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1.  The translational landscape of the splicing factor SRSF1 and its role in mitosis 
eLife  2014;3:e02028.
The shuttling serine/arginine rich (SR) protein SRSF1 (previously known as SF2/ASF) is a splicing regulator that also activates translation in the cytoplasm. In order to dissect the gene network that is translationally regulated by SRSF1, we performed a high-throughput deep sequencing analysis of polysomal fractions in cells overexpressing SRSF1. We identified approximately 1500 mRNAs that are translational targets of SRSF1. These include mRNAs encoding proteins involved in cell cycle regulation, such as spindle, kinetochore, and M phase proteins, which are essential for accurate chromosome segregation. Indeed, we show that translational activity of SRSF1 is required for normal mitotic progression. Furthermore, we found that mRNAs that display alternative splicing changes upon SRSF1 overexpression are also its translational targets, strongly suggesting that SRSF1 couples pre-mRNA splicing and translation. These data provide insights on the complex role of SRSF1 in the control of gene expression at multiple levels and its implications in cancer.
DOI: http://dx.doi.org/10.7554/eLife.02028.001
eLife digest
Genes contain the instructions to make proteins. These instructions are first transcribed to produce an intermediate molecule called a messenger RNA (mRNA), which is then translated to produce the protein. However, gene sequences are often interrupted by ‘introns’, sections of DNA that do not code for protein, and these introns must be removed from the mRNA molecules via a process called ‘splicing’ before the protein is produced.
Splicing can also be used to ‘mix and match’ sections of gene sequences to produce slightly different versions of the same protein in a process called ‘alternative splicing’. SRSF1 is one of a family of proteins that control both types of gene splicing but also promotes the translation of specific mRNAs. To date only a few of the genes whose translation is regulated by SRSF1 have been identified.
Here, Maslon, Heras et al. have used human cells that artificially produce more SRSF1 protein than normal to identify those genes whose translation is regulated by SRSF1. Over 1500 ‘target genes’ were found; many of which encoded proteins that are involved in cell division—and cells with less SRSF1 than normal failed to divide properly. Maslon, Heras et al. also found a link between alternative splicing and protein translation: many of the mRNAs that were spliced differently in cells that over-produced SRSF1 were also genes whose translation was affected by SRSF1.
Since uncontrolled cell division, or defects in mRNA splicing or protein synthesis are all often linked to cancer, these discoveries might provide new insights into the mechanisms underlying this disease.
DOI: http://dx.doi.org/10.7554/eLife.02028.002
doi:10.7554/eLife.02028
PMCID: PMC4027812  PMID: 24842991
translation; splicing; SR proteins; human
2.  The RNA-binding landscapes of two SR proteins reveal unique functions and binding to diverse RNA classes 
Genome Biology  2012;13(3):R17.
Background
The SR proteins comprise a family of essential, structurally related RNA binding proteins. The complexity of their RNA targets and specificity of RNA recognition in vivo is not well understood. Here we use iCLIP to globally analyze and compare the RNA binding properties of two SR proteins, SRSF3 and SRSF4, in murine cells.
Results
SRSF3 and SRSF4 binding sites mapped to largely non-overlapping target genes, and in vivo consensus binding motifs were distinct. Interactions with intronless and intron-containing mRNAs as well as non-coding RNAs were detected. Surprisingly, both SR proteins bound to the 3' ends of the majority of intronless histone transcripts, implicating SRSF3 and SRSF4 in histone mRNA metabolism. In contrast, SRSF3 but not SRSF4 specifically bound transcripts encoding numerous RNA binding proteins. Remarkably, SRSF3 was shown to modulate alternative splicing of its own as well as three other transcripts encoding SR proteins. These SRSF3-mediated splicing events led to downregulation of heterologous SR proteins via nonsense-mediated decay.
Conclusions
SRSF3 and SRSF4 display unique RNA binding properties underlying diverse cellular regulatory mechanisms, with shared as well as unique coding and non-coding targets. Importantly, CLIP analysis led to the discovery that SRSF3 cross-regulates the expression of other SR protein family members.
doi:10.1186/gb-2012-13-3-r17
PMCID: PMC3439968  PMID: 22436691
3.  Proteasome-Mediated Proteolysis of SRSF5 Splicing Factor Intriguingly Co-occurs with SRSF5 mRNA Upregulation during Late Erythroid Differentiation 
PLoS ONE  2013;8(3):e59137.
SR proteins exhibit diverse functions ranging from their role in constitutive and alternative splicing, to virtually all aspects of mRNA metabolism. These findings have attracted growing interest in deciphering the regulatory mechanisms that control the tissue-specific expression of these SR proteins. In this study, we show that SRSF5 protein decreases drastically during erythroid cell differentiation, contrasting with a concomitant upregulation of SRSF5 mRNA level. Proteasome chemical inhibition provided strong evidence that endogenous SRSF5 protein, as well as protein deriving from stably transfected SRSF5 cDNA, are both targeted to proteolysis as the cells undergo terminal differentiation. Consistently, functional experiments show that overexpression of SRSF5 enhances a specific endogenous pre-mRNA splicing event in proliferating cells, but not in differentiating cells, due to proteasome-mediated targeting of both endogenous and transfection-derived SRSF5. Further investigation of the relationship between SRSF5 structure and its post-translation regulation and function, suggested that the RNA recognition motifs of SRSF5 are sufficient to activate pre-mRNA splicing, whereas proteasome-mediated proteolysis of SRSF5 requires the presence of the C-terminal RS domain of the protein. Phosphorylation of SR proteins is a key post-translation regulation that promotes their activity and subcellular availability. We here show that inhibition of the CDC2-like kinase (CLK) family and mutation of the AKT phosphorylation site Ser86 on SRSF5, have no effect on SRSF5 stability. We reasoned that at least AKT and CLK signaling pathways are not involved in proteasome-induced turnover of SRSF5 during late erythroid development.
doi:10.1371/journal.pone.0059137
PMCID: PMC3594168  PMID: 23536862
4.  SRSF10 Regulates Alternative Splicing and Is Required for Adipocyte Differentiation 
Molecular and Cellular Biology  2014;34(12):2198-2207.
