Mechanisms of Decay of Toxic CUGn RNA in DM1 Patients
In patients with DM1, the mutant DMPK mRNA containing expanded CUG repeats forms nuclear foci. It is expected that the disassembly of these foci will reverse the disease phenotype. It is also expected that degradation of mutant DMPK mRNA would reduce toxicity of CUG repeats. However, recent data suggest that the short products of degradation of the mutant DMPK mRNA are more toxic than un-degraded RNA containing long CUG repeats. (1) It has been recently found that the accumulation of multiple copies of short CUG repeats of normal size produces DM1-like phenotype (myotonia, muscular dystrophy and cardiac conduction defects), while mice with low number of copies of long CUG repeats were asymptomatic; (2) Our recent data on the mechanism of DM2, which is associated with expansion of untranslated CCTG repeats showed that accumulation of normal size of RNA CCUG repeats leads to the DM2-specific abnormalities. Thus, toxicity of CUG repeat expansion in DM1 patients might be associated with the products of degradation of the mutant DMPK mRNA, while undegraded mutant RNA might be not toxic.
To examine this hypothesis, we propose to compare the effect of un-degraded (stable) and decayed CUG transcripts on DM1-specific pattern of RNA processing (translation and splicing). Time curve for mutant CUGn RNA decay will be determined after transcription pulse of CUGn-containing transcripts using tet-responsive promoter. We will examine whether nuclear CUG foci are formed by non-degraded or degraded mutant CUG repeats and whether CUG foci formation increases or reduces toxicity of CUGn repeats. We will determine the period of life of CUGn mutant transcript during which it has highest toxicity. We will also address the mechanisms regulating stability and decay of normal and mutant DMPK mRNA. Regulatory regions within the 3' UTR of DMPK controlling DMPK mRNA turnover will be determined. Protein factors regulating normal and mutant DMPK mRNA stability and decay will be identified. The results of this study will show when mutant CUGn repeats become toxic and will help to develop approach to reduce CUGn RNA toxicity by controlling decay of the mutant DMPK mRNA.
Relevance of the project to IDDRC mission:
Identification of the stage at which RNA CUG expansion becomes toxic will help to develop the approaches to reduce toxicity of CUG RNA in muscle and, perhaps, in brain of DM1 patients. If nuclear CUG foci are protective, then the approaches stabilizing these foci will be developed. If the products of degradation of CUG RNA are toxic, then the approaches preventing degradation of mutant DMPK mRNA will be developed.