Serine-arginine Rich Splicing Factor Research Articles

Identification of SRSF10 as a promising prognostic biomarker with functional significance among SRSFs for glioma

AimsThe serine/arginine-rich splicing factor (SRSF) protein family members are essential mediators of the alternative splicing (AS) regulatory network, which is tightly implicated in cancer progression. However, the expression, clinical correlation, immune infiltration, and prognostic value of SRSFs in gliomas remain unclear. Materials and methodsGlioma samples were extracted from The Cancer Genome Atlas (TCGA) and Chinese Glioma Genome Atlas (CGGA) datasets. Several databases, such as HPA, DAVID, UALCAN were used to comprehensively explore the roles of SRSFs. In addition, experimental validation of SRSF10 was also conducted. Key findingsHere, we found the expression alterations of the SRSF family in glioma samples using data from the TCGA and CGGA_325 datasets. Among the 12 genes, most were found to be closely associated with glioma clinical features, which linked to poor prognosis in glioma patients. Interestingly, survival analysis identified only SRSF10 as a potential independent risk prognostic biomarker for glioma patients. Immune analysis indicated that glioma patients with high SRSF10 expression may respond well to immunotherapies targeting immune checkpoint (ICP) genes. Finally, knocking down SRSF10 reduced glioma cell viability, induced G1 cell cycle arrest, and induced the exclusion of bcl-2-associated transcription factor 1 (BCLAF1) exon 5a. SignificanceOverall, this study uncovers the oncogenic roles of most SRSF family members in glioma, with the exception of SRSF5, while highlighting SRSF10 as a potential novel independent prognostic biomarker for glioma.

Targeting alternative splicing as a new cancer immunotherapy-phosphorylation of serine arginine-rich splicing factor (SRSF1) by SR protein kinase 1 (SRPK1) regulates alternative splicing of PD1 to generate a soluble antagonistic isoform that prevents T cell exhaustion

BackgroundRegulation of alternative splicing is a new therapeutic approach in cancer. The programmed cell death receptor 1 (PD-1) is an immunoinhibitory receptor expressed on immune cells that binds to its ligands, PD-L1 and PD-L2 expressed by cancer cells forming a dominant immune checkpoint pathway in the tumour microenvironment. Targeting this pathway using blocking antibodies (nivolumab and pembrolizumab) is the mainstay of anti-cancer immunotherapies, restoring the function of exhausted T cells. PD-1 is alternatively spliced to form isoforms that are either transmembrane signalling receptors (flPD1) that mediate T cell death by binding to the ligand, PD-L1 or an alternatively spliced, soluble, variant that lacks the transmembrane domain.MethodsWe used PCR and western blotting on primary peripheral blood mononuclear cells (PBMCs) and Jurkat T cells, IL-2 ELISA, flow cytometry, co-culture of melanoma and cholangiocarcinoma cells, and bioinformatics analysis and molecular cloning to examine the mechanism of splicing of PD1 and its consequence.ResultsThe soluble form of PD-1, generated by skipping exon 3 (∆Ex3PD1), was endogenously expressed in PBMCs and T cells and prevents cancer cell-mediated T cell repression. Multiple binding sites of SRSF1 are adjacent to PD-1 exon 3 splicing sites. Overexpression of phosphomimic SRSF1 resulted in preferential expression of flPD1. Inhibition of SRSF1 phosphorylation both by SRPK1 shRNA knockdown and by a selective inhibitor, SPHINX31, resulted in a switch in splicing to ∆Ex3PD1. Cholangiocarcinoma cell-mediated repression of T cell IL-2 expression was reversed by SPHINX31 (equivalent to pembrolizumab).ConclusionsThese results indicate that switching of the splicing decision from flPD1 to ∆Ex3PD1 by targeting SRPK1 could represent a potential novel mechanism of immune checkpoint inhibition in cancer.

Increased Expression of SRSF1 Predicts Poor Prognosis in Multiple Myeloma.

