Summary Transcription of expanded microsatellite repeats is associated with multiple human diseases, including myotonic dystrophy, Fuchs' endothelial corneal dystrophy, and C9orf72-ALS/FTD. Reducing production of RNA and proteins arising from these expanded loci holds therapeutic benefit. Here, we tested the hypothesis that deactivated Cas9 enzyme impedes transcription across expanded microsatellites. We observed a repeat length-, PAM-, and strand-dependent reduction of repeat-containing RNAs upon targeting dCas9 directly to repeat sequences; targeting the non-template strand was more effective. Aberrant splicing patterns were rescued in DM1 cells, and production of RAN peptides characteristic of DM1, DM2, and C9orf72-ALS/FTD cells was drastically decreased. Systemic delivery of dCas9/gRNA by adeno-associated virus led to reductions in pathological RNA foci, rescue of chloride channel 1 protein expression, and decreased myotonia. These observations suggest that transcription of microsatellite repeat-containing RNAs is more sensitive to perturbation than transcription of other RNAs, indicating potentially viable strategies for therapeutic intervention.

A CTG repeat expansion in the DMPK gene is the causative mutation of myotonic dystrophy type 1 (DM1). Transcription of the expanded CTG repeat produces toxic gain-of-function CUG RNA, leading to disease symptoms. A screening platform that targets production or stability of the toxic CUG RNA in a selective manner has the potential to provide new biological and therapeutic insights. A DM1 HeLa cell model was generated that stably expresses a toxic r(CUG)480 and an analogous r(CUG)0 control from DMPK and was used to measure the ratio-metric level of r(CUG)480 versus r(CUG)0. This DM1 HeLa model recapitulates pathogenic hallmarks of DM1, including CUG ribonuclear foci and missplicing of pre-mRNA targets of the muscleblind (MBNL) alternative splicing factors. Repeat-selective screening using this cell line led to the unexpected identification of multiple microtubule inhibitors as hits that selectively reduce r(CUG)480 levels and partially rescue MBNL-dependent missplicing. These results were validated by using the Food and Drug Administration-approved clinical microtubule inhibitor colchicine in DM1 mouse and primary patient cell models. The mechanism of action was found to involve selective reduced transcription of the CTG expansion that we hypothesize to involve the LINC (linker of nucleoskeleton and cytoskeleton) complex. The unanticipated identification of microtubule inhibitors as selective modulators of toxic CUG RNA opens research directions for this form of muscular dystrophy and may shed light on the biology of CTG repeat expansion and inform therapeutic avenues. This approach has the potential to identify modulators of expanded repeat-containing gene expression for over 30 microsatellite expansion disorders.

The muscleblind-like (MBNL) family of proteins are key developmental regulators of alternative splicing. Sequestration of MBNL proteins by expanded CUG/CCUG repeat RNA transcripts is a major pathogenic mechanism in the neuromuscular disorder myotonic dystrophy (DM). MBNL1 contains four zinc finger (ZF) motifs that form two tandem RNA binding domains (ZF1–2 and ZF3–4) which each bind YGCY RNA motifs. In an effort to determine the differences in function between these domains, we designed and characterized synthetic MBNL proteins with duplicate ZF1–2 or ZF3–4 domains, referred to as MBNL-AA and MBNL-BB, respectively. Analysis of splicing regulation revealed that MBNL-AA had up to 5-fold increased splicing activity while MBNL-BB had 4-fold decreased activity compared to a MBNL protein with the canonical arrangement of zinc finger domains. RNA binding analysis revealed that the variations in splicing activity are due to differences in RNA binding specificities between the two ZF domains rather than binding affinity. Our findings indicate that ZF1–2 drives splicing regulation via recognition of YGCY RNA motifs while ZF3–4 acts as a general RNA binding domain. Our studies suggest that synthetic MBNL proteins with improved or altered splicing activity have the potential to be used as both tools for investigating splicing regulation and protein therapeutics for DM and other microsatellite diseases.

The Muscleblind (MBL) protein family is a deeply conserved family of RNA binding proteins that regulate alternative splicing, alternative polyadenylation, RNA stability and RNA localization. Their inactivation due to sequestration by expanded CUG repeats causes symptoms in the neuromuscular disease myotonic dystrophy. MBL zinc fingers are the most highly conserved portion of these proteins, and directly interact with RNA. We identified putative MBL homologs in Ciona intestinalis and Trichoplax adhaerens, and investigated their ability, as well as that of MBL homologs from human/mouse, fly and worm, to regulate alternative splicing. We found that all homologs can regulate alternative splicing in mouse cells, with some regulating over 100 events. The cis-elements through which each homolog exerts its splicing activities are likely to be highly similar to mammalian Muscleblind-like proteins (MBNLs), as suggested by motif analyses and the ability of expanded CUG repeats to inactivate homolog-mediated splicing. While regulation of specific target exons by MBL/MBNL has not been broadly conserved across these species, genes enriched for MBL/MBNL binding sites in their introns may play roles in cell adhesion, ion transport and axon guidance, among other biological pathways, suggesting a specific, conserved role for these proteins across a broad range of metazoan species.