SRSF3: Newly discovered functions and roles in human health and diseases

The serine/arginine rich proteins (SR proteins) are members of a family of RNA binding proteins involved in regulating various features of RNA metabolism, including pre-mRNA constitutive and alternative splicing. In humans, a total of 12 SR splicing factors (SRSFs) namely SRSF1-SRSF12 have been reported. SRSF3, the smallest member of the SR family and the focus of this review, regulates critical steps in mRNA metabolism and has been shown to have mRNA-independent functions as well. Recent studies on SRSF3 have uncovered its role in a wide array of complex biological processes. We have also reviewed the involvement of SRSF3 in disease conditions like cancer, ageing, neurological and cardiac disorders. Finally, we have discussed in detail the autoregulation of SRSF3 and its implications in cancer and commented on the potential of SRSF3 as a therapeutic target, especially in the context of cancer.     

Splicing factor SRSF3 represses the translation of programmed cell death 4 mRNA by associating with the 5′-UTR region

Serine/arginine-rich splicing factor 3 (SRSF3), a member of the serine/arginine (SR)-rich family of proteins, regulates both alternative splicing of pre-mRNA and export of mature mRNA from the nucleus. Although its role in nuclear mRNA processing is well understood, the mechanism by which it alters the fate of cytoplasmic mRNA molecules remains elusive. Here, we provide evidence that SRSF3 not only regulates the alternative splicing pattern of programmed cell death 4 (PDCD4) mRNA, but also modulates its translational efficiency in the cytoplasm by lowering translation levels. We observed a marked increase in PDCD4 mRNA in translating polysome fractions upon silencing of SRSF3, and, conversely, ectopic overexpression of SRSF3 shifted PDCD4 mRNA into non-translating ribosomal fractions. In live cells, SRSF3 colocalized with PDCD4 mRNA in P-bodies (PBs), where translationally silenced mRNAs are deposited, and this localization was abrogated upon SRSF3 silencing. Furthermore, using two different reporter systems, we showed that SRSF3 interacts directly with PDCD4 mRNA and mediates translational repression by binding to the 5′-untranslated region (5′-UTR). In summary, our data suggest that the oncogenic potential of SRSF3 might be realized, in part, through the translational repression of PDCD4 mRNA.     

SRSF1和Survivin在口腔鳞状细胞癌中的表达及临床意义

目的:研究人丝氨酸/精氨酸富有剪接因子1(SRSF1)和凋亡抑制因子(Survivin)在口腔鳞状细胞癌(OSCC)组织和正常口腔黏膜(NOM)中的表达,探究两因子在OSCC中的相关性及其临床意义。方法:采用免疫组织化学SP法,检测两因子在60例OSCC组织、20例NOM组织中的表达,并结合组织学分级、淋巴结转移及临床分期等相关的信息进行相关性分析。结果:在NOM中SRSF1和Survivin低表达,在OSCC组织中均高表达,比例分别为68.3%和60%。SRSF1和Survivin与淋巴结转移、组织学分级以及临床分期之间存在统计学差异(P0.05);并且在OSCC组织中二者之间表达存在着显著正相关性,(r=0.541,P0.05)。结论:SRSF1和Survivin在OSCC中均高表达,与口腔鳞癌的发生、发展密切相关并具有协同作用。     

Acetylation and phosphorylation of SRSF2 control cell fate decision in response to cisplatin

SRSF2 is a serine/arginine-rich protein belonging to the family of SR proteins that are crucial regulators of constitutive and alternative pre-mRNA splicing. Although it is well known that phosphorylation inside RS domain controls activity of SR proteins, other post-translational modifications regulating SRSF2 functions have not been described to date. In this study, we provide the first evidence that the acetyltransferase Tip60 acetylates SRSF2 on its lysine 52 residue inside the RNA recognition motif, and promotes its proteasomal degradation. We also demonstrate that the deacetylase HDAC6 counters this acetylation and acts as a positive regulator of SRSF2 protein level. In addition, we show that Tip60 downregulates SRSF2 phosphorylation by inhibiting the nuclear translocation of both SRPK1 and SRPK2 kinases. Finally, we demonstrate that this acetylation/phosphorylation signalling network controls SRSF2 accumulation as well as caspase-8 pre-mRNA splicing in response to cisplatin and determines whether cells undergo apoptosis or G(2)/M cell cycle arrest. Taken together, these results unravel lysine acetylation as a crucial post-translational modification regulating SRSF2 protein level and activity in response to genotoxic stress.     