Multiple myeloma (MM) is a clonal plasma cell disorder which still lacks sufficient prognostic factors. The serine/arginine-rich splicing factor (SRSF) family serves as an important splicing regulator in organ development. Among all members, SRSF1 plays an important role in cell proliferation and renewal. However, the role of SRSF1 in MM is still unknown. SRSF1 was selected from the primary bioinformatics analysis of SRSF family members, and then we integrated 11 independent datasets and analyzed the relationship between SRSF1 expression and MM clinical characteristics. Gene set enrichment analysis (GSEA) was conducted to explore the potential mechanism of SRSF1 in MM progression. ImmuCellAI was used to estimate the abundance of immune infiltrating cells between the SRSF1high and SRSF1low groups. The ESTIMATE algorithm was used to evaluate the tumor microenvironment in MM. The expression of immune-related genes was compared between the groups. Additionally, SRSF1 expression was validated in clinical samples. SRSF1 knockdown was conducted to explore the role of SRSF1 in MM development. SRSF1 expression showed an increasing trend with the progression of myeloma. Besides, SRSF1 expression increased as the age, ISS stage, 1q21 amplification level, and relapse times increased. MM patients with higher SRSF1 expression had worse clinical features and poorer outcomes. Univariate and multivariate analysis indicated that upregulated SRSF1 expression was an independent poor prognostic factor for MM. Enrichment pathway analysis confirmed that SRSF1 takes part in the myeloma progression via tumor-associated and immune-related pathways. Several checkpoints and immune-activating genes were significantly downregulated in the SRSF1high groups. Furthermore, we detected that SRSF1 expression was significantly higher in MM patients than that in control donors. SRSF1 knockdown resulted in proliferation arrest in MM cell lines. The expression value of SRSF1 is positively associated with myeloma progression, and high SRSF1 expression might be a poor prognostic biomarker in MM patients.

Abstract 5093: Chemically inducible splice-neoantigens for cancer immunotherapy

Abstract Although immune checkpoint blockade therapies have dramatically improved the survival rate of patients with cancer compared with other treatment approaches, a substantial number of patients show poor response or resistance to these therapies. Meta-analyses for programmed cell death 1 (PD-1) blockade responses highlight the importance of neoantigens, derived from non-synonymous mutations in cancer cells, for antitumor immune responses. Intriguingly, recent transcriptome and genomics studies predict the presence of putative neoantigens as a result of an irregular pre-mRNA splicing regulation in cancer cells, indicating that alterations in RNA splicing in cancer cells might induce antitumor immune responses. Here, we report that induction of serine/arginine-rich splicing factor (SRSF)-dependent splicing boosts the production of splicing-associated neoantigens (splice-neoantigens) and potentiates the response to PD-1 blockade. Administration of a synthetic SRSF activator RECTAS suppressed tumor growth in a host CD8+ T cell- and tumor major histocompatibility complex class I-dependent manner and promoted the antitumor effect of anti-PD-L1 antibody without detectable autoimmunity. Subsequent transcriptome analysis and validation for immunogenicity identified six splice-neoantigen candidates whose expression was induced by RECTAS treatment. Importantly, vaccination of the identified neoepitopes elicited T cell responses capable of killing cancer cells in vitro, in addition to suppression of tumor growth in vivo upon sensitization with RECTAS. Collectively, these results provide support for the further development of splice variant-inducing treatments for cancer immunotherapy. Citation Format: Shingo Matsushima, Masahiko Ajiro, Kei Iida, Kenji Chamoto, Tasuku Honjo, Masatoshi Hagiwara. Chemically inducible splice-neoantigens for cancer immunotherapy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5093.

SRSF1 promotes ASMC proliferation in asthma by competitively binding CCND2 with miRNA-135a

BackgroundAsthma is an inflammatory syndrome characterized by airway hyperresponsiveness, bronchial inflammation, and airway remodeling. Abnormal proliferation of airway smooth muscle cells (ASMCs) is the main pathological feature of asthma. This study investigated the function and mechanism of serine arginine-rich splicing factor 1 (SRSF1) in ASMC proliferation in asthma. MethodsSRSF1 expressions in the bronchi of ovalbumin-induced asthmatic mice and IgE-treated mouse ASMCs (mASMCs) were evaluated using quantitative real-time PCR and Western blot. The localization and expression of SRSF1 in the bronchi of asthmatic mice were assessed by immunohistochemistry. Functionally, gain- and loss-of-function assays, flow cytometry, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays were conducted. Mechanistically, RNA degradation assay, RNA immunoprecipitation, RNA pull-down, and dual-luciferase reporter gene assays were carried out. ResultsSRSF1 was highly expressed in the bronchi of ovalbumin-induced asthma mice and IgE-treated mASMCs and was mainly located in the nucleus. Experiments on the function of SRSF1 showed that the silencing of SRSF1 induced the cell cycle of mASMC arrest and restrained mASMC proliferation. Investigations into the mechanism of SRSF1 revealed that SRSF1 and miR-135a are competitively bound to the 3′UTR region of Cyclin D2 (CCND2). SRSF1 overexpression repressed the degradation of CCND2 mRNA, and miR-135a negatively regulated CCND2 expression. Furthermore, SRSF1 knockdown inhibited ASMC proliferation in asthma mouse models by regulating the levels of miR-135a and CCND2. ConclusionSRSF1 knockdown repressed ASMC proliferation in asthma by regulating miR-135a/CCND2 levels.

