卵细胞成熟相关蛋白MARF1的结构与底物识别机制研究

The abnormality of oogenic process is one of the major reasons of female infertility. Oogenic process is essential for producing fertilization-competent oocyte, including accurate completion of two-step meiosis, cytoplasmic maturation and maintenance of genomic integrity. Su et al. found that MARF1 (meiosis arrest female 1) is required for these process and mutation of Marf1 cause meiotic arrest and female infertility. Further study demonstrated that MARF1 regulates cytoplasmic maturation and retrotransposon silencing by suppressing levels of specific transcripts. However, its underlying molecular mechanism is unknown. This projects will focus on three aspects of structure and molecular mechanism of MARF1: (1) elucidating the crystal structures of the novel domains (NYN, RRM and LOTUS domains) and fragments of MARF1 using X-ray crystallography; (2) assessing the RNA binding and nuclease activity of MARF1 by the biochemical study, and obtaining the specific RNA binding motif by an in vitro selection method combined SELEX (Systematic Evolution of Ligands by exponential enrichment) and NGS (Next-generation sequencing); (3) studying the structures of full-length MARF1 protein and its complex with substrates by a combination of X-ray crystallography and Cryo-EM techniques, and revealing the molecular mechanism of substrate recognition and degradation. The aim of this project is to illuminate the molecular structure of MARF1 and its recognition of substrates, and to provide basis for further study of regulation mechanism of MARF1 for oogenic process, which will be of significance for prevention and treatment of female infertility and development of more effective medicine for birth control.

卵细胞成熟是一个复杂的生理过程，包括细胞核成熟和细胞质成熟，并通过两次减数分裂过程完成。苏有强等发现Marf1（meiosis arrest female 1）基因突变表现为卵细胞减数分裂停滞和雌性不育的表型，但其调控的分子机制还不清楚。本项目将围绕MARF1的结构和机制开展以下三方面研究：（1）通过X射线晶体学解析MARF1蛋白含有的结构域（NYN，RRM和LOTUS结构域）或者片段的晶体结构；（2）通过生化实验研究MARF1结合和降解RNA底物的活性，获得MARF1底物特异性结合的核酸序列；（3）结合X射线晶体学和冷冻电镜技术研究全长MARF1蛋白及其结合RNA底物复合物的结构，揭示其识别底物并降解的分子机制。本项目的目标是首次阐明MARF1的结构以及作用底物的分子机制，将为深入研究MARF1对卵细胞成熟的调控机制打下基础，对防治女性不孕不育以及研制新型更有效的女性避孕药具有重要意义。

卵细胞成熟是一个复杂的生理过程，包括细胞核成熟和细胞质成熟，并通过两次减数分裂过程完成。本项目合作者苏有强等发现Marf1(meiosis arrest female 1)基因突变表现为卵细胞减数分裂停滞和雌性不育的表型。进一步的研究表明MARF1可能通过抑制特定的转录本调控逆转座子的沉默和细胞质成熟。但其调控的分子机制还不清楚。本项目围绕MARF1的结构和作用机制展开了以下三方面的研究：（1）通过X射线晶体学解析MARF1蛋白含有的结构域(NYN，RRM和LOTUS结构域)或者片段的晶体结构;（2）通过生化实验研究MARF1结合和降解 RNA底物的活性，获得MARF1底物特异性结合的核酸序列；（3）结合X射线晶体学和冷冻电镜技术研究全长MARF1蛋白及其结合RNA底物复合物的结构，揭示其识别底物并降解的分子机制。项目按计划基本完成，解析了MARF1的三个结构域NYN、RRM和LOTUS的晶体结构；通过体外活性和结合实验阐明了MARF1的NYN结构域作为核酸酶和结合RNA底物的结构特征；通过CLIP-seq的方法获得了MARF1的LOTUS结构域结合底物RNA的序列特征，并通过体外结合实验进行了验证；对于全长MARF1蛋白，通过分析多个相互作用蛋白（EDC4和DDX6），为完整揭示MARF1结合底物的分子机制奠定基础。上述部分结果已总结发表，其余结果正在总结，等待合作者共同发表。本项目的研究成果对防治女性不孕不育以及研制新型更有效的女性避孕药具有可能的应用价值。