During adipocyte differentiation, significant alternative splicing changes occur in association with the adipogenic process. However, little is known about roles played by splicing factors in this process. We observed that mice deficient for the splicing factor SRSF10 exhibit severely impaired development of subcutaneous white adipose tissue (WAT) as a result of defects in adipogenic differentiation. To identify splicing events responsible for this, transcriptome sequencing (RNA-seq) analysis was performed using embryonic fibroblast cells. Several SRSF10-affected splicing events that are implicated in adipogenesis have been identified. Notably, lipin1, known as an important regulator during adipogenesis, was further investigated. While lipin1β is mainly involved in lipogenesis, its alternatively spliced isoform lipin1α, generated through the skipping of exon 7, is primarily required for initial adipocyte differentiation. Skipping of exon 7 is controlled by an SRSF10-regulated cis element located in the constitutive exon 8. The activity of this element depends on the binding of SRSF10 and correlates with the relative abundance of lipin1α mRNA. A series of experiments demonstrated that SRSF10 controls the production of lipin1α and thus promotes adipocyte differentiation. Indeed, lipin1α expression could rescue SRSF10-mediated adipogenic defects. Taken together, our results identify SRSF10 as an essential regulator for adipocyte differentiation and also provide new insights into splicing control by SRSF10 in lipin1 pre-mRNA splicing.
doi:10.1128/MCB.01674-13
PMCID: PMC4054296  PMID: 24710272
5.  A new function of the splicing factor SRSF2 in the control of E2F1-mediated cell cycle progression in neuroendocrine lung tumors 
Cell Cycle  2013;12(8):1267-1278.
The transcription factor E2F1 belongs to the E2F family and plays a crucial role during cell cycle progression and apoptosis. Ser/Arg-Rich (SR) proteins are a family of RNA-binding phosphoproteins that control both constitutive and alternative pre-mRNA splicing events. We previously identified the SR protein SRSF2 as a new transcriptional target of E2F1 and demonstrated that both proteins cooperate to induce apoptosis in non-small cell lung carcinoma. In this study, we postulated that SRSF2 is also involved in the proliferative functions of E2F1. Using IHC, we first demonstrate that SRSF2 and its phosphorylated form (P-SRSF2) are overexpressed in neuroendocrine lung tumors that are highly proliferative tumors expressing high levels of E2F1. Importantly, we show a direct correlation between cyclin E, an E2F1-target gene controlling S phase, and P-SRSF2 proteins levels (p = 0.0083), suggesting a role of SRSF2 in E2F1-mediated cellular proliferation. Accordingly, using neuroendocrine lung carcinoma cell lines, we demonstrate that SRSF2 is a cell cycle-regulated protein involved in entry and progression into S phase. We also provide evidence that SRSF2 interacts with E2F1 and stimulates its transcriptional control of cell cycle target genes such as cyclin E. Finally, we show that inhibition of AKT signaling pathway prevents SRSF2 phosphorylation and activity toward E2F1 transcriptional function. Taken together, these results identify a new role of SRSF2 in the control of cell cycle progression and reinforce the functional link between SRSF2 and E2F1 proteins.
doi:10.4161/cc.24363
PMCID: PMC3674091  PMID: 23518498
AKT; cellular proliferation; E2F1; neuroendocrine lung tumors; p45SKP2; SRSF2
6.  Abnormal Expression of the Pre-mRNA Splicing Regulators SRSF1, SRSF2, SRPK1 and SRPK2 in Non Small Cell Lung Carcinoma 
PLoS ONE  2012;7(10):e46539.
Splicing abnormalities frequently occur in cancer. A key role as splice site choice regulator is played by the members of the SR (Ser/Arg-rich) family of proteins. We recently demonstrated that SRSF2 is involved in cisplatin-mediated apoptosis of human lung carcinoma cell lines. In this study, by using immunohistochemistry, we demonstrate that the SR proteins SRSF1 and SRSF2 are overexpressed in 63% and 65% of lung adenocarcinoma (ADC) as well as in 68% and 91% of squamous cell lung carcinoma (SCC), respectively, compared to normal lung epithelial cells. In addition, we show that SRSF2 overexpression correlates with high level of phosphorylated SRSF2 in both ADC (p<0.0001) and SCC (p = 0.02), indicating that SRSF2 mostly accumulates under a phosphorylated form in lung tumors. Consistently, we further show that the SR-phosphorylating kinases SRPK1 and SRPK2 are upregulated in 92% and 94% of ADC as well as in 72% and 68% of SCC, respectively. P-SRSF2 and SRPK2 scores are correlated in ADC (p = 0.01). Using lung adenocarcinoma cell lines, we demonstrate that SRSF1 overexpression leads to a more invasive phenotype, evidenced by activation of PI3K/AKT and p42/44MAPK signaling pathways, increased growth capacity in soft agar, acquisition of mesenchymal markers such as E cadherin loss, vimentin and fibronectin gain, and increased resistance to chemotherapies. Finally, we provide evidence that high levels of SRSF1 and P-SRSF2 proteins are associated with extensive stage (III–IV) in ADC. Taken together, these results indicate that a global deregulation of pre-mRNA splicing regulators occurs during lung tumorigenesis and does not predict same outcome in both Non Small Cell Lung Carcinoma histological sub-types, likely contributing to a more aggressive phenotype in adenocarcinoma.
doi:10.1371/journal.pone.0046539
PMCID: PMC3468597  PMID: 23071587
7.  Psip1/Ledgf p52 Binds Methylated Histone H3K36 and Splicing Factors and Contributes to the Regulation of Alternative Splicing 
PLoS Genetics  2012;8(5):e1002717.
