Deciphering pachytene piRNA function

破译粗线期 piRNA 功能

• 批准号: 9902461
• 负责人: ZISSIMOS MOURELATOS
• 金额: $ 32.2万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-08-01 至 2022-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9902461
• 关键词: Adult; Affect; Animals; Arginine; Binding; Binding Proteins; Binding Sites; Biochemical; Biological; Cell Nucleus; Cellular biology; Cytoplasmic Granules; DNA Polymerase II; Development; Disease; Exhibits; Germ; Germ Cells; Housekeeping; In Vitro; Kinetics; Knock-in Mouse; Knowledge; Laboratories; Lead; Length; Liquid substance; Male Infertility; Mammals; Maps; Mediating; Meiosis; Messenger RNA; MicroRNAs; Modeling; Mus; Outcome; Pachytene Stage; Phase; Phenocopy; Play; Population; Process; Property; Protein Family; Proteins; RNA; RNA Binding; Reproduction; Retrotransposon; Ribonucleoproteins; Role; Site; Small RNA; Sorting - Cell Movement; Specificity; Spermatids; Spermatocytes; Spermatogenesis; Spermiogenesis; Structure; Testing; Testis; Time; Transcript; Translating; Translations; Untranslated RNA; Wild Type Mouse; in vivo; in vivo evaluation; insight; male; male fertility; messenger ribonucleoprotein; metaplastic cell transformation; mouse model; mutant; novel; predictive modeling; transcriptome

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.

破译粗线期皮尔纳功能 项目总结/摘要 哺乳动物减数分裂Ⅰ粗线期出现一类神秘的小RNA 精母细胞它们由长的非编码RNA加工而成，与Piwil 1结合（在小鼠中通常 已知为Miwi）蛋白，并且称为粗线期piwi相互作用（pi）RNA。Miwi/piRNA是 对精子生成和男性生育力至关重要。我们的实验室发现，在不同的物种中， 装载有piRNA的蛋白质在特定的寡核苷酸上被对称二甲基化， Tdrd蛋白是一种与Tudor结构域（Tdrd）蛋白相互作用的蛋白质。Miwi绑定 Tdrd 6是一种含有六个典型Tudor结构域的蛋白质，它们共同构成了 类染色体是一种大的、细胞质的、非膜结合的结构， mRNA沿着粗线期piRNA。 粗线期piRNA非常丰富;它们甚至在密切相关的物种中也不保守; 它们的序列多样性是巨大的;它们的功能仍然是一个谜。假说 它们的作用需要协调两个看似矛盾的特性：像microRNA，粗线期， piRNA被加载到Argonaute蛋白Miwi上，并且可以作为结合RNA靶标的向导。 与microRNA不同，它们的序列多样性是如此巨大，以至于它们可以以多种方式结合任何mRNA。 位点，从而失去序列驱动的特异性。在本申请中，我们提出了一种全新的概念， 框架来破解粗线期皮尔纳功能之谜。我们将检验这个假设， 粗线期piRNA通过动态捕获非精子生成的转录物在分选转录物中起关键作用， Miwi-piRNA-Tdrd 6组装体中的mRNA，其形成拟染色体体的核心。在我们的模型中， Miwi/piRNA之间的多价相互作用，其以部分互补性与mRNA结合， 在Miwi和Tdrd 6的多个Tudor结构域之间，使拟染色体体成核， 螯合捕获的mRNA，以便在精子形成过程中最终消除。该模型预测 较长的mRNA优先被捕获，因为它们含有更多的piRNA结合位点，而 需要翻译以驱动精子细胞分化的生精mRNAs应该更短， 避免陷入困境。我们还将测试Tdrd 6-Miwi/piRNA-mRNA中的多价相互作用是否与Tdrd 6-Miwi/piRNA-mRNA中的多价相互作用有关。 组装导致液-液相分离，这是类染色体形成的基础。 我们有信心，我们提出的多个，正交，在体外和体内的方法， 将阐明预期和意外的结果，并真正揭示难以捉摸的功能， 哺乳动物粗线期piRNA。