跨膜转录因子MYRF控制miRNA lin-4在发育中表达上调的机制研究

MicroRNAs（miRNAs）are an abundant class of small (~22 nt) noncoding regulatory RNAs. The abnormal expression of miRNAs usually correlates with developmental defect and serious diseases, thus it is necessary to uncover the regulatory mechanism underlying miRNA expression. lin-4, the founding member of miRNA superfamily discovered 20 years ago, is important for epidermis differentiation, gonad morphogenesis, neuronal development, normal life span, etc. The expression of lin-4 in C. elegans increases sharply at late Larval stage 1 (L1). It is unclear what factors are responsible for up-regulating the lin-4 at late L1. MYRF (myelin-regulatory transcription factor) family proteins are recently identified, conserved, membrane-bound transcription factors. MYRF are critical for embryonic development, and MYRFs' transcriptional targets remain unknown during early development. We found that dysfunction of MYRF eliminated the increased expression of lin-4 at late L1, suggesting that MYRF plays an important role in up-regulating lin-4 expression at late L1. Here we propose to study the genetic interaction between lin-4 and myrf, by examining whether MYRF regulates lin-4 in cell-autonomous manner and whether tissue specific ablation of MYRF will produce reiterated cell-division similar to lin-4 mutant. We further examine whether MYRF regulates lin-4 directly, and identify MYRF's DNA-binding sequence in lin-4 promoter. To explore whether MYRF regulates the expression of a series of miRNAs during L1, we will compare miRNAs expression between wild type and MYRF mutants via miRNA sequencing. Our proposed work will elucidate regulatory mechanisms of miRNA expression and provide support in developing therapeutics for diseases associated with miRNA abnormality.

MicroRNAs（miRNA）是一类～22nt的非编码RNA，其异常表达常常与发育异常和重大疾病相关，揭示其表达的调控机制显得尤为重要。作为首次发现并被熟知的miRNA lin-4，与生长发育，细胞分化等密切相关。lin-4的表达在L1后期迅速上调，但其调控因子未见报道。MYRF是一类新型的跨膜转录因子，在胚胎发育中不可或缺，其调控发育的作用靶点未知。我们发现线虫中MYRF功能缺失后lin-4在L1后期表达未见上调，推测MYRF对此时期lin-4的表达起重要作用。本项目拟系统探讨MYRF对lin-4表达是否起关键调控作用，进而确定MYRF对lin-4是否有直接的转录调控，鉴别MYRF的结合DNA的位点序列，建立MYRF对 lin-4表达的调控机理。我们将进一步检测MYRF是否对同时期表达的一系列miRNA具有转录调控。该项目为治疗miRNA异常导致的重大疾病具有重要科学意义和参考价值。

MicroRNAs（miRNA）是一类～22nt的非编码RNA，其异常表达常常与发育异常和重大疾病 相关，揭示其表达的调控机制显得尤为重要。作为首次发现并被熟知的miRNA lin-4，与生长发育，细胞分化等密切相关。lin-4的表达在L1后期迅速上调，但其调控因子未见报道。MYRF 是一类新型的跨膜转录因子，在胚胎发育中不可或缺，其调控发育的作用靶点未知。我们发现 线虫中MYRF功能缺失后lin-4在L1后期表达未见上调，推测MYRF对此时期lin-4的表达起重要作用。本项目使用CRISPR-Cas9技术引入生物素诱导的降解系统（AID系统）在不同发育阶段降解MYRF-1，发现MYRF-1在每个幼虫发育阶段促进Lin-4的转录上调。通过转基因方式，制作组织特异性回补和敲除MYRF-1突变体，发现lin-4的转录也随着特定的回补和敲除发生组织特异性的改变，推测MYRF-1以细胞自主的方式调控转录。之后我们制作了MYRF-1 N端提前入核突变体，结果显示lin-4在发育早期甚至胚胎期就发生了转录，说明MYRF-1提前发挥转录因子活性驱动了lin-4转录过早上调。通过在细胞内引入大量的DNA质粒造成MYRF-1蛋白异常大量聚集，暗示MYRF-1通过N端结合启动子区域调控转录。使用MYRF-1突变体进行了miRNA测序分析，并结合转录报告荧光蛋白验证，结果显示MYRF-1 调控一系列发育时序相关miRNA的转录表达。该研究系统的探讨了MYRF-1蛋白在调控miRNA中发挥着关键作用，发现了MYRF-1调控miRNA转录的新功能，同时miRNA的转录调控也取得了突破性进展，为治疗miRNA异常导致 的重大疾病具有重要科学意义和参考价值。