Increasing evidence suggests that chromatin modifications have important roles in modulating constitutive or alternative splicing. Here we demonstrate that the PWWP domain of the chromatin-associated protein Psip1/Ledgf can specifically recognize tri-methylated H3K36 and that, like this histone modification, the Psip1 short (p52) isoform is enriched at active genes. We show that the p52, but not the long (p75), isoform of Psip1 co-localizes and interacts with Srsf1 and other proteins involved in mRNA processing. The level of H3K36me3 associated Srsf1 is reduced in Psip1 mutant cells and alternative splicing of specific genes is affected. Moreover, we show altered Srsf1 distribution around the alternatively spliced exons of these genes in Psip1 null cells. We propose that Psip1/p52, through its binding to both chromatin and splicing factors, might act to modulate splicing.
Author Summary
The regulated processing of mRNAs by splicing of exons and introns has the potential to increase the information content of the genome. Various splicing factors have been identified whose binding to cis-acting sequences can influence whether an alternative exon is included or excluded (skipped) in the mature mRNA. However, increasing evidence suggests that the chromatin template also has an important role in modulating splicing. Here we identify a chromatin-associated protein Psip1/Ledgf that can bind to a histone modification enriched at active genes and that can also interact with other proteins involved in mRNA splicing. Loss of Psip1 reduces the chromatin association of specific splicing proteins and alters the pattern of alternative splicing. We propose that Psip1, through its binding to both chromatin and splicing factors, might act to modulate splicing.
doi:10.1371/journal.pgen.1002717
PMCID: PMC3355077  PMID: 22615581
8.  Regulation of Mcl-1 by SRSF1 and SRSF5 in Cancer Cells 
PLoS ONE  2012;7(12):e51497.
Up-regulation of the apoptosis-regulatory gene Mcl-1 (myeloid cell leukemia-1) occurs in different cancer types and is linked with drug resistance to cancer therapies. It is well known that Mcl-1 pre-mRNA undergoes alternative splicing events to produce two functionally distinct proteins, Mcl-1S (pro-apoptotic) and Mcl-lL (anti-apoptotic); the latter isoform is predominant in different cancers including breast and ovarian cancer cells. In the present study we report that the RNA-binding protein (RBP) and proto-oncogene SRSF1 (serine and arginine-rich splicing factor 1) influences splicing of Mcl-1 in both MCF-7 and MDA-MB-231 breast cancer cells and JAR choriocarcinoma cells; we also show for the first time that another RBP SRSF5 affects splicing of Mcl-1 in the MCF-7 cells. Moreover, we report that SRSF1 is involved in other aspects of Mcl-1 regulation with knockdown of SRSF1, by RNAi, resulting in a significant decrease in Mcl-1 protein levels in MCF-7 cells but an increase in JAR cells, respectively, by potentially affecting protein stability and translation of Mcl-l. The key findings from this study highlight the importance of the cellular context of different cancer cells for the function of multifunctional RBPs like SRSF1 and have implications for therapeutic approaches employed to target Mcl-1.
doi:10.1371/journal.pone.0051497
PMCID: PMC3524227  PMID: 23284704
9.  Expression of Tra2β in Cancer Cells as a Potential Contributory Factor to Neoplasia and Metastasis 
The splicing regulator proteins SRSF1 (also known as ASF/SF2) and SRSF3 (also known as SRP20) belong to the SR family of proteins and can be upregulated in cancer. The SRSF1 gene itself is amplified in some cancer cells, and cancer-associated changes in the expression of MYC also increase SRSF1 gene expression. Increased concentrations of SRSF1 protein promote prooncogenic splicing patterns of a number of key regulators of cell growth. Here, we review the evidence that upregulation of the SR-related Tra2β protein might have a similar role in cancer cells. The TRA2B gene encoding Tra2β is amplified in particular tumours including those of the lung, ovary, cervix, stomach, head, and neck. Both TRA2B RNA and Tra2β protein levels are upregulated in breast, cervical, ovarian, and colon cancer, and Tra2β expression is associated with cancer cell survival. The TRA2B gene is a transcriptional target of the protooncogene ETS-1 which might cause higher levels of expression in some cancer cells which express this transcription factor. Known Tra2β splicing targets have important roles in cancer cells, where they affect metastasis, proliferation, and cell survival. Tra2β protein is also known to interact directly with the RBMY protein which is implicated in liver cancer.
doi:10.1155/2013/843781
PMCID: PMC3723085  PMID: 23935626
10.  ONCOGENIC SPLICING FACTOR SRSF1 IS A CRITICAL TRANSCRIPTIONAL TARGET OF MYC 
Cell reports  2012;1(2):110-117.
