In vivo discovery of a peptide that prevents CUG–RNA hairpin formation and reverses RNA toxicity in myotonic dystrophy models

Abstract

Myotonic dystrophy type 1 (DM1) is caused by the expansion of noncoding CTG repeats in the dystrophia myotonica-protein kinase gene. Mutant transcripts form CUG hairpins that sequester RNAbinding factors into nuclear foci, including Muscleblind-like-1 protein (MBNL1), which regulate alternative splicing and gene expression. To identify molecules that target toxic CUG transcripts in vivo, we performed a positional scanning combinatorial peptide library screen using a Drosophila model of DM1. The screen identified a D-amino acid hexapeptide (ABP1) that reduced CUG foci formation and suppressed CUG-induced lethality and muscle degeneration when administered orally. Transgenic expression of natural, L-amino acid ABP1 analogues reduced CUG-induced toxicity in fly eyes and muscles. Furthermore, ABP1 reversed muscle histopathology and splicing misregulation of MBNL1 targets in DM1 model mice. In vitro, ABP1 bound to CUG hairpins and induced a switch to a single-stranded conformation. Our findings demonstrate that ABP1 shows antimyotonic dystrophy activity by targeting the core of CUG toxicity.disease model | drug discovery | non-coding RNA disease | medicinal chemistry | RNA secondary structure M yotonic dystrophy type 1 (DM1, OMIM #160900) is an autosomal dominant disease caused by the expansion of a CTG trinucleotide repeat in the 3′ untranslated region (UTR) of the dystrophia myotonica-protein kinase (DMPK) gene. Characteristic symptoms include myotonia, progressive muscle wasting, and cardiac conduction defects, among other systemic manifestations. The molecular mechanisms underlying DM1 pathogenesis are complex and affect a large number of cellular processes (1). However, most data suggest that the main triggering event is a toxic gain-of-function of the expanded CUG RNA. CUG-repeat expansions form double-stranded hairpins that are retained as inclusions within the nucleus. These hairpins recruit a number of transcription and splicing factors, including Muscleblind-like 1 (MBNL1) (2-6). Sequestration of MBNL1 originates a loss of function, which plays a key role in the development of DM1 symptoms. Mbnl1 knockout mice reproduced typical features of DM1, and overexpression of Mbnl1 in a mouse model that expressed CTG repeats reversed these phenotypes (7,8). CTGrepeat expression in mice caused misregulation of at least 156 alternative splicing events. Of these, 128 also occurred in Mbnl1 knockout animals (9-12). The splicing factor CUG-binding protein 1 (CUGBP1) is another key component in the development of DM1 phenotypes. CUGBP1 antagonizes MBNL1 activity in the regulated use of alternative exons in a number of transcripts and is abnormally upregulated in patients with DM1, further contributing to splicing misregulation (13-15).Mahadevan et al. provided the first in vivo proof-of-principle for a therapeutic strategy based on ablating toxic RNA molecules in DM1 (16). They demonstrated that expanded CTG-induced effects could be reverted if CTG-repeat transgene expression was interrupted in a DM1 mouse model. Several ...

RNA-binding proteins in neurodegeneration: mechanisms in aggregate

“…Establishing the significance of such correlations will depend on the development of simpler, more accurate methods for measuring repeat length (Yum et al 2017). Along with these endeavors, ongoing DM research has a broad curative focus, from developing DM gene silencing therapies (Thornton et al 2017) to understanding the structure and role of extended hairpins and loops formed by the repeat sequences (Dere et al 2004;Yuan et al 2007;deLorimier et al 2017) and how their modular structure may be therapeutically targeted (Garcia-Lopez et al 2011;Childs-Disney et al 2012, 2014.…”

RNase H-mediated degradation of toxic RNA in myotonic dystrophy type 1

“…A potential alternative approach is to use combined administration of CAG morpholinos with gapmers targeting different sequences within the DMPK mRNA to avoid binding competition. In addition, several reports have identified small molecules and peptides with the ability to disrupt RNA foci formation (31,32). This opens up the possibility of combining ASOs with other nonantisense strategies for DM1 therapy.…”

The RNA binding KH domain of Spoonbill depletes pathogenic non-coding spinocerebellar ataxia 8 transcripts and suppresses neurodegeneration in Drosophila

“…Earlier reports have identified peptides which can induce formation of single stranded conformation of hairpin structure of expanded DM1 transcripts, leading to reversal of the degenerative phenotype (37). We argued that Spoon might be either depleting toxic SCA8 …”

“…DM1 is associated with CTG repeat expansion in 3'UTR and is known to cause muscle degeneration (37). We investigated whether pathogenic SCA8(CTG112) transcripts also causes toxicity in non-neuronal tissue like muscles.…”