Myotonic dystrophy (DM) is a genetic disorder caused by the expression (as RNA) of expanded CTG or CCTG repeats. The alternative splicing factor MBNL1 is sequestered to the expanded RNA repeats, resulting in missplicing of a subset of pre-mRNAs linked to symptoms found in DM patients. Current data suggest that if MBNL1 is released from sequestration, disease symptoms may be alleviated. We identified the small molecules pentamidine and neomycin B as compounds that disrupt MBNL1 binding to CUG repeats in vitro. We show in cell culture that pentamidine was able to reverse the missplicing of 2 pre-mRNAs affected in DM, whereas neomycin B had no effect. Pentamidine also significantly reduced the formation of ribonuclear foci in tissue culture cells, releasing MBNL1 from the foci in the treated cells. Furthermore, pentamidine partially rescued splicing defects of 2 pre-mRNAs in mice expressing expanded CUG repeats.alternative splicing ͉ triplet repeats ͉ MBNL1 ͉ muscleblind-like 1 ͉ RNA repeats M yotonic dystrophy (DM) is an autosomal dominant genetic disorder that is characterized by a variety of symptoms. There are 2 types of myotonic dystrophy, type 1 (DM1) and type 2 (DM2). DM1 is linked to a (CTG) n repeat expansion in the 3Ј untranslated region of the DMPK gene, whereas DM2 is linked to a (CCTG) n repeat expansion in intron 1 of the ZNF9 gene. The current model is that the expanded repeats are toxic on the RNA level, where either repeat can form a stable structured RNA that aberrantly interacts with proteins in the nucleus (for review see refs. 1, 2).One proposed molecular mechanism that may account for the disease symptoms is that, upon transcription of the expansions, either the CUG or CCUG repeats sequester RNA binding proteins from their normal cellular functions. The protein MBNL1 (Muscleblind-like 1) has been shown to bind both expanded CUG and CCUG repeats in vitro (3-5), and colocalize with these expanded repeats in vivo (6-10). MBNL1 is an alternative splicing factor and its sequestration leads to the missplicing of multiple pre-mRNAs in DM, which is thought to give rise to many of the symptoms of the disease. In support of this model, it has been shown that the disruption of the MBNL1 gene or expression of CUG repeats in mice causes symptoms and missplicing similar to those seen in DM patients (11)(12)(13)(14). Furthermore, disease symptoms can be rescued and missplicing of many pre-mRNAs can be reversed in mice expressing CUG repeats by overexpression of MBNL1 (15).Aside from the overexpression of MBNL1, another possible approach to overcoming the sequestration of MBNL1 is to identify small molecules that specifically bind the CUG repeats and competitively release the sequestered MBNL1. As a step toward identifying a small molecule therapeutic for DM, a small library of molecules known to bind structured nucleic acid were screened for their ability to disrupt an MBNL1-CUG repeat interaction in vitro. Two molecules were identified that strongly disrupted the complex in vitro. Further testing showed tha...

Myotonic dystrophy (DM) is a genetic disorder with multisystemic symptoms that is caused by expression (as RNA) of expanded repeats of CTG or CCTG in the genome. It is hypothesized that the RNA splicing factor muscleblind-like (MBNL) is sequestered to the expanded CUG or CCUG RNAs. Mislocalization of MBNL results in missplicing of a subset of pre-mRNAs that are linked to the symptoms found in DM patients. We demonstrate that MBNL can bind short structured CUG and CCUG repeats with high affinity and specificity. Only 6 base pairs are necessary for MBNL binding: two pyrimidine mismatches and four guanosinecytosine base pairs in a stem. MBNL also has a preference for pyrimidine mismatches, but many other mismatches are tolerated with decreased affinity. We also demonstrate that MBNL binds the helical region of a stem-loop in the endogenous pre-mRNA target, the cardiac troponin T (cTNT) pre-mRNA. The stem-loop contains two mismatches and resembles both CUG and CCUG repeats. In vivo splicing results indicate that MBNL-regulated splicing is dependent upon the formation of stem-loops recognized by MBNL. These results suggest that MBNL may bind all of its RNA substrates, both normal and pathogenic, as structured stem-loops containing pyrimidine mismatches.

Pre-mRNA splicing is the process of removing non-coding introns from RNA transcripts and is carried out by the spliceosome, along with other auxiliary factors. In general, research in splicing has focused on the sequences within the pre-mRNA, without taking into account the structures that these sequences might form. However, we propose that the role of RNA structure deserves more consideration when thinking about splicing mechanisms. RNA structures can inhibit or aid binding of spliceosomal components to the pre-mRNA, or can increase splicing efficiency by bringing important sequences into close proximity. Recent reports have also identified proteins and small molecules that can regulate splicing by modulating RNA structures, thus expanding our knowledge of the mechanisms used to regulate splicing.

