Myotonic dystrophy (DM) is a dominantly-inherited myo- and neuro- degenerative disease. The genetic basis is an expanded CTG repeat in the 3' untranslated region of the DMPK gene. The expanded CUG repeat triggers the nuclear retention of mutant DMPK transcripts, but the resulting underexpression of DMPK does not fully account for the severe phenotype. One proposed disease mechanism is that nuclear accumulation of expanded CUG repeats interferes with nuclear function. In support of this hypothesis, we show that the manifestations of DM in muscle, including the retention of mutant transcripts in myonuclei, are largely reproduced by the expression of an expanded, untranslated CTG repeat in transgenic mice. To investigate the mechanism for this effect, we studied the structural features and protein interactions of CUG repeat RNAs in vitro. CUG repeats form highly stable hairpins that interact with double-stranded RNA binding proteins (dsRBPs). Furthermore, pathologically expanded CUG repeats can activate PKR, the dsRNA-activated protein kinase. These observations suggest a novel mechanism whereby an imperfect RNA duplex triggers a pathologic state via length-dependent interactions with RNA binding proteins.
The aim of this proposal is to demonstrate the feasibility of ligand-inducible expression of untranslated CTG repeats. Transgenic mice will be developed in which untranslated CTG repeats can be induced/de-induced specifically in skeletal muscle or non-specifically in all tissues. This system will be used subsequently to study the nuclear accumulation and clearance of expanded CUG repeats, the activation of dsRBPs, and the susceptibility of muscle and other organs to (CUG)n-mediated toxicity at different developmental stages. A parallel system for transfer of ligand-inducible transgenes into cultured myogenic cells will be developed to determine the length- threshold for nuclear retention of CUG repeats, and to identify other sequence motifs that may cause similar phenomena of nuclear accumulation. These experimental systems will provide useful tools for studying the disease mechanism in myotonic dystrophy and other muscular dystrophies.
