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Background The Mnk2 kinase, encoded by MKNK2 gene, plays critical roles in MAPK signaling and was involved in oncogenesis. Human MKNK2 pre-mRNA can be alternatively spliced into two splicing isoforms, the MKNK2a and MKNK2b , thus yielding Mnk2a and Mnk2b proteins with different domains. The involvement of Mnk2 alternative splicing in colon cancer has been implicated based on RNA-sequencing data from TCGA database. This study aimed at investigating the upstream modulators and clinical relevance of Mnk2 alternative splicing in colon adenocarcinoma (CAC). Methods PCR, western blotting and immunohistochemistry (IHC) were performed to assess the expression of Mnk2 and upstream proteins in CAC. The function of Mnk2 and its regulators were demonstrated in different CAC cell lines as well as in xenograft models. Two independent cohorts of CAC patients were used to reveal the clinical significance of MKNK2 alternative splicing. Results Comparing with adjacent nontumorous tissue, CAC specimen showed a decreased MKNK2a level and an increased MKNK2b level, which were correlated with KRAS mutation and tumor size. The SRSF1 (serine/arginine-rich splicing factor 1) was further confirmed to be the major splicing factor targeting MKNK2 in CAC cells. Higher expression of SRPK1/2 or decreased activity of PP1α were responsible for enhancing SRSF1 phosphorylation and nucleus translocation, subsequently resulted in a switch of MKNK2 alternative splicing. Conclusions Our data showed that phosphorylation and subcellular localization of SRSF1 were balanced by SRPK1/2 and PP1α in CAC cells. High nucleus SRSF1 promoted MKNK2 splicing into MKNK2b instead of MNK2a , consequently enhanced tumor proliferation.

Background Intensive evidence has highlighted the effect of aberrant alternative splicing (AS) events on cancer progression when triggered by dysregulation of the SR protein family. Nonetheless, the underlying mechanism in breast cancer (BRCA) remains elusive. Here we sought to explore the molecular function of SRSF1 and identify the key AS events regulated by SRSF1 in BRCA. Methods We conducted a comprehensive analysis of the expression and clinical correlation of SRSF1 in BRCA based on the TCGA dataset, Metabric database and clinical tissue samples. Functional analysis of SRSF1 in BRCA was conducted in vitro and in vivo. SRSF1-mediated AS events and their binding motifs were identified by RNA-seq, RNA immunoprecipitation-PCR (RIP-PCR) and in vivo crosslinking followed by immunoprecipitation (CLIP), which was further validated by the minigene reporter assay. PTPMT1 exon 3 (E3) AS was identified to partially mediate the oncogenic role of SRSF1 by the P-AKT/C-MYC axis. Finally, the expression and clinical significance of these AS events were validated in clinical samples and using the TCGA database. Results SRSF1 expression was consistently upregulated in BRCA samples, positively associated with tumor grade and the Ki-67 index, and correlated with poor prognosis in a hormone receptor-positive (HR+) cohort, which facilitated proliferation, cell migration and inhibited apoptosis in vitro and in vivo. We identified SRSF1-mediated AS events and discovered the SRSF1 binding motif in the regulation of splice switching of PTPMT1. Furthermore, PTPMT1 splice switching was regulated by SRSF1 by binding directly to its motif in E3 which partially mediated the oncogenic role of SRSF1 by the AKT/C-MYC axis. Additionally, PTPMT1 splice switching was validated in tissue samples of BRCA patients and using the TCGA database. The high-risk group, identified by AS of PTPMT1 and expression of SRSF1, possessed poorer prognosis in the stage I/II TCGA BRCA cohort. Conclusions SRSF1 exerts oncogenic roles in BRCA partially by regulating the AS of PTPMT1, which could be a therapeutic target candidate in BRCA and a prognostic factor in HR+ BRCA patient.

