SFRS1 Research Articles

SRSF1 promotes vascular smooth muscle cell proliferation through a \u0394133p53/EGR1/KLF5 pathway

Though vascular smooth muscle cell (VSMC) proliferation underlies all cardiovascular hyperplastic disorders, our understanding of the molecular mechanisms responsible for this cellular process is still incomplete. Here we report that SRSF1 (serine/arginine-rich splicing factor 1), an essential splicing factor, promotes VSMC proliferation and injury-induced neointima formation. Vascular injury in vivo and proliferative stimuli in vitro stimulate SRSF1 expression. Mice lacking SRSF1 specifically in SMCs develop less intimal thickening after wire injury. Expression of SRSF1 in rat arteries enhances neointima formation. SRSF1 overexpression increases, while SRSF1 knockdown suppresses the proliferation and migration of cultured human aortic and coronary arterial SMCs. Mechanistically, SRSF1 favours the induction of a truncated p53 isoform, Δ133p53, which has an equal proliferative effect and in turn transcriptionally activates Krüppel-like factor 5 (KLF5) via the Δ133p53-EGR1 complex, resulting in an accelerated cell-cycle progression and increased VSMC proliferation. Our study provides a potential therapeutic target for vascular hyperplastic disease.

Mutational analysis of RNA splicing machinery genes SF3B1, U2AF1 and SRSF2 in 118 patients with myelodysplastic syndromes and related diseases

目的探讨RNA剪接体复合物编码基因SF3B1、U2AF1和SRSF2突变在MDS及相关疾病中的突变率、突变特点及临床意义。方法以118例MDS及相关疾病患者为研究对象，采用PCR扩增产物直接测序法分别检测SF3B1（K700E）、U2AF1（S34、Q157P）和SRSF2（P95）突变情况。结果118例MDS患者中，男76例，女42例，中位年龄53.5（13~84）岁。对全部患者均进行SF3B1（K700E）基因突变分析，SF3B1（K700E）的突变率为19.49%（118例中23例）。在突变的22例MDS患者中，14例环形铁粒幼红细胞（RS）比例≥15%，其中难治性贫血伴环状铁粒幼红细胞（RARS）7例、难治性血细胞减少伴有多系发育异常（RCMD）6例、难治性贫血（RA）1例。与未突变组相比，突变组患者的年龄偏大[58（32~78）岁对51（13~84）岁，z=−1.981，P=0.048]、PLT偏高[121（22~888）×109/L对59（6~1 561）× 109/L，z=−3.305，P=0.001]、骨髓原始细胞比例偏低[0.007（0~0.122）对0.017（0~0.268），z=−2.885，P=0.004]、RS比例偏高[0（0~64%）对0（0~58%），z=−4.664，P<0.001]，HGB偏低[63（40~95）g/L对77（34~144）g/L，z=−3.192，P=0.001]。对105例患者进行U2AF1（S34、Q157P）基因突变分析，突变率为21.90%（105例中23例）。突变组患者各临床特征与未突变组相比差异均无统计学意义。107例患者有SRSF2（P95）突变结果，8例突变，突变率为7.48%，突变组患者初诊时年龄偏高，中位年龄63（50~84）岁，其中难治性贫血伴有原始细胞增多-1（RAEB-1）4例，突变率为14.29%（28例中4例）；MDS转化的急性髓系白血病3例。1例患者同时出现SF3B1（K700E）和SRSF2（P95H）突变，2例患者同时出现SF3B1（K700E）和U2AF1（S34Y）突变。结论SF3B1、U2AF1和SRSF2剪接体复合物蛋白编码基因中，仅SF3B1基因突变与环状铁粒幼红细胞增多性贫血密切相关，是该亚型的主要致病基因。

HNRNPA1, a Splicing Regulator, Is an Effective Target Protein for Cervical Cancer Detection: Comparison With Conventional Tumor Markers.

