Abstract
Myotonic dystrophy type 1 (DM1) is a neuromuscular disorder caused by an expansion of CUG repeats in the 3' UTR of the DMPK gene. The CUG repeats form aggregates of mutant mRNA, which cause misregulation and/or sequestration of RNA-binding proteins, causing aberrant alternative splicing in cells. Previously, we showed that the multi-functional RNA-binding protein Staufen1 (Stau1) was increased in skeletal muscle of DM1 mouse models and patients. We also showed that Stau1 rescues the alternative splicing profile of pre-mRNAs, e.g. the INSR and CLC1, known to be aberrantly spliced in DM1. In order to explore further the potential of Stau1 as a therapeutic target for DM1, we first investigated the mechanism by which Stau1 regulates pre-mRNA alternative splicing. We report here that Stau1 regulates the alternative splicing of exon 11 of the human INSR via binding to Alu elements located in intron 10. Additionally, using a high-throughput RT-PCR screen, we have identified numerous Stau1-regulated alternative splicing events in both WT and DM1 myoblasts. A number of these aberrant ASEs in DM1, including INSR exon 11, are rescued by overexpression of Stau1. However, we find other ASEs in DM1 cells, where overexpression of Stau1 shifts the splicing patterns away from WT conditions. Moreover, we uncovered that Stau1-regulated ASEs harbour Alu elements in intronic regions flanking the alternative exon more than non-Stau1 targets. Taken together, these data highlight the broad impact of Stau1 as a splicing regulator and suggest that Stau1 may act as a disease modifier in DM1.
Highlights
Alternative splicing of pre-mRNAs is a phenomenon allowing multiple mRNA transcripts to be produced from a single pre-mRNA
JC was the recipient of a Canada Research Chair (Tier II) in RNA Metabolism funded through CIHR
Human Insulin Receptor (INSR) alternative exon 11 inclusion is regulated by Stau1
Summary
Alternative splicing of pre-mRNAs is a phenomenon allowing multiple mRNA transcripts to be produced from a single pre-mRNA. Recent reports estimate that 95–100% of human multiexon genes produce two or more mRNA splice variants, with a majority yielding an average of eight variants [1,2,3,4,5] Generation of these variants by alternative splicing is a major mechanism responsible for the complexity of the transcriptome and proteome observed in eukaryotes [6]. There is a variety of conserved RNA secondary structures that interfere with the recognition of splicing signals and influence splice site selection [8, 9] The binding of these regulatory elements by RNA-binding proteins can inhibit or enhance the use of core splice sites and results in alternative splicing. Regulation of alternative splicing is mediated through the intricate interplay between these cis-acting and trans-acting regulatory elements
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