scholarly journals SRPK1/2 and PP1α exert opposite functions by modulating SRSF1-guided MKNK2 alternative splicing in colon adenocarcinoma

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
Hongda Liu ◽  
Zheng Gong ◽  
Kangshuai Li ◽  
Qun Zhang ◽  
Zekuan Xu ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Colon Adenocarcinoma ◽  
Mapk Signaling ◽  
Splicing Factor ◽  
Tumor Proliferation ◽  
Sequencing Data ◽  
Alternatively Spliced ◽  
Xenograft Models ◽  
Splicing Isoforms ◽  
Splicing Factor 1

Abstract 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.

scholarly journals Phosphorylation status of the Kep1 protein alters its affinity for its protein binding partner alternative splicing factor ASF/SF2

2006 ◽  
Vol 400 (1) ◽  
pp. 91-97 ◽  
Author(s):  
Cécile Robard ◽  
Alex Daviau ◽  
Marco Di Fruscio
Keyword(s):  
Alternative Splicing ◽  
Protein Binding ◽  
Cell Line ◽  
Topoisomerase I ◽  
Target Genes ◽  
Rna Binding ◽  
Splicing Factor ◽  
Alternatively Spliced ◽  
Alternative Splicing Factor

Mutations in the Drosophila kep1 gene, encoding a single maxi KH (K homology) domain-containing RNA-binding protein, result in a reduction of fertility in part due to the disruption of the apoptotic programme during oogenesis. This disruption is concomitant with the appearance of an alternatively spliced mRNA isoform encoding the inactive caspase dredd. We generated a Kep1 antibody and have found that the Kep1 protein is present in the nuclei of both the follicle and nurse cells during all stages of Drosophila oogenesis. We have shown that the Kep1 protein is phosphorylated in ovaries induced to undergo apoptosis following treatment with the topoisomerase I inhibitor camptothecin. We have also found that the Kep1 protein interacts specifically with the SR (serine/arginine-rich) protein family member ASF/SF2 (alternative splicing factor/splicing factor 2). This interaction is independent of the ability of Kep1 to bind RNA, but is dependent on the phosphorylation of the Kep1 protein, with the interaction between Kep1 and ASF/SF2 increasing in the presence of activated Src. Using a CD44v5 alternative splicing reporter construct, we observed 99% inclusion of the alternatively spliced exon 5 following kep1 transfection in a cell line that constitutively expresses activated Src. This modulation in splicing was not observed in the parental NIH 3T3 cell line in which we obtained 7.5% exon 5 inclusion following kep1 transfection. Our data suggest a mechanism of action in which the in vivo phosphorylation status of the Kep1 protein affects its affinity towards its protein binding partners and in turn may allow for the modulation of alternative splice site selection in Kep1–ASF/SF2-dependent target genes.

scholarly journals Identification of Recurrent Alternative RNA Splicing in Adverse-Risk Acute Myeloid Leukemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 457-457
Author(s):  
Govardhan Anande ◽  
Ashwin Unnikrishnan ◽  
Nandan Deshpande ◽  
Sylvain Mareschal ◽  
Aarif M. N. Batcha ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Board Of Directors ◽  
Rna Splicing ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Splicing Factor ◽  
Rna Seq ◽  
Advisory Committees ◽  
Alternatively Spliced ◽  
Acute Myeloid