Background Circular RNAs (circRNAs) act as gene expression regulators and are involved in cancer progression. However, their functions have not been sufficiently investigated in nasopharyngeal carcinoma (NPC). Methods The expression profiles of circRNAs in NPC cells within different metastatic potential were reanalyzed. Quantitative reverse transcription PCR and in situ hybridization were used to detect the expression level of circPVT1 in NPC cells and tissue samples. The association of expression level of circPVT1 with clinical properties of NPC patients was evaluated. Then, the effects of circPVT1 expression on NPC metastasis were investigated by in vitro and in vivo functional experiments. RNA immunoprecipitation, pull-down assay and western blotting were performed to confirm the interaction between circPVT1 and β-TrCP in NPC cells. Co-immunoprecipitation and western blotting were performed to confirm the interaction between β-TrCP and c-Myc in NPC cells. Results We find that circPVT1 , a circular RNA, is significantly upregulated in NPC cells and tissue specimens. In vitro and in vivo experiments showed that circPVT1 promotes the invasion and metastasis of NPC cells. Mechanistically, circPVT1 inhibits proteasomal degradation of c-Myc by binding to β-TrCP, an E3 ubiquiting ligase. Stablization of c-Myc by circPVT1 alters the cytoskeleton remodeling and cell adhesion in NPC, which ultimately promotes the invasion and metastasis of NPC cells. Furthermore, c-Myc transcriptionally upregulates the expression of SRSF1, an RNA splicing factor, and recruits SRSF1 to enhance the biosynthesis of circPVT1 through coupling transcription with splicing, which forms a positive feedback for circPVT1 production. Conclusions Our results revealed the important role of circPVT1 in the progression of NPC through the β-TrCP/c-Myc/SRSF1 positive feedback loop, and circPVT1 may serve as a prognostic biomarker or therapeutic target in patients with NPC.

Background Glioma is the most common and malignant tumor of central nervous system. The tumor initiation, self-renewal, and multi-lineage differentiation abilities of glioma stem cells (GSCs) are responsible for glioma proliferation and recurrence. Although circular RNAs (circRNAs) play vital roles in the progression of glioma, the detailed mechanisms remain unknown. Methods qRT-PCR, western blotting, immunohistochemistry, and bioinformatic analysis were performed to detect the expression of circATP5B, miR-185-5p, HOXB5, and SRSF1. Patient-derived GSCs were established, and MTS, EDU, neurosphere formation, and limiting dilution assays were used to detect the proliferation of GSCs. RNA-binding protein immunoprecipitation, RNA pull-down, luciferase reporter assays, and chromatin immunoprecipitation assays were used to detect these molecules’ regulation mechanisms. Results We found circATP5B expression was significantly upregulated in GSCs and promoted the proliferation of GSCs. Mechanistically, circATP5B acted as miR-185-5p sponge to upregulate HOXB5 expression. HOXB5 was overexpressed in glioma and transcriptionally regulated IL6 expression and promoted the proliferation of GSCs via JAK2/STAT3 signaling. Moreover, RNA binding protein SRSF1 could bind to and promote circATP5B expression and regulate the proliferation of GSCs, while HOXB5 also transcriptionally regulated SRSF1 expression. Conclusions Our study identified the SRSF1/circATP5B/miR-185-5p/HOXB5 feedback loop in GSCs. This provides an effective biomarker for glioma diagnosis and prognostic evaluation.

Alternative splicing (AS) plays significant roles in a multitude of fundamental biological activities. AS is prevalent in the testis, but the regulations of AS in spermatogenesis is only little explored. Here, we report that Serine/arginine-rich splicing factor 1 (SRSF1) plays critical roles in alternative splicing and male reproduction. Male germ cell-specific deletion of Srsf1 led to complete infertility by affecting spermatogenesis. Mechanistically, by combining RNA-seq data with LACE-seq data, we showed that SRSF1 affected the AS of Stra8 in a direct manner and Dazl, Dmc1, Mre11a, Syce2 and Rif1 in an indirect manner. Our findings demonstrate that SRSF1 has crucial functions in spermatogenesis and male fertility by regulating alternative splicing.Alternative splicing (AS) plays significant roles in a multitude of fundamental biological activities. AS is prevalent in the testis, but the regulations of AS in spermatogenesis is only little explored. Here, we report that Serine/arginine-rich splicing factor 1 (SRSF1) plays critical roles in alternative splicing and male reproduction. Male germ cell-specific deletion of Srsf1 led to complete infertility by affecting spermatogenesis. Mechanistically, by combining RNA-seq data with LACE-seq data, we showed that SRSF1 affected the AS of Stra8 in a direct manner and Dazl, Dmc1, Mre11a, Syce2 and Rif1 in an indirect manner. Our findings demonstrate that SRSF1 has crucial functions in spermatogenesis and male fertility by regulating alternative splicing.

