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 HnRNPA1 and Its Effect in the Expression of ABCC (ABCC4 and ABCC6) Transporter in Glioma Cell Lines

2020 ◽  
Author(s):  
Tundup Namgail ◽  
Dinesh Kumar ◽  
Vidhi Vashistha ◽  
Ahmed Aquib ◽  
Ajay Yadav
Keyword(s):  
Drug Resistance ◽  
Brain Tumor ◽  
Alternative Splicing ◽  
Cell Line ◽  
Reverse Transcription ◽  
Glioma Cell ◽  
Glioma Cell Line ◽  
Splicing Factor ◽  
Glioma Cell Lines ◽  
Alternative Splicing Factor

Glioblastoma multiforme (GBM) is classified as WHO grade IV Astrocytoma & is the most common highly aggressive form of primary brain tumor. Garde IV tumor are highly recurrent even after treatment, with patient survival rate is less than two years from the time of diagnosis. This might be due to overexpression of one of the factor such as ATP-binding cassette transporters (ABC transporters) responsible for drug resistance. ABCC transporter family a member of ABC transporter was found to mostly responsible for multi drug resistance (MDR) in cancer cells. On the other hand, heterogeneous nuclear ribonucleoprotein (hnRNP) an alternative splicing factor play different role in various cellular process such as nucleic acid metabolism, transcription and translation regulation, among them hnRNPA1 is best studied and its aberrant deregulation favor development of cancer. This study was focused on to study the function of hnRNPA1 in the expression analysis of ABCC transporter (responsible for MDR) in glioma cell lines. The expression of ABCC transporter (ABCC4 and ABCC6) gene was examined in two glioma cell line i.e. U87MG and T98G in normal and knockdown two alternative variants of hnRNPA1 by Quantitative Realtime PCR and Reverse Transcription PCR. We found that ABCC4 was significantly overexpressed in hnRNPA1 Variant 2 knockdown cells (si hnRNPA1 V2) in U87 (3-fold) and in T98G (18.34-fold), While hnRNPA1 Variant 1 knockdown cells (hnRNPA1 V1i) does not shows any significant effect. Further, the expression of ABCC6 was decreased in both hnRNPA1 V1i (0.40-fold) and hnRNPA1 V2i (0.48-fold) in U87. Reverse transcription based result were complemented with normal PCR based detection strategy after running in agarose gel for U87MG and T98G glioma cell line. Finally, this result indicates that hnRNPA1 an alternative splicing factor regulate the expression of ABCC4 and ABCC6 transporter which are responsible for multiple drug resistance in cancer. This information will help in future for the development of an alternative method for the treatment of drug resistance cases in brain tumor and other tumors by targeting hnRNPA1 splicing factor.

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.

scholarly journals Distinctive Features of Drosophila Alternative Splicing Factor RS Domain: Implication for Specific Phosphorylation, Shuttling, and Splicing Activation

2001 ◽  
Vol 21 (4) ◽  
pp. 1345-1359 ◽  
Author(s):  
Eric Allemand ◽  
Renata Gattoni ◽  
Henri-Marc Bourbon ◽  
James Stevenin ◽  
Javier F. Cáceres ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Structural Features ◽  
Splicing Factor ◽  
Sr Protein ◽  
Developmental Defects ◽  
Distinctive Features ◽  
Carboxyl Terminal ◽  
Alternative Splicing Factor ◽  
Transgenic Flies

ABSTRACT The human splicing factor 2, also called human alternative splicing factor (hASF), is the prototype of the highly conserved SR protein family involved in constitutive and regulated splicing of metazoan mRNA precursors. Here we report that the Drosophila homologue of hASF (dASF) lacks eight repeating arginine-serine dipeptides at its carboxyl-terminal region (RS domain), previously shown to be important for both localization and splicing activity of hASF. While this difference has no effect on dASF localization, it impedes its capacity to shuttle between the nucleus and cytoplasm and abolishes its phosphorylation by SR protein kinase 1 (SRPK1). dASF also has an altered splicing activity. While being competent for the regulation of 5′ alternative splice site choice and activation of specific splicing enhancers, dASF fails to complement S100-cytoplasmic splicing-deficient extracts. Moreover, targeted overexpression of dASF in transgenic flies leads to higher deleterious developmental defects than hASF overexpression, supporting the notion that the distinctive structural features at the RS domain between the two proteins are likely to be functionally relevant in vivo.