RNA splicing is a fundamental biological process that generates protein diversity from a finite set of genes. Recurrent somatic mutations of genes involved in RNA splicing are present at high frequency in Myelodysplasia (up to 70%) but less so in Acute Myeloid Leukemia (AML; less than 20%). To investigate whether there were aberrant and recurrent RNA splicing events in the AML transcriptome that were associated with poor prognosis in the absence of splicing factor mutations, we developed a bioinformatics pipeline to systematically annotate and quantify alternative splicing events from RNA-sequencing data (Fig A). We first analysed publicly available RNA-seq data from The Cancer Genome Atlas (TCGA, n=170). We focussed on non-M3 AML patients with no splicing factor mutations (based on reported genomic sequencing and verified by re-analysis of RNA-seq data from all patients) who had received intensive chemotherapy. We segregated these patients based on their European Leukaemia Net (ELN) risk classification and identified 1290 alternatively spliced events impacting 910 genes that were significantly different (FDR<0.05) between all ELNAdv (n=41) versus all ELNFav patients (n=21, Fig B). The majority were exon skipping events (716 events, 62%, Fig B-C), followed by intron retention (201 events, 15.6%, Fig B). We next used RNA-seq data from a second non-M3 AML patient cohort (ClinSeq- Sweden; ELNAdv, n=75 and ELNFav, n=47), detecting 2507 events mapping to 1566 genes. Comparing across the two cohorts, 222 shared genes were detected to be affected by alternative splicing (Fig D). Ingenuity pathway analysis associated these genes with pathways related to protein translation. In order to prioritise those alternatively spliced events most likely to have a deleterious function, we developed an analytical framework to predict their impact on protein structure (Fig E). 87 alternatively spliced events, 25.81% of the commonly shared splicing events, relating to 78 genes (35.13% of all genes) were predicted to directly alter highly conserved protein domains within the affected genes, leading to either a complete (~25%, Fig E) or a partial loss of a domain (20%, Fig E). These in silico predictions are likely to be an underestimate of the true impact, as splicing alterations mapping to poorly annotated domains or affecting the tertiary structure of proteins would be missed. A number of splicing factors themselves were differentially spliced, with the alternative splicing predicted to have functional consequences. This was exemplified by hnRNPA1, a factor with well-established roles in splicing, is itself alternatively spliced in patients and predicted to be deleterious. Consistent with this, motif scanning analyses indicated that a number of mis-spliced transcripts had hnRNPA1 binding motifs (Fig F). To assess the impact of these alternatively spliced events (that were predicted to also disrupt highly conserved protein domains) on the transcriptome, we simultaneously quantified differential gene expression. IPA analysis of the 602 genes that were differentially expressed between ELNAdv and ELNFav patients and shared between both TCGA and ClinSeq cohorts indicated that they were associated with pathways (Fig G) that were distinct from those associated with aberrantly spliced genes (Fig D). A number of pathways related to inflammation were enriched amongst the genes observed to be upregulated in ELNAdv patients (Fig G). Network analyses integrating the alternatively spliced genes with differentially expressed genes revealed strong interactions (Fig H), indicating functional associations between these biological events. Given these strong network interactions, we investigated the potential prognostic significance of these alternatively spliced events. To this end, we utilised machine-learning methods to derive a "splicing signature" of four mis-spliced genes with a predictive capacity equivalent to the ELN (Fig I). The splicing signature further refined existing risk prediction algorithms to improve the classification of patients (Fig J). Taken together, we report the presence of extensive deregulation of RNA splicing in AML patients even in the absence of splicing factor mutations. Many of these events were shared in patients with adverse outcomes and their impact on the AML transcriptome points towards vulnerabilities that could be targeted. Figure Disclosures Unnikrishnan: Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Lehmann:TEVA: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Abbive: Membership on an entity's Board of Directors or advisory committees. Pimanda:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

The interplay between microRNA and alternative splicing of linear and circular RNAs in eleven plant species

2019 ◽  
Vol 35 (17) ◽  
pp. 3119-3126 ◽  
Author(s):  
Huiyuan Wang ◽  
Huihui Wang ◽  
Hangxiao Zhang ◽  
Sheng Liu ◽  
Yongsheng Wang ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Plant Species ◽  
Single Molecule ◽  
Mirna Target ◽  
Supplementary Information ◽  
Circular Rnas ◽  
Sequencing Data ◽  
Genomic Locus ◽  
Target Sites ◽  
Alternatively Spliced

Abstract Motivation MicroRNA (miRNA) and alternative splicing (AS)-mediated post-transcriptional regulation has been extensively studied in most eukaryotes. However, the interplay between AS and miRNAs has not been explored in plants. To our knowledge, the overall profile of miRNA target sites in circular RNAs (circRNA) generated by alternative back splicing has never been reported previously. To address the challenge, we identified miRNA target sites located in alternatively spliced regions of the linear and circular splice isoforms using the up-to-date single-molecule real-time (SMRT) isoform sequencing (Iso-Seq) and Illumina sequencing data in eleven plant species. Results In total, we identified 399 401 and 114 574 AS events from linear and circular RNAs, respectively. Among them, there were 64 781 and 41 146 miRNA target sites located in linear and circular AS region, respectively. In addition, we found 38 913 circRNAs to be overlapping with 45 648 AS events of its own parent isoforms, suggesting circRNA regulation of AS of linear RNAs by forming R-loop with the genomic locus. Here, we present a comprehensive database of miRNA targets in alternatively spliced linear and circRNAs (ASmiR) and a web server for deposition and identification of miRNA target sites located in the alternatively spliced region of linear and circular RNAs. This database is accompanied by an easy-to-use web query interface for meaningful downstream analysis. Plant research community can submit user-defined datasets to the web service to search AS regions harboring small RNA target sites. In conclusion, this study provides an unprecedented resource to understand regulatory relationships between miRNAs and AS in both gymnosperms and angiosperms. Availability and implementation The readily accessible database and web-based tools are available at http://forestry.fafu.edu.cn/bioinfor/db/ASmiR. Supplementary information Supplementary data are available at Bioinformatics online.

