Deciphering pachytene piRNA function

破译粗线期piRNA功能 项目摘要/摘要 在哺乳动物减数分裂I的粗线期出现了一类神秘的小RNA 精母细胞。它们是从长的、非编码的RNA中加工出来的，与Piwil1结合(通常在小鼠中 称为Miwi)蛋白，被称为粗线期Piwi相互作用(Pi)RNA。Miwi/piRNA是 对精子发生和男性生育是必不可少的。我们的实验室发现，在不同的物种中，Piwi 装载了piRNAs的蛋白质在特定的精氨酸上对称地二甲基化 与含Tudor结构域(Tdrd)蛋白的相互作用。Miwi绑定 直接与Tdrd6结合，Tdrd6是一种含有六个标准都铎结构域的蛋白质，共同构成了 类染色体是一种大的、细胞质的、无膜结合的结构，它包含许多 MRNAs和粗线期piRNAs。 粗线虫的piRNAs非常丰富，即使在亲缘关系密切的物种中也不保守； 它们的序列多样性是巨大的；它们的功能仍然是一个谜。关于以下方面的假设 它们的作用需要调和两个看似矛盾的属性：像microRNA、粗线虫 PiRNAs被加载到ArgAerte蛋白Miwi上，并可以作为结合RNA靶标的向导。 与microRNAs不同，它们的序列多样性如此之大，以至于它们可以多次结合任何mrna。 因此失去了序列驱动的特异性。在本申请中，我们提出了一个全新的概念 破解粗线虫piRNA功能之谜的框架。我们将检验这一假设 粗线虫piRNAs通过动态捕获非生精作用的转录本，在转录本的分选中起着关键作用。 Miwi-piRNA-Tdrd6组件中的mRNAs，形成类染色体的核心。在我们的模型中， Miwi/piRNAs之间的多价相互作用，与mRNAs具有部分互补性， 而在Miwi和Tdrd6的多个Tudor结构域之间，核化了 隔离剂捕获mRNAs，最终在受精过程中消除。该模型预测， 较长的mRNAs优先被捕获，因为它们包含更多的piRNAs结合位点，而 需要翻译以驱动精子细胞分化的生精mRNAs应该缩短到 避免设置陷阱。我们还将测试Tdrd6-Miwi/piRNA-mRNA中的多价相互作用 组装导致液-液相分离，而液-液分离是形成类色谱体的基础。 我们相信，我们提出的多种、正交、体外和体内的方法 将阐明预期和意想不到的结果，并真正揭示 哺乳动物粗线期piRNAs。

Deciphering pachytene piRNA function PROJECT SUMMARY / ABSTRACT An enigmatic class of small RNAs appears at the pachynema of Meiosis I of mammalian spermatocytes. They are processed from long, non-coding RNAs, bind to Piwil1 (in mouse commonly known as Miwi) protein, and are termed pachytene piwi-interacting (pi) RNAs. Miwi/piRNAs are essential for spermiogenesis and male fertility. Our laboratory discovered that in diverse species, Piwi proteins loaded with piRNAs are symmetrically dimethylated on specific arginines by the methylosome, and mediate interaction with Tudor domain containing (Tdrd) proteins. Miwi binds directly to Tdrd6, a protein that contains six canonical Tudor domains, and together form the core of the chromatoid body, a large, cytoplasmic, non-membrane bound structure that contains numerous mRNAs along with pachytene piRNAs. Pachytene piRNAs are very abundant; they are not conserved even among closely related species; their sequence diversity is enormous; and their function still remains a mystery. Hypotheses about their roles need to reconcile two seemingly contradictory properties: like microRNAs, pachytene piRNAs are loaded to an Argonaute protein, Miwi, and can serve as guides to bind RNA targets. Unlike microRNAs, their sequence diversity is so enormous that they can bind any mRNA at multiple sites, thus losing sequence-driven specificity. In this application we propose a radically new conceptual framework to crack the enigma of pachytene piRNA function. We will test the hypothesis that pachytene piRNAs play a critical role in sorting transcripts, by dynamically trapping non-spermiogenic mRNAs in Miwi-piRNA-Tdrd6 assemblies, which form the core of the chromatoid body. In our model, multivalent interactions between Miwi/piRNAs, which bind with partial complementarity to mRNAs, and between Miwi and the multiple Tudor domains of Tdrd6, nucleate the chromatoid body that sequesters trapped mRNAs for eventual elimination during spermiation. The model predicts that longer mRNAs are preferentially trapped as they contain more binding sites for piRNAs, while spermiogenic mRNAs that need to be translated to drive spermatid differentiation should be shorter to avoid trapping. We will also test whether the multivalent interactions in Tdrd6-Miwi/piRNA-mRNA assemblies lead to liquid-liquid phase separations that underlie the formation of the chromatoid body. We are confident that the multiple, orthogonal, in vitro and in vivo approaches that we propose will illuminate expected and unexpected outcomes and truly uncover the elusive function of mammalian pachytene piRNAs.