Heterogeneous nuclear ribonucleoprotein A1 (HNRNPA1), serine/arginine-rich splicing factor 1 (SRSF1), and SRSF3 are splicing regulators associated with oncogenesis. However, the alterations of SF proteins and their diagnostic values in cervical cancer are unclear. To apply SFs clinically, effective marker selection and characterization of the target organ properties are essential. We concurrently analyzed HNRNPA1, SRSF1, SRSF3, and the conventional tumor markers squamous cell carcinoma antigen (SCCA) and carcinoembryonic antigen (CEA) in cervical tissue samples (n = 127) using semiquantitative immunoblotting. In addition, we compared them with p16 (cyclin-dependent kinase inhibitor 2A [CDKN2A]), which has shown high diagnostic efficacy in immunohistochemical staining studies and has been proposed as a candidate protein for point-of-care screening biochemical tests of cervical neoplasia. HNRNPA1, higher molecular weight forms of SRSF1 (SRSF1-HMws), SRSF3, CEA, and p16 levels were higher (P < 0.05) in cervical carcinoma tissue samples than in nontumoral cervical tissue samples. However, the levels of SRSF1-Total (sum of SRSF1-HMws and a lower molecular weight form of SRSF1) and SCCA, a commonly used cervical tumor marker, were not different between carcinoma and nontumoral tissue samples. In paired sample comparisons, HNRNPA1 (94%) showed the highest incidence of up-regulation (carcinoma/nontumor, >1.5) in cervical carcinoma, followed by p16 (84%), SRSF1-HMws (69%), SRSF3 (66%), CEA (66 %), SCCA (32%), and SRSF1-Total (31%). HNRNPA1 (92%) and p16 (91%) presented the two highest diagnostic accuracies for cervical carcinoma, which were superior to those of SRSF3 (75%), SRSF1-HMws (72%), CEA (72%), SCCA (59%), and SRSF1-Total (55%). Our results identified that HNRNPA1 is the best diagnostic marker among the SFs and conventional markers given its excellent diagnostic efficacy for cervical carcinoma, and it has a p16-comparable diagnostic value. We suggest that HNRNPA1 is an additional effective target protein for developing cervical cancer detection tools.

Targeting the pro-angiogenic forms of VEGF or inhibiting their expression as anti-cancer strategies.

Tumor growth relies on oxygen and blood supply depending on neo-vascularization. This process is mediated by the Vascular Endothelial Growth Factor (VEGF) in many tumors. This paradigm has led to the development of specific therapeutic approaches targeting VEGF or its receptors. Despite their promising effects, these strategies have not improved overall survival of patients suffering from different cancers compared to standard therapies. We hypothesized that the existence of anti-angiogenic forms of VEGF VEGFxxxb which are still present in many tumors limit the therapeutic effects of the anti-VEGF antibodies bevacizumab/Avastin (BVZ). To test this hypothesis, we generated renal cell carcinoma cells (RCC) expressing VEGF165b. The incidence of tumors xenografts generated in nude mice and their growth were inferior to those obtained with control cells. Whereas BVZ had no effect on control tumors, it slowed-down the growth of tumor generated with VEGF165b expressing cells. A prophylactic immunization against the domain discriminating VEGF from VEGFxxxb isoforms inhibited the growth of tumor generated with two different syngenic tumor cell lines (melanoma (B16 cells) and RCC (RENCA cells)). Purified immunoglobulins from immunized mice also slowed-down tumor growth of human RCC xenografts in nude mice, producing a potent effect compared to BVZ in this model. Furthermore, down-regulating the serine-arginine-rich splicing factor 1 (SRSF1) or masking SRSF1 binding sites by 2’O-Methyl RNA resulted in the increase of the VEGFxxxb/VEGF ratio. Therefore, a vaccine approach, specific antibodies against pro-angiogenic forms of VEGF, or increasing the VEGFxxxb/VEGF ratio may represent new prophylactic or pro-active anti-cancer strategies.

