MBNL splicing factors regulate the microtranscriptome of skeletal muscles (mouse, skeletal muscle, developmental panel)

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. MicroRNA (miRNA) sequencing from skeletal muscles (quadriceps) of WT postnatal day 1 (n=3), postnatal day 5 (n=3), postnatal day 14 (n=3) and postnatal day 90 (n=3) mice肌盲样蛋白（Muscleblind-like proteins, MBNLs）可调控多种RNA加工过程，包括可变剪接、多聚腺苷酸化、RNA稳定性以及mRNA的细胞内定位。1型肌强直性营养不良（Myotonic dystrophy type 1, DM1）是成人最常见的肌肉萎缩症，患者体内的MBNLs会被携带有扩增CUG重复序列的毒性RNA捕获，进而破坏MBNL调控的生理过程，引发DM1的疾病特征。本研究阐明了MBNLs在骨骼肌出生后发育过程中调控微小转录组动态变化，以及在DM1小鼠模型与患者体内出现的microRNA (miRNA)失调中的重要作用。我们筛选出多种对MBNL蛋白不足敏感的miRNA，并发现其中多数在出生后阶段受到调控，这与该过程中MBNL活性的升高密切相关。在成年Mbnl1基因敲除小鼠中，miRNA的表达谱呈现出从成年向新生小鼠的转变。我们明确了MBNLs影响miRNA表达水平的两种机制：其一，MBNL缺失会引发miRNA前体的转录变化；其二，MBNLs通过调控miRNA初级转录本的可变剪接，影响miRNA的生物发生过程。我们提出，由同一基因簇转录产生的miR-23b、miR-27b以及miR-24-1的表达，依赖于MBNL介导的、包含miRNA序列的可变外显子的保留。本研究结果表明，DM1中MBNL的捕获现象是导致miRNA活性异常的部分原因。本研究为MBNL蛋白作为骨骼肌miRNA表达调控因子的生物学功能提供了全新的研究视角。本研究的测序数据来源于野生型（Wild Type, WT）小鼠出生后第1天（n=3）、第5天（n=3）、第14天（n=3）及第90天（n=3）的骨骼肌（股四头肌）的miRNA测序结果。