syndrome. At the tailbud stage, morphant embryos had decreased sox10 and tfap2a expression in the pharyngeal arches, indicative of a reduced number of neural crest cells. Later in development, Sf3b4-depleted tadpoles exhibited hypoplasia of neural crest-derived craniofacial cartilages, phenocopying aspects of the craniofacial skeletal defects seen in Nager syndrome patients. With this animal model we are now poised to gain important insights into the etiology and pathogenesis of Nager type MFD, and to identify the molecular targets of Sf3b4. PMID:26874011

Molecular analysis of alternative transcripts of equine AXL receptor tyrosine kinase gene.

PubMed

Park, Jeong-Woong; Song, Ki-Duk; Kim, Nam Young; Choi, Jae-Young; Hong, Seul A; Oh, Jin Hyeog; Kim, Si Won; Lee, Jeong Hyo; Park, Tae Sub; Kim, Jin-Kyoo; Kim, Jong Geun; Cho, Byung-Wook

2017-10-01

Since athletic performance is a most importance trait in horses, most research focused on physiological and physical studies of horse athletic abilities. In contrast, the molecular analysis as well as the regulatory pathway studies remain insufficient for evaluation and prediction of horse athletic abilities. In our previous study, we identified AXL receptor tyrosine kinase ( AXL ) gene which was expressed as alternative spliced isoforms in skeletal muscle during exercise. In the present study, we validated two AXL alternative splicing transcripts (named as AXLa for long form and AXLb for short form) in equine skeletal muscle to gain insight(s) into the role of each alternative transcript during exercise. We validated two isoforms of AXL transcripts in horse tissues by reverse transcriptase polymerase chain reaction (RT-PCR), and then cloned the transcripts to confirm the alternative locus and its sequences. Additionally, we examined the expression patterns of AXLa and AXLb transcripts in horse tissues by quantitative RT-PCR (qRT-PCR). Both of AXLa and AXLb transcripts were expressed in horse skeletal muscle and the expression levels were significantly increased after exercise. The sequencing analysis showed that there was an alternative splicing event at exon 11 between AXLa and AXLb transcripts. 3-dimentional (3D) prediction of the alternative protein structures revealed that the structural distance of the connective region between fibronectin type 3 (FN3) and immunoglobin (Ig) domain was different between two alternative isoforms. It is assumed that the expression patterns of AXLa and AXLb transcripts would be involved in regulation of exercise-induced stress in horse muscle possibly through an NF-κB signaling pathway. Further study is necessary to uncover biological function(s) and significance of the alternative splicing isoforms in race horse skeletal muscle.

Transcriptome and proteome analyses and the role of atypical calpain protein and autophagy in the spliced leader silencing pathway in Trypanosoma brucei.

PubMed

Hope, Ronen; Egarmina, Katarina; Voloshin, Konstantin; Waldman Ben-Asher, Hiba; Carmi, Shai; Eliaz, Dror; Drori, Yaron; Michaeli, Shulamit

2016-10-01

Under persistent ER stress, Trypanosoma brucei parasites induce the spliced leader silencing (SLS) pathway. In SLS, transcription of the SL RNA gene, the SL donor to all mRNAs, is extinguished, arresting trans-splicing and leading to programmed cell death (PCD). In this study, we investigated the transcriptome following silencing of SEC63, a factor essential for protein translocation across the ER membrane, and whose silencing induces SLS. The proteome of SEC63-silenced cells was analyzed with an emphasis on SLS-specific alterations in protein expression, and modifications that do not directly result from perturbations in trans-splicing. One such protein identified is an atypical calpain SKCRP7.1/7.2. Co-silencing of SKCRP7.1/7.2 and SEC63 eliminated SLS induction due its role in translocating the PK3 kinase. This kinase initiates SLS by migrating to the nucleus and phosphorylating TRF4 leading to shut-off of SL RNA transcription. Thus, SKCRP7.1 is involved in SLS signaling and the accompanying PCD. The role of autophagy in SLS was also investigated; eliminating autophagy through VPS34 or ATG7 silencing demonstrated that autophagy is not essential for SLS induction, but is associated with PCD. Thus, this study identified factors that are used by the parasite to cope with ER stress and to induce SLS and PCD. © 2016 John Wiley & Sons Ltd.