The SR protein splicing factor SRSF1 is a potent proto-oncogene that is frequently upregulated in cancer. Here we show that SRSF1 is a direct target of the transcription-factor oncoprotein MYC. These two oncogenes are significantly co-expressed in lung carcinomas, and MYC knockdown downregulates SRSF1 expression in lung-cancer cell lines. MYC directly activates transcription of SRSF1 through two non-canonical E-boxes in its promoter. The resulting increase in SRSF1 protein is sufficient to modulate alternative splicing of a subset of transcripts. In particular, MYC induction leads to SRSF1-mediated alternative splicing of the signaling kinase MKNK2 and the transcription factor TEAD1. SRSF1 knockdown reduces MYC’s oncogenic activity, decreasing proliferation and anchorage-independent growth. These results suggest a mechanism for SRSF1 upregulation in tumors with elevated MYC, and identify SRSF1 as a critical MYC target that contributes to its oncogenic potential by enabling MYC to regulate the expression of specific protein isoforms through alternative splicing.
doi:10.1016/j.celrep.2011.12.001
PMCID: PMC3334311  PMID: 22545246
11.  RRP1B is a Metastasis Modifier That Regulates the Expression of Alternative mRNA Isoforms Through Interactions with SRSF1 
Oncogene  2013;33(14):1818-1827.
RRP1B (ribosomal RNA processing 1 homolog B) was first identified as a metastasis susceptibility gene in breast cancer through its ability to modulate gene expression in a manner that can be used to accurately predict prognosis in breast cancer. However, the mechanism(s) by which RRP1B modulates gene expression is currently unclear. Many RRP1B binding candidates are involved in alternative splicing, a mechanism of gene expression regulation that is increasingly recognized to be involved in cancer progression and metastasis. One such target is SRSF1 (SF2/ASF), an essential splicing regulator that also functions as an oncoprotein. Earlier studies demonstrated that splicing and transcription occur concurrently and are coupled processes. Given that RRP1B regulates transcriptional activity, we hypothesized that RRP1B also regulates the expression of alternative mRNA isoforms through its interaction with SRSF1. Interaction between RRP1B and SRSF1 was verified by co-immunoprecipitation and co-immunofluorescence. Treatment of cells with transcriptional inhibitors significantly increased this interaction, demonstrating that the association of these two proteins is transcriptionally regulated. To assess the role of RRP1B in the regulation of alternative isoform expression, RNA-seq data were generated from control and Rrp1b-knockdown cells. Knockdown of Rrp1b induced a significant change in isoform expression in over 600 genes compared to control cell lines. This was verified by qRT-PCR using isoform-specific primers. Pathway enrichment analyses identified cell cycle and checkpoint regulation to be those most affected by Rrp1b knockdown. These data suggest that RRP1B suppresses metastatic progression by altering the transcriptome through its interaction with splicing regulators such as SRSF1.
doi:10.1038/onc.2013.133
PMCID: PMC3925194  PMID: 23604122
Metastasis; RRP1B; germline modifiers; RNA-seq; alternative splicing; breast cancer
12.  Protein Kinase A–Dependent Phosphorylation of Serine 119 in the Proto-Oncogenic Serine/Arginine-Rich Splicing Factor 1 Modulates Its Activity as a Splicing Enhancer Protein 
Genes & Cancer  2011;2(8):841-851.
Serine/arginine-rich splicing factor 1 (SRSF1), previously designated SF2/ASF, belongs to a family of SR proteins that regulate constitutive and alternative splicing. SRSF1 expression is increased in tumors from several tissues and elicits changes in key target genes involved in tumor genesis. Several protein kinases phosphorylate SRSF1, which regulates its localization and function. It is previously reported that protein kinase A (PKA) phosphorylates SRSF1, but the importance of this modification is not well characterized. Here, we show that PKA phosphorylates SRSF1 on serine 119 in vitro. Phosphorylation of SRSF1 on this site enhanced the RNA binding capacity of SRSF1 in vivo and reduced the protein’s capacity to activate splicing of the Minx transcript in vitro. We also confirm an interaction between SRSF1 and PKA Cα1 and demonstrate that this interaction is not dependent on serine 119 phosphorylation but requires active PKA Cα1. We conclude that PKA phosphorylation of SRSF1 at serine 119 regulates SFRS1-dependent RNA binding and processing but not its interaction with PKA.
doi:10.1177/1947601911430226
PMCID: PMC3278900  PMID: 22393468
pre-mRNA splicing regulation; SRSF1; PKA; phosphorylation
13.  hnRNP A1 and hnRNP F Modulate the Alternative Splicing of Exon 11 of the Insulin Receptor Gene 
PLoS ONE  2011;6(11):e27869.
Exon 11 of the insulin receptor gene (INSR) is alternatively spliced in a developmentally and tissue-specific manner. Linker scanning mutations in a 5′ GA-rich enhancer in intron 10 identified AGGGA sequences that are important for enhancer function. Using RNA-affinity purification and mass spectrometry, we identified hnRNP F and hnRNP A1 binding to these AGGGA sites and also to similar motifs at the 3′ end of the intron. The hnRNPs have opposite functional effects with hnRNP F promoting and hnRNP A1 inhibiting exon 11 inclusion, and deletion of the GA-rich elements eliminates both effects. We also observed specific binding of hnRNP A1 to the 5′ splice site of intron 11. The SR protein SRSF1 (SF2/ASF) co-purified on the GA-rich enhancer and, interestingly, also competes with hnRNP A1 for binding to the splice site. A point mutation -3U→C decreases hnRNP A1 binding, increases SRSF1 binding and renders the exon constitutive. Lastly, our data point to a functional interaction between hnRNP F and SRSF1 as a mutant that eliminates SRSF1 binding to exon 11, or a SRSF1 knockdown, which prevents the stimulatory effect of hnRNP F over expression.
doi:10.1371/journal.pone.0027869
PMCID: PMC3223206  PMID: 22132154
14.  SRSF1 regulates the assembly of pre-mRNA processing factors in nuclear speckles 
Molecular Biology of the Cell  2012;23(18):3694-3706.
