Myotonic dystrophy: molecular pathomechanism and therapeutic strategies
Myotonic dystrophies (DMs) are autosomal dominant disorders caused by an expansion of either CTG or CCTG nucleotide repeats in two different genes. The mutated transcripts containing toxic, expanded CUG repeats (CUGexp) or CCUG repeats (CCUGexp) accumulate in nuclei forming RNA foci and sequester some nuclear proteins regulating RNA metabolism, mostly muscleblind-like proteins (MBNLs). MBNLs function as factors regulating RNA metabolism at multiple developmental stages. Using microscopic techniques we showed that MBNL-CUGexp complexes are highly dynamic structures composed of tightly packed, although mobile, MBNL proteins that modulate RNA foci morphology. We also showed that sequestration of MBNL proteins in DM1 and DM2 results in aberrant alternative splicing of hundreds of pre-mRNAs. Some of them showed graded changes that correlated with strength of muscle weakness in patients. They may serve as biomarkers of disease severity and therapeutic response in DM. We also found that among alternative exons significantly misregulated in DM are exons forming alternative 3’ untranslated regions (3’UTRs). Depletion of MBNL proteins in DM leads to misregulation of thousands of alternative polyadenylation events. These findings reveal an additional developmental function for MBNL proteins and demonstrate that DM is characterized by misregulation of pre-mRNA processing at multiple levels. We also tested several strategies to eliminate or reduce the toxic effect of CUGexp in different DM1 models. They include the siRNA-induced degradation of CUGexp and inhibition of nuclear protein sequestration by antisense oligomers (AONs) which specifically bind to CUGexp. Therapeutic AONs and siRNAs induce 1) reduction of the number and size of CUGexp foci, 2) reduction of MBNL sequestration and correction of MBNL-dependent alternative splicing and 3) significant reduction of myotonia. Our data showed that short AONs and siRNA are potential therapeutic agent in DM1 treatment. FINANCIAL SUPPORT: This work was supported by: the Polish National Science Centre [grants 2011/01/B/ NZ1/01603 and 2014/15/NZ2/02453], and the Ministry of Science and Higher Education of the Republic of Poland under the Leading National Research Centre programme [KNOW RNA Research Centre in Poznan (No. 01/ KNOW2/2014)].