Metastasis associated lung adenocarcinoma transcript 1(MALAT1) is a long non‐coding RNA, broadly expressed in mammalian tissues including kidney and up‐regulated in a variety of cancer cells. To date, its functions in podocytes are largely unknown. β‐catenin is a key mediator in the canonical and non‐canonical Wnt signalling pathway; its aberrant expression promotes podocyte malfunction and albuminuria, and contributes to kidney fibrosis. In this study, we found that MALAT1 levels were increased in kidney cortices from C57BL/6 mice with streptozocin (STZ)‐induced diabetic nephropathy, and dynamically regulated in cultured mouse podocytes stimulated with high glucose, which showed a trend from rise to decline. The decline of MALAT1 levels was accompanied with β‐catenin translocation to the nuclei and enhanced expression of serine/arginine splicing factor 1 (SRSF1), a MALAT1 RNA‐binding protein. Further we showed early interference with MALAT1 siRNA partially restored podocytes function and prohibited β‐catenin nuclear accumulation and SRSF1 overexpression. Intriguingly, we showed that β‐catenin was involved in MALAT1 transcription by binding to the promotor region of MALAT1; β‐catenin knock‐down also decreased MALAT1 levels, suggesting a novel feedback regulation between MALAT1 and β‐catenin. Notably, β‐catenin deletion had limited effects on SRSF1 expression, demonstrating β‐catenin might serve as a downstream signal of SRSF1. These findings provided evidence for a pivotal role of MALAT1 in diabetic nephropathy and high glucose‐induced podocyte damage.

Circular RNAs (circRNAs) are a large class of RNAs with regulatory functions within cells. We recently showed that circSMARCA5 is a tumor suppressor in glioblastoma multiforme (GBM) and acts as a decoy for Serine and Arginine Rich Splicing Factor 1 (SRSF1) through six predicted binding sites (BSs). Here we characterized RNA motifs functionally involved in the interaction between circSMARCA5 and SRSF1. Three different circSMARCA5 molecules (Mut1, Mut2, Mut3), each mutated in two predicted SRSF1 BSs at once, were obtained through PCR-based replacement of wild-type (WT) BS sequences and cloned in three independent pcDNA3 vectors. Mut1 significantly decreased its capability to interact with SRSF1 as compared to WT, based on the RNA immunoprecipitation assay. In silico analysis through the “Find Individual Motif Occurrences” (FIMO) algorithm showed GAUGAA as an experimentally validated SRSF1 binding motif significantly overrepresented within both predicted SRSF1 BSs mutated in Mut1 (q-value = 0.0011). U87MG and CAS-1, transfected with Mut1, significantly increased their migration with respect to controls transfected with WT, as revealed by the cell exclusion zone assay. Immortalized human brain microvascular endothelial cells (IM-HBMEC) exposed to conditioned medium (CM) harvested from U87MG and CAS-1 transfected with Mut1 significantly sprouted more than those treated with CM harvested from U87MG and CAS-1 transfected with WT, as shown by the tube formation assay. qRT-PCR showed that the intracellular pro- to anti-angiogenic Vascular Endothelial Growth Factor A (VEGFA) mRNA isoform ratio and the amount of total VEGFA mRNA secreted in CM significantly increased in Mut1-transfected CAS-1 as compared to controls transfected with WT. Our data suggest that GAUGAA is the RNA motif responsible for the interaction between circSMARCA5 and SRSF1 as well as for the circSMARCA5-mediated control of GBM cell migration and angiogenic potential.