Serine/Arginine-Rich Splicing Factor 1 (SRSF1) Is Required for Adult and Embryonic Hematopoiesis and Has Non-Overlapping Roles with SRSF2 in Hematopoiesis

The discovery that mRNA splicing factors are frequently mutated in clonal blood disorders represented one of the most unexpected findings from cancer genome sequencing projects. While initial functional studies of these mutations have suggested that alterations in constitutive and alternative splicing may directly contribute to malignant hematopoiesis, they also highlighted limitations in our knowledge of how splicing factors regulate differential splicing and usage of mRNA isoforms in normal hematopoietic development. Interestingly, the splicing factor SRSF2, a member of the serine/arginine-rich (SR) protein family that regulates alternative splicing in a tissue-specific manner, is recurrently mutated only in myeloid leukemias, whereas other splicing factors are also often mutated in lymphoid leukemias and epithelial cancers. These data suggest a specific role for SRSF2 in hematopoiesis and/or myelopoiesis and previous studies have shown that Srsf2 is required for T-lymphopoiesis and hematopoietic stem and progenitor cell (HSPC) function. At the same time, SRSF2s functions in regulating splicing have been suggested to be at least partially overlapping with those of SRSF1, the founding member of the SR protein family. We therefore set out to understand the role and function of SRSF1 in normal fetal and adult hematopoiesis and to directly compare SRSF1s role in normal hematopoiesis to that of SRSF2.To understand the role of Srsf1 in adult hematopoiesis, we utilized mice for conditional deletion of 1 or 2 copies of Srsf1 or Srsf2 and performed non-competitive and competitive bone marrow transplantation (BMT) assays. BM from adult Mx1-Cre/Srsf1+/+, Mx1-Cre/Srsf1fl/+, Mx1-Cre/Srsf1fl/fl and Mx1-Cre/Srsf2fl/fl mice (together with the same number of wildtype CD45.1 marrow in competitive BMT) were transplanted into lethally irradiated CD45.1 recipients, followed by polyinosinic:polycytidylic acid (pIpC) administration 4 weeks after reconstitution to induce Srsf1 deletion. In both competitive and non-competitive BMT, peripheral blood (PB) chimerism showed that while heterozygous deletion of Srsf1 did not affect PB chimerism, homozygous deletion of Srsf1 resulted in compromised multi-lineage reconstitution similar to homozygous Srsf2 deletion (Figure 1A). Analysis of hematopoietic organs 20 weeks post BMT revealed no contribution to hematopoiesis by Srsf1 or Srsf2 homozygous knockout cells. More specifically, the contribution of HSPC-enriched Lineage- Sca-1+ c-Kit+ (LSK) and myeloid progenitor-enriched Lineage- Sca-1- c-Kit+ (LK) fractions were significantly reduced in mice transplanted with Srsf1- and Srsf2-deficient BM (Figure 1B). These observations identify an absolute requirement for both splicing factors in HSPC function in adult mice.To determine if Srsf1 or Srsf2 are required for fetal hematopoiesis, we crossed Vav-CreTg/Srsf1fl/+ to Srsf1fl/fl mice and Vav-CreTg/Srsf2fl/+ to Srsf2fl/fl mice followed by HSPC analysis at embryonic day 13~14. At this time point, both Vav-CreTg/Srsf1fl/fl and Vav-CreTg/Srsf2fl/fl embryos were found at the approximate expected Mendelian ratio (p=0.77 for Srsf1 crosses and p=0.22 for Srsf2 crosses; Fishers exact test). Interestingly, the numbers of HSPCs and erythroid progenitors (determined by flow cytometry using Ter119 and CD71 antibodies) were similar in Vav-CreTg/Srsf1fl/fl fetal livers compared to control embryos, while Vav-CreTg/Srsf2fl/fl fetal livers had drastically reduced fetal liver cellularity, HSPC, and erythroid progenitors relative to control embryos. However, both Vav-CreTg/Srsf1fl/fl and Vav-CreTg/Srsf2fl/fl HSPCs had reduced colony-forming capacities in vitro relative to control, suggesting severe functional defects in homozygous mutants that are otherwise not observed in heterozygous mice.The above data identify that both Srsf1 and Srsf2 are essential for normal hematopoietic function in both embryonic and adult life. Although SRSF1 and SRSF2 have been suggested to regulate splicing by binding to similar sequences of pre-mRNA to promote splicing, here we find that both are absolutely required for hematopoiesis in a haplosufficient, non-overlapping manner. We are now performing transcriptomic analyses to delineate the common as well as distinct molecular targets of how SR proteins regulate HSPC function. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

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

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.

Prolyl hydroxylation regulates protein degradation, synthesis, and splicing in human induced pluripotent stem cell-derived cardiomyocytes.

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. 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. 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.