How do messenger RNA splicing alterations drive myelodysplasia?

PubMed Central

2017-01-01

Mutations in RNA splicing factors are the single most common class of genetic alterations in myelodysplastic syndrome (MDS) patients. Although much has been learned about how these mutations affect splicing at a global- and transcript-specific level, critical questions about the role of these mutations in MDS development and maintenance remain. Here we present the questions to be addressed in order to understand the unique enrichment of these mutations in MDS. PMID:28348147

CryoEM structure of the spliceosome immediately after branching

PubMed Central

Galej, Wojciech P.; Wilkinson, Max E.; Fica, Sebastian M.; Oubridge, Chris; Newman, Andrew J.; Nagai, Kiyoshi

2016-01-01

Pre-mRNA splicing proceeds by two consecutive trans-esterification reactions via a lariat-intron intermediate. We present the 3.8Å cryoEM structure of the spliceosome immediately after lariat formation. The 5’-splice site is cleaved but remains close to the catalytic Mg2+ site in the U2/U6 snRNA triplex, and the 5’-phosphate of the intron nucleotide G(+1) is linked to the branch adenosine 2’OH. The 5’-exon is held between the Prp8 N-terminal and Linker domains, and base-pairs with U5 snRNA loop 1. Non-Watson-Crick interactions between the branch helix and 5’-splice site dock the branch adenosine into the active site, while intron nucleotides +3 to +6 base-pair with the U6 snRNA ACAGAGA sequence. Isy1 and the step one factors Yju2 and Cwc25 stabilise docking of the branch helix. The intron downstream of the branch site emerges between the Prp8 RT and Linker domains and extends towards Prp16 helicase, suggesting a plausible mechanism of remodelling before exon ligation. PMID:27459055

The MTL1 Pentatricopeptide Repeat Protein Is Required for Both Translation and Splicing of the Mitochondrial NADH DEHYDROGENASE SUBUNIT7 mRNA in Arabidopsis.

PubMed

Haïli, Nawel; Planchard, Noelya; Arnal, Nadège; Quadrado, Martine; Vrielynck, Nathalie; Dahan, Jennifer; des Francs-Small, Catherine Colas; Mireau, Hakim

2016-01-01

Mitochondrial translation involves a complex interplay of ancient bacteria-like features and host-derived functionalities. Although the basic components of the mitochondrial translation apparatus have been recognized, very few protein factors aiding in recruiting ribosomes on mitochondria-encoded messenger RNA (mRNAs) have been identified in higher plants. In this study, we describe the identification of the Arabidopsis (Arabidopsis thaliana) MITOCHONDRIAL TRANSLATION FACTOR1 (MTL1) protein, a new member of the Pentatricopeptide Repeat family, and show that it is essential for the translation of the mitochondrial NADH dehydrogenase subunit7 (nad7) mRNA. We demonstrate that mtl1 mutant plants fail to accumulate the Nad7 protein, even though the nad7 mature mRNA is produced and bears the same 5' and 3' extremities as in wild-type plants. We next observed that polysome association of nad7 mature mRNA is specifically disrupted in mtl1 mutants, indicating that the absence of Nad7 results from a lack of translation of nad7 mRNA. These findings illustrate that mitochondrial translation requires the intervention of gene-specific nucleus-encoded PPR trans-factors and that their action does not necessarily involve the 5' processing of their target mRNA, as observed previously. Interestingly, a partial decrease in nad7 intron 2 splicing was also detected in mtl1 mutants, suggesting that MTL1 is also involved in group II intron splicing. However, this second function appears to be less essential for nad7 expression than its role in translation. MTL1 will be instrumental to understand the multifunctionality of PPR proteins and the mechanisms governing mRNA translation and intron splicing in plant mitochondria. © 2016 American Society of Plant Biologists. All Rights Reserved.