Splicing factor SRSF1 is essential for CD8 T cell function and host antigen-specific viral immunity.

Cytotoxic CD8 T cells are crucial for the host antigen-specific immune response to viral pathogens. Here we report the identification of an essential role for the serine/arginine-rich splicing factor (SRSF) 1 in CD8 T cell homeostasis and function. Specifically, SRSF1 is necessary for the maintenance of normal CD8 T lymphocyte numbers in the lymphoid compartment, and for the proliferative capacity and cytotoxic function of CD8 T cells. Furthermore, SRSF1 is required for antigen-specific IFN-γ cytokine responses in a viral infection challenge in mice. Transcriptomics analyses of Srsf1-deficient T cells reveal that SRSF1 controls proliferation, MAP kinase signaling and IFN signaling pathways. Mechanistically, SRSF1 controls the expression and activity of the Mnk2/p38-MAPK axis at the molecular level. Our findings reveal previously unrecognized roles for SRSF1 in the physiology and function of cytotoxic CD8 T lymphocytes and a potential molecular mechanism in viral immunopathogenesis.

Hepatocyte Deletion of IGF2 Prevents DNA Damage and Tumor Formation in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide. Serine‐arginine rich splicing factor 3 (SRSF3) plays a critical role in hepatocyte function and its loss in mice promotes chronic liver damage and leads to HCC. Hepatocyte‐specific SRSF3 knockout mice (SKO mice) also overexpress insulin‐like growth factor 2 (IGF2). In the present study, double deletion of Igf2 and Srsf3 (DKO mice) prevents hepatic fibrosis and inflammation, and completely prevents tumor formation, and is associated with decreased proliferation, apoptosis and DNA damage, and restored DNA repair enzyme expression. This is confirmed in vitro, where IGF2 treatment of HepG2 hepatoma cells decreases DNA repair enzyme expression and causes DNA damage. Tumors from the SKO mice also show mutational signatures consistent with homologous recombination and mismatch repair defects. Analysis of frozen human samples shows that SRSF3 protein is decreased sixfold in HCC compared to normal liver tissue but SRSF3 mRNA is increased. Looking at public TCGA data, HCC patients having high SRSF3 mRNA expression show poor survival, as do patients with alterations in known SRSF3‐dependent splicing events. The results indicate that IGF2 overexpression in conjunction with reduced SRSF3 splicing activity could be a major cause of DNA damage and driver of liver cancer.

The RNA-binding protein SRSF3 has an essential role in megakaryocyte maturation and platelet production

AbstractRNA processing is increasingly recognized as a critical control point in the regulation of different hematopoietic lineages including megakaryocytes responsible for the production of platelets. Platelets are anucleate cytoplasts that contain a rich repertoire of RNAs encoding proteins with essential platelet functions derived from the parent megakaryocyte. It is largely unknown how RNA binding proteins contribute to the development and functions of megakaryocytes and platelets. We show that serine-arginine–rich splicing factor 3 (SRSF3) is essential for megakaryocyte maturation and generation of functional platelets. Megakaryocyte-specific deletion of Srsf3 in mice led to macrothrombocytopenia characterized by megakaryocyte maturation arrest, dramatically reduced platelet counts, and abnormally large functionally compromised platelets. SRSF3 deficient megakaryocytes failed to reprogram their transcriptome during maturation and to load platelets with RNAs required for normal platelet function. SRSF3 depletion led to nuclear accumulation of megakaryocyte mRNAs, demonstrating that SRSF3 deploys similar RNA regulatory mechanisms in megakaryocytes as in other cell types. Our study further suggests that SRSF3 plays a role in sorting cytoplasmic megakaryocyte RNAs into platelets and demonstrates how SRSF3-mediated RNA processing forms a central part of megakaryocyte gene regulation. Understanding SRSF3 functions in megakaryocytes and platelets provides key insights into normal thrombopoiesis and platelet pathologies as SRSF3 RNA targets in megakaryocytes are associated with platelet diseases.

