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）尾长的细胞质变化，其影响稳定性和翻译。这种调节对于生殖系干细胞生物学和早期胚胎发生是必不可少的，这取决于母体mRNA的调节。聚腺苷酸尾缩短（去腺苷酸化）导致mRNA衰减和/或翻译抑制。CCR 4-NOT复合物作为最重要的去腺苷酶，可以在RNA结合蛋白或小的非编码RNA的帮助下调节特定的mRNA。此外，CCR 4-NOT复合物还参与poly（A）非依赖性调节机制。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生成的工具和数据。 我们以前已经开发了一个无细胞系统来自早期果蝇胚胎，复制nanos（NOS）mRNA，这是胚胎发育所必需的转录后调控。根据阻遏物Smaug的结合位点，适当的RNA构建体在体外被去腺苷化和抑制。 我们还鉴定了一种抑制机制，其中piRNA引起CCR 4-NOT依赖性去腺苷化。piRNA主要由转座因子序列产生，其主要功能是抑制生殖系中的转座因子。我们已经揭示了piRNA在胚胎基因调控中的作用：piRNA通过与Smaug一起募集阻遏物复合物来靶向nos mRNA，该阻遏物复合物导致胚胎体细胞部分中的nos的CCR 4-N 0 T依赖性去腺苷化和翻译阻遏，这是胚胎图案化所需的。目前的数据表明，皮尔纳途径在基因调控中的这种功能是广泛的，并且可能在许多生物过程中产生重大影响，包括疾病，就像miRNA的调控一样。 该项目旨在解决piRNA和RNA结合蛋白的mRNA调控的分子机制。我们将研究mRNA衰减和翻译抑制，以及我们最近发现的皮尔纳途径在mRNA稳定中的相反作用。该合作项目将涉及创新方法，包括果蝇中的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-蛋白质相互作用图谱。通过建立mRNA调控的特定机制与其在发育过程中的功能之间的联系，该项目应该对我们理解转录后水平的基因调控和小的非编码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.