Simple and reliable light launch from a conventional single-mode fiber into a helical-core fiber through an adiabatically tapered splice.

PubMed

Kim, Hyuntai; Kim, Jongki; Jung, Yongmin; Vazquez-Zuniga, Luis Alonso; Lee, Seung Jong; Choi, Geunchang; Oh, Kyunghwan; Wang, Pu; Clarkson, W A; Jeong, Yoonchan

2012-11-05

We propose a simple and efficient light launch scheme for a helical-core fiber (HCF) by using an adiabatically tapered splice technique, through which we overcome its inherent difficulty with light launch owing to the large lateral offset and angular tilt of its core. We experimentally demonstrate single-mode excitation in the HCF in this configuration, which yields the coupling efficiency of around -5.9 dB (26%) for a ~1.1-μm light input when the splice joint is tapered down to 30 μm in diameter. To our knowledge, this is the first proof-of-principle report on the fusion-splice coupling between an HCF and a conventional single-mode fiber.

Role of the DNA Damage Response in Human Papillomavirus RNA Splicing and Polyadenylation.

PubMed

Nilsson, Kersti; Wu, Chengjun; Schwartz, Stefan

2018-06-12

Human papillomaviruses (HPVs) have evolved to use the DNA repair machinery to replicate its DNA genome in differentiated cells. HPV activates the DNA damage response (DDR) in infected cells. Cellular DDR factors are recruited to the HPV DNA genome and position the cellular DNA polymerase on the HPV DNA and progeny genomes are synthesized. Following HPV DNA replication, HPV late gene expression is activated. Recent research has shown that the DDR factors also interact with RNA binding proteins and affects RNA processing. DDR factors activated by DNA damage and that associate with HPV DNA can recruit splicing factors and RNA binding proteins to the HPV DNA and induce HPV late gene expression. This induction is the result of altered alternative polyadenylation and splicing of HPV messenger RNA (mRNA). HPV uses the DDR machinery to replicate its DNA genome and to activate HPV late gene expression at the level of RNA processing.

PTP1B-dependent regulation of receptor tyrosine kinase signaling by the actin-binding protein Mena.

PubMed

Hughes, Shannon K; Oudin, Madeleine J; Tadros, Jenny; Neil, Jason; Del Rosario, Amanda; Joughin, Brian A; Ritsma, Laila; Wyckoff, Jeff; Vasile, Eliza; Eddy, Robert; Philippar, Ulrike; Lussiez, Alisha; Condeelis, John S; van Rheenen, Jacco; White, Forest; Lauffenburger, Douglas A; Gertler, Frank B

2015-11-01

During breast cancer progression, alternative mRNA splicing produces functionally distinct isoforms of Mena, an actin regulator with roles in cell migration and metastasis. Aggressive tumor cell subpopulations express Mena(INV), which promotes tumor cell invasion by potentiating EGF responses. However, the mechanism by which this occurs is unknown. Here we report that Mena associates constitutively with the tyrosine phosphatase PTP1B and mediates a novel negative feedback mechanism that attenuates receptor tyrosine kinase signaling. On EGF stimulation, complexes containing Mena and PTP1B are recruited to the EGFR, causing receptor dephosphorylation and leading to decreased motility responses. Mena also interacts with the 5' inositol phosphatase SHIP2, which is important for the recruitment of the Mena-PTP1B complex to the EGFR. When Mena(INV) is expressed, PTP1B recruitment to the EGFR is impaired, providing a mechanism for growth factor sensitization to EGF, as well as HGF and IGF, and increased resistance to EGFR and Met inhibitors in signaling and motility assays. In sum, we demonstrate that Mena plays an important role in regulating growth factor-induced signaling. Disruption of this attenuation by Mena(INV) sensitizes tumor cells to low-growth factor concentrations, thereby increasing the migration and invasion responses that contribute to aggressive, malignant cell phenotypes. © 2015 Hughes, Oudin, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

30 CFR 7.406 - Flame test apparatus.

Code of Federal Regulations, 2012 CFR

2012-07-01

... Cable Splice Kits § 7.406 Flame test apparatus. The principal parts of the apparatus used to test for... extend to the sample end location. These are used to energize the electric cable and splice specimens. They are not used, but may stay in place, when testing signaling cables. (b) Specimen holder (support...

