Myotonic dystrophy type 2 (DM2) is an autosomal dominant form of muscular dystrophy, resulting from expansion of a CCTG repeat in CNBP, the gene encoding Cellular Nucleic Acid Binding Protein. The discovery of the DM2 mutation provided critical support for the concept that expanded repeats in non-coding regions give rise to dominant acting RNAs. A unifying model for RNA toxicity in myotonic dystrophy type 1 (DM1), DM2, and potentially other repeat expansion diseases is that RNA binding proteins are sequestered by expanded RNA repeats, giving rise to pervasive changes in the transcriptome when the functions of RNA binding proteins are lost. However, 3 of the 4 commonest RNA dominant diseases result from repeat expansions in introns. Once transcribed, introns are rapidly excised and degraded, therefore the concept that intronic repeats act as a sink for RNA binding proteins seems implausible. More surprisingly, recent studies also indicate that intronic expanded repeats in patients with DM2 or familial ALS undergo translation in the central nervous system, through a process of Repeat Associated Non-AUG (RAN) translation, resulting in production of deleterious tetra- or di-peptide repeat proteins. Whether this occurs in skeletal muscle has not been determined. Therapeutic development for DM1 has advanced rapidly, but has hardly begun for DM2, partly due to the lack of animal models. DM1 and DM2 many share many clinical features, but also have important differences. For example, DM1 often effects skeletal muscle in infants, leading to congenital myopathy, but this does not occur in DM2.
In Aim 1 of this proposal we have developed transgenic mouse models of DM2, that express expanded CCUG repeat (CCUGexp) RNA in an intron or in the 3? untranslated region. To better understand differences between DM1 and DM2, we will use these models, and previous models for DM1, to compare toxicities and transcriptomic effects of expanded RNA repeats in introns versus exons, and the relative toxicities of CCUG versus CUG repeats. We will also study the metabolism of intronic RNA repeats. In particular, we will follow up on preliminary data suggesting that processing of an intron containing expanded CCUG repeats is altered, leading to intron retention.
In Aim 2 we will determine the conditions under which RAN translation of intronic CCUGexp RNA occurs in skeletal muscle in vivo. Using transgenic mice, and an electroporation model for transient expression of CCUGexp RNA, we will quantify RAN translation, and test the hypothesis that RAN translation is promoted or pre-conditioned by muscle stress. Finally in Aim 3 we will study therapeutic effects in the new mouse models, using antisense oligonucleotide and small molecule drugs. Overall, this project will clarify mechanisms important to DMpathogenesis and initiate the process of therapeutic development for DM2.
| Sznajder, ?ukasz J; Thomas, James D; Carrell, Ellie M et al. (2018) Intron retention induced by microsatellite expansions as a disease biomarker. Proc Natl Acad Sci U S A 115:4234-4239 |
| Pinto, Belinda S; Saxena, Tanvi; Oliveira, Ruan et al. (2017) Impeding Transcription of Expanded Microsatellite Repeats by Deactivated Cas9. Mol Cell 68:479-490.e5 |