AB125. The correlation of SRSF1 and survivin in prostate cancer

ObjectiveTo test the expression of serine/arginine rich splicing factor 1 (SRSF1) and survivin [poptosis inhibiting factor (survivin)] in prostate cancer organizations, and study their correlation with the pathological features of prostate cancer, thus put forward the new targets in the treatment of prostate cancer.MethodsSRSF1, survivin protein was analyzed in 20 prostate tissue samples including prostate cancer and benign prostatic hyperplasia by immunohistochemical SP method. SRSF1, survivin was correlated to pathological features, and both the relevance was analyzed (no related reports at home and abroad).ResultsThe positive expression rate of SRSF1 protein in prostate cancer tissue cells was 76.37%±5.06%, significantly higher than that of benign prostatic hyperplasia 11.30%+1.09% (P<0.05); the positive expression rate of survivin protein in prostate cancer tissue cells was 86.93%±3.21%, significantly higher than that of benign prostatic hyperplasia 17.67%±1.99% (P<0.05); SRSF1 and survivin protein expressed in prostate cancer organizations and were positively correlated to pathological Gleason grading, and the second Have significant correlation (P<0.05).ConclusionsSRSF1 and survivin protein were highly expressed in adenocarcinoma tissue, significantly increased than that of benign hyperplasia of prostate tissue. The positive expression SRSF1 and survivin protein were positively correlated to pathological Gleason grading. The expression of survivin protein was elevated with the expression of survivin SRSF1protein in prostate cancer. Preliminary evidence about the role of SRSF1 may be through up-regulation the expression of survivin, thus promote the occurrence and development of prostate cancer.

Modulation of LMNA splicing as a strategy to treat prelamin A diseases.

The alternatively spliced products of LMNA, lamin C and prelamin A (the precursor to lamin A), are produced in similar amounts in most tissues and have largely redundant functions. This redundancy suggests that diseases, such as Hutchinson-Gilford progeria syndrome (HGPS), that are caused by prelamin A-specific mutations could be treated by shifting the output of LMNA more toward lamin C. Here, we investigated mechanisms that regulate LMNA mRNA alternative splicing and assessed the feasibility of reducing prelamin A expression in vivo. We identified an exon 11 antisense oligonucleotide (ASO) that increased lamin C production at the expense of prelamin A when transfected into mouse and human fibroblasts. The same ASO also reduced the expression of progerin, the mutant prelamin A protein in HGPS, in fibroblasts derived from patients with HGPS. Mechanistic studies revealed that the exon 11 sequences contain binding sites for serine/arginine-rich splicing factor 2 (SRSF2), and SRSF2 knockdown lowered lamin A production in cells and in murine tissues. Moreover, administration of the exon 11 ASO reduced lamin A expression in wild-type mice and progerin expression in an HGPS mouse model. Together, these studies identify ASO-mediated reduction of prelamin A as a potential strategy to treat prelamin A-specific diseases.

The Involvement of Splicing Factor hnRNP A1 in UVB-induced Alternative Splicing of hdm2.

Human homolog double minute 2 (hdm2), an oncoprotein, which binds to tumor suppressor p53 to facilitate its degradation, has been known to contribute to tumorigenesis. Its splicing variants are reported to be highly expressed in many cancers and can be induced by ultraviolet B light (UVB). However, the mechanisms of how UVB radiation induces hdm2 alternative splicing still remain unclear. In this study, we investigated the roles of two common splicing factors, heterogeneous nuclear ribonucleoproteins (hnRNP) A1 and serine/arginine-rich splicing factor 1 (SRSF1), in regulating UVB-induced hdm2 splicing. Our study indicated that while the expression of both hnRNP A1 and SRSF1 are induced, only hnRNP A1 is involved in hdm2 alternative splicing upon UVB irradiation. Overexpression of hnRNP A1 resulted in decrease of full-length hdm2 (hdm2-FL) and increase of hdm2B, one of hdm2 alternate-splicing forms; while down-regulated hnRNP A1 expression led to the decrease of the hdm2-FL and hdm2B in HaCaT cells. Protein-mRNA binding assay confirmed that UVB irradiation could increase the binding of hnRNP A1 to hdm2 pre-mRNA. In conclusion, we elucidated that UVB induces alternative splicing of hdm2 by increasing the expression and the binding of hnRNP A1 to hdm2 full-length mRNA.