30 CFR 7.406 - Flame test apparatus.

Code of Federal Regulations, 2013 CFR

2013-07-01

... Cable Splice Kits § 7.406 Flame test apparatus. The principal parts of the apparatus used to test for... extend to the sample end location. These are used to energize the electric cable and splice specimens. They are not used, but may stay in place, when testing signaling cables. (b) Specimen holder (support...

30 CFR 7.406 - Flame test apparatus.

Code of Federal Regulations, 2014 CFR

2014-07-01

... Cable Splice Kits § 7.406 Flame test apparatus. The principal parts of the apparatus used to test for... extend to the sample end location. These are used to energize the electric cable and splice specimens. They are not used, but may stay in place, when testing signaling cables. (b) Specimen holder (support...

Effective delivery of large genes to the retina by dual AAV vectors

PubMed Central

Trapani, Ivana; Colella, Pasqualina; Sommella, Andrea; Iodice, Carolina; Cesi, Giulia; de Simone, Sonia; Marrocco, Elena; Rossi, Settimio; Giunti, Massimo; Palfi, Arpad; Farrar, Gwyneth J; Polishchuk, Roman; Auricchio, Alberto

2014-01-01

Retinal gene therapy with adeno-associated viral (AAV) vectors is safe and effective in humans. However, AAV's limited cargo capacity prevents its application to therapies of inherited retinal diseases due to mutations of genes over 5 kb, like Stargardt's disease (STGD) and Usher syndrome type IB (USH1B). Previous methods based on ‘forced’ packaging of large genes into AAV capsids may not be easily translated to the clinic due to the generation of genomes of heterogeneous size which raise safety concerns. Taking advantage of AAV's ability to concatemerize, we generated dual AAV vectors which reconstitute a large gene by either splicing (trans-splicing), homologous recombination (overlapping), or a combination of the two (hybrid). We found that dual trans-splicing and hybrid vectors transduce efficiently mouse and pig photoreceptors to levels that, albeit lower than those achieved with a single AAV, resulted in significant improvement of the retinal phenotype of mouse models of STGD and USH1B. Thus, dual AAV trans-splicing or hybrid vectors are an attractive strategy for gene therapy of retinal diseases that require delivery of large genes. PMID:24150896

Identification of Lectins from Metastatic Cancer Cells through Magnetic Glyconanoparticles

PubMed Central

Kavunja, Herbert W.; Voss, Patricia G.

2016-01-01

Cancer cells can have characteristic carbohydrate binding properties. Previously, it was shown that a highly metastatic melanoma cell line B16F10 bound to galacto-side-functionalized nanoparticles much stronger than the corresponding less metastatic B16F1 cells. To better understand the carbohydrate binding properties of cancer cells, herein, we report the isolation and characterization of endogenous galactose binding proteins from B16F10 cells using magnetic glyconanoparticles. The galactose-coated magnetic glyconanoparticles could bind with lectins present in the cells and be isolated through magnet-mediated separation. Through Western blot and mass spectrometry, the arginine/serine rich splicing factor Sfrs1 was identified as a galactose-selective endogenous lectin, overexpressed in B16F10 cells, compared with B16F1 cells. In addition, galactin-3 was found in higher amounts in B16F10 cells. Finally, the glyconanoparticles exhibited a superior efficiency in lectin isolation, from both protein mixtures and live cells, than the corresponding more traditional microparticles functionalized with carbohydrates. Thus, the magnetic glyconanoparticles present a useful tool for discovery of endogenous lectins, as well as binding partners of lectins, without prior knowledge of protein identities. PMID:27110035

RBFox1-mediated RNA splicing regulates cardiac hypertrophy and heart failure.