Myotonic dystrophy type 1 (DM1) is a genetic disorder linked to a (CTG)n repeat expansion in the 3 untranslated region of the DMPK gene. Upon transcription in the nucleus, the CUG repeats form a stable RNA stem-loop that sequesters the RNA-binding protein MBNL1 from its normal function in the cell. MBNL1 regulates the alternative splicing of many pre-mRNAs, and upon MBNL1's sequestration, the alternative splicing of many genes is misregulated, leading to disease symptoms. MBNL1 is known to bind directly to at least 3 of the pre-mRNAs that it regulates, but how MBNL1 binding mechanistically regulates alternative splicing is unclear. Here, we demonstrate that MBNL1 controls the splicing of exon 5 in the cardiac troponin T (cTNT) pre-mRNA by competing directly with the essential splicing factor U2AF65 for binding at the 3 end of intron 4. When U2AF65 is prevented from binding to the pre-mRNA, the U2 snRNP can no longer be recruited and the following exon is skipped. Furthermore, MBNL1 and U2AF65 appear to compete by binding to mutually exclusive RNA structures. When bound by splicing factors, the 3 end of intron 4 can form either a stem-loop or a single-stranded structure. MBNL1 binds a portion of the intron as a stem-loop, whereas U2AF65 binds the same region in a single-strand structure. Mutations that strengthen the stem-loop decrease U2AF65 binding affinity and also repress exon 5 inclusion, independently of MBNL1. Thus, U2AF65 binding can be blocked either by MBNL1 binding or by the stabilization of RNA secondary structure.muscleblind-like 1 ͉ myotonic dystrophy ͉ alternative splicing A lternative splicing is a fundamentally important process that many organisms use to increase proteomic diversity. Many diseases are known to arise from the mis-regulation of alternative splicing (1). Myotonic dystrophy (DM) is an example of a disease where the alternative splicing of many pre-mRNAs is mis-regulated. The mis-splicing seen in DM is thought to be caused, at least in part, by the mis-localization of the splicing factor MBNL1 (muscleblind-like 1) (2-4). MBNL1 is known to interact directly with 3 pre-mRNAs that are mis-regulated in DM, the cardiac troponin T (cTNT) (5, 6), fast troponin T (7), and SERCA1 (8). It is unclear how many more of the misregulated pre-mRNAs observed in DM may also be directly bound and regulated by MBNL1.We have recently shown that MBNL1 binds a stem-loop within intron 4 of the cardiac troponin T (cTNT) pre-mRNA. This stem-loop is located directly upstream of exon 5, which is mis-regulated in this disease (5, 6). The mechanism used by MBNL1 to repress exon 5 remains undetermined. One mechanism might entail a direct binding competition between MBNL1 and other splicing factors. One of the best articulated models of the regulation of alternative splicing is sex determination in Drosophila melanogaster, where the protein Sex Lethal (Sxl) competes with the splicing factor U2AF65 at the 3Ј end of certain introns [reviewed in ref. 9]. U2AF65 is thought to be 1 of the first splicing factors to bind ...

MicroRNAs (miRNAs) are small regulatory RNAs that are essential in all studied metazoans. Research has focused on the prediction and identification of novel miRNAs, while little has been done to validate, annotate, and characterize identified miRNAs. Using Illumina sequencing,~20 million small RNA sequences were obtained from Caenorhabditis elegans. Of the 175 miRNAs listed on the miRBase database, 106 were validated as deriving from a stem-loop precursor with hallmark characteristics of miRNAs. This result suggests that not all sequences identified as miRNAs belong in this category of small RNAs. Our large data set of validated miRNAs facilitated the determination of general sequence and structural characteristics of miRNAs and miRNA precursors. In contrast to previous observations, we did not observe a preference for the 59 nucleotide of the miRNA to be unpaired compared to the 59 nucleotide of the miRNA*, nor a preference for the miRNA to be on either the 59 or 39 arm of the miRNA precursor stem-loop. We observed that steady-state pools of miRNAs have fairly homogeneous termini, especially at their 59 end. Nearly all mature miRNA-miRNA* duplexes had two nucleotide 39 overhangs, and there was a preference for a uracil in the first and ninth position of the mature miRNA. Finally, we observed that specific nucleotides and structural distortions were overrepresented at certain positions adjacent to Drosha and Dicer cleavage sites. Our study offers a comprehensive data set of C. elegans miRNAs and their precursors that significantly decreases the uncertainty associated with the identity of these molecules in existing databases.