scholarly journals Oncogenic Alternative Splicing Switches: Role in Cancer Progression and Prospects for Therapy

2013 ◽  
Vol 2013 ◽  
pp. 1-17 ◽  
Author(s):  
Serena Bonomi ◽  
Stefania Gallo ◽  
Morena Catillo ◽  
Daniela Pignataro ◽  
Giuseppe Biamonti ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Cancer Progression ◽  
Cancer Treatments ◽  
Splicing Regulators ◽  
Transcriptional Regulatory Mechanism ◽  
Transcriptional Regulatory ◽  
Anti Cancer ◽  
Alternatively Spliced ◽  
Splicing Isoforms

Alterations in the abundance or activities of alternative splicing regulators generate alternatively spliced variants that contribute to multiple aspects of tumor establishment, progression and resistance to therapeutic treatments. Notably, many cancer-associated genes are regulated through alternative splicing suggesting a significant role of this post-transcriptional regulatory mechanism in the production of oncogenes and tumor suppressors. Thus, the study of alternative splicing in cancer might provide a better understanding of the malignant transformation and identify novel pathways that are uniquely relevant to tumorigenesis. Understanding the molecular underpinnings of cancer-associated alternative splicing isoforms will not only help to explain many fundamental hallmarks of cancer, but will also offer unprecedented opportunities to improve the efficacy of anti-cancer treatments.

scholarly journals Quantifying full-length circular RNAs in cancer

2021 ◽  
Author(s):  
Ken Hung-On Yu ◽  
Christina Huan Shi ◽  
Bo Wang ◽  
Savio Ho-Chit Chow ◽  
Grace Tin-Yun Chung ◽  
...  
Keyword(s):  
Experimental Data ◽  
Alternative Splicing ◽  
Nasopharyngeal Carcinoma ◽  
Computational Efficiency ◽  
Full Length ◽  
Circular Rnas ◽  
Sequencing Data ◽  
Response Elements ◽  
Different Types ◽  
Alternatively Spliced

AbstractCircular RNAs (circRNAs) are abundantly expressed in cancer. Their resistance to exonucleases enables them to have potentially stable interactions with different types of biomolecules. Alternative splicing can create different circRNA isoforms that have different sequences and unequal interaction potentials. The study of circRNA function thus requires knowledge of complete circRNA sequences. Here we describe psirc, a method that can identify full-length circRNA isoforms and quantify their expression levels from RNA sequencing data. We confirm the effectiveness and computational efficiency of psirc using both simulated and actual experimental data. Applying psirc on transcriptome profiles from nasopharyngeal carcinoma and normal nasopharynx samples, we discover and validate circRNA isoforms differentially expressed between the two groups. Compared to the assumed circular isoforms derived from linear transcript annotations, some of the alternatively spliced circular isoforms have 100 times higher expression and contain substantially fewer microRNA response elements, demonstrating the importance of quantifying full-length circRNA isoforms.

scholarly journals Quantifying full-length circular RNAs in cancer

2021 ◽  
pp. gr.275348.121
Author(s):  
Ken Hung-On Yu ◽  
Christina Huan Shi ◽  
Bo Wang ◽  
Savio Ho-Chit Chow ◽  
Grace Tin-Yun Chung ◽  
...  
Keyword(s):  
Experimental Data ◽  
Alternative Splicing ◽  
Nasopharyngeal Carcinoma ◽  
Computational Efficiency ◽  
Full Length ◽  
Circular Rnas ◽  
Sequencing Data ◽  
Response Elements ◽  
Different Types ◽  
Alternatively Spliced