The Pan-Clk/Dyrk Inhibitor Cirtuvivint (SM08502) Exposes Mechanistic Underpinnings of Alternative Splicing As a Therapeutic Vulnerability in Heme Malignancies

Mutations in spliceosomal genes are one of the most common classes of somatic alterations in patients with Myelodysplastic Syndrome (MDS) and occur across the entire spectrum of myeloid malignancies, including 10‒25% of patients with acute myeloid leukemia (AML). These mutations occur in higher proportions in AML subjects greater than 60 years of age, or when AML has transformed from an antecedent MDS. Spliceosomal gene mutations are implicated in the production of pathological RNA splicing patterns that block cell differentiation and maintain a myeloid precursor phenotype. This suggests deranged pre-mRNA splicing is a mechanistic determinant of many heme malignancies and, as such, has provoked interest in therapeutic modulation of pre-mRNA splicing as a treatment paradigm. The CLK/DYRK family of protein kinases has been recognized as an integration hub for signal transduction-dependent modulation of alternative pre-mRNA splice junction selection through direct phosphorylation of the serine/arginine-rich splicing factor (SRSF) splice junction enhancer-binding proteins. Thus, these kinases potentially represent a druggable intervention point in alternative splicing-dependent cancers. The isoquinoline SM08502 (cirtuvivint) is a potent ATP-competitive inhibitor of the Cdc2-like kinases (CLK1-4) and the dual specificity tyrosine phosphorylation-regulated kinases (DYRK1-4) with activity against only a minimal number of the remaining members of the CMGC-family kinases and the kinome as a whole. Here the consequence of pan-CLK/DYRK kinase inhibition on cell viability and tumorigenicity was evaluated across a panel of human tumor-derived AML, DLBCL, MCL, myeloma, T-ALL, and CML/CLL models. EC 50s in response to cirtuvivint ranged from 0.014 μM‒0.495 μM in 4-day in vitro cell viability assays, with low-dose responders enriched in subsets of AML, myeloma, DLBCL, MCL and T-ALL. Cell viability EC 50s were associated with induction of programed cell death at drug exposures that inhibited accumulation of phosphorylated SRSF proteins and the anti-apoptotic protein MCL-1. To directly evaluate the contribution of CLK/DYRK kinases to alternative splicing profiles, high-depth RNAseq analysis was performed across 4 cell lines (3 acute myeloid leukemia cell lines and 1 mantle cell lymphoma cell line) +/- a 6-hour exposure to 1µM cirtuvivint. Both baseline and drug-induced changes in alternative splicing events (ASEs) were measured using a multivariate analysis of splicing transcripts (rMATS). The frequency of cirtuvivint-induced ASEs was approximately 20% of total detected ASEs. Concordant drug-induced ASEs among the tested cell lines were in genes enriched in pathways known to drive oncogenesis in hematopoietic lineages, including the MAP kinase and mTOR signaling pathways. Tumor growth inhibition assays in immunocompromised mice showed a range of model-specific responses, including tumor stasis and partial to complete tumor regression at clinically relevant exposures. A cell-based synthetic-lethal screen of cirtuvivint across 36 small molecule inhibitors identified multiple BCL-2 inhibitors among the most prominent synergistic combinations. Consistent with this, combination of the BCL-2 inhibitor venetoclax with cirtuvivint was sufficient to induce tumor regressions in AML xenograft models (KG-1 and HL-60) that were resistant to either single-agent drug at the same concentrations. These observations support further evaluation of CLK/DYRK inhibitors as a therapeutic strategy for heme malignancies dependent upon alternative pre-mRNA splicing. DisclosuresBossard: Biosplice Therapeutics: Current Employment. McMillan: Prizer: Ended employment in the past 24 months. Beaupre: Pfizer: Ended employment in the past 24 months. White: Pfizer: Ended employment in the past 24 months.

SRSF protein kinase 1 modulates RAN translation and suppresses CGG repeat toxicity.

Transcribed CGG repeat expansions cause neurodegeneration in Fragile X‐associated tremor/ataxia syndrome (FXTAS). CGG repeat RNAs sequester RNA‐binding proteins (RBPs) into nuclear foci and undergo repeat‐associated non‐AUG (RAN) translation into toxic peptides. To identify proteins involved in these processes, we employed a CGG repeat RNA‐tagging system to capture repeat‐associated RBPs by mass spectrometry in mammalian cells. We identified several SR (serine/arginine‐rich) proteins that interact selectively with CGG repeats basally and under cellular stress. These proteins modify toxicity in a Drosophila model of FXTAS. Pharmacologic inhibition of serine/arginine protein kinases (SRPKs), which alter SRSF protein phosphorylation, localization, and activity, directly inhibits RAN translation of CGG and GGGGCC repeats (associated with C9orf72 ALS/FTD) and triggers repeat RNA retention in the nucleus. Lowering SRPK expression suppressed toxicity in both FXTAS and C9orf72 ALS/FTD model flies, and SRPK inhibitors suppressed CGG repeat toxicity in rodent neurons. Together, these findings demonstrate roles for CGG repeat RNA binding proteins in RAN translation and repeat toxicity and support further evaluation of SRPK inhibitors in modulating RAN translation associated with repeat expansion disorders.

