Deciphering targeting rules of splicing modulator compounds: case of TG003.

Maki Sakuma,Kei Iida,Masatoshi Hagiwara

doi:10.1186/s12867-015-0044-6

Abstract

BackgroundRecent advances in the development of small chemical compounds that can modulate RNA splicing brought excitement to the field of splicing-targeting therapy. Splicing-targeting therapy tries to ameliorate the disease by altering the exon combination of transcripts to reduce the undesired effect of genetic mutations. However, the knowledge and tools to understand factors contributing to splicing modulator compound sensitivity have been lacking. Our goal was to establish a method to characterize sequence features found in compound sensitive exons.ResultsHere we developed a comparative transcriptomic approach to explore features that make an exon sensitive to a chemical compound. In this study, we chose TG003, a potential drug for Duchenne muscular dystrophy, and performed RNA-sequencing on samples from human and mouse skeletal muscle cells, with and without TG003 treatments. We compared TG003 responsiveness between homologous exon pairs and identified 21 pairs in which human exons were skip-enhanced but not mouse exons. We compared the sequence features; splice site scores, number of splicing factor binding sites, and properties of branch sequence and polypyrimidine tracts, and found that polypyrimidine tracts were stronger (longer stretches and richer content of consecutive polypyrimidine) in the mouse TG003 insensitive exons. We also compared the features between TG003 skip-enhanced and insensitive exons within the species, and discovered that human TG003 skip-enhanced exons were shorter and had less splicing factor binding sites than the group of human TG003 insensitive exons. Mouse insensitive exons homologous to human TG003 skip-enhanced exons shared these properties. Our results suggested that these features are prerequisites for TG003 skip-enhanced exons and weak polypyrimidine tracts are defining features, which were supported by a decision tree analysis on all cassette exons in human.ConclusionsIn this study we established a comparative transcriptomic approach, which shed lights on how small chemical compounds modulate RNA splicing. The results described here was the first attempt to decipher the targeting rules of a splicing modulator compound. We expect that this approach would contribute to the precise understanding of the mechanism of TG003-induced splicing modulation, expand target diseases of splicing modulators in general, as well as the development of new splicing modulators.Electronic supplementary materialThe online version of this article (doi:10.1186/s12867-015-0044-6) contains supplementary material, which is available to authorized users.

Highlights

Recent advances in the development of small chemical compounds that can modulate RNA splicing brought excitement to the field of splicing-targeting therapy
This study opened the possibility of treatment of Duchenne muscular dystrophy (DMD) with TG003, and we further identified a patient whose mutated exon 27 can be enhanced skipping by TG003, whereas none of the wild type exons are affected by TG003 [11]
Validation of RNA‐seq data In order to investigate the TG003 sensitive exons in the two species, we performed RNA-seq on human skeletal muscle cells and mouse C2C12 cells treated with 20 μM of TG003 or no treatment (0.4 % DMSO) for 4 h

Summary

Introduction

Recent advances in the development of small chemical compounds that can modulate RNA splicing brought excitement to the field of splicing-targeting therapy. In order to compensate for the loss of the functional SMN1 gene, the therapy of Spinal Muscular Atrophy intends to increase the expression of SMN2 gene by enhancing the inclusion of a normally skipped exon 7, which is necessary to produce a functional transcript of SMN2 gene [7]. Another example is one of the therapy strategies of Duchenne muscular dystrophy (DMD), which is to induce the skipping of exons mutated to be poison exons in the dystrophin gene [8]. Other possible target disorders of this exon-skipping strategy include pseudo-exon diseases [9], which are diseases caused by an emergence of an exon in the intronic regions due to genetic mutations that create a de novo splice site

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