SRSF1 splicing factor and nuclear-localized MALAT1 RNA influence the assembly of nuclear speckles. Depletion of SRSF1 compromises the association of splicing factors to nuclear speckles and influences the levels of other SR proteins. SRSF1 regulates RNA polymerase II–mediated transcription.
The mammalian cell nucleus is compartmentalized into nonmembranous subnuclear domains that regulate key nuclear functions. Nuclear speckles are subnuclear domains that contain pre-mRNA processing factors and noncoding RNAs. Many of the nuclear speckle constituents work in concert to coordinate multiple steps of gene expression, including transcription, pre-mRNA processing and mRNA transport. The mechanism that regulates the formation and maintenance of nuclear speckles in the interphase nucleus is poorly understood. In the present study, we provide evidence for the involvement of nuclear speckle resident proteins and RNA components in the organization of nuclear speckles. SR-family splicing factors and their binding partner, long noncoding metastasis-associated lung adenocarcinoma transcript 1 RNA, can nucleate the assembly of nuclear speckles in the interphase nucleus. Depletion of SRSF1 in human cells compromises the association of splicing factors to nuclear speckles and influences the levels and activity of other SR proteins. Furthermore, on a stably integrated reporter gene locus, we demonstrate the role of SRSF1 in RNA polymerase II–mediated transcription. Our results suggest that SR proteins mediate the assembly of nuclear speckles and regulate gene expression by influencing both transcriptional and posttranscriptional activities within the cell nucleus.
doi:10.1091/mbc.E12-03-0206
PMCID: PMC3442416  PMID: 22855529
15.  iCLIP Predicts the Dual Splicing Effects of TIA-RNA Interactions 
PLoS Biology  2010;8(10):e1000530.
Transcriptome-wide analysis of protein-RNA interactions predicts the dual splicing effects of TIA proteins, showing that their local enhancing function is associated with diverse distal splicing silencing effects.
The regulation of alternative splicing involves interactions between RNA-binding proteins and pre-mRNA positions close to the splice sites. T-cell intracellular antigen 1 (TIA1) and TIA1-like 1 (TIAL1) locally enhance exon inclusion by recruiting U1 snRNP to 5′ splice sites. However, effects of TIA proteins on splicing of distal exons have not yet been explored. We used UV-crosslinking and immunoprecipitation (iCLIP) to find that TIA1 and TIAL1 bind at the same positions on human RNAs. Binding downstream of 5′ splice sites was used to predict the effects of TIA proteins in enhancing inclusion of proximal exons and silencing inclusion of distal exons. The predictions were validated in an unbiased manner using splice-junction microarrays, RT-PCR, and minigene constructs, which showed that TIA proteins maintain splicing fidelity and regulate alternative splicing by binding exclusively downstream of 5′ splice sites. Surprisingly, TIA binding at 5′ splice sites silenced distal cassette and variable-length exons without binding in proximity to the regulated alternative 3′ splice sites. Using transcriptome-wide high-resolution mapping of TIA-RNA interactions we evaluated the distal splicing effects of TIA proteins. These data are consistent with a model where TIA proteins shorten the time available for definition of an alternative exon by enhancing recognition of the preceding 5′ splice site. Thus, our findings indicate that changes in splicing kinetics could mediate the distal regulation of alternative splicing.
Author Summary
Studies of splicing regulation have generally focused on RNA elements located close to alternative exons. Recently, it has been suggested that splicing of alternative exons can also be regulated by distal regulatory sites, but the underlying mechanism is not clear. The TIA proteins are key splicing regulators that enhance the recognition of 5′ splice sites, and their distal effects have remained unexplored so far. Here, we use a new method to map the positions of TIA-RNA interactions with high resolution on a transcriptome-wide scale. The identified binding positions successfully predict the local enhancing and distal silencing effects of TIA proteins. In particular, we show that TIA proteins can regulate distal alternative 3′ splice sites by binding at the 5′ splice site of the preceding exon. This result suggests that alternative splicing is affected by the timing of alternative exon definition relative to the recognition of the preceding 5′ splice site. These findings highlight the importance of analysing distal regulatory sites in order to fully understand the regulation of alternative splicing.
doi:10.1371/journal.pbio.1000530
PMCID: PMC2964331  PMID: 21048981
16.  hnRNP L regulates the tumorigenic capacity of lung cancer xenografts in mice via caspase-9 pre-mRNA processing 
The Journal of Clinical Investigation  2010;120(11):3923-3939.
Caspase-9 is involved in the intrinsic apoptotic pathway and suggested to play a role as a tumor suppressor. Little is known about the mechanisms governing caspase-9 expression, but post-transcriptional pre-mRNA processing generates 2 splice variants from the caspase-9 gene, pro-apoptotic caspase-9a and anti-apoptotic caspase-9b. Here we demonstrate that the ratio of caspase-9 splice variants is dysregulated in non–small cell lung cancer (NSCLC) tumors. Mechanistic analysis revealed that an exonic splicing silencer (ESS) regulated caspase-9 pre-mRNA processing in NSCLC cells. Heterogeneous nuclear ribonucleoprotein L (hnRNP L) interacted with this ESS, and downregulation of hnRNP L expression induced an increase in the caspase-9a/9b ratio. Although expression of hnRNP L lowered the caspase-9a/9b ratio in NSCLC cells, expression of hnRNP L produced the opposite effect in non-transformed cells, suggesting a post-translational modification specific for NSCLC cells. Indeed, Ser52 was identified as a critical modification regulating the caspase-9a/9b ratio. Importantly, in a mouse xenograft model, downregulation of hnRNP L in NSCLC cells induced a complete loss of tumorigenic capacity that was due to the changes in caspase-9 pre-mRNA processing. This study therefore identifies a cancer-specific mechanism of hnRNP L phosphorylation and subsequent lowering of the caspase-9a/9b ratio, which is required for the tumorigenic capacity of NSCLC cells.