Long Noncoding RNA FENDRR Inhibits Lung Fibroblast Proliferation via a Reduction of β-Catenin

Idiopathic Pulmonary Fibrosis (IPF) is a chronic, progressive, and usually lethal lung disease and it has been widely accepted that fibroblast proliferation is one of the key characteristics of IPF. Long noncoding RNAs (lncRNAs) play vital roles in the pathogenesis of many diseases. In this study, we investigated the role of lncRNA FENDRR on fibroblast proliferation. Human lung fibroblasts stably overexpressing FENDRR showed a reduced cell proliferation compared to those expressing the control vector. On the other hand, FENDRR silencing increased fibroblast proliferation. FENDRR bound serine-arginine rich splicing factor 9 (SRSF9) and inhibited the phosphorylation of p70 ribosomal S6 kinase 1 (PS6K), a downstream protein of the mammalian target of rapamycin (mTOR) signaling. Silencing SRSF9 reduced fibroblast proliferation. FENDRR reduced β-catenin protein, but not mRNA levels. The reduction of β-catenin protein levels in lung fibroblasts by gene silencing or chemical inhibitor decreased fibroblast proliferation. Adenovirus-mediated FENDRR transfer to the lungs of mice reduced asbestos-induced fibrotic lesions and collagen deposition. RNA sequencing of lung tissues identified 7 cell proliferation-related genes that were up-regulated by asbestos but reversed by FENDRR. In conclusion, FENDRR inhibits fibroblast proliferation and functions as an anti-fibrotic lncRNA.

Alternative splicing of Bax controls cell death and innate immune responses in macrophages

Abstract To defend against invading pathogens, the macrophage innate immune response requires rapid, robust gene expression reprogramming. Innate immune genes that are induced as part of this response are regulated at many steps. While transcriptional activation of innate immune genes has been extensively studied, the mechanisms driving their post-transcriptional regulation (i.e. alternative splicing) needs further investigation. To begin learning how pre-mRNA splicing changes impact the innate immune response in macrophages, we examined how loss of a splicing regulatory protein called SRSF6 (serine arginine rich splicing factor 6) impacts gene expression. We discovered a novel phenotype whereby loss of SRSF6 increased basal expression of many interferon stimulated genes (ISGs). Consistent with mitochondrial dysfunction, a phenotype that we and others have previously associated high basal ISG expression, we measured an increase in apoptosis and macrophage depolarization in SRSF6 knockdown macrophages compared to scramble controls. Subsequent bioinformatic analysis of RNA-seq data from SRSF6 knockdown macrophages identified an alternative splicing event in the apoptosis activation factor BAX. While Bax isoforms have previously been linked to cell death regulation, no one has reported a role for Bax isoforms in modulating mtDNA-driven innate immune responses. Our data is consistent with a model whereby upregulation of a Bax isoform promotes pore formation in mitochondria which 1) causes leakage of mtDNA into the cytosol where it is detected by cGAS, eliciting type I interferon production and 2) primes macrophages to undergo programmed cell death.

Noncanonical functions of the serine-arginine-rich splicing factor (SR) family of proteins in development and disease.

Members of the serine/arginine (SR)-rich protein family of splicing factors play versatile roles in RNA processing steps and are often essential for normal development. Dynamic changes in RNA processing and turnover allow fast cellular adaptions to a changing microenvironment and thereby closely cooperate with transcription factor networks that establish cell identity within tissues. SR proteins play fundamental roles in the processing of pre-mRNAs by regulating constitutive and alternative splicing. More recently, SR proteins have also been implicated in other aspects of RNA metabolism such as mRNA stability, transport and translation. The- emerging noncanonical functions highlight the multifaceted functions of these SR proteins and identify them as important coordinators of gene expression programmes. Accordingly, most SR proteins are essential for normal cell function and their misregulation contributes to human diseases such as cancer.

