Proteomic analysis of murine Piwi proteins reveals a role for arginine methylation in specifying interaction with Tudor family members

In germ cells, Piwi proteins interact with a specific class of small non-coding RNAs, piwi-interacting RNAs (piRNAs). Together, these form a pathway that represses transposable elements, thus safeguarding germ cell genomes. While basic models describe the operation of piRNA pathways, neither the protein compositions of Piwi complexes, the critical protein-protein interactions that drive small RNA production and target recognition, or the precise molecular consequences of conserved localization to germline structures, call nuage, is well understood. We purified the three murine Piwi family proteins, Mili, Miwi, and Miwi2, from mouse germ cells and characterized their interacting protein partners. Piwi proteins were found in complex with Prmt5/Wdr77, an enzyme that di-methylates arginine residues. By immunoprecipitation with specific antibodies and by mass spectrometry, we found that Piwi proteins are arginine methylated at conserved positions in their amino termini. These modifications are essential to direct complex formation with specific Tudor-domain proteins, whose interactions with Piwis can be required for localization of RNP complexes in cytoplasmic nuage, proper piRNA expression, and transposon silencing. Considered together, our findings indicate that arginine methylation drives the assembly of multi-protein machines whose integrity and specific sub-cellular localization is necessary for efficient function of the piRNA pathway. Keywords: gene regulation study Total small RNA in embryonic and post-birth mouse testes of tdrd1 and tdrd6 mutants在生殖细胞中，Piwi蛋白(Piwi)与一类特定的小型非编码RNA——Piwi互作RNA（piRNAs，piwi-interacting RNAs）——发生相互作用。二者共同构成一条可抑制转座因子的通路，从而保护生殖细胞基因组的完整性。尽管已有基础模型阐释了piRNA通路的运作机制，但目前对于Piwi复合物的蛋白组成、驱动小型RNA生成与靶标识别的关键蛋白-蛋白相互作用，以及这类蛋白保守定位于被称为nuage的生殖细胞结构的精确分子效应，仍缺乏充分认知。本研究从小鼠生殖细胞中纯化得到三种鼠源Piwi家族蛋白——Mili、Miwi及Miwi2，并鉴定了其互作蛋白。研究发现Piwi蛋白与Prmt5/Wdr77形成复合物，后者是一种可对精氨酸残基进行二甲基化修饰的酶。通过特异性抗体免疫沉淀与质谱分析技术，我们发现Piwi蛋白的氨基末端保守位点存在精氨酸甲基化修饰。这类修饰对于介导Piwi蛋白与特定Tudor结构域蛋白(Tudor-domain)形成复合物至关重要；而Piwi蛋白与Tudor结构域蛋白的相互作用，对于核糖核蛋白(Ribonucleoprotein, RNP)复合物在细胞质nuage中的定位、正常piRNA表达以及转座因子沉默均不可或缺。综合来看，本研究结果表明，精氨酸甲基化驱动了多蛋白复合体的组装，而这类多蛋白复合体的完整性与特定亚细胞定位，是piRNA通路高效发挥功能的必要条件。关键词：基因调控研究；tdrd1与tdrd6突变小鼠胚胎及出生后睾丸中的总小型RNA