生殖细胞的RNA解旋酶GLH家族及其调控Piwi-piRNA依赖的基因表观遗传沉默的研究

生殖发育研究领域的一个基本问题是核中转录和剪接事件如何与胞质mRNA翻译事件紧密偶联，Argonaute与非编码小RNA在该过程中起着重要调节作用。piRNAs与Argonaute PRG-1形成复合物，通过miRNA样的“种子”碱基配对原则，靶向识别基因，触发WAGO依赖的基因沉默，抑制靶向基因表达。意外的是，申请人发现piRNAs可以靶向表达基因，暗示piRNA信号通路可能募集不同的下游因子来调节其靶向mRNA的命运。本项目将综合遗传、生信和生化等学科的方法，辅以显微镜和深度核酸测序等技术，系统研究RNA解旋酶GLH家族的生物学功能。重点探索GLH-1和GLH-4对piRNA信号通路特异性沉默基因的分子机制；探明GLH-2和GLH-3与其它Argonaute相互作用的生物学功能；并构建GLHs家族互作的分子网络。本项目将阐明piRNA调控基因特异性的机制，揭示GLHs的新生物学功能。

A long-standing question in eukaryotic gene regulation is how, and to what extent, are the nuclear events of transcription and splicing coupled to the subsequent cytoplasmic events controlling mRNA translation. Argonaute systems provide potential links between these fundamental steps in gene expression. In C. elegans, Argonautes engage both mature cytoplasmic transcripts and nascent nuclear transcripts. For example, the Argonaute CSR-1 binds chromatin and mRNAs of active genes, while WAGO Argonautes target silent genes and mRNAs. This connection is best understood for the WAGO/silencing pathway wherein chromatin factors, including homologs of Polycomb and heterochromatin protein 1 (HP1), are required for WAGO-mediated silencing. Thus, mRNAs produced within silent chromatin are thought to feed the amplification and maintenance of silencing, while mRNAs produced within euchromatin are somehow protected. Recently, I analyzed mRNA targeting by a third Argonaute system in the germline, the piRNA/Piwi Argonaute pathway. Targeting by piRNAs, which are genomically encoded, triggers WAGO-dependent silencing. My analysis revealed that piRNA targeting follows micro-RNA-like “seed” and supplementary base pairing rules. Surprisingly, even expressed mRNAs, although protected from piRNA-induced silencing, are abundantly targeted by piRNAs within both their open reading frames (ORFs) and untranslated regions (UTRs). I hypothesize that the piRNA pathway senses features of mRNAs that reflect their origin within euchromatin or heterochromatin and, in response to these cues, recruits distinct downstream factors that regulate the fates of their target mRNAs. ..We have found that GLH-1 and GLH-4, DEAD-box RNA helicases, interact with PIWI Argonaute PRG-1 mostly through single-strand RNA. In this study, we will continue to explore whether these two DEAD-box proteins govern the fates of mRNA targeted by piRNAs and provide specificity in piRNA pathway in reproductive cells of animals. Interestingly, all of four GLH proteins are expressed in germline nuage, this suggests that they might impart diversity to different Argonaute proteins. We will determine whether GLH-2 and GLH-3 interact with other Argonaute proteins and their roles in regulating germline gene expression and fertility in animals. We will also study the complex cross-talk between these four different GLH proteins. This study will uncover fundamental mechanisms that link transcription and translation in the germline, and perhaps other tissues, and will broaden our understanding of piRNA function in human reproductive cells, and may uncover strategies to treat human disorders, such as infertility and cancer.