Molecular interplay between T-Antigen and splicing factor, arginine/serine-rich 1 (SRSF1) controls JC virus gene expression in glial cells.

BackgroundHuman polyomavirus JCV is the etiologic agent of progressive multifocal leukoencephalopathy (PML), a fatal demyelinating disease characterized by lytic infection of glial cells in the central nervous system. PML is seen primarily in immunosuppressed patients and is mainly classified as an AIDS-defining disease. In addition to structural capsid proteins, JCV encodes multiple regulatory proteins, including T-antigen and agnoprotein, which are required for functional lytic infection. Previous studies have suggested that molecular interaction between viral proteins and host factors play an important role in reactivation of JCV and progression of the viral life cycle in glial cells. Recently, serine/arginine rich splicing factor 1 (SRSF1), a cellular alternative splicing factor, was identified as a strong negative regulator of JCV in glial cells. SRSF1 inhibits JCV gene expression and viral replication by directly interacting with viral promoter sequences. Here, we have investigated possible impact of JCV regulatory proteins, T-antigen and agnoprotein, on SRSF1-mediated suppression of JCV gene expression in glial cells.ResultsReporter gene analysis has suggested that T-antigen rescues viral transcriptional suppression mediated by SRSF1. Further analyses have revealed that T-antigen promotes viral gene expression by suppressing SRSF1 gene transcription in glial cells. A subsequent ChIP analysis revealed that T-antigen associates with the promoter region of SRSF1 to induce the transcriptional suppression.ConclusionsThese findings have revealed a molecular interplay between cellular SRSF1 and viral T-antigen in controlling JCV gene expression, and may suggest a novel mechanism of JCV reactivation in patients who are at risk of developing PML.

Mutational Hotspot of TET2, IDH1, IDH2, SRSF2, SF3B1, KRAS, and NRAS from Human Systemic Mastocytosis Are Not Conserved in Canine Mast Cell Tumors.

IntroductionBoth canine cutaneous mast cell tumor (MCT) and human systemic mastocytosis (SM) are characterized by abnormal proliferation and accumulation of mast cells in tissues and, frequently, by the presence of activating mutations in the receptor tyrosine kinase V-Kit Hardy-Zuckerman 4 Feline Sarcoma Viral Oncogene Homolog (c-KIT), albeit at different incidence (>80% in SM and 10–30% in MCT). In the last few years, it has been discovered that additional mutations in other genes belonging to the methylation system, the splicing machinery and cell signaling, contribute, with c-KIT, to SM pathogenesis and/or phenotype. In the present study, the mutational profile of the Tet methylcytosine dioxygenase 2 (TET2), the isocitrate dehydrogenases 1 and 2 (IDH1 and IDH2), the serine/arginine-rich splicing factor 2 (SRSF2), the splicing factor 3b subunit 1 (SF3B1), the Kirsten rat sarcoma viral oncogene homolog (KRAS) and the neuroblastoma RAS viral oncogene homolog (NRAS), commonly mutated in human myeloid malignancies and mastocytosis, was investigated in canine MCTs.MethodsUsing the Sanger sequencing method, a cohort of 75 DNA samples extracted from MCT biopsies already investigated for c-KIT mutations were screened for the “human-like” hot spot mutations of listed genes.ResultsNo mutations were ever identified except for TET2 even if with low frequency (2.7%). In contrast to what is observed in human TET2 no frame-shift mutations were found in MCT samples.ConclusionResults obtained in this preliminary study are suggestive of a substantial difference between human SM and canine MCT if we consider some target genes known to be involved in the pathogenesis of human SM.