Effects of SRSF1 on subnuclear localization of topoisomerase I

Subnuclear localization of topoisomerase I (top I) is determined by its DNA relaxation activity and a net of its interactions with in majority unidentified nucleolar and nucleoplasmic elements. Here, we recognized SR protein SRSF1 (Serine/arginine-rich splicing factor 1, previously known as SF2/ASF) as a new element of the net. In HeLa cells, overexpression of SRSF1 recruited top I to the nucleoplasm whereas its silencing concentrated it in the nucleolus. Effect of SRSF1 was independent of top I relaxation activity and was the best pronounced for the mutant inactive in relaxation reaction. In HCT116 cells where top I was not released from the nucleolus upon halting relaxation activity, it was also not relocated by elevated level of SRSF1. Out of remaining SR proteins, SRSF5, SRSF7, and SRSF9 did not influence the localization of top I in HeLa cells whereas overexpression of SRSF2, SRSF3, SRSF6, and partly SRSF4 concentrated top I in the nucleolus, most possibly due to the reduction of the SRSF1 accessibility. Specific effect of SRSF1 was exerted because of its distinct RS domain. Silencing of SRSF1 compensated the deletion of the top I N-terminal region, individually responsible for nucleoplasmic localization of the mutant, and restored the wild-type phenotype of deletion mutant localization. SRSF1 was essential for the camptothecin-induced clearance from the nucleolus. These results suggest a possible role of SRSF1 in establishing partition of top I between the nucleolus and the nucleoplasm in some cell types with distinct combinations of SR proteins levels.     

Hepatocyte Growth Factor Enhances Alternative Splicing of the Kruppel-like Factor 6 (KLF6) Tumor Suppressor to Promote Growth through SRSF1

Alternative splicing of the Krüppel-like factor 6 (KLF6) tumor suppressor into an antagonistic splice variant 1 (SV1) is a pathogenic event in several cancers including hepatocellular carcinoma (HCC) because elevated SV1 is associated with increased tumor metastasis and mortality. Ras activation is one factor that can enhance KLF6 splicing in cancer cells, however pathways driving KLF6 splicing are unknown. Splice site selection is regulated by splice factors that include serine/arginine-rich (SR) proteins such as SRSF1 (ASF-SF2), which in turn is controlled by phosphoinositide 3-kinase (PI3K)/Akt and the mitogen-activated protein kinase (MAPK) signaling pathway. Because signaling pathways downstream of the liver mitogen hepatocyte growth factor (HGF) include Akt, we explored whether HGF induces KLF6 alternative splicing. In HepG2 cells, HGF (25 ng/mL) significantly increases the ratio of SV1/KLF6 full by 40% through phosphorylation of Akt and subsequent downregulation of two splicing regulators, SRSF3 (SRp20) and SRSF1. Decreased SRSF3 levels regulate SRSF1 levels by alternative splicing associated with the nonsense-mediated mRNA decay pathway (AS-NMD), which stimulates cell growth by decreasing p21 levels. Enhanced cell replication through increased KLF6 alternative splicing is a novel growth-promoting pathway of HGF that could contribute to the molecule's mitogenic activity in physiologic liver growth and hepatocellular carcinoma. Mol Cancer Res; 10(9); 1216-27. ?2012 AACR.     

