miR-140 mutation and skeletal dysplasia [seCLIP]. miR-140 mutation and skeletal dysplasia [seCLIP]

MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression. Heterozygous loss-of-function point mutations of miRNA genes are associated with several human congenital disorders, but neomorphic (gain-of-new-function) mutations in miRNAs due to nucleotide substitutions have not been reported. Here we describe a neomorphic seed region mutation in the chondrocyte-specific, super-enhancer-associated MIR140 gene encoding microRNA-140 (miR-140) in a novel autosomal dominant human skeletal dysplasia. Mice with the corresponding single nucleotide substitution show skeletal abnormalities similar to those of the patients but distinct from those of miR-140-null mice. This mutant miRNA gene yields abundant mutant miR-140-5p expression without miRNA-processing defects. In chondrocytes, the mutation causes widespread derepression of wild-type miR-140-5p targets and repression of mutant miR-140-5p targets, indicating that the mutation produces both loss-of-function and gain-of-function effects. Furthermore, the mutant miR-140-5p seed competes with the conserved RNA-binding protein Ybx1 for overlapping binding sites. This finding may explain the potent target repression and robust in vivo effect by this mutant miRNA even in the absence of evolutionary selection of miRNA–target RNA interactions, which contributes to the strong regulatory effects of conserved miRNAs. Our study presents the first case of a pathogenic gain-of-function miRNA mutation and provides molecular insight into neomorphic actions of emerging and/or mutant miRNAs. Overall design: seCLIP analysis for Ago2 and Ybx1 in chondrocytes with miR-140 G mutation

微小RNA（MicroRNAs，miRNAs）是基因表达的转录后调控因子。miRNA基因的杂合型功能丧失型点突变与多种人类先天性疾病相关，但由核苷酸替换导致的miRNA新效型（获得新功能）突变尚未见报道。本研究在一例新型常染色体显性遗传性人类骨骼发育不良患者中，发现软骨细胞特异性、超级增强子关联的MIR140基因（编码微小RNA-140（microRNA-140，miR-140））存在新效型种子区突变。携带该对应单核苷酸替换的小鼠，呈现出与患者相似的骨骼异常，但与miR-140基因敲除小鼠的表型存在显著差异。该突变型miRNA基因可丰度较高地产生成熟突变型miR-140-5p，且未出现miRNA加工缺陷。在软骨细胞中，该突变可导致野生型miR-140-5p靶基因的广泛去抑制，同时对突变型miR-140-5p的靶基因产生抑制效应，表明该突变同时兼具功能丧失与功能获得双重效应。此外，突变型miR-140-5p的种子区可与保守RNA结合蛋白Ybx1竞争重叠结合位点。这一发现或可解释，为何该突变型miRNA即使在缺乏miRNA-靶RNA相互作用的进化选择（该过程是保守miRNA发挥强效调控作用的核心基础）的情况下，仍能实现对靶基因的强效抑制以及显著的体内生物学效应。本研究首次报道了致病性功能获得型miRNA突变，并为新兴及突变型miRNA的新效型作用机制提供了分子层面的全新见解。实验整体设计：对携带miR-140 G突变的软骨细胞开展Ago2与Ybx1的seCLIP分析。