scholarly journals Splicing-associated chromatin signatures: a combinatorial and position-dependent role for histone marks in splicing definition

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
E. Agirre ◽  
A. J. Oldfield ◽  
N. Bellora ◽  
A. Segelle ◽  
R. F. Luco
Keyword(s):  
Alternative Splicing ◽  
Rna Binding ◽  
Embryonic Stem ◽  
Chromatin Modifications ◽  
Histone Marks ◽  
Splicing Regulators ◽  
Machine Learning Approach ◽  
Alternatively Spliced ◽  
Rna Motif ◽  
Regulation Changes

AbstractAlternative splicing relies on the combinatorial recruitment of splicing regulators to specific RNA binding sites. Chromatin has been shown to impact this recruitment. However, a limited number of histone marks have been studied at a global level. In this work, a machine learning approach, applied to extensive epigenomics datasets in human H1 embryonic stem cells and IMR90 foetal fibroblasts, has identified eleven chromatin modifications that differentially mark alternatively spliced exons depending on the level of exon inclusion. These marks act in a combinatorial and position-dependent way, creating characteristic splicing-associated chromatin signatures (SACS). In support of a functional role for SACS in coordinating splicing regulation, changes in the alternative splicing of SACS-marked exons between ten different cell lines correlate with changes in SACS enrichment levels and recruitment of the splicing regulators predicted by RNA motif search analysis. We propose the dynamic nature of chromatin modifications as a mechanism to rapidly fine-tune alternative splicing when necessary.

scholarly journals The Combined Human Genotype of Truncating TTN and RBM20 Mutations Is Associated with Severe and Early Onset of Dilated Cardiomyopathy

Genes ◽  
2021 ◽  
Vol 12 (6) ◽  
pp. 883
Author(s):  
Anna Gaertner ◽  
Julia Bloebaum ◽  
Andreas Brodehl ◽  
Baerbel Klauke ◽  
Katharina Sielemann ◽  
...  
Keyword(s):  
Heart Failure ◽  
Dilated Cardiomyopathy ◽  
Early Onset ◽  
Target Genes ◽  
Frameshift Mutation ◽  
Rna Binding ◽  
Splicing Factor ◽  
Binding Motif ◽  
Rich Domain ◽  
Generation Sequencing

A major cause of heart failure is cardiomyopathies, with dilated cardiomyopathy (DCM) as the most common form. Over 40 genes are linked to DCM, among them TTN and RBM20. Next Generation Sequencing in clinical DCM cohorts revealed truncating variants in TTN (TTNtv), accounting for up to 25% of familial DCM cases. Mutations in the cardiac splicing factor RNA binding motif protein 20 (RBM20) are also known to be associated with severe cardiomyopathies. TTN is one of the major RBM20 splicing targets. Most of the pathogenic RBM20 mutations are localized in the highly conserved arginine serine rich domain (RS), leading to a cytoplasmic mislocalization of mutant RBM20. Here, we present a patient with an early onset DCM carrying a combination of (likely) pathogenic TTN and RBM20 mutations. We show that the splicing of RBM20 target genes is affected in the mutation carrier. Furthermore, we reveal RBM20 haploinsufficiency presumably caused by the frameshift mutation in RBM20.

scholarly journals RBMX suppresses tumorigenicity and progression of bladder cancer by interacting with the hnRNP A1 protein to regulate PKM alternative splicing

Oncogene ◽  
2021 ◽  
Author(s):  
Qiuxia Yan ◽  
Peng Zeng ◽  
Xiuqin Zhou ◽  
Xiaoying Zhao ◽  
Runqiang Chen ◽  
...  
Keyword(s):  
Bladder Cancer ◽  
Alternative Splicing ◽  
Rna Binding ◽  
Aerobic Glycolysis ◽  
Histological Grade ◽  
Migration And Invasion ◽  
Binding Motif ◽  
Hnrnp A1

