Abstract
Myotonic dystrophy type 1 (DM1) is a complex neuromuscular disorder caused by expansion of a CTG repeat in the 3'-untranslated region (UTR) of the DMPK gene. Mutant DMPK transcripts form aberrant structures and anomalously associate with RNA-binding proteins (RBPs). As a first step toward better understanding of the involvement of abnormal DMPK mRNA folding in DM1 manifestation, we used SHAPE, DMS, CMCT, and RNase T1 structure probing in vitro for modeling of the topology of the DMPK 3'-UTR with normal and pathogenic repeat lengths of up to 197 CUG triplets. The resulting structural information was validated by disruption of base-pairing with LNA antisense oligonucleotides (AONs) and used for prediction of therapeutic AON accessibility and verification of DMPK knockdown efficacy in cells. Our model for DMPK RNA structure demonstrates that the hairpin formed by the CUG repeat has length-dependent conformational plasticity, with a structure that is guided by and embedded in an otherwise rigid architecture of flanking regions in the DMPK 3'-UTR. Evidence is provided that long CUG repeats may form not only single asymmetrical hairpins but also exist as branched structures. These newly identified structures have implications for DM1 pathogenic mechanisms, like sequestration of RBPs and repeat-associated non-AUG (RAN) translation.