Disease-associated mutation in SRSF2 misregulates splicing by altering RNA-binding affinities

Serine/arginine-rich splicing factor 2 (SRSF2) is an RNA-binding protein that plays important roles in splicing of mRNA precursors. SRSF2 mutations are frequently found in patients with myelodysplastic syndromes and certain leukemias, but how these mutations affect SRSF2 function has only begun to be examined. We used clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 nuclease to introduce the P95H mutation to SRSF2 in K562 leukemia cells, generating an isogenic model so that splicing alterations can be attributed solely to mutant SRSF2. We found that SRSF2 (P95H) misregulates 548 splicing events (<1% of total). Of these events, 374 involved the inclusion of cassette exons, and the inclusion was either increased (206) or decreased (168). We detected a specific motif (UCCA/UG) enriched in the more-included exons and a distinct motif (UGGA/UG) in the more-excluded exons. RNA gel shift assays showed that a mutant SRSF2 derivative bound more tightly than its wild-type counterpart to RNA sites containing UCCAG but bound less tightly to UGGAG sites. Thus in most cases the pattern of exon inclusion or exclusion correlated with stronger or weaker RNA binding, respectively. We further show that the P95H mutation does not affect other functions of SRSF2, i.e., protein-protein interactions with key splicing factors. Our results thus demonstrate that the P95H mutation positively or negatively alters the binding affinity of SRSF2 for cognate RNA sites in target transcripts, leading to misregulation of exon inclusion. Our findings shed light on the mechanism of the disease-associated SRSF2 mutation in splicing regulation and also reveal a group of misspliced mRNA isoforms for potential therapeutic targeting.

Abstract 2124: Analysis of the functional relevance of novel alternative splicing events in non-small cell lung cancer

Abstract Lung cancer is the most common cause of death by cancer worldwide. Deregulation of alternative splicing (AS) is becoming increasingly relevant for the understanding of the molecular mechanisms of lung tumor progression. Genome-wide microarrays containing probes in exons and junctions can be used to identify differential AS events. We developed an algorithm, called ExonPointer, which was optimized to detect AS events using this kind of platforms. Using this technique, we successfully detected splicing events regulated by the oncogene serine/arginine-rich splicing factor 1 (SRSF1) in lung cancer cell lines. Moreover, four of these genes, ATP11C, IQCB1, TUBD1 and PRRC2C, showed a significantly different pattern of AS in primary non-small cell lung cancer (NSCLC) compared with normal lung tissue. In the case of PRRC2C, we also demonstrated that the deregulation of the splicing event was important for tumor progression. We next applied the same technique to target differential AS events in NSCLC primary tissues (13 lung squamous cell carcinomas and 9 lung adenocarcinomas). Their paired normal lung tissues were used as reference. The validation rate for the top 20 events identified by ExonPointer was 70%. Gene cluster analyses showed an enrichment of clusters implicated in cancer development such as Cellular Growth and Proliferation, or Cell Death and Survival. Among others, we observed accumulation of a tumor-specific splice variant in Mitogen-Activated Protein Kinase 9 (MAPK9 or JNK2). The tumor-specific variant for this gene contains an alternative exon 2 which causes a frame shift introducing a premature stop codon. Functional analyses using isoform-specific siRNAs showed that this variant did not affect tumor growth or progression. However, its expression significantly correlated with the expression of Regulator of Nonsense Transcripts 3A and B (UPF3A and UPF3B) in 42 lung cancer cell lines. These proteins belong to the Nonsense Mediated Decay (NMD) pathway, which eliminates aberrant mRNA transcripts, suggesting that the presence of some aberrant AS variants in lung cancer are caused by a malfunction of NMD. In conclusion, we have developed a reliable tool for the high throughput analysis of AS, and have identified new splicing events in lung cancer that can be studied as potential diagnostic markers and therapeutic targets. Citation Format: Fernando J. de Miguel, Ravi D. Sharma, Pablo Reclusa, María J. Pajares, Angel Rubio, Ruben Pio, Luis M. Montuenga. Analysis of the functional relevance of novel alternative splicing events in non-small cell lung cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2124. doi:10.1158/1538-7445.AM2015-2124

Serine Arginine-Rich Splicing Factor 1 (SRSF1) Contributes to the Transcriptional Activation of CD3ζ in Human T Cells.

