Abstract
CUG repeat expansions in the 3′ UTR of dystrophia myotonica protein kinase (DMPK) cause myotonic dystrophy type 1 (DM1). As RNA, these repeats elicit toxicity by sequestering splicing proteins, such as MBNL1, into protein–RNA aggregates. Structural studies demonstrate that CUG repeats can form A-form helices, suggesting that repeat secondary structure could be important in pathogenicity. To evaluate this hypothesis, we utilized structure-stabilizing RNA modifications pseudouridine (Ψ) and 2′-O-methylation to determine if stabilization of CUG helical conformations affected toxicity. CUG repeats modified with Ψ or 2′-O-methyl groups exhibited enhanced structural stability and reduced affinity for MBNL1. Molecular dynamics and X-ray crystallography suggest a potential water-bridging mechanism for Ψ-mediated CUG repeat stabilization. Ψ modification of CUG repeats rescued mis-splicing in a DM1 cell model and prevented CUG repeat toxicity in zebrafish embryos. This study indicates that the structure of toxic RNAs has a significant role in controlling the onset of neuromuscular diseases.
Highlights
Myotonic dystrophy is a genetic disorder that is the most common adult-onset form of muscular dystrophy, affecting ∼1 in 8000 people
Pseudouridine and 2 -O-methyl modifications increase the thermal stability of short (CUG)4 stem-loops
The modifications were targeted to the uridines because these were proposed as dynamic mismatches [23]. (CUG)4 was synthesized with one, two and four s in place of uridines (Figure 1B)
Summary
Myotonic dystrophy is a genetic disorder that is the most common adult-onset form of muscular dystrophy, affecting ∼1 in 8000 people. A crystal structure by Teplova et al showed how a single MBNL1 zinc finger domain interacts with an RNA containing a YGCY binding site primarily via the Watson–Crick face of the nucleotides [24]. This result suggested that MBNL proteins will not bind or bind weakly to CUG repeats when they are in double-stranded or helical conformation, as the Watson– Crick faces of the bases form hydrogen bonds with the opposite strand, leaving them unavailable for interaction with MBNL. We found that incorporation of into a toxic CUG repeat RNA in a zebrafish model of DM1 ameliorated defects in motor function and enhanced viability
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