PubMed

Gao, Chen; Ren, Shuxun; Lee, Jae-Hyung; Qiu, Jinsong; Chapski, Douglas J; Rau, Christoph D; Zhou, Yu; Abdellatif, Maha; Nakano, Astushi; Vondriska, Thomas M; Xiao, Xinshu; Fu, Xiang-Dong; Chen, Jau-Nian; Wang, Yibin

2016-01-01

RNA splicing is a major contributor to total transcriptome complexity; however, the functional role and regulation of splicing in heart failure remain poorly understood. Here, we used a total transcriptome profiling and bioinformatic analysis approach and identified a muscle-specific isoform of an RNA splicing regulator, RBFox1 (also known as A2BP1), as a prominent regulator of alternative RNA splicing during heart failure. Evaluation of developing murine and zebrafish hearts revealed that RBFox1 is induced during postnatal cardiac maturation. However, we found that RBFox1 is markedly diminished in failing human and mouse hearts. In a mouse model, RBFox1 deficiency in the heart promoted pressure overload-induced heart failure. We determined that RBFox1 is a potent regulator of RNA splicing and is required for a conserved splicing process of transcription factor MEF2 family members that yields different MEF2 isoforms with differential effects on cardiac hypertrophic gene expression. Finally, induction of RBFox1 expression in murine pressure overload models substantially attenuated cardiac hypertrophy and pathological manifestations. Together, this study identifies regulation of RNA splicing by RBFox1 as an important player in transcriptome reprogramming during heart failure that influence pathogenesis of the disease.

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.

RBFox1-mediated RNA splicing regulates cardiac hypertrophy and heart failure

PubMed Central

Gao, Chen; Ren, Shuxun; Lee, Jae-Hyung; Qiu, Jinsong; Chapski, Douglas J.; Rau, Christoph D.; Zhou, Yu; Abdellatif, Maha; Nakano, Astushi; Vondriska, Thomas M.; Xiao, Xinshu; Fu, Xiang-Dong; Chen, Jau-Nian; Wang, Yibin

2015-01-01

RNA splicing is a major contributor to total transcriptome complexity; however, the functional role and regulation of splicing in heart failure remain poorly understood. Here, we used a total transcriptome profiling and bioinformatic analysis approach and identified a muscle-specific isoform of an RNA splicing regulator, RBFox1 (also known as A2BP1), as a prominent regulator of alternative RNA splicing during heart failure. Evaluation of developing murine and zebrafish hearts revealed that RBFox1 is induced during postnatal cardiac maturation. However, we found that RBFox1 is markedly diminished in failing human and mouse hearts. In a mouse model, RBFox1 deficiency in the heart promoted pressure overload–induced heart failure. We determined that RBFox1 is a potent regulator of RNA splicing and is required for a conserved splicing process of transcription factor MEF2 family members that yields different MEF2 isoforms with differential effects on cardiac hypertrophic gene expression. Finally, induction of RBFox1 expression in murine pressure overload models substantially attenuated cardiac hypertrophy and pathological manifestations. Together, this study identifies regulation of RNA splicing by RBFox1 as an important player in transcriptome reprogramming during heart failure that influence pathogenesis of the disease. PMID:26619120

RBFOX and PTBP1 proteins regulate the alternative splicing of micro-exons in human brain transcripts

