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MBNL splicing factors regulate the microtranscriptome of skeletal muscles (mouse, skeletal muscle, Mbnl-KO panel)

ABSTRACT: Muscleblind-like proteins (MBNLs) regulate various RNA-processing steps, including alternative splicing, polyadenylation, RNA stability, and mRNA intracellular localization. In myotonic dystrophy type 1 (DM1), the most common muscular dystrophy in adults, MBNLs are sequestered on toxic RNA containing expanded CUG repeats, which leads to disruption of MBNL-regulated processes and disease features of DM1. Herein, we showed the significance of MBNLs in the regulation of microtranscriptome dynamics during postnatal development of skeletal muscles and in microRNA (miRNA) misregulation observed in mouse models and patients with DM1. We identified multiple miRNAs sensitive to insufficiency of MBNL proteins and revealed that many of them were postnatally regulated, which was correlated with increases in the activity of these proteins during this process. In adult Mbnl1-knockout mice, miRNA expression exhibited an adult-to-newborn shift. We identified two mechanisms through which MBNLs influence miRNA levels. First, MBNL loss induces transcriptional changes in miRNA precursors. Second, MBNLs affect miRNA biogenesis by regulating the alternative splicing of miRNA primary transcripts. We propose that the expression of miR-23b, miR-27b and miR-24-1, produced from the same cluster, depends on the MBNL-sensitive inclusion of alternative exons containing miRNA sequences. Our findings suggest that MBNL sequestration in DM1 is partially responsible for altered miRNA activity. This study provides new insights into the biological roles and functions of MBNL proteins as regulators of miRNA expression in skeletal muscles.

ORGANISM(S): Mus musculus

PROVIDER: GSE245814 | GEO | 2024/08/22

REPOSITORIES: GEO

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Project description:For some neurological disorders, disease is primarily RNA-mediated due to expression of non-coding microsatellite expansion RNAs (RNAexp). Toxicity is thought to result from enhanced binding of proteins to these expansions and depletion from their normal cellular targets. However, experimental evidence for this sequestration model is lacking. Here, we use HITS-CLIP and pre-mRNA processing analysis of human control versus myotonic dystrophy (DM) brains to provide compelling evidence for this RNA toxicity model. MBNL2 binds directly to DM repeat expansions in the brain resulting in depletion from its normal RNA targets with downstream effects on alternative splicing and polyadenylation. Similar RNA processing defects were detected in Mbnl compound knockout mice, highlighted by dysregulation of Mapt splicing and fetal tau isoform expression in adults. These results demonstrate that MBNL proteins are directly sequestered by RNAexp in the DM brain and introduce a powerful experimental tool to evaluate RNA-mediated toxicity in other expansion diseases. HITS-CLIP analysis was performed to identify RNA binding sites of MBNL2 in control, DM type 1 (DM1), and DM type 2 (DM2) autopsy-derived brain (n=3). Two regions of the brain selected for the study included the frontal cortex and hippocampus. Libraries were sequenced and wiggle files were generated for each biological replicate as well as three pooled biological replicates (3BRs) per group (control, DM1, and DM2). In addition, differential CLIP analysis (dCLIP) was performed to normalize binding data between groups and identify statistically significant changes in binding. The dCLIP analysis generated bedgraph files representing normalized binding profiles of MBNL2 in each group (control, DM1, and DM2) for visualization and comparative analysis. PolyA-seq was performed on control, DM1, and DM2 autopsy-derived brain samples (hippocampus and frontal cortex, n=3) as well as wild-type (WT) and Mbnl1; Mbnl2 conditional double knockout (Mbnl1-/-; Mbnl2c/c; Nestin-Cre+/- or DKO) brain (n=3). Libraries were sequenced and the resultant files were processed and aligned to the reference genomes (hg19 and mm10). Further computational processing was performed to remove internal oligo(dT) mis-priming events, identify valid polyA sites, and trim to the exact polyA sites. BedGraph files were generated for each group in human (control, DM1, and DM2) and mouse (WT and Mbnl DKO) for comparative visualization.

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MBNL splicing factors regulate the microtranscriptome of skeletal muscles (mouse, skeletal muscle, Mbnl-KO panel)

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