The splicing factor SRSF1 stabilizes the mRNA of TSLP to enhance acute lung injury

Acute lung injury (ALI) is a severe disease with a high rate of morbidity and mortality, characterized by excessive and uncontrolled inflammatory response in lung. Recent studies demonstrated that serine arginine-rich splicing factor 1 (SRSF1) is involved in inflammation. However, whether SRSF1 modulates ALI remains to be determined. In this study, we established an ALI mouse model that induced by lipopolysaccharide (LPS), with or without the treatment of SRSF1 antibody. Our result showed that SRSF1 expression was elevated in LPS-induced ALI. Importantly, treatment with SRSF1 antibody notably ameliorated ALI in mice, as determined by reduction in lung W/D ratios, histopathological changes, lung inflammation and TSLP expression. Besides, exposure of human alveolar epithelial A549 cells to LPS enhanced the expression of both SRSF1 and TSLP, while knockdown or overexpression of SRSF1 significantly lowered or upregulated the expression of TSLP induced by LPS. Interestingly, the expression of SRSF1 and TSLP showed a positive correlation in normal human lung tissues. Mechanistically, we found that SRSF1 directly bound with the mRNA of TSLP and may exert its function by stabilizing the mRNA of TSLP in LPS-induced ALI. Therefore, our results indicated that SRSF1 may be an important contributor in lung inflammation of LPS-induced ALI and SRSF1 signaling blocking may serve as a potential treatment of ALI.

Abstract 2466: A novel role for the transcription factor SNAIL in alternative splicing

Abstract The transcription factor SNAIL is a master regulator of the Epithelial to Mesenchymal Transition (EMT), a hallmark event during cancer metastasis. SNAIL repressed transcription of several epithelial cell-specific genes such as E-cadherin, leading to a mesenchymal phenotype that is less attached and more migratory. As a DNA-binding transcription factor, SNAIL binds consensus E-box motifs in gene promoters, and recruits epigenetic modifiers to repress or activate gene expression. Previous studies from our lab and others have indicated that SNAIL and its family member SLUG have several gene targets in common but also bind to unique gene targets. Whether SNAIL or SLUG share protein-interacting partners is unknown. To identify interactome partners of SNAIL and SLUG, we performed co-immunoprecipitation followed by iTRAQ mass spectrometry. The protein interactome of SNAIL, but not SLUG, was highly enriched for splicing proteins, of which the top hit was the Serine-Arginine Rich-Splicing Factor, SRSF1. SRSF1 participates in regulation of alternative splicing, a mechanism through which a cell can generate multiple proteins from the same gene. Alternative splicing events play a central role in EMT progression, and SRSF1 is overexpressed in many cancers. We explored the role of SNAIL and SRSF1 in driving alternative splicing events during EMT. First, we confirmed the association of SNAIL and SRSF1 by co-immunofluorescence, PLA assay and co-immunoprecipitation experiments during TGF-beta induction of EMT. RNA-IP (RIP) experiments demonstrated association of SNAIL with mesenchymal specific but not epithelial-specific splice isoforms, further suggesting an RNA binding role for SNAIL. Lastly, RNA-EMSA experiments suggest specificity for some but not all four Zinc fingers of SNAIL in binding to RNA. Based on these observations, we hypothesize that SNAIL regulates alternative splicing events during EMT at least in part through association with SRSF1 on RNA. As transcription and splicing are closely coupled, we predict that the regulation of splicing by transcription factors through RNA binding is universally utilized by other biological systems to alter phenotype in response to external stimuli. We are currently working on identifying the genome-wide changes in alternative splicing regulated by SNAIL, and the mechanism by which SNAIL interacts with the spliceosome to regulate alternative splicing. Citation Format: Atrayee Ray, Smruthi Rudraraju, Shawn Krueger, Janani kumar, Danielle Perley, Adam Scheidegger, Archana Dhasarathy. A novel role for the transcription factor SNAIL in alternative splicing [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2466.

Splicing factor SRSF1 controls T cell homeostasis and its decreased levels are linked to lymphopenia in systemic lupus erythematosus.

