Tudor Domain Proteins in Germline Genome Defense

项目摘要 转座因子是“基因组寄生虫”，可以复制和重新整合 插入宿主细胞基因组中。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，PIWI- 皮尔纳途径使用小RNA作为引导来沉默移动的TE以保护基因组 完整性和可持续生育能力。piRNA的适当产生对于TE沉默至关重要 和精子发生。然而，控制皮尔纳生物合成的机制并不 很好理解。特别是，许多RNA结合蛋白在皮尔纳过程中的作用 生物起源仍然难以捉摸。通过研究Tudor结构域蛋白的一个亚组， 我们发现TDRD 5是一个新的RNA结合蛋白， 对于小鼠中的皮尔纳生物合成至关重要。引人注目的是，我们发现了一种新的RNA 在细菌、植物和动物中保守的LOTUS结构域的结合特性。 这种结合特性不同于已报道的某些动物蛋白质的结合特性 LOTUS域名。我们假设动物LOTUS结构域既有RNA， 蛋白结合活性，并且LOTUS组的Tudor结构域蛋白一起 在哺乳动物皮尔纳生物发生中起关键作用。为了验证这个假设，我们将使用 生物化学方法和小鼠模型：1）阐明RNA和蛋白质结合 LOTUS结构域超家族的活性; 2）确定特定的 LOTUS结构域-RNA相互作用;和3）定义LOTUS的功能参与 结构域蛋白在皮尔纳生物合成和Vasa调节中的作用。这些研究将 为皮尔纳生物合成机制提供了有价值的新见解， 生殖系基因组的完整性，以维持男性生育能力，并扩大我们在更广泛的知识 RNA生物学和发育生物学。

PROJECT SUMMARY 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. The proper production of piRNAs is critical for TE silencing and spermatogenesis. However, the mechanisms governing piRNA biogenesis are not well understood. In particular, the roles of many RNA binding proteins during piRNA biogenesis remain elusive. By studying a subgroup of Tudor domain proteins that also harbor LOTUS domains, we discovered that TDRD5 is a novel RNA binding protein critical for piRNA biogenesis in mice. Strikingly, we have discovered a novel RNA binding property of LOTUS domains that is conserved in bacteria, plants and animals. This binding property is different from reported protein binding feature of some animal LOTUS domains. We hypothesize that animal LOTUS domains have both RNA and protein binding activities and that the LOTUS group of Tudor domain proteins together play critical roles in mammalian piRNA biogenesis. To test this hypothesis, we will use biochemical approaches and mouse models to: 1) Clarify the RNA and protein binding activities of the LOTUS domain superfamily; 2) Determine the mechanism of a specific LOTUS domain-RNA interaction; and 3) Define the functional involvement of LOTUS domain proteins in piRNA biogenesis and Vasa regulation in mice. These studies will provide valuable new insights into the mechanisms of piRNA biogenesis that safeguard germline genome integrity to sustain male fertility and expand our knowledge in broader RNA biology and developmental biology.