CircPLCE1 facilitates the malignant progression of colorectal cancer by repressing the SRSF2-dependent PLCE1 pre-RNA splicing

Studies have demonstrated that circular RNAs (circRNAs) play important roles in various types of cancer; however, the mechanisms of circRNAs located in the nucleus have rarely been explored. Here, we report a novel circular RNA circPLCE1 (hsa_circ_0019230) that facilitates the malignant progression of colorectal cancer (CRC) by repressing serine/arginine-rich splicing factor 2 (SRSF2)-dependent phospholipase C epsilon 1 (PLCE1) pre-RNA splicing. Quantitative real-time polymerase chain reaction was used to determine the expression of circPLCE1 in CRC tissues and cells. Cell Counting Kit-8, Transwell and flow cytometric assays were used to assess the role of circPLE1 in CRC cell proliferation, migration and apoptosis, respectively. An animal study was conducted to test the role of circPLCE1 in vivo. Furthermore, catRAPID and RPISeq were used to predict the possible binding proteins of circPLCE1. RNA fractionation and RNA immunoprecipitation assays were used to confirm the RNA-protein interaction. In this study, we found that circPLCE1 was more significantly down-regulated in CRC tissues compared with that in adjacent normal tissues. However, circPLCE1 knockdown suppressed CRC cell proliferation, migration and invasion and increased apoptosis. Nude mouse experiments showed that ectopic expression of circPLCE1 dramatically increased tumour growth in vivo. Mechanistically, circPLCE1 directly bound to the SRSF2 protein, repressing SRSF2-dependent PLCE1 pre-RNA splicing, resulting in the progression of CRC. Individually mutating the binding sites of circPLCE1 abolished the inhibition of PLCE1 mRNA production. Our study revealed a novel molecular mechanism in the regulation of PLCE1 and suggested a new function of circular RNA.     

DNA methylation-regulated miR-193a-3p dictates resistance of hepatocellular carcinoma to 5-fluorouracil via repression of SRSF2 expression

Chemoresistance prevents effective cancer therapy and is rarely predictable prior to treatment, particularly for hepatocellular carcinoma (HCC). Following the chemoresistance profiling of eight HCC cell lines to each of nine chemotherapeutics, two cell lines (QGY-7703 as a sensitive and SMMC-7721 as a resistant cell line to 5-fluorouracil (5-FU) treatment) were systematically studied for mechanistic insights underpinning HCC 5-FU chemoresistance. Genomic profiling at both DNA methylation and microRNA (miR) levels and subsequent mechanistic studies illustrate a new mechanism for how DNA methylation-regulated miR-193a-3p dictates the 5-FU resistance of HCC cells via repression of serine/arginine-rich splicing factor 2 (SRSF2) expression. In turn, SRSF2 preferentially up-regulates the proapoptotic splicing form of caspase 2 (CASP2L) and sensitizes HCC cells to 5-FU. Forced changes of miR-193a-3p level reverse all of the phenotypic features examined, including cell proliferation, cell cycle progression, and 5-FU sensitivity, in cell culture and in nude mice. Importantly, the siRNA-mediated repression of SRSF2 phenocopies all of the miR-193a-3p mimic-triggered changes in QGY-7703. This newly identified miR-193a-3p-SRSF2 axis highlights a new set of companion diagnostics required for optimal 5-FU therapy of HCC, which involve assaying both the DNA methylation state of the miR-193a gene and the expression of miR-193a-3p and SRSF2 and the relative level of the proapoptotic versus antiapoptotic splicing forms of caspase 2 in clinical samples.     