Circular RNAs (circRNAs) are abundantly expressed in cancer. Their resistance to exonucleases enables them to have potentially stable interactions with different types of biomolecules. Alternative splicing can create different circRNA isoforms that have different sequences and unequal interaction potentials. The study of circRNA function thus requires knowledge of complete circRNA sequences. Here we describe psirc, a method that can identify full-length circRNA isoforms and quantify their expression levels from RNA sequencing data. We confirm the effectiveness and computational efficiency of psirc using both simulated and actual experimental data. Applying psirc on transcriptome profiles from nasopharyngeal carcinoma and normal nasopharynx samples, we discover and validate circRNA isoforms differentially expressed between the two groups. Compared to the assumed circular isoforms derived from linear transcript annotations, some of the alternatively spliced circular isoforms have 100 times higher expression and contain substantially fewer microRNA response elements, demonstrating the importance of quantifying full-length circRNA isoforms.

scholarly journals Isoform-specific characterization implicates alternative splicing in APOBEC3B as a mechanism restricting APOBEC-mediated mutagenesis

2020 ◽  
Author(s):  
A. Rouf Banday ◽  
Olusegun O. Onabajo ◽  
Seraph Han-Yin Lin ◽  
Adeola Obajemu ◽  
Joselin M. Vargas ◽  
...  
Keyword(s):  
Bladder Cancer ◽  
Alternative Splicing ◽  
Mutagenic Activity ◽  
Progression Free Survival ◽  
Splicing Factor ◽  
Free Survival ◽  
Alternatively Spliced ◽  
Intron 4 ◽  
Weak Branch

ABSTRACTAPOBEC3A (A3A) and APOBEC3B (A3B) enzymes drive APOBEC-mediated mutagenesis, but the understanding of the regulation of their mutagenic activity remains limited. Here, we showed that mutagenic and non-mutagenic A3A and A3B enzymes are produced by canonical and alternatively spliced A3A and A3B isoforms, respectively. Notably, increased expression of the canonical A3B isoform, which encodes the mutagenic A3B enzyme, predicted shorter progression-free survival of bladder cancer patients. Expression of the mutagenic A3B isoform was reduced by exon 5 skipping, generating a non-mutagenic A3B isoform. The exon 5 skipping, which was dependent on the interaction between SF3B1 splicing factor and weak branch point sites in intron 4, could be enhanced by an SF3B1 inhibitor, decreasing the production of the mutagenic A3B enzyme. Thus, our results underscore the role of A3B, especially in bladder cancer, and implicate alternative splicing of A3B as a mechanism and therapeutic target to restrict APOBEC-mediated mutagenesis.

scholarly journals Hemimetabolous insects elucidate the origin of sexual development via alternative splicing

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Judith Wexler ◽  
Emily Kay Delaney ◽  
Xavier Belles ◽  
Coby Schal ◽  
Ayako Wada-Katsumata ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Alternative Splicing ◽  
Sexual Development ◽  
Sexual Differentiation ◽  
Splicing Factor ◽  
Male And Female ◽  
Hemimetabolous Insect ◽  
Alternatively Spliced

Insects are the only known animals in which sexual differentiation is controlled by sex-specific splicing. The doublesex transcription factor produces distinct male and female isoforms, which are both essential for sex-specific development. dsx splicing depends on transformer, which is also alternatively spliced such that functional Tra is only present in females. This pathway has evolved from an ancestral mechanism where dsx was independent of tra and expressed and required only in males. To reconstruct this transition, we examined three basal, hemimetabolous insect orders: Hemiptera, Phthiraptera, and Blattodea. We show that tra and dsx have distinct functions in these insects, reflecting different stages in the changeover from a transcription-based to a splicing-based mode of sexual differentiation. We propose that the canonical insect tra-dsx pathway evolved via merger between expanding dsx function (from males to both sexes) and narrowing tra function (from a general splicing factor to dedicated regulator of dsx).

scholarly journals Structural evolution of the tissue-specific U2AF2 paralog and alternative splicing factor LS2

2020 ◽  
Author(s):  
Ashish Ashok Kawale ◽  
J. Matthew Taliaferro ◽  
Hyun-Seo Kang ◽  
Christoph Hartmüller ◽  
Arie Geerlof ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Rna Binding ◽  
Mutational Analysis ◽  
Splicing Factor ◽  
Rna Recognition Motif ◽  
Sequencing Data ◽  
Tissue Specific ◽  
Molecular Features ◽  
G Quadruplex

