Tudor Domain Proteins in Germline Genome Defense

种系基因组防御中的 Tudor 结构域蛋白

• 批准号: 8942455
• 负责人: Chen Chen
• 金额: $ 33万
• 依托单位: MICHIGAN STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-01 至 2019-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8942455
• 关键词: Arginine; Binding; Biochemical; Cells; Complex; DNA Damage; DNA Transposable Elements; Data; Development; Electrophoretic Mobility Shift Assay; Epigenetic Process; Failure; Family; Fertility; Gametogenesis; Genes; Genetic Transcription; Genome; Genomics; Genus - Lotus; Germ Cells; In Vitro; Infertility; Lead; Length; Light; Male Contraceptive Agents; Male Infertility; Mammals; Mediating; Meiosis; Messenger RNA; Methylation; Modeling; Modification; Molecular; Mus; Mutate; Mutation; Parasites; Pathway interactions; Phenocopy; Play; Property; Protein Family; Proteins; R7 Virus; RNA; RNA Binding; RNA Recognition Motif; Recombinants; Recruitment Activity; Retrotransposon; Ribonucleoproteins; Role; Seminal; Slice; Small RNA; Spermatogenesis; Staging; Structure; System; Tertiary Protein Structure; Testing; cell type; crosslink; endonuclease; genome integrity; insight; male; mammalian genome; mouse model; novel; nuclear reprogramming; piRNA; protein complex; protein protein interaction; public health relevance

 DESCRIPTION (provided by applicant): Transposable elements (TEs) are "genomic parasites" that can replicate and re-integrate into the host cell genome. Uncontrolled TE activity in germ cells leads to DNA damage, disruption of gametogenesis, and infertility. In mammalian male germ cells, the Piwi- piRNA pathway uses small RNAs as a guide to silence mobile TEs to protect genome integrity and sustain fertility. A unique family of proteins, the Tudor domain containing proteins (TDRDs), directly bind to Piwi proteins and facilitate their roles in TE silencing and spermatogenesis. The mechanistic involvement of TDRD proteins in conjunction with Piwi proteins in meiotic TE silencing is currently unknown. Mutation of TDRD proteins TDRD5 or TDRD7 alone leads to L1 (the most abundant TE in mammals) activation and male infertility. However, the mechanism by which they are integrated into the Piwi-piRNA pathway to suppress TE expression remains elusive. In addition to the Tudor domain, TDRD5 and TDRD7 also contain the Lotus domain, a novel putative RNA binding domain of unknown function. The overall objective of this proposal is to define the molecular mechanism whereby TDRD5 and TDRD7 post transcriptionally suppress TE activation during meiosis. Our preliminary data indicate that the Lotus domain is an RNA binding domain. We hypothesize that TDRD5 and TDRD7 act as "Tudor adaptors" to directly recruit TE mRNAs to the Piwi protein complex for degradation or sequestration. This is likely achieved by virtue of their Tudor and Lotus domains binding to Piwi proteins and TE mRNAs, respectively. To test this hypothesis, we will use biochemical approaches and mouse models to: 1) Determine the Lotus domain RNA binding properties; 2) Determine the differential binding mechanisms of TDRD5 and TDRD7 with Piwi proteins Miwi and Mili; 3) Define the precise role of TDRD5 in meiotic TE silencing in mice. These studies will provide valuable new insight into the mechanisms that safeguard germline genome integrity and sustain male fertility.

 描述(申请人提供)：转座元件(TES)是一种“基因组寄生虫”，可以复制并重新整合到宿主细胞基因组中。生殖细胞中不受控制的TE活性会导致DNA损伤、配子发生中断和不育。在哺乳动物雄性生殖细胞中，Piwi-piRNA途径使用小RNA作为向导，使移动的TE沉默，以保护基因组完整性和维持生育。一个独特的蛋白质家族，Tudor结构域包含蛋白(TDRD)，直接与Piwi蛋白结合，促进它们在TE沉默和精子发生中的作用。TDRD蛋白和Piwi蛋白共同参与减数分裂TE沉默的机制目前尚不清楚。仅TDRD蛋白TDRD5或TDRD7的突变就会导致L1(哺乳动物中最丰富的TE)激活和男性不育。然而，它们被整合到Piwi-piRNA途径以抑制TE表达的机制仍然不清楚。除了Tudor结构域，TDRD5和TDRD7还含有一个新的未知功能的RNA结合域--莲花结构域。该提案的总体目标是确定TDRD5和TDRD7在减数分裂过程中转录后抑制TE激活的分子机制。我们的初步数据表明，Lotus域是一个RNA结合域。我们假设TDRD5和TDRD7作为“Tudor适配器”，直接将TERNAs招募到Piwi蛋白复合体进行降解或隔离。这可能是通过它们的Tudor和Lotus结构域分别与Piwi蛋白和TE mRNAs结合实现的。为了验证这一假说，我们将使用生化方法和小鼠模型：1)确定莲花结构域RNA的结合特性；2)确定TDRD5和TDRD7与Piwi蛋白Miwi和Mili的不同结合机制；3)确定TDRD5在小鼠减数分裂TE沉默中的确切作用。这些研究将为保护生殖系基因组完整性和维持男性生育能力的机制提供有价值的新见解。