PubMed Central

Sanchez-Pulido, Luis; Haerty, Wilfried

2015-01-01

Ninety-four percent of mammalian protein-coding exons exceed 51 nucleotides (nt) in length. The paucity of micro-exons (≤ 51 nt) suggests that their recognition and correct processing by the splicing machinery present greater challenges than for longer exons. Yet, because thousands of human genes harbor processed micro-exons, specialized mechanisms may be in place to promote their splicing. Here, we survey deep genomic data sets to define 13,085 micro-exons and to study their splicing mechanisms and molecular functions. More than 60% of annotated human micro-exons exhibit a high level of sequence conservation, an indicator of functionality. While most human micro-exons require splicing-enhancing genomic features to be processed, the splicing of hundreds of micro-exons is enhanced by the adjacent binding of splice factors in the introns of pre-messenger RNAs. Notably, splicing of a significant number of micro-exons was found to be facilitated by the binding of RBFOX proteins, which promote their inclusion in the brain, muscle, and heart. Our analyses suggest that accurate regulation of micro-exon inclusion by RBFOX proteins and PTBP1 plays an important role in the maintenance of tissue-specific protein–protein interactions. PMID:25524026

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.

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

49 CFR Appendix G to Subchapter B... - Minimum Periodic Inspection Standards

Code of Federal Regulations, 2011 CFR

2011-10-01

...). (2) Any portion of the drum or rotor missing or in danger of falling away. d. Brake Hose. (1) Hose... a reinforcement ply). (Thermoplastic nylon may have braid reinforcement or color difference between... pressure is applied. (3) Any audible leaks. (4) Two hoses improperly joined (such as a splice made by...

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.

The relationship between RNA catalytic processes

NASA Astrophysics Data System (ADS)

Cedergren, Robert; Lang, B. Franz; Gravel, Denis

1988-09-01

Proposals that an RNA-based genetic system preceeded DNA, stem from the ability of RNA to store genetic information and to promote simple catalysis. However, to be a valid basis for the RNA world, RNA catalysis must demonstrate or be related to intrinsic chemical properties which could have existed in primordial times. We analyze this question by first classifying RNA catalysis and related processes according to their mechanism. We define: (A) thedisjunct nucleophile class which leads to 5'-phosphates. These include Group I and II intron splicing, nuclear mRNA splicing and RNase P reactions. Although Group I introns and its excision mechanism is likely to have existed in primordial times, present-day examples have arisen independently in different phyla much more recently. Comparative methodology indicates that RNase P catalysis originated before the divergence of the major kingdoms. In addition, alldisjunct nucleophile reactions can be interrelated by a proposed mechanism involving a distant 2-OH nucleophile. (B) theconjunct nucleophile class leading to 3'-phosphates. This class is composed of self-cleaving RNAs found in plant viruses and the newt. We propose that tRNA splicing is related to this mechanism rather than the previous one. The presence of introns in tRNA genes of eukaryotes and archaebacteria supports the idea that tRNA splicing predates the divergence of these cell types.

Site-Selective RNA Splicing Nanozyme: DNAzyme and RtcB Conjugates on a Gold Nanoparticle.

PubMed

Petree, Jessica R; Yehl, Kevin; Galior, Kornelia; Glazier, Roxanne; Deal, Brendan; Salaita, Khalid

2018-01-19

Modifying RNA through either splicing or editing is a fundamental biological process for creating protein diversity from the same genetic code. Developing novel chemical biology tools for RNA editing has potential to transiently edit genes and to provide a better understanding of RNA biochemistry. Current techniques used to modify RNA include the use of ribozymes, adenosine deaminase, and tRNA endonucleases. Herein, we report a nanozyme that is capable of splicing virtually any RNA stem-loop. This nanozyme is comprised of a gold nanoparticle functionalized with three enzymes: two catalytic DNA strands with ribonuclease function and an RNA ligase. The nanozyme cleaves and then ligates RNA targets, performing a splicing reaction that is akin to the function of the spliceosome. Our results show that the three-enzyme reaction can remove a 19 nt segment from a 67 nt RNA loop with up to 66% efficiency. The complete nanozyme can perform the same splice reaction at 10% efficiency. These splicing nanozymes represent a new promising approach for gene manipulation that has potential for applications in living cells.