AbstractThe Drosophila melanogaster LS2 protein is a tissue-specific paralog of U2AF2 that mediates testis-specific alternative splicing. In order to understand the structural mechanisms underlying the distinct RNA binding specificity we determined the solution structures of the LS2 RNA recognition motif (RRM) domains and characterized their interaction with cis-regulatory guanosine-rich RNA motifs found in intron regions upstream of alternatively spliced exons. We show that the guanosine-rich RNA adopts a G quadruplex (G4) fold in vitro. The LS2 tandem RRMs adopt canonical RRM folds that are connected by a 38-residue linker that harbors a small helical motif α0. The LS2 RRM2 domain and the α0 helix in the interdomain linker mediate interactions with the G4 RNA. The functional importance of these unique molecular features in LS2 is validated by mutational analysis in vitro and RNA splicing assays in vivo. RNA sequencing data confirm the enrichment of G4-forming LS2 target motifs near LS2-affected exons. Our data indicate a role of G quadruplex structures as cis-regulatory motifs in introns for the regulation of alternative splicing, that engage non-canonical interactions with a tandem RRM protein. These results highlight the intriguing molecular evolution of a tissue-specific splicing factor from its conserved U2AF2 paralog as a result of (retro-) gene duplication in D. melanogaster.

scholarly journals SRSF1 Is a Mediator of Radiation-Induced Alternative Splicing in B-Lymphocytes

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1341-1341
Author(s):  
Niema Razavian ◽  
Vivian Cheung
Keyword(s):  
Alternative Splicing ◽  
B Lymphocytes ◽  
Cellular Response ◽  
Rna Binding ◽  
Radiation Treatment ◽  
Exon Skipping ◽  
Splicing Factor ◽  
Splicing Factors ◽  
Alternatively Spliced ◽  
Radiation Induced

Abstract Ionizing radiation is used in the treatment of Hodgkin and non-Hodgkin lymphomas (Spetch et al. 2014; Illidge et al. 2014). Despite its effectiveness, radiation is a "blunt" therapy that damages indiscriminately both cancer and normal cells, and can result in secondary malignancies (Dores et al. 2002). To better understand cellular response to radiation, we examined alternative splicing, and its regulation, in irradiated human cells. To accomplish this, we exposed cultured B-lymphocytes from 10 individuals to 10 Gy of ionizing radiation, and performed RNA sequencing before, and two and six hours after radiation treatment. With these data, we first identified alternative splicing events. From about 60 million reads per sample, we detected over 20,000 alternatively spliced events. In total, 1,600 events were radiation responsive (ANOVA, FDR<5%). The splicing events in irradiated cells belong mainly to three categories, each of which occurred in genes with distinct biological functions. Cassette exons, which were the most numerous splicing event, were found primarily in DNA damage response and apoptotic genes, while alternative first or last exons were found in chromatin assembly genes; retained introns occurred in genes involved in RNA processing and translation. Using these radiation-responsive events, we then examined the kinetics of this response. We found that changes in alternative splicing were acute, occurring within two hours of radiation treatment. Overall, the splicing events produced shorter transcripts in irradiated cells. Next, we looked for how alternative splicing is regulated in response to radiation. To identify putative mediators of this response, we determined the expression levels of over 60 trans-acting splicing factors. We found that the expression level of 26 splicing factors changed significantly in irradiated cells (ANOVA, FDR<5%). Of these putative mediators, we further examined serine/arginine-rich splicing factor (SRSF1)'s role in radiation response. First, we assessed SRSF1 expression in irradiated cells. Following radiation exposure, the transcript and protein expressions of SRSF1 decreased. Next, we performed motif enrichment analysis to identify target genes. SRSF1 RNA-binding sites were enriched in skipped cassette exons: specifically, of the 362 skipped cassette exons in irradiated cells, 93 (26%) contained SRSF1 binding motifs. Finally, we examined the link between SRSF1 and exon skipping in irradiated cells more closely. For example, in the transcripts for RNA-binding protein 3 (RBM3), SRSF1 binds an RBM3 exon that contains a premature termination codon (Sanford et al. 2009), and mediates exclusion of this exon. We confirmed experimentally that this results in an increase in RBM3 protein expression. Together, our data demonstrate that alternative splicing is a key part of cellular response to radiation, and SRSF1 plays a role in mediating exon skipping. In this presentation, we will describe radiation-induced alternative splicing by discussing the genes that are alternatively spliced, and its regulation by the splicing factor SRSF1. Disclosures No relevant conflicts of interest to declare.

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