Muscleblind-like (MBNL) proteins are key regulators of precursor and mature mRNA metabolism in mammals. Based on published and novel data, we explore models of tissue-specific MBNL interaction with RNA. We portray MBNL domains critical for RNA binding and splicing regulation, and the structure of MBNL's normal and pathogenic RNA targets, particularly in the context of myotonic dystrophy (DM), in which expanded CUG or CCUG repeat transcripts sequester several nuclear proteins including MBNLs. We also review the properties of MBNL/RNA complex, including recent data obtained from UV cross-linking and immunoprecipitation (CLIP-Seq), and discuss how this interaction shapes normal MBNL-dependent alternative splicing regulation. Finally, we review how this acquired knowledge about the pathogenic RNA structure and nature of MBNL sequestration can be translated into the design of therapeutic strategies against DM.

The ability to control pre-mRNA splicing with small molecules could facilitate the development of therapeutics or cell-based circuits that control gene function. Myotonic dystrophy type 1 (DM1) is caused by the dysregulation of alternative pre-mRNA splicing due to sequestration of muscleblind-like 1 protein (MBNL1) by expanded, non-coding r(CUG) repeats (r(CUG)exp). Here we report two small molecules that induce or ameliorate alternative splicing dysregulation. The thiophene-containing small molecule (1) inhibits the interaction of MBNL1 with its natural pre-mRNA substrates. Compound (2), a substituted naphthyridine, binds r(CUG)exp and displaces MBNL1. Structural models show that 1 binds MBNL1 in the Zn-finger domain and that 2 interacts with UU loops in r(CUG)exp. This study provides a structural framework for small molecules that target MBNL1 by mimicking r(CUG)exp and shows that targeting MBNL1 causes dysregulation of alternative splicing, suggesting that MBNL1 is thus not a suitable therapeutic target for the treatment of DM1.

Myotonic dystrophy type 1 (DM1) is an autosomal dominant multisystemic disorder caused by expansion of CTG triplet repeats in 3′-untranslated region of DMPK gene. The pathomechanism of DM1 is driven by accumulation of toxic transcripts containing expanded CUG repeats (CUGexp) in nuclear foci which sequester several factors regulating RNA metabolism, such as Muscleblind-like proteins (MBNLs). In this work, we utilized very short chemically modified antisense oligonucleotides composed exclusively of locked nucleic acids (all-LNAs) complementary to CUG repeats, as potential therapeutic agents against DM1. Our in vitro data demonstrated that very short, 8- or 10-unit all-LNAs effectively bound the CUG repeat RNA and prevented the formation of CUGexp/MBNL complexes. In proliferating DM1 cells as well as in skeletal muscles of DM1 mouse model the all-LNAs induced the reduction of the number and size of CUGexp foci and corrected MBNL-sensitive alternative splicing defects with high efficacy and specificity. The all-LNAs had low impact on the cellular level of CUGexp-containing transcripts and did not affect the expression of other transcripts with short CUG repeats. Our data strongly indicate that short all-LNAs complementary to CUG repeats are a promising therapeutic tool against DM1.

Muscleblind-like proteins (MBNLs) are regulators of RNA metabolism. During tissue differentiation the level of MBNLs increases, while their functional insufficiency plays a crucial role in myotonic dystrophy (DM). Deep sequencing of RNA molecules cross-linked to immunoprecipitated protein particles (CLIP-seq) revealed that MBNL1 binds to MBNL1 exon 1 (e1) encoding both the major part of 5΄UTR and an amino-terminal region of MBNL1 protein. We tested several hypotheses regarding the possible autoregulatory function of MBNL1 binding to its own transcript. Our data indicate that MBNLs induce skipping of e1 from precursor MBNL1 mRNA and that e1 exclusion may impact transcript association with polysomes and translation. Furthermore, e1-deficient protein isoform lacking the first two zinc fingers is highly unstable and its EGFP fusion protein has severely compromised splicing activity. We also show that MBNL1 can be transcribed from three different promoters and that the transcription initiation site determines the mode of e1 regulation. Taken together, we demonstrate that MBNL proteins control steady-state levels of MBNL1 through an interaction with e1 in its precursor mRNA. Insights from our study open a new avenue in therapies against DM based on manipulation of the transcription initiation site and e1 splicing of MBNL1 mRNA.

Muscleblind-like (MBNL) proteins bind to hundreds of pre- and mature mRNAs to regulate their alternative splicing, alternative polyadenylation, stability and subcellular localization. Once MBNLs are withheld from transcript regulation, cellular machineries generate products inapt for precise embryonal/adult developmental tasks and myotonic dystrophy, a devastating multi-systemic genetic disorder, develops. We have recently demonstrated that all three MBNL paralogs are capable of fine-tuning cellular content of one of the three MBNL paralogs, MBNL1, by binding to the first coding exon (e1) of its pre-mRNA. Intriguingly, this autoregulatory feedback loop grounded on alternative splicing of e1 appears to play a crucial role in delaying the onset of myotonic dystrophy. Here, we describe this process in the context of other autoregulatory and regulatory loops that maintain the content and diverse functions of MBNL proteins at optimal level in health and disease, thus supporting the overall cellular homeostasis.