doi:10.1172/JCI43552
PMCID: PMC2964989  PMID: 20972334
17.  Correlation of SRSF1 and PRMT1 expression with clinical status of pediatric acute lymphoblastic leukemia 
Background
Acute lymphoblastic leukemia (ALL) is the most frequently-occurring malignant neoplasm in children, but the pathogenesis of the disease remains unclear. In a microarray assay using samples from 100 children with ALL, SFRS1 was found to be up-regulated. Serine/arginine-rich splicing factor 1 (SRSF1, also termed SF2/ASF), encoded by the SFRS1 gene, had been shown to be a pro-oncoprotein. Our previous study indicated that SRSF1 can be methylated by protein arginine methyltransferase 1 (PRMT1) in vitro; however, the biological function of SRSF1 and PRMT1 in pediatric ALL are presently unknown.
Methods
Matched, newly diagnosed (ND), complete remission (CR) and relapse (RE) bone marrow samples from 57 patients were collected in order to evaluate the expression patterns of SRSF1 and PRMT1. The potential oncogenic mechanism of SRSF1 and PRMT1 in leukemogenesis was also investigated.
Results
We identified significant up-regulation of SRSF1 and PRMT1 in the ND samples. Importantly, the expression of SRSF1 and PRMT1 returned to normal levels after CR, but rebounded in the RE samples. Our observation that SRSF1 could predict disease relapse was of particular interest, although the expression patterns of SRSF1 and PRMT1 were independent of the cytogenetic subtypes. In pre-B-cell lines, both SRSF1 and PRMT1 expression could be efficiently attenuated by the clinical chemotherapy agents arabinoside cytosine (Ara-c) or vincristine (VCR). Moreover, SRSF1 and PRMT1 were associated with each other in leukemia cells in vivo. Knock-down of SRSF1 resulted in an increase in early apoptosis, which could be further induced by chemotherapeutics.
Conclusions
Our results indicate that SRSF1 serves as an anti-apoptotic factor and potentially contributes to leukemogenesis in pediatric ALL patients by cooperating with PRMT1.
doi:10.1186/1756-8722-5-42
PMCID: PMC3459738  PMID: 22839530
Acute lymphoblastic leukemia; Splicing factor SRSF1; Protein arginine methyltransferase 1 (PRMT1); Alternative splicing; Arginine methylation
18.  The structure and selectivity of the SR protein SRSF2 RRM domain with RNA 
Nucleic Acids Research  2011;40(7):3232-3244.
SRSF2 is a prototypical SR protein which plays important roles in the alternative splicing of pre-mRNA. It has been shown to be involved in regulatory pathways for maintaining genomic stability and play important roles in regulating key receptors in the heart. We report here the solution structure of the RNA recognition motifs (RRM) domain of free human SRSF2 (residues 9–101). Compared with other members of the SR protein family, SRSF2 structure has a longer L3 loop region. The conserved aromatic residue in the RNP2 motif is absent in SRSF2. Calorimetric titration shows that the RNA sequence 5′AGCAGAGUA3′ binds SRSF2 with a Kd of 61 ± 1 nM and a 1:1 stoichiometry. NMR and mutagenesis experiments reveal that for SFSF2, the canonical β1 and β3 interactions are themselves not sufficient for effective RNA binding; the additional loop L3 is crucial for RNA complex formation. A comparison is made between the structures of SRSF2–RNA complex with other known RNA complexes of SR proteins. We conclude that interactions involving the L3 loop, N- and C-termini of the RRM domain are collectively important for determining selectivity between the protein and RNA.
doi:10.1093/nar/gkr1164
PMCID: PMC3326313  PMID: 22140111
19.  Splicing factor SRSF6 promotes hyperplasia of sensitized skin 
Summary
Many biological processes involve gene-expression regulation by alternative splicing. Here, we identify the splicing factor SRSF6 as a regulator of wound healing and tissue homeostasis in skin. We show that SRSF6 is a proto-oncogene that is frequently overexpressed in human skin cancer. Overexpressing it in transgenic mice induces hyperplasia of sensitized skin and promotes aberrant alternative splicing. We identify 139 target genes of SRSF6 in skin, and show that this SR protein binds to alternative exons of the extracellular-matrix protein tenascin C pre-mRNA, promoting the expression of isoforms characteristic of invasive and metastatic cancer in a cell-type-independent manner. SRSF6 overexpression additionally results in depletion of Lgr6+ stem cells, and excessive keratinocyte proliferation and response to injury. Furthermore, the effects of SRSF6 in wound healing assayed in vitro depend on the TNC isoforms. Thus, abnormal SR-protein expression can perturb tissue homeostasis.
doi:10.1038/nsmb.2756
PMCID: PMC4118672  PMID: 24440982
20.  A dedicated microarray for in-depth analysis of pre-mRNA splicing events: application to the study of genes involved in the response to targeted anticancer therapies 
Molecular Cancer  2014;13:9.
