Failure of MBNL1-dependent post-natal splicing transitions in myotonic dystrophy.
ABSTRACT In myotonic dystrophy (DM), expression of RNA containing expanded CUG or CCUG repeats leads to misregulated alternative splicing of pre-mRNA. The repeat-bearing transcripts accumulate in nuclear foci, together with proteins in the muscleblind family, MBNL1 and MBNL2. In transgenic mice that express expanded CUG repeats, we show that the splicing defect selectively targets a group of exons that share a common temporal pattern of developmental regulation. These exons undergo a synchronized splicing switch between post-natal day 2 and 20 in wild-type mice. During this post-natal interval, MBNL1 protein translocates from a predominantly cytoplasmic to nuclear distribution. In the absence of MBNL1, these physiological splicing transitions do not occur. The splicing defect induced by expanded CUG repeats in mature muscle fibers is closely reproduced by deficiency of MBNL1 but not by deficiency of MBNL2. A parallel situation exists in human DM type 1 and type 2. MBNL1 is depleted from the muscle nucleoplasm because of sequestration in nuclear foci, and the associated splicing defects are remarkably similar to those observed in MBNL1 knockout mice. These results indicate that MBNL1 participates in the post-natal remodeling of skeletal muscle by controlling a key set of developmentally regulated splicing switches. Sequestration of MBNL1, and failure to maintain these splicing transitions, has a pivotal role in the pathogenesis of muscle disease in DM.
Article: From dynamic combinatorial 'hit' to lead: in vitro and in vivo activity of compounds targeting the pathogenic RNAs that cause myotonic dystrophy.[show abstract] [hide abstract]
ABSTRACT: The myotonic dystrophies (DM) are human diseases in which the accumulation of toxic RNA (CUG or CCUG) repeats in the cell causes sequestration of splicing factors, including MBNL1, leading to clinical symptoms such as muscle wasting and myotonia. We previously used Dynamic Combinatorial Chemistry to identify the first compounds known to inhibit (CUG)-MBNL1 binding in vitro. We now report transformation of those compounds into structures with activity in vivo. Introduction of a benzo[g]quinoline substructure previously unknown in the context of RNA recognition, as well as other modifications, provided several molecules with enhanced binding properties, including compounds with strong selectivity for CUG repeats over CAG repeats or CAG-CUG duplex RNA. Compounds readily penetrate cells, and improve luciferase activity in a mouse myoblast assay in which enzyme function is coupled to a release of nuclear CUG-RNA retention. Most importantly, two compounds are able to partially restore splicing in a mouse model of DM1.Nucleic Acids Research 04/2012; 40(13):6380-90. · 8.03 Impact Factor
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ABSTRACT: This review presents detailed information about the structure of triplet repeat RNA and addresses the simple sequence repeats of normal and expanded lengths in the context of the physiological and pathogenic roles played in human cells. First, we discuss the occurrence and frequency of various trinucleotide repeats in transcripts and classify them according to the propensity to form RNA structures of different architectures and stabilities. We show that repeats capable of forming hairpin structures are overrepresented in exons, which implies that they may have important functions. We further describe long triplet repeat RNA as a pathogenic agent by presenting human neurological diseases caused by triplet repeat expansions in which mutant RNA gains a toxic function. Prominent examples of these diseases include myotonic dystrophy type 1 and fragile X-associated tremor ataxia syndrome, which are triggered by mutant CUG and CGG repeats, respectively. In addition, we discuss RNA-mediated pathogenesis in polyglutamine disorders such as Huntington's disease and spinocerebellar ataxia type 3, in which expanded CAG repeats may act as an auxiliary toxic agent. Finally, triplet repeat RNA is presented as a therapeutic target. We describe various concepts and approaches aimed at the selective inhibition of mutant transcript activity in experimental therapies developed for repeat-associated diseases.Nucleic Acids Research 09/2011; 40(1):11-26. · 8.03 Impact Factor
Article: New function for the RNA helicase p68/DDX5 as a modifier of MBNL1 activity on expanded CUG repeats.[show abstract] [hide abstract]
ABSTRACT: Myotonic Dystrophy type I (DM1) is caused by an abnormal expansion of CTG triplets in the 3' UTR of the dystrophia myotonica protein kinase (DMPK) gene, leading to the aggregation of the mutant transcript in nuclear RNA foci. The expanded mutant transcript promotes the sequestration of the MBNL1 splicing factor, resulting in the misregulation of a subset of alternative splicing events. In this study, we identify the DEAD-box RNA helicase p68 (DDX5) in complexes assembled onto in vitro-transcribed CUG repeats. We showed that p68 colocalized with RNA foci in cells expressing the 3'UTR of the DMPK gene containing expanded CTG repeats. We found that p68 increased MBNL1 binding onto pathological repeats and the stem-loop structure regulatory element within the cardiac Troponin T (TNNT2) pre-mRNA, splicing of which is misregulated in DM1. Mutations in the helicase core of p68 prevented both the stimulatory effect of the protein on MBNL1 binding and the colocalization of p68 with CUG repeats, suggesting that remodeling of RNA secondary structure by p68 facilitates MBNL1 binding. We also found that the competence of p68 for regulating TNNT2 exon 5 inclusion depended on the integrity of MBNL1 binding sites. We propose that p68 acts as a modifier of MBNL1 activity on splicing targets and pathogenic RNA.Nucleic Acids Research 12/2011; 40(7):3159-71. · 8.03 Impact Factor