Dose-Dependent Regulation of Alternative Splicing by MBNL Proteins Reveals Biomarkers for Myotonic Dystrophy.

Stacey D Wagner,Eric T Wang,Riti Gupta,Amy E Mahady,Katy Eichinger,Charles A Thornton,Dylan R Farnsworth,Adam J Struck,J Andrew Berglund,Gregory A Cox

doi:10.1371/journal.pgen.1006316

Abstract

Alternative splicing is a regulated process that results in expression of specific mRNA and protein isoforms. Alternative splicing factors determine the relative abundance of each isoform. Here we focus on MBNL1, a splicing factor misregulated in the disease myotonic dystrophy. By altering the concentration of MBNL1 in cells across a broad dynamic range, we show that different splicing events require different amounts of MBNL1 for half-maximal response, and respond more or less steeply to MBNL1. Motifs around MBNL1 exon 5 were studied to assess how cis-elements mediate the MBNL1 dose-dependent splicing response. A framework was developed to estimate MBNL concentration using splicing responses alone, validated in the cell-based model, and applied to myotonic dystrophy patient muscle. Using this framework, we evaluated the ability of individual and combinations of splicing events to predict functional MBNL concentration in human biopsies, as well as their performance as biomarkers to assay mild, moderate, and severe cases of DM.

Highlights

Alternative splicing increases the coding potential of a gene and importantly, allows for regulation of expression of specific isoforms in a developmental and tissue-specific manner
We focused on alternative splicing regulation by MBNL1, an RNA binding protein involved in muscle, heart, and CNS development [8,9]
MBNL proteins bind to YGCY motifs using Zn finger RNA binding motifs [10,11], including sequences found in the toxic RNAs that cause both myotonic dystrophy type 1 (DM1) and myotonic dystrophy type 2 (DM2) [12,13,14,15,16]

Summary

Introduction

Alternative splicing increases the coding potential of a gene and importantly, allows for regulation of expression of specific isoforms in a developmental and tissue-specific manner. Active areas of investigation in the alternative splicing field include how concentrations of alternative splicing factors affect isoform ratios, and what properties determine whether an isoform is responsive to a broad range of splicing factor concentrations or sensitive to a threshold level of activity. To address these questions, we focused on alternative splicing regulation by MBNL1, an RNA binding protein involved in muscle, heart, and CNS development [8,9]. MBNL splicing targets are differentially affected by the disease; DM1 patient samples exhibit a broad range of alternative exon inclusion levels, as compared to control samples containing low numbers of repeats [17,18,19], and MBNL1-dependent splicing events were shown to behave differently in response to different doses of MBNL1 protein [20]

Methods

Results

Discussion

Conclusion