AbstractThe prognosis for patients with metastatic bladder cancer (BCa) is poor, and it is not improved by current treatments. RNA-binding motif protein X-linked (RBMX) are involved in the regulation of the malignant progression of various tumors. However, the role of RBMX in BCa tumorigenicity and progression remains unclear. In this study, we found that RBMX was significantly downregulated in BCa tissues, especially in muscle-invasive BCa tissues. RBMX expression was negatively correlated with tumor stage, histological grade and poor patient prognosis. Functional assays demonstrated that RBMX inhibited BCa cell proliferation, colony formation, migration, and invasion in vitro and suppressed tumor growth and metastasis in vivo. Mechanistic investigations revealed that hnRNP A1 was an RBMX-binding protein. RBMX competitively inhibited the combination of the RGG motif in hnRNP A1 and the sequences flanking PKM exon 9, leading to the formation of lower PKM2 and higher PKM1 levels, which attenuated the tumorigenicity and progression of BCa. Moreover, RBMX inhibited aerobic glycolysis through hnRNP A1-dependent PKM alternative splicing and counteracted the PKM2 overexpression-induced aggressive phenotype of the BCa cells. In conclusion, our findings indicate that RBMX suppresses BCa tumorigenicity and progression via an hnRNP A1-mediated PKM alternative splicing mechanism. RBMX may serve as a novel prognostic biomarker for clinical intervention in BCa.

Abstract 12192: MBNL1 Directly Regulates the Alternative Splicing of mRNAs That Promote Myofibroblast Differentiation

Circulation ◽  
2014 ◽  
Vol 130 (suppl_2) ◽  
Author(s):  
Jennifer Davis ◽  
Michelle Sargent ◽  
Jianjian Shi ◽  
Lei Wei ◽  
Maurice S Swanson ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Molecular Mechanisms ◽  
Rna Binding ◽  
Transforming Growth Factor ◽  
Ventricular Remodeling ◽  
Cardiac Injury ◽  
Myofibroblast Differentiation ◽  
Genome Wide ◽  
A Genome

Rationale: During the cardiac injury response fibroblasts differentiate into myofibroblasts, a cell type that enhances extracellular matrix production and facilitates ventricular remodeling. To better understand the molecular mechanisms whereby myofibroblasts are generated in the heart we performed a genome-wide screen with 18,000 cDNAs, which identified the RNA-binding protein muscleblind-like splicing regulator 1 (MBNL1), suggesting a novel association between mRNA alternative splicing and the regulation of myofibroblast differentiation. Objective: To determine the mechanism whereby MBNL1 regulates myofibroblast differentiation and the cardiac fibrotic response. Methods and Results: Confirming the results from our genome wide screen, adenoviral-mediated overexpression of MBNL1 promoted transformation of rat cardiac fibroblasts and mouse embryonic fibroblasts (MEFs) into myofibroblasts, similar to the level of conversion obtained by the profibrotic agonist transforming growth factor β (TGFβ). Antithetically, Mbnl1 -/- MEFs were refractory to TGFβ-induced myofibroblast differentiation. MBNL1 expression is induced in transforming fibroblasts in response to TGFβ and angiotensin II. These results were extended in vivo by analysis of dermal wound healing, a process dependent on myofibroblast differentiation and their proper activity. By day 6 control mice had achieved 82% skin wound closure compared with only 40% in Mbnl1 -/- mice. Moreover, Mbnl1 -/- mice had reduced survival following myocardial infarction injury due to defective fibrotic scar formation and healing. High throughput RNA sequencing (RNAseq) and RNA immunoprecipitation revealed that MBNL1 directly regulates the alternative splicing of transcripts for myofibroblast signaling factors and cytoskeletal-assembly elements. Functional analysis of these factors as mediators of MBNL1 activity is also described here. Conclusions: Collectively, our data suggest that MBNL1 coordinates myofibroblast transformation by directly mediating the alternative splicing of an array of mRNAs encoding differentiation-specific signaling transcripts, which then alter the fibroblast proteome for myofibroblast structure and function.

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