Alternative pre-mRNA splicing (AS) widely expands proteome diversity through the combinatorial assembly of exons. The analysis of AS on a large scale, by using splice-sensitive microarrays, is a highly efficient method to detect the majority of known and predicted alternative transcripts for a given gene. The response to targeted anticancer therapies cannot easily be anticipated without prior knowledge of the expression, by the tumor, of target proteins or genes. To analyze, in depth, transcript structure and levels for genes involved in these responses, including AKT1-3, HER1-4, HIF1A, PIK3CA, PIK3R1-2, VEGFA-D and PIR, we engineered a dedicated gene chip with coverage of an average 185 probes per gene and, especially, exon-exon junction probes. As a proof of concept, we demonstrated the ability of such a chip to detect the effects of over-expressed SRSF2 RNA binding protein on the structure and abundance of mRNA products in H358 lung cancer cells conditionally over-expressing SRSF2. Major splicing changes were observed, including in HER1/EGFR pre-mRNA, which were also seen in human lung cancer samples over-expressing the SRSF2 protein. In addition, we showed that variations in HER1/EGFR pre-mRNA splicing triggered by SRSF2 overexpression in H358 cells resulted in a drop in HER1/EGFR protein level, which correlated with increased sensitivity to gefitinib, an EGFR tyrosine kinase inhibitor. We propose, therefore, that this novel tool could be especially relevant for clinical applications, with the aim to predict the response before treatment.
doi:10.1186/1476-4598-13-9
PMCID: PMC3899606  PMID: 24428911
DNA chip; Targeted anticancer therapies; Pre-mRNA splicing; SRSF2
21.  Discovery and Analysis of Evolutionarily Conserved Intronic Splicing Regulatory Elements 
PLoS Genetics  2007;3(5):e85.
Knowledge of the functional cis-regulatory elements that regulate constitutive and alternative pre-mRNA splicing is fundamental for biology and medicine. Here we undertook a genome-wide comparative genomics approach using available mammalian genomes to identify conserved intronic splicing regulatory elements (ISREs). Our approach yielded 314 ISREs, and insertions of ~70 ISREs between competing splice sites demonstrated that 84% of ISREs altered 5′ and 94% altered 3′ splice site choice in human cells. Consistent with our experiments, comparisons of ISREs to known splicing regulatory elements revealed that 40%–45% of ISREs might have dual roles as exonic splicing silencers. Supporting a role for ISREs in alternative splicing, we found that 30%–50% of ISREs were enriched near alternatively spliced (AS) exons, and included almost all known binding sites of tissue-specific alternative splicing factors. Further, we observed that genes harboring ISRE-proximal exons have biases for tissue expression and molecular functions that are ISRE-specific. Finally, we discovered that for Nova1, neuronal PTB, hnRNP C, and FOX1, the most frequently occurring ISRE proximal to an alternative conserved exon in the splicing factor strongly resembled its own known RNA binding site, suggesting a novel application of ISRE density and the propensity for splicing factors to auto-regulate to associate RNA binding sites to splicing factors. Our results demonstrate that ISREs are crucial building blocks in understanding general and tissue-specific AS regulation and the biological pathways and functions regulated by these AS events.
Author Summary
During RNA splicing, sequences (introns) in a pre-mRNA are excised and discarded, and the remaining sequences (exons) are joined to form the mature RNA. Splicing is regulated not only by the binding of the basic splicing machinery to splice sites located at the exon–intron boundaries, but also by the combined effects of various other splicing factors that bind to a multitude of sequence elements located both in the exons as well as the flanking introns. Instances of alternative splicing, where usage of splice site(s) is incomplete or different between tissues, cell types, or lineages, can be created by the interaction of sequence elements and tissue, cell type, and stage-specific splicing factors. To better understand constitutive and alternative pre-mRNA splicing, the authors describe a comparative genomics approach, using available mammalian genomes, to systematically identify splicing regulatory elements located in the introns proximal to exons. A quarter of the elements were tested experimentally, and most of them altered splicing in human cells. The authors also showed that that the intronic elements are close to tissue-specific alternative exons and are more likely to be located in specific positions in the introns, suggestive of potential regulatory function. These elements are also frequently found in tissue-specific genes, suggesting a coupling between expression and alternative splicing of these genes. Finally, the authors propose a strategy using the elements to identify the binding sites of several splicing factors.
doi:10.1371/journal.pgen.0030085
PMCID: PMC1877881  PMID: 17530930
22.  Mutually exclusive splicing regulates the Nav 1.6 sodium channel function through a combinatorial mechanism that involves three distinct splicing regulatory elements and their ligands 
Nucleic Acids Research  2012;40(13):6255-6269.
Mutually exclusive splicing is a form of alternative pre-mRNA processing that consists in the use of only one of a set of two or more exons. We have investigated the mechanisms involved in this process for exon 18 of the Nav 1.6 sodium channel transcript and its significance regarding gene-expression regulation. The 18N exon (neonatal form) has a stop codon in phase and although the mRNA can be detected by amplification methods, the truncated protein has not been observed. The switch from 18N to 18A (adult form) occurs only in a restricted set of neural tissues producing the functional channel while other tissues display the mRNA with the 18N exon also in adulthood. We demonstrate that the mRNA species carrying the stop codon is subjected to Nonsense-Mediated Decay, providing a control mechanism of channel expression. We also map a string of cis-elements within the mutually exclusive exons and in the flanking introns responsible for their strict tissue and temporal specificity. These elements bind a series of positive (RbFox-1, SRSF1, SRSF2) and negative (hnRNPA1, PTB, hnRNPA2/B1, hnRNPD-like JKTBP) splicing regulatory proteins. These splicing factors, with the exception of RbFox-1, are ubiquitous but their levels vary during development and differentiation, ensuing unique sets of tissue and temporal levels of splicing factors. The combinatorial nature of these elements is highlighted by the dominance of the elements that bind the ubiquitous factors over the tissue specific RbFox-1.
doi:10.1093/nar/gks249
PMCID: PMC3401437  PMID: 22434879
23.  Heat Shock-Induced SRSF10 Dephosphorylation Displays Thermotolerance Mediated by Hsp27▿  
Molecular and Cellular Biology  2010;31(3):458-465.