T lymphocytes from many patients with systemic lupus erythematosus (SLE) express decreased levels of the T cell receptor (TCR)-associated CD3 zeta (ζ) signaling chain, a feature directly linked to their abnormal phenotype and function. Reduced mRNA expression partly due to defective alternative splicing, contributes to the reduced expression of CD3ζ chain. We previously identified by oligonucleotide pulldown and mass spectrometry approaches, the serine arginine-rich splicing factor 1 (SRSF1) binding to the 3’ untranslated region (UTR) of CD3ζ mRNA. We showed that SRSF1 regulates alternative splicing of the 3’UTR of CD3ζ to promote expression of the normal full length 3`UTR over an unstable splice variant in human T cells. In this study we show that SRSF1 regulates transcriptional activation of CD3ζ. Specifically, overexpression and silencing of SRSF1 respectively increases and decreases CD3ζ total mRNA and protein expression in Jurkat and primary T cells. Using promoter-luciferase assays, we show that SRSF1 enhances transcriptional activity of the CD3ζ promoter in a dose dependent manner. Chromatin immunoprecipitation assays show that SRSF1 is recruited to the CD3ζ promoter. These results indicate that SRSF1 contributes to transcriptional activation of CD3ζ. Thus our study identifies a novel mechanism whereby SRSF1 regulates CD3ζ expression in human T cells and may contribute to the T cell defect in SLE.

Sam68 Regulates S6K1 Alternative Splicing during Adipogenesis.

The requirement for alternative splicing during adipogenesis is poorly understood. The Sam68 RNA binding protein is a known regulator of alternative splicing, and mice deficient for Sam68 exhibit adipogenesis defects due to defective mTOR signaling. Sam68 null preadipocytes were monitored for alternative splicing imbalances in components of the mTOR signaling pathway. Herein, we report that Sam68 regulates isoform expression of the ribosomal S6 kinase gene (Rps6kb1). Sam68-deficient adipocytes express Rps6kb1-002 and its encoded p31S6K1 protein, in contrast to wild-type adipocytes that do not express this isoform. Sam68 binds an RNA sequence encoded by Rps6kb1 intron 6 and prevents serine/arginine-rich splicing factor 1 (SRSF1)-mediated alternative splicing of Rps6kb1-002, as assessed by cross-linking and immunoprecipitation (CLIP) and minigene assays. Depletion of p31S6K1 with small interfering RNAs (siRNAs) partially restored adipogenesis of Sam68-deficient preadipocytes. The ectopic expression of p31S6K1 in wild-type 3T3-L1 cells resulted in adipogenesis differentiation defects, showing that p31S6K1 is an inhibitor of adipogenesis. Our findings indicate that Sam68 is required to prevent the expression of p31S6K1 in adipocytes for adipogenesis to occur.

Increase of a group of PTC+ transcripts by curcumin through inhibition of the NMD pathway

Nonsense-mediated mRNA decay (NMD), an mRNA surveillance mechanism, eliminates premature termination codon-containing (PTC+) transcripts. For instance, it maintains the homeostasis of splicing factors and degrades aberrant transcripts of human genetic disease genes. Here we examine the inhibitory effect on the NMD pathway and consequent increase of PTC+ transcripts by the dietary compound curcumin. We have found that several PTC+ transcripts including that of serine/arginine-rich splicing factor 1 (SRSF1) were specifically increased in cells by curcumin. We also observed a similar curcumin effect on the PTC+ mutant transcript from a Tay–Sachs-causing HEXA allele or from a beta-globin reporter gene. The curcumin effect was accompanied by significantly reduced expression of the NMD factors UPF1, 2, 3A and 3B. Consistently, in chromatin immunoprecipitation assays, curcumin specifically reduced the occupancy of acetyl-histone H3 and RNA polymerase II at the promoter region (−376 to −247nt) of human UPF1, in a time- and dosage-dependent way. Importantly, knocking down UPF1 abolished or substantially reduced the difference of PTC+ transcript levels between control and curcumin-treated cells. The disrupted curcumin effect was efficiently rescued by expression of exogenous Myc–UPF1 in the knockdown cells. Together, our data demonstrate that a group of PTC+ transcripts are stabilized by a dietary compound curcumin through the inhibition of UPF factor expression and the NMD pathway.

Non-t(6;9) and Non-Inv(3) Balanced Chromosomal Rearrangements Are Associated With Poor Survival Outcomes in Myelodysplastic Syndromes.

