Tudor Domain Proteins in Germline Genome Defense

 描述（由申请人提供）：转座因子（TE）是“基因组寄生物”，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，皮尔纳途径使用小RNA作为引导使移动的TE沉默以保护基因组完整性并维持生育力.一个独特的蛋白质家族，Tudor结构域包含蛋白质（TDRD），直接结合Piwi蛋白，并促进其在TE沉默和精子发生中的作用。TDRD蛋白与Piwi蛋白结合参与减数分裂TE沉默的机制目前尚不清楚。TDRD蛋白TDRD 5或TDRD 7的突变单独导致L1（哺乳动物中最丰富的TE）激活和雄性不育。然而，它们整合到Piwi-piRNA途径中以抑制TE表达的机制仍然难以捉摸。除了Tudor结构域，TDRD 5和TDRD 7还含有Lotus结构域，一种功能未知的新型推定RNA结合结构域。本提案的总体目标是确定TDRD 5和TDRD 7转录后抑制减数分裂期间TE激活的分子机制。我们的初步数据表明Lotus结构域是一个RNA结合结构域。我们假设TDRD 5和TDRD 7作为“Tudor衔接子”直接将TE mRNA募集到Piwi蛋白复合物中进行降解或螯合。这可能是由于它们的Tudor和Lotus结构域分别与Piwi蛋白和TE mRNA结合而实现的。为了验证这一假设，我们将使用生物化学方法和小鼠模型来：1）确定Lotus结构域RNA结合特性; 2）确定TDRD 5和TDRD 7与Piwi蛋白Miwi和Mili的差异结合机制; 3）确定TDRD 5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力的机制提供有价值的新见解。

 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.