Gene regulation in response to environmental stress is critical for the survival of all organisms. From Saccharomyces cerevisiae to humans, it has been observed that splicing of mRNA precursors is repressed upon heat shock. However, a mild heat pretreatment often prevents splicing inhibition in response to a subsequent and more severe heat shock, a phenomenon called splicing thermotolerance. We have shown previously that the splicing regulator SRSF10 (formerly SRp38) is specifically dephosphorylated by the phosphatase PP1 in response to heat shock and that dephosphorylated SRSF10 is responsible for splicing repression caused by heat shock. Here we report that a mild heat shock protects SRSF10 from dephosphorylation during a second and more severe heat shock. Furthermore, this “thermotolerance” of SRSF10 phosphorylation, like that of splicing, requires de novo protein synthesis, specifically the synthesis of heat shock proteins. Indeed, overexpression of one of these proteins, Hsp27, inhibits SRSF10 dephosphorylation in response to heat shock and does so by interaction with SRSF10. Our data thus provide evidence that splicing thermotolerance is acquired through maintenance of SRSF10 phosphorylation and that this is mediated at least in part by Hsp27.
doi:10.1128/MCB.01123-10
PMCID: PMC3028621  PMID: 21135127
24.  Drosha Promotes Splicing of a Pre-microRNA-like Alternative Exon 
PLoS Genetics  2014;10(5):e1004312.
The ribonuclease III enzyme Drosha has a central role in the biogenesis of microRNA (miRNA) by binding and cleaving hairpin structures in primary RNA transcripts into precursor miRNAs (pre-miRNAs). Many miRNA genes are located within protein-coding host genes and cleaved by Drosha in a manner that is coincident with splicing of introns by the spliceosome. The close proximity of splicing and pre-miRNA biogenesis suggests a potential for co-regulation of miRNA and host gene expression, though this relationship is not completely understood. Here, we describe a cleavage-independent role for Drosha in the splicing of an exon that has a predicted hairpin structure resembling a Drosha substrate. We find that Drosha can cleave the alternatively spliced exon 5 of the eIF4H gene into a pre-miRNA both in vitro and in cells. However, the primary role of Drosha in eIF4H gene expression is to promote the splicing of exon 5. Drosha binds to the exon and enhances splicing in a manner that depends on RNA structure but not on cleavage by Drosha. We conclude that Drosha can function like a splicing enhancer and promote exon inclusion. Our results reveal a new mechanism of alternative splicing regulation involving a cleavage-independent role for Drosha in splicing.
Author Summary
MicroRNAs (miRNAs) are short non-coding RNAs that function in gene silencing and are produced by cleavage from a larger primary RNA transcript through a reaction that is carried out by the Microprocessor. Primary miRNA transcripts are often located within the introns of genes. Thus, both the Microprocessor and the spliceosome, which is responsible for pre-mRNA splicing, interact with the same sequences, though little is known about how these two processes influence each other. In this study, we discovered that the alternatively spliced eIF4H exon 5 is predicted to form an RNA hairpin that resembles a Microprocessor substrate. We found that the Microprocessor can bind and cleave exon 5, which precludes inclusion of the exon in the mRNA. However, we find that Drosha, a component of the Microprocessor, primarily functions to enhance exon 5 splicing both in vitro and in cells, rather than to cleave the RNA. Our results suggest that the Microprocessor has a role in splicing that is distinct from its role in miRNA biogenesis. This Microprocessor activity represents a new function for the complex that may be an important mechanism for regulating alternative splicing.
doi:10.1371/journal.pgen.1004312
PMCID: PMC4006729  PMID: 24786770
25.  Transcriptome analysis of alternative splicing events regulated by SRSF10 reveals position-dependent splicing modulation 
Nucleic Acids Research  2014;42(6):4019-4030.
Splicing factor SRSF10 is known to function as a sequence-specific splicing activator. Here, we used RNA-seq coupled with bioinformatics analysis to identify the extensive splicing network regulated by SRSF10 in chicken cells. We found that SRSF10 promoted both exon inclusion and exclusion. Motif analysis revealed that SRSF10 binding to cassette exons was associated with exon inclusion, whereas the binding of SRSF10 within downstream constitutive exons was associated with exon exclusion. This positional effect was further demonstrated by the mutagenesis of potential SRSF10 binding motifs in two minigene constructs. Functionally, many of SRSF10-verified alternative exons are linked to pathways of stress and apoptosis. Consistent with this observation, cells depleted of SRSF10 expression were far more susceptible to endoplasmic reticulum stress-induced apoptosis than control cells. Importantly, reconstituted SRSF10 in knockout cells recovered wild-type splicing patterns and considerably rescued the stress-related defects. Together, our results provide mechanistic insight into SRSF10-regulated alternative splicing events in vivo and demonstrate that SRSF10 plays a crucial role in cell survival under stress conditions.
doi:10.1093/nar/gkt1387
PMCID: PMC3973337  PMID: 24442672

Results 1-25 (1140734)