Muscleblind-like 1 (Mbnl1) promotes insulin receptor exon 11 inclusion via binding to a downstream evolutionarily conserved intronic enhancer.
ABSTRACT The insulin receptor exists as two isoforms, IR-A and IR-B, which result from alternative splicing of exon 11 in the primary transcript. These two isoforms show a cell-specific distribution, and their relative proportions also vary during development, aging, and in different disease states. We have previously demonstrated that both intron 10 and the alternatively spliced exon 11 contain regulatory sequences that affect insulin receptor splicing both positively and negatively and that these sequences bind the serine/arginine-rich (SR) proteins SRp20 and SF2/ASF and the CELF protein CUG-BP1. In this study, we describe a new intronic splicing element within intron 11 that is highly conserved across species. Using minigenes carrying deletion mutations within intron 11, we demonstrated that this sequence functions as an intronic splicing enhancer. We subsequently used RNA affinity chromatography to identify Mbnl1 as a splicing factor that recognizes this enhancer. By ribonucleoprotein immunoprecipitation, we also established that Mbnl1 binds specifically to the INSR (insulin receptor gene) RNA. Overexpression or knockdown of Mbnl1 in hepatoma and embryonic kidney cells altered the levels of exon 11 inclusion. Finally, we showed that deletion of the intronic enhancer eliminates the ability of Mbnl1 to promote exon inclusion. Collectively, these findings demonstrate a role for Mbnl1 in controlling insulin receptor exon 11 inclusion via binding to a downstream intronic enhancer element.
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ABSTRACT: CELF/Bruno-like proteins play multiple roles, including the regulation of alternative splicing and translation. These RNA-binding proteins contain two RNA recognition motif (RRM) domains at the N-terminus and another RRM at the C-terminus. CUGBP2 is a member of this family of proteins that possesses several alternatively spliced exons. The present study investigated the expression of exon 14, which is an alternatively spliced exon and encodes the first half of the third RRM of CUGBP2. The ratio of exon 14 skipping product (R3δ) to its inclusion was reduced in neuronal cells induced from P19 cells and in the brain. Although full length CUGBP2 and the CUGBP2 R3δ isoforms showed a similar effect on the inclusion of the smooth muscle (SM) exon of the ACTN1 gene, these isoforms showed an opposite effect on the skipping of exon 11 in the insulin receptor gene. In addition, examination of structural changes in these isoforms by molecular dynamics simulation and NMR spectrometry suggested that the third RRM of R3δ isoform was flexible and did not form an RRM structure. Our results suggest that CUGBP2 regulates the splicing of ACTN1 and insulin receptor by different mechanisms. Alternative splicing of CUGBP2 exon 14 contributes to the regulation of the splicing of the insulin receptor. The present findings specifically show how alternative splicing events that result in three-dimensional structural changes in CUGBP2 can lead to changes in its biological activity.BMC Biochemistry 03/2012; 13:6. · 1.99 Impact Factor
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ABSTRACT: An increasing body of evidence connects alterations in the process of alternative splicing with cancer development and progression. However, a direct role of splicing factors as drivers of cancer development is mostly unknown. We analyzed the gene copy number of several splicing factors in colon and lung tumors and found that the gene encoding for the splicing factor SRSF6 is amplified and overexpressed in these cancers. Moreover, overexpression of SRSF6 in immortal lung epithelial cells enhanced proliferation, protected them from chemotherapy-induced cell death and converted them to be tumorigenic in mice. In contrast, knockdown of SRSF6 in lung and colon cancer cell lines inhibited their tumorigenic abilities. SRSF6 up- or down regulation altered the splicing of several tumor suppressors and oncogenes to generate the oncogenic isoforms and reduce the tumor suppressive isoforms. Our data suggests that the splicing factor SRSF6 is an oncoprotein which regulates proliferation and survival of lung and colon cancer cells. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.The Journal of Pathology 11/2012; · 6.32 Impact Factor
Chapter: hnRNP and hnRNP‑Like Proteins in Splicing Control: Molecular Mechanisms and Implication in Human Pathologies[show abstract] [hide abstract]
ABSTRACT: The heterogeneous nuclear ribonucleoprotein (hnRNP) family includes a diverse group of RNA binding proteins to which we are affiliating here other structurally and functionally related proteins (e.g., Nova, Sam68, ESRP, Fox, TDP‑43, Hu, CUG‑BP, MBNL and TIA proteins). These hnRNP and hnRNP‑like proteins make important contributions to protein diversity and activity by modulating the alternative splicing of a large repertoire of pre‑mRNAs. They achieve this function through a variety of molecular strategies ranging from directly preventing the recognition of splice sites, antagonizing or helping the assembly of positive regulatory complexes, interfering with spliceosome assembly and changing the conformation of pre‑mRNAs. In addition to regulating key splicing events, defects in the expression of these proteins are now being documented for a growing number of human diseases including cancer. hnRNP and hnRNP‑like proteins therefore represent a group of proteins whose roles in the control of splicing pervade all areas of biology and human health.01/2011: pages 1-25;