METTL14介导m6A RNA甲基化调控pri-miRNA加工影响鸭成肌细胞增殖分化的机制研究

越来越多的研究表明m6A RNA甲基化动态可逆修饰调控pri-miRNA的加工。项目组前期明确了鸭胚胎期胸肌生长发育规律，分离和鉴定了其发育相关的miRNA，克隆了鸭m6A相关酶，并在组织和细胞上分析了其表达，检测了整体RNA m6A水平，初步证明m6A调控pri-miRNA加工影响成肌细胞增殖和分化，但具体调控机制尚不清楚。本项目拟通过MeRIP-seq、免疫荧光、motif定点突变等技术挖掘鸭成肌细胞发育中受m6A调控的pri-miRNA（m6A-pri-miRNA）；研究METTL14对m6A-pri-miRNA和骨骼肌卫星细胞增殖分化的影响；m6A-pri-miRNA成熟体对METTL14干扰或过表达的表型补偿验证；挖掘METTL14调控pri-miRNA的m6A motif序列及其功能。结果将明确m6A对pri-miRNA加工的表观遗传学机制，并为鸭转录后调控提供新的靶点。

More and more studies have shown that m6A RNA methylation dynamically reversible modification regulated the processing of primay microRNAs splicing. Our study group has found the myogenesis regular pattern of breast muscle during embryonic stage, and the development-related microRNAs were isolated and identified in the early stage. The m6A-related enzymes of duck and their expression were cloned and analyzed in tissues and cells. The overall level of RNA m6A was detected. It was preliminarily proved that m6A regulated the processing of primay microRNAs splicing and affected the proliferation and differentiation of myoblasts, However the specific regulatory mechanism is still unclear. By methylated RNA immunoprecipitation sequening, immunofluorescence and motif site-directed mutagenesis techniques, this study will excavate pri-microNA regulated by m6A (m6A-pri-microNA) during the development of duck myoblasts; explore the effects of METTL14 on the proliferation and differentiation of m6A-pri-microNA in skeletal muscle satellite cells; verify the phenotypic compensation of interference or overexpression of METTL14 by m6A-pri-microNA mature bodies; and dig the sequence and function of m6A motif of primay microRNAs regulated by METTL14 The results will clarify the epigenetic mechanism of m6A regular primay microRNAs splicing, and provide new post-transcriptional regulatory sites of duck.