Lymphopenia is a frequent clinical manifestation and risk factor for infections in SLE, but the underlying mechanisms are not fully understood. We previously identified novel roles for the RNA-binding protein serine arginine-rich splicing factor 1 (SRSF1) in the control of genes involved in signalling and cytokine production in human T cells. SRSF1 is decreased in T cells from patients with SLE and associates with severe disease. Because SRSF1 controls the expression of apoptosis-related genes, we hypothesized that SRSF1 controls T cell homeostasis and, when reduced, leads to lymphopenia. We evaluated SRSF1 expression in T cells from SLE patients by immunoblots and analysed its correlation with clinical parameters. T cell conditional Srsf1 knockout mice were used to evaluate lymphoid cells and apoptosis by flow cytometry. Quantitative PCR and immunoblots were used to assess Bcl-xL mRNA and protein expression. SRSF1 overexpression was performed by transient transfections by electroporation. We found that low SRSF1 levels correlated with lymphopenia in SLE patients. Selective deletion of Srsf1 in T cells in mice led to T cell lymphopenia, with increased apoptosis and decreased expression of the anti-apoptotic Bcl-xL. Lower SRSF1 expression correlated with low Bcl-xL levels in T cells and lower Bcl-xL levels associated with lymphopenia in SLE patients. Importantly, overexpression of SRSF1 rescued survival of T cells from patients with SLE. Our studies uncovered a previously unrecognized role for SRSF1 in the control of T cell homeostasis and its reduced expression as a molecular defect that contributes to lymphopenia in systemic autoimmunity.

TIMP1 intron 3 retention is a marker of colon cancer progression controlled by hnRNPA1.

We previously reported a 40-transcripts signature marking the normal mucosa to colorectal adenocarcinoma transition. Eight of these mRNAs also showed splicing alterations, including a specific intron 3 retention in tissue metalloprotease inhibitor I (TIMP1), which decreased during the early steps of colorectal cancer progression. To decipher the mechanism of intron 3 retention/splicing, we first searched for putative RNA binding protein binding sites onto the TIMP1 sequence. We identified potential serine arginine rich splicing factor 1 (SRSF1) and heterogeneous nuclear RiboNucleoProtein A1 (hnRNPA1) binding sites at the end of intron 3 and the beginning of exon 4, respectively. RNA immunoprecipitation showed that hnRNPA1, but not SRSF1 could bind to the corresponding region in TIMP1 pre-mRNA in live cells. Furthermore, using a TIMP1-based ex vivo minigene approach, together with a plasmon resonance in vitro RNA binding assay, we confirmed that hnRNPA1 could indeed bind to wild type TIMP1 exon 4 pre-mRNA and control TMP1 intron 3 splicing, the interaction being abolished in presence of a mutant sequence that disrupted this site. These results indicated that hnRNPA1, upon binding to TIMP1 exon 4, was a positive regulator of intron 3 splicing. We propose that this TIMP1-intron 3 + transcript belongs to the class of nuclear transcripts with "detained" introns, an abundant molecular class, including in cancer.

An activator of G protein-coupled receptor and MEK1/2-ERK1/2 signaling inhibits HIV-1 replication by altering viral RNA processing.

The ability of HIV-1 to evolve resistance to combined antiretroviral therapies (cARTs) has stimulated research into alternative means of controlling this infection. We assayed >60 modulators of RNA alternative splicing (AS) to identify new inhibitors of HIV-1 RNA processing-a segment of the viral lifecycle not targeted by current drugs-and discovered compound N-[4-chloro-3-(trifluoromethyl)phenyl]-7-nitro-2,1,3-benzoxadiazol-4-amine (5342191) as a potent inhibitor of both wild-type (Ba-L, NL4-3, LAI, IIIB, and N54) and drug-resistant strains of HIV-1 (IC50: ~700 nM) with no significant effect on cell viability at doses tested. 5342191 blocks expression of four essential HIV-1 structural and regulatory proteins (Gag, Env, Tat, and Rev) without affecting total protein synthesis of the cell. This response is associated with altered unspliced (US) and singly-spliced (SS) HIV-1 RNA accumulation (~60% reduction) and transport to the cytoplasm (loss of Rev) whereas parallel analysis of cellular RNAs revealed less than a 0.7% of host alternative splicing (AS) events (0.25-0.67% by ≥ 10-20%), gene expression (0.01-0.46% by ≥ 2-5 fold), and protein abundance (0.02-0.34% by ≥ 1.5-2 fold) being affected. Decreased expression of Tat, but not Gag/Env, upon 5342191 treatment was reversed by a proteasome inhibitor, suggesting that this compound alters the synthesis/degradation of this key viral factor. Consistent with an affect on HIV-1 RNA processing, 5342191 treatment of cells altered the abundance and phosphorylation of serine/arginine-rich splicing factor (SRSF) 1, 3, and 4. Despite the activation of several intracellular signaling pathways by 5342191 (Ras, MEK1/2-ERK1/2, and JNK1/2/3), inhibition of HIV-1 gene expression by this compound could be reversed by pre-treatment with either a G-protein α-subunit inhibitor or two different MEK1/2 inhibitors. These observations demonstrate enhanced sensitivity of HIV-1 gene expression to small changes in host RNA processing and highlights the potential of modulating host intracellular signaling as an alternative approach for controlling HIV-1 infection.

