title: Colocalization of muscleblind with RNA foci is separable from mis-regulation of alternative splicing in myotonic dystrophy.
abstract: 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.
authors.affiliation: Department of Molecular and Cellular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA.
authors.forename: Thai H
authors.initials: TH
authors.lastname: Ho
authors.forename: Rajesh S
authors.initials: RS
authors.lastname: Savkur
authors.forename: Michael G
authors.initials: MG
authors.lastname: Poulos
authors.forename: Michael A
authors.initials: MA
authors.lastname: Mancini
authors.forename: Maurice S
authors.initials: MS
authors.lastname: Swanson
authors.forename: Thomas A
authors.initials: TA
authors.lastname: Cooper
mesh_terms: D000328:Adult; D017398:Alternative Splicing; D002460:Cell Line; D002467:Cell Nucleus / Q000235:genetics* / Q000378:metabolism; D005091:Exons; D005347:Fibroblasts / Q000378:metabolism / Q000648:ultrastructure; D015870:Gene Expression; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D009223:Myotonic Dystrophy / Q000145:classification / Q000235:genetics* / Q000378:metabolism*; D012313:RNA / Q000235:genetics* / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D013997:Time Factors; D019680:Trinucleotide Repeat Expansion / Q000235:genetics
chemical_list: C493663:MBNL1 protein, human; D016601:RNA-Binding Proteins; D049452:Green Fluorescent Proteins; D012313:RNA