Phosphorylation regulates arginine-rich RNA-binding protein solubility and oligomerization

Posttranslational modifications (PTMs) such as phosphorylation of RNA-binding proteins (RBPs) regulate several critical steps in RNA metabolism, including spliceosome assembly, alternative splicing, and mRNA export. Notably, serine-/arginine- (SR)-rich RBPs are densely phosphorylated compared with the remainder of the proteome. Previously, we showed that dephosphorylation of the splicing factor SRSF2 regulated increased interactions with similar arginine-rich RBPs U1-70K and LUC7L3. However, the large-scale functional and structural impact of these modifications on RBPs remains unclear. In this work, we dephosphorylated nuclear extracts using phosphatase in vitro and analyzed equal amounts of detergent-soluble and -insoluble fractions by mass-spectrometry-based proteomics. Correlation network analysis resolved 27 distinct modules of differentially soluble nucleoplasm proteins. We found classes of arginine-rich RBPs that decrease in solubility following dephosphorylation and enrich the insoluble pelleted fraction, including the SR protein family and the SR-like LUC7L RBP family. Importantly, increased insolubility was not observed across broad classes of RBPs. We determined that phosphorylation regulated SRSF2 structure, as dephosphorylated SRSF2 formed high-molecular-weight oligomeric species in vitro. Reciprocally, phosphorylation of SRSF2 by serine/arginine protein kinase 2 (SRPK2) in vitro decreased high-molecular-weight SRSF2 species formation. Furthermore, upon pharmacological inhibition of SRPKs in mammalian cells, we observed SRSF2 cytoplasmic mislocalization and increased formation of cytoplasmic granules as well as cytoplasmic tubular structures that associated with microtubules by immunocytochemical staining. Collectively, these findings demonstrate that phosphorylation may be a critical modification that prevents arginine-rich RBP insolubility and oligomerization.     

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

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.     

Long noncoding RNA DGCR5 involves in tumorigenesis of esophageal squamous cell carcinoma via SRSF1-mediated alternative splicing of Mcl-1

Long noncoding RNAs (lncRNAs) emerge as essential roles in the regulation of alternative splicing (AS) in various malignancies. Serine- and arginine-rich splicing factor 1 (SRSF1)-mediated AS events are the most important molecular hallmarks in cancer. Nevertheless, the biological mechanism underlying tumorigenesis of lncRNAs correlated with SRSF1 in esophageal squamous cell carcinoma (ESCC) remains elusive. In this study, we found that lncRNA DiGeorge syndrome critical region gene 5 (DGCR5) was upregulated in ESCC clinical samples, which associated with poor prognosis. Through RNA interference and overexpression approaches, we confirmed that DGCR5 contributed to promote ESCC cell proliferation, migration, and invasion while inhibited apoptosis in vitro. Mechanistically, DGCR5 could directly bind with SRSF1 to increase its stability and thus stimulate alternative splicing events. Furthermore, we clarified that SRSF1 regulated the aberrant splicing of myeloid cell leukemia-1 (Mcl-1) and initiated a significant Mcl-1L (antiapoptotic) isoform switch, which contributed to the expression of the full length of Mcl-1. Moreover, the cell-derived xenograft (CDX) model was validated that DGCR5 could facilitate the tumorigenesis of ESCC in vivo. Collectively, our findings identified that the key biological role of lncRNA DGCR5 in alternative splicing regulation and emphasized DGCR5 as a potential biomarker and therapeutic target for ESCC.Subject terms: Tumour biomarkers, Apoptosis     

Ubiquitination Regulates Expression of the Serine/Arginine-rich Splicing Factor 1 (SRSF1) in Normal and Systemic Lupus Erythematosus (SLE) T Cells

