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

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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.