Molecular aspects of mRNA regulation by piRNAs and RNA binding proteins in Drosophila

转录后基因调控的一个重要机制涉及mRNA poly(A)尾巴长度的细胞质变化，这影响了稳定性和翻译。这种调节对生殖系干细胞生物学和早期胚胎发生至关重要，这取决于母体mrna的调节。Poly(A)尾部缩短（deadenylation）导致mRNA衰变和/或翻译抑制。CCR4-NOT复合体作为最重要的deadenylase，可以借助RNA结合蛋白或小的非编码RNA调控特异性mrna。此外，CCR4-NOT复合物还参与了不依赖于聚(A)的调控机制。我们使用果蝇胚胎作为模型系统来研究转录后调控。我们的多学科项目是基于我们在遗传学（Simonelig组）和生物化学（Wahle组）方面的互补专业知识以及我们已经产生的工具和数据。我们之前已经开发了一种来自早期果蝇胚胎的无细胞系统，该系统可以复制纳米（nos） mRNA的转录后调控，这对胚胎发育至关重要。根据抑制因子Smaug的结合位点，适当的RNA结构在体外被死基化和翻译。我们还发现了一种抑制机制，其中pirna导致ccr4非依赖性死基化。pirna主要由转座因子序列产生，其主要功能是抑制种系转座因子。我们已经发现了pirna在胚胎基因调控中的作用：pirna通过与Smaug（一种抑制复合物）一起募集nos mRNA，导致胚胎体细胞部分的ccr4非依赖性死蛋白化和nos的翻译抑制，这是胚胎模式形成所必需的。目前的数据表明，piRNA通路在基因调控中的这种功能是广泛存在的，可能对许多生物过程（包括疾病）产生重大影响，mirna的调控也是如此。本项目旨在研究pirna和RNA结合蛋白调控mRNA的分子机制。我们将研究mRNA衰变和翻译抑制，以及我们最近发现的piRNA通路在mRNA稳定中的相反作用。该合作项目将涉及创新方法，包括果蝇CRISPR-Cas9编辑系统、超分辨率成像、用于复杂分析的质谱和蛋白质-蛋白质相互作用制图。通过建立mRNA调控的具体机制及其在发育过程中的功能之间的联系，该项目将对我们理解转录后水平的基因调控和小非编码rna的功能产生重大影响。

An important mechanism of post-transcriptional gene regulation involves cytoplasmic variations of mRNA poly(A) tail length, which affects both stability and translation. This regulation is essential for germline stem cell biology and early embryogenesis, which depends on regulation of maternal mRNAs. Poly(A) tail shortening (deadenylation) leads to mRNA decay and/or translational repression. As the most important deadenylase, the CCR4-NOT complex can regulate specific mRNAs with the help of RNA binding proteins or small non-coding RNAs. In addition, the CCR4-NOT complex is also involved in poly(A)-independent regulatory mechanisms. We use the Drosophila embryo as a model system to investigate post-transcriptional regulation. Our multidisciplinary project is based on our complementary expertise in genetics (Simonelig group) and biochemistry (Wahle group) and the tools and data we have already generated. We have previously developed a cell-free system derived from early Drosophila embryos that reproduces the post-transcriptional regulation of nanos (nos) mRNA, which is essential for embryonic development. Depending on binding sites for the repressor Smaug, appropriate RNA constructs are both deadenylated and translationally in vitro. We have also identified a repression mechanism in which piRNAs cause CCR4-NOT-dependent deadenylation. piRNAs are mostly produced from transposable element sequences, and their primary function is the repression of transposable elements in the germline. We have uncovered a role of piRNAs in gene regulation in the embryo: piRNAs target the nos mRNA by recruiting, together with Smaug, a repressor complex leading to the CCR4-NOT-dependent deadenylation and translational repression of nos in the somatic part of the embryo, which is required for embryonic patterning. Current data indicate that this function of the piRNA pathway in gene regulation is widespread and likely to have a major impact in many biological processes, including diseases, as is the case for regulation by miRNAs. This project aims at addressing the molecular mechanisms underlying mRNA regulation by piRNAs and RNA binding proteins. We will investigate both mRNA decay and translational repression, as well as an opposite role of the piRNA pathway in mRNA stabilization that we have uncovered recently. This collaborative project will involve innovative approaches including the CRISPR-Cas9 editing system in Drosophila, super-resolution imaging, mass spectrometry for complex analysis and protein-protein interaction mapping. By establishing a link between specific mechanisms of mRNA regulation and their function in developmental processes, this project should have a major impact for our understanding of both gene regulation at the post-transcriptional level and the function of small non-coding RNAs.