Cytogenetics is an important predictor of survival in patients with myeloid malignancies including myelodysplastic syndromes (MDS). The roles of balanced chromosomal rearrangements (BCR) specifically balanced translocations and inversions in MDS are less established. We hypothesized that BCR are commonly found in MDS and might confer important prognostic and therapeutic effect. Cytogenetic, hematologic, clinical, and survival data from a total of 302 MDS patients seen at the Cleveland Clinic between the years 2002 and 2011 were collected. Direct sequencing for genes relevant in MDS pathophysiology was performed on patients with BCR. Categorical data were analyzed using the χ2 test and survival outcomes were analyzed using the Kaplan-Meier method. Twenty-three patients (8%) carried non-t(6;9) and non-inv(3) BCR. These patients had short overall survival (OS) similar to patients with poor risk cytogenetics, defined according to the International Prognostic Scoring System, which was not altered by new single-nucleotide polymorphism arrays or acquired somatic uniparental disomy lesions. Among the detected somatic mutations, only patients harboring mutations in the RNA splicing gene serine/arginine-rich splicing factor 2 (SRSF2) conferred worse outcomes in this group of patients. Therapeutically, patients who received allogeneic hematopoietic cell transplantations had better OS compared with patients treated with pharmacologic therapies or best supportive care only. BCR are not common in MDS and are associated with poor survival, which might be influenced by the presence of SRSF2 mutation.

SRSF1 facilitates cytosolic DNA-induced production of type I interferons recognized by RIG-I.

BackgroundEvidence has shown that psoriasis is closely associated with infection; however, the mechanism of this association remains unclear. In mammalian cells, viral or bacterial infection is accompanied by the release of cytosolic DNA, which in turn triggers the production of type-I interferons (IFNs). Type I IFNs and their associated genes are significantly upregulated in psoriatic lesions. RIG-I is also highly upregulated in psoriatic lesions and is responsible for IFN production. However, RIG-I mediated regulatory signaling in psoriasis is poorly understood.MethodsWe screened a cDNA library and identified potential RIG-I interacting partners that may play a role in psoriasis.ResultsWe found that serine/arginine-rich splicing factor 1 (SRSF1) could specifically interact with RIG-I to facilitate RIG-I mediated production of type-I IFN that is triggered by cytosolic DNA. We found SRSF1 associates with RNA polymerase III and RIG-I in a DNA-dependent manner. In addition, treatment with a TNFα inhibitor downregulated SRSF1 expression in peripheral blood mononuclear cells (PBMCs) from psoriasis vulgaris patients.DiscussionBased on the abundance of pathogenic cytosolic DNA that is detected in psoriatic lesions, our finding that RIG-I interacts with SRSF1 to regulate type-I IFN production reveals a critical link regarding how cytosolic DNA specifically activates aberrant IFN expression. These data may provide new therapeutic targets for the treatment of psoriasis.

Detection of SRSF2-P95 mutation by high-resolution melting curve analysis and its effect on prognosis in myelodysplastic syndrome.

Hotspot mutations of serine/arginine-rich splicing factor 2 (SRSF2) gene have been identified in a proportion of hematologic malignancies including myelodysplastic syndrome (MDS). The aim of the present study was to develop a new approach to screen SRSF2 mutation and analyze the clinical relevance of SRSF2 mutations in Chinese MDS. A protocol based on high-resolution melting analysis (HRMA) was established to screen SRSF2-P95 mutation in 108 MDS patients and was compared with Sanger sequencing. The clinical relevance of SRSF2 mutations was further evaluated. HRMA identified five (4.6%) cases with SRSF2 mutation, completely validated by Sanger sequencing without false positive or negative results. The sensitivities of HRMA and Sanger sequencing were 10% and 25% for the detection of SRSF2-P95H mutation, respectively, against the background of wild-type DNA. Patients with SRSF2 mutation had shorter overall survival time than those with wild-type SRSF2 in both the whole cohort of cases and those with normal karyotype (P = 0.069 and 0.023, respectively). Multivariate analysis confirmed SRSF2 mutation as an independent risk factor in both patient populations. We established a fast, high-throughput, and inexpensive HRMA-based method to screen SRSF2 mutation, which could be used in clinical diagnostic laboratories. SRSF2 mutations were significantly associated with mortality rate in the MDS affected Chinese.