Ovarian granulosa cell (GC) apoptosis is the major cause of follicular atresia. Regulation of non-coding RNAs (ncRNAs) was proved to be involved in regulatory mechanisms of GC apoptosis. circRNAs have been recognized to play important roles in cellular activity. However, the regulatory network of circRNAs in follicular atresia has not been fully validated. In this study, we report a new circRNA, circSLC41A1, which has higher expression in healthy follicles compared to atretic follicles, and confirm its circular structure using RNase R treatment. The resistant function of circSLC41A1 during GC apoptosis was detected by si-RNA transfection and the competitive binding of miR-9820-5p by circSLC41A1 and SRSF1 was detected with a dual-luciferase reporter assay and co-transfection of their inhibitors or siRNA. Additionally, we predicted the protein-coding potential of circSLC41A1 and analyzed the structure of circSLC41A1-134aa. Our study revealed that circSLC41A1 enhanced SRSF1 expression through competitive binding of miR-9820-5p and demonstrated a circSLC41A1–miR-9820-5p–SRSF1 regulatory axis in follicular GC apoptosis. The study adds to knowledge of the post-transcriptional regulation of follicular atresia and provides insight into the protein-coding function of circRNA.

Gastric cancer (GC) is one of the malignancies with the highest incidence and mortality worldwide. Evasion of apoptosis is a hallmark of cancer that drives tumor progression. The splicing factor SRSF1 and antiapoptotic protein Mcl‐1, including its isoforms Mcl‐1L and Mcl‐1S, play significant roles in cancer development; however, the regulatory mechanisms of the SRSF1‐Mcl‐1 axis in GC remain unclear. This study systematically evaluated the function of SRSF1 in GC by integrating multidatabase analyses (TIMER, UALCAN, and KM‐Plotter), in vitro experiments (qRT‐PCR, Western blot, Transwell migration/invasion, and apoptosis assays), in vivo xenograft models, and bioinformatic approaches (single‐cell RNA sequencing, hdWGCNA, cell interaction analysis, and mutational analysis). SRSF1 is significantly overexpressed in GC tissues and cell lines, correlating with poor prognosis in patients. Through comprehensive multiomics analysis, we first revealed that SRSF1‐positive malignant epithelial cells possess a unique coexpression network and exhibit significantly enhanced interactions with fibroblasts, reshaping the tumor microenvironment (TME). Subsequently, functional assays demonstrated that SRSF1 overexpression enhances cell invasion, migration, and apoptosis resistance by inhibiting the proapoptotic isoform Mcl‐1S and suppressing the mitochondrial apoptosis pathway (Bak/caspase‐9/caspase‐3). In conclusion, the SRSF1‐Mcl‐1 axis serves as a dual key regulator of invasion/migration and apoptosis evasion in GC, providing a new strategy for targeted therapy in advanced GC.

Phase separation plays crucial roles in both sustaining cellular function and perpetuating disease states. Despite extensive studies, our understanding of this process is hindered by low solubility of phase-separating proteins. One example of this is found in SR and SR-related proteins. These proteins are characterized by domains rich in arginine and serine (RS domains), which are essential to alternative splicing and in vivo phase separation. However, they are also responsible for a low solubility that has made these proteins difficult to study for decades. Here, we solubilize the founding member of the SR family, SRSF1, by introducing a peptide mimicking RS repeats as a co-solute. We find that this RS-mimic peptide forms interactions similar to those of the protein’s RS domain. Both interact with a combination of surface-exposed aromatic residues and acidic residues on SRSF1’s RNA Recognition Motifs (RRMs) through electrostatic and cation-pi interactions. Analysis of RRM domains from human SR proteins indicates that these sites are conserved across the protein family. In addition to opening an avenue to previously unavailable proteins, our work provides insight into how SR proteins phase separate and participate in nuclear speckles.