Colocalization of muscleblind with RNA foci is separable from mis-regulation of alternative splicing in myotonic dystrophy

• Published in: Journal of cell science Vol. 118; no. 13; pp. 2923 - 2933
• Main Authors: Ho, Thai H, Savkur, Rajesh S, Poulos, Michael G, Mancini, Michael A, Swanson, Maurice S, Cooper, Thomas A
• Format: Journal Article
• Language: English
• Published: England 01.07.2005
• Subjects: Adult; Alternative Splicing; Cell Line; Cell Nucleus - genetics; Cell Nucleus - metabolism; Exons; Fibroblasts - metabolism; Fibroblasts - ultrastructure; Gene Expression; Green Fluorescent Proteins - genetics; Green Fluorescent Proteins - metabolism; Humans; In Situ Hybridization, Fluorescence; Myotonic Dystrophy - classification; Myotonic Dystrophy - genetics; Myotonic Dystrophy - metabolism; RNA - genetics; RNA - metabolism; RNA-Binding Proteins - genetics; RNA-Binding Proteins - metabolism; Time Factors; Trinucleotide Repeat Expansion - genetics
• ISSN: 0021-9533; 1477-9137
• DOI: 10.1242/jcs.02404

Myotonic dystrophy type I (DM1), which is caused by a non-coding CTG-repeat expansion in the dystrophia myotonica-protein kinase (DMPK) gene, is an RNA-mediated disease. Expanded CUG repeats in transcripts of mutant DMPK form nuclear foci that recruit muscleblind-like (MBNL) proteins, a family of alternative splicing factors. Although transcripts of mutant DMPK and MBNL proteins accumulate in nuclear RNA foci, it is not clear whether foci formation is required for splicing mis-regulation. Here, we use a co-transfection strategy to show that both CUG and CAG repeats form RNA foci that colocalize with green fluorescent protein (GFP)-MBNL1 and endogenous MBNL1. However, only CUG repeats alter splicing of the two tested pre-mRNAs, cardiac troponin T (cTNT) and insulin receptor (IR). Using FRAP, we demonstrate that GFP-MBNL1 in CUG and CAG foci have similar half-times of recovery and fractions of immobile molecules, suggesting that GFP-MBNL1 is bound by both CUG and CAG repeats. We also find an immobile fraction of GFP-MBNL1 in DM1 fibroblasts and a similar rapid exchange in endogenous CUG RNA foci. Therefore, formation of RNA foci and disruption of MBNL1-regulated splicing are separable events.