First Identification of RNA-Binding Proteins That Regulate Alternative Exons in the Dystrophin Gene.

Julie Miro,Sylvie Tuffery-Giraud,Mireille Claustres,Emmanuelle Beyne,Michel Koenig,Eva Murauer,Anne-Laure Bougé,Dylan Da Cunha

doi:10.3390/ijms21207803

Julie Miro, Sylvie Tuffery-Giraud + Show 6 more

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https://doi.org/10.3390/ijms21207803

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Abstract

The Duchenne muscular dystrophy (DMD) gene has a complex expression pattern regulated by multiple tissue-specific promoters and by alternative splicing (AS) of the resulting transcripts. Here, we used an RNAi-based approach coupled with DMD-targeted RNA-seq to identify RNA-binding proteins (RBPs) that regulate splicing of its skeletal muscle isoform (Dp427m) in a human muscular cell line. A total of 16 RBPs comprising the major regulators of muscle-specific splicing events were tested. We show that distinct combinations of RBPs maintain the correct inclusion in the Dp427m of exons that undergo spatio-temporal AS in other dystrophin isoforms. In particular, our findings revealed the complex networks of RBPs contributing to the splicing of the two short DMD exons 71 and 78, the inclusion of exon 78 in the adult Dp427m isoform being crucial for muscle function. Among the RBPs tested, QKI and DDX5/DDX17 proteins are important determinants of DMD exon inclusion. This is the first large-scale study to determine which RBP proteins act on the physiological splicing of the DMD gene. Our data shed light on molecular mechanisms contributing to the expression of the different dystrophin isoforms, which could be influenced by a change in the function or expression level of the identified RBPs.

Highlights

The Duchenne muscular dystrophy (DMD) gene, the largest human gene spanning 2.2 Mb on the X chromosome, is composed of 79 exons separated by introns whose size varies widely (107 to 248,401 nucleotides)
SRSF1, SRSF2, hTra2ß, hnRNPA1, hnRNPA2/B1, DAZAP1 and the TAR DNA-binding protein 43 (TDP-43), a protein involved in different aspects of RNA processing that was recently shown to play a role in the expression of muscle-specific genes [27,28,29], were included in the functional screen
We report the first study aimed at determining which RNA-binding proteins (RBPs) regulate splicing of the DMD pre-mRNA in skeletal muscle under physiological conditions

Summary

Introduction

The Duchenne muscular dystrophy (DMD) gene, the largest human gene spanning 2.2 Mb on the X chromosome, is composed of 79 exons separated by introns whose size varies widely (107 to 248,401 nucleotides). In striated skeletal and cardiac muscle, a full-length transcript of 14 kb encodes dystrophin, a subsarcolemmal 427 kDa rod-shaped protein incorporated into a multimolecular membrane-associated protein complex that connects intracellular cytoskeleton to extracellular matrix. This complex ensures membrane stability during muscle contraction and mediates cellular signaling [1,2]. The products of four alternative promoters in downstream introns encode N-terminally truncated proteins, named according to their molecular weight as Dp260, Dp140, Dp116 and Dp71, that are expressed in various organs [4,5]

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