Modifications to toxic CUG RNAs induce structural stability, rescue mis-splicing in a myotonic dystrophy cell model and reduce toxicity in a myotonic dystrophy zebrafish model

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.

Pseudouridine Modification Inhibits Muscleblind-like 1 (MBNL1) Binding to CCUG Repeats and Minimally Structured RNA through Reduced RNA Flexibility

“…Based on the distribution of base separation distances, unmodified RNA also tends to form stacking interactions, but the population of those conformations is much less abundant than the range of open configurations. These data and our previous work (20) suggest that ⌿ modifications can generally be used to shift YGCY motifs into conformations with increased base stacking that reduce binding by MBNL proteins.…”

“…These results show that ⌿ has a modest inhibitory effect on MBNL1 binding to the YGCYbinding sites in the CCUG repeats. This effect is significantly less than that observed with ⌿ modification of CUG repeats (20).…”

“…Replacing Uridine with ⌿ in CCUG Repeat RNA Increases Structural Stability-Previous studies showed that ⌿ increased the thermal stability of CUG repeat RNA in a helical conformation (20). Here, we determined whether ⌿ had a similar effect on stabilization of CCUG repeats.…”

“…In contrast, stabilization of CUG repeats with two or four ⌿s resulted in a larger increase in thermal stability (13 or 19°C for two or four ⌿ modifications, respectively) (20) and displayed a more dramatic effect on MBNL1 binding (Ͼ20-fold loss of binding) compared with CCUG repeat binding. A possible explanation for the difference in MBNL1 binding observed between CUG and CCUG repeats with ⌿ modification is that CCUG repeats are relatively less stable compared with CUG repeats (the observed T m of (CUG) 4 was ϳ59°C (20) and that of CCUG repeat RNA 1 was ϳ47°C) and the increased stability provided by the ⌿ modification to the CCUG repeats was not sufficient to strongly inhibit MBNL1 binding. Another possibility is that modifying both pyrimidines of the YGCY motif is necessary for robust inhibition of MBNL1 binding.…”

“…X-ray crystallography data suggested that ⌿ stabilized the CUG repeats through water bridging via hydrogen bonding of the N1-H of ⌿ with the uridine on the opposing strand (20). In this work, we have extended our studies to determine that ⌿ can stabilize CCUG repeat RNA and minimally structured RNA containing YGCY motifs and that this modification inhibits MBNL1 binding.…”