T cells from patients with systemic lupus erythematosus (SLE) exhibit reduced expression of the critical T cell receptor (TCR)-associated CD3ζ signaling chain and are poor producers of the vital cytokine IL-2. By oligonucleotide pulldown and mass spectrometry discovery approaches, we identified the splicing regulator serine/arginine-rich splicing factor (SRSF) 1 or splicing factor 2/alternative splicing factor (SF2/ASF) to be important in the expression of CD3ζ chain. Importantly, increases in the expression of SRSF1 rescued IL-2 production in T cells from patients with SLE. In this study, we investigated the regulation of SRSF1 expression in resting and activated human T cells. We found that T cell stimulation induced a rapid and significant increase in mRNA expression of SRSF1; however, protein expression levels did not correlate with this increase. Co-engagement of CD28 induced a similar mRNA induction and reduction in protein levels. Proteasomal but not lysosomal degradation was involved in this down-regulation as evidenced by blocking with specific inhibitors MG132 and bafilomycin, respectively. Immunoprecipitation studies showed increased ubiquitination of SRSF1 in activated T cells. Interestingly, T cells from patients with SLE showed increased ubiquitination of SRSF1 when compared with those from healthy individuals. Our results demonstrate a novel mechanism of regulation of the splicing factor SRSF1 in human T cells and a potential molecular mechanism that controls its expression in SLE.     

Proteasome-Mediated Proteolysis of SRSF5 Splicing Factor Intriguingly Co-occurs with SRSF5 mRNA Upregulation during Late Erythroid Differentiation

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.     

SRSF2 Is Essential for Hematopoiesis,and Its Myelodysplastic Syndrome-Related Mutations Dysregulate Alternative Pre-mRNA Splicing

Myelodysplastic syndromes (MDS) are a group of neoplasms characterized by ineffective myeloid hematopoiesis and various risks for leukemia. SRSF2, a member of the serine/arginine-rich (SR) family of splicing factors, is one of the mutation targets associated with poor survival in patients suffering from myelodysplastic syndromes. Here we report the biological function of SRSF2 in hematopoiesis by using conditional knockout mouse models. Ablation of SRSF2 in the hematopoietic lineage caused embryonic lethality, and Srsf2-deficient fetal liver cells showed significantly enhanced apoptosis and decreased levels of hematopoietic stem/progenitor cells. Induced ablation of SRSF2 in adult Mx1-Cre Srsf2^flox/flox mice upon poly(I):poly(C) injection demonstrated a significant decrease in lineage⁻ Sca⁺ c-Kit⁺ cells in bone marrow. To reveal the functional impact of myelodysplastic syndromes-associated mutations in SRSF2, we analyzed splicing responses on the MSD-L cell line and found that the missense mutation of proline 95 to histidine (P95H) and a P95-to-R102 in-frame 8-amino-acid deletion caused significant changes in alternative splicing. The affected genes were enriched in cancer development and apoptosis. These findings suggest that intact SRSF2 is essential for the functional integrity of the hematopoietic system and that its mutations likely contribute to development of myelodysplastic syndromes.     

Relative strength of 5’ splice-site strength defines functions of SRSF2 and SRSF6 in alternative splicing of Bcl-x pre-mRNA

Bcl-x, a member of the Bcl-2 family, plays a key role in apoptosis. Alternative splicing of Bcl-x pre-mRNA through alternative 5’ splice-site selection produces an anti-apoptotic mRNA isoform that includes exon 2b and a pro-apoptotic Bcl-x mRNA isoform that excludes exon 2b. Here we used Bcl-x minigene and identified SRSF2 and SRSF6 as two regulatory factors of 5’ splice-site selection of Bcl-x pre-mRNA. We selected binding clusters closer to 5’ splice-sites from multiple potential binding sites of SRSF2 and SRSF6 to perform loss of functions analysis through site-directed mutagenesis. Our results demonstrated that these mutations did not abolish regulatory functions of SRSF2 or SRSF6, indicating that a single binding motif or a cluster was not a functional target of these proteins in Bcl-x pre-mRNA splicing. Random deletion mutagenesis did not disrupt the role of SRSF2 and SRSF6. Importantly, mutagenesis of 5’ splice-site to a conserved or a weaker score demonstrated that the weaker strength of the target 5’ splice-site or higher strength of the other 5’ splice-site strength limited the role of SRSF2 and SRSF6 in 5’ splice-site activation.     

Abnormal Expression of the Pre-mRNA Splicing Regulators SRSF1, SRSF2, SRPK1 and SRPK2 in Non Small Cell Lung Carcinoma

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.