Cellular stressors may alter islet hormone cell proportions by moderation of alternative splicing patterns.

Changes to islet cell identity in response to type 2 diabetes (T2D) have been reported in rodent models, but are less well characterized in humans. We assessed the effects of aspects of the diabetic microenvironment on hormone staining, total gene expression, splicing regulation and the alternative splicing patterns of key genes in EndoC-βH1 human beta cells. Genes encoding islet hormones [somatostatin (SST), insulin (INS), Glucagon (GCG)], differentiation markers [Forkhead box O1 (FOXO1), Paired box 6, SRY box 9, NK6 Homeobox 1, NK6 Homeobox 2] and cell stress markers (DNA damage inducible transcript 3, FOXO1) were dysregulated in stressed EndoC-βH1 cells, as were some serine arginine rich splicing factor splicing activator and heterogeneous ribonucleoprotein particle inhibitor genes. Whole transcriptome analysis of primary T2D islets and matched controls demonstrated dysregulated splicing for ~25% of splicing events, of which genes themselves involved in messenger ribonucleic acid processing and regulation of gene expression comprised the largest group. Approximately 5% of EndoC-βH1 cells exposed to these factors gained SST positivity in vitro. An increased area of SST staining was also observed ex vivo in pancreas sections recovered at autopsy from donors with type 1 diabetes (T1D) or T2D (9.3% for T1D and 3% for T2D, respectively compared with 1% in controls). Removal of the stressful stimulus or treatment with the AKT Serine/Threonine kinase inhibitor SH-6 restored splicing factor expression and reversed both hormone staining effects and patterns of gene expression. This suggests that reversible changes in hormone expression may occur during exposure to diabetomimetic cellular stressors, which may be mediated by changes in splicing regulation.

Cellular stress and AMPK activation as a common mechanism of action linking the effects of metformin and diverse compounds that alleviate accelerated aging defects in Hutchinson-Gilford progeria syndrome

Hutchinson-Gilford progeria syndrome (HGPS) is a rare genetic disorder characterized by an accelerated aging phenotype that typically leads to death via stroke or myocardial infarction at approximately 14.6 years of age. Most cases of HGPS have been linked to the extensive use of a cryptic splice donor site located in the LMNA gene due to a de novo mutation, generating a truncated and toxic protein known as progerin. Progerin accumulation in the nuclear membrane and within the nucleus distorts the nuclear architecture and negatively effects nuclear processes including DNA replication and repair, leading to accelerated cellular aging and premature senescence. The serine-arginine rich splicing factor SRSF1 (also known as ASF/SF2) has recently been shown to modulate alternative splicing of the LMNA gene, with SRSF1 inhibition significantly reducing progerin at both the mRNA and protein levels. In 2014, we hypothesized for the first time that compounds including metformin that induce activation of AMP-activated protein kinase (AMPK), a master metabolic regulator activated by cellular stress (e.g. increases in intracellular calcium, reactive oxygen species, and/or an AMP(ADP)/ATP ratio increase, etc.), will beneficially alter gene splicing in progeria cells by inhibiting SRSF1, thus lowering progerin levels and altering the LMNA pre-mRNA splicing ratio. Recent evidence has substantiated this hypothesis, with metformin significantly reducing the mRNA and protein levels of both SRSF1 and progerin, activating AMPK, and alleviating pathological defects in HGPS cells. Metformin has also recently been shown to beneficially alter gene splicing in normal humans. Interestingly, several chemically distinct compounds, including rapamycin, methylene blue, all-trans retinoic acid, MG132, 1α,25-dihydroxyvitamin D3, sulforaphane, and oltipraz have each been shown to alleviate accelerated aging defects in patient-derived HGPS cells. Each of these compounds has also been independently shown to induce AMPK activation. Because these compounds improve accelerated aging defects in HGPS cells either by enhancing mitochondrial functionality, increasing Nrf2 activity, inducing autophagy, or by altering gene splicing and because AMPK activation beneficially modulates each of the aforementioned processes, it is our hypothesis that cellular stress-induced AMPK activation represents an indirect yet common mechanism of action linking such chemically diverse compounds with the beneficial effects of those compounds observed in HGPS cells. As normal humans also produce progerin at much lower levels through a similar mechanism, compounds that safely induce AMPK activation may have wide-ranging implications for both normal and pathological aging.