DNA Damage Signaling and Transcriptome Regulation in Drosophila Embryogenesis

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BACKGROUND: The faithful execution of embryonic development relies on the capacity of organisms to adapt to stress. In early stage Drosophila embryos, a process of nuclear fallout enables the selective elimination of nuclei with replication defects or excessive DNA damage during the midblastula transition. The molecular mechanisms controlling nuclear fallout have long remained poorly understood. In a recent study, our group defined a novel mechanism through which DNA-damage signalling mediated by the Chk2 kinase leads to the selective retention of transcribed mRNAs in fallout nuclei, blocking the translation of transcripts encoding key proteins. Our work identified the stem loop binding protein (SLBP), an RNA binding protein (RBP) required for histone mRNA nuclear export, as a direct target Chk2. While SLBP is a key target explaining histone mRNA nuclear retention in fallout nuclei, several other mRNAs are retained through SLBP-independent mechanisms. Thus, we hypothesize that Chk2 signalling selectively inhibits the function of other RBPs normally implicated in mRNA nuclear export and that these events are crucial in coordinating the nuclear fallout process. We will pursue the following specific aims:******Aim 1: Identify RBPs that regulate nuclear retained mRNAs and nuclear fallout. We will employ RNA capture assays combined with mass spectrometry to identify RBPs that specifically interact with nuclear retained mRNAs. We will then perform in vivo loss-of-function studies to assess which RBPs impact nuclear fallout under normal or stress conditions. ***Aim 2: Define how candidate RBPs function in relation to the Chk2. To define the regulatory links between candidate RBPs and Chk2, we will conduct genetic epistasis studies to contrast the phenotypes embryos mutant for chk2 and/or RBPs. We test whether select RBPs are Chk2 targets by performing in vitro kinase assays and in vivo phospho-proteomics studies on wild-type and chk2 mutant embryos. ***Aim 3: Define the RNA binding properties of candidate RBPs in normal or stress conditions. To define the binding properties of candidate RBPs, we will conduct RBP immuno-precipitation combined with RNA deep sequencing (RIP-seq), using extracts of embryos grown under normal or stress conditions. We will also use RNA deep sequencing to evaluate the trancriptomic impact of mutations in candidate RBPs.******SIGNIFICANCE: While the DNA damage response signalling pathways have been intensely investigated, their influence on transcriptome regulatory mechanisms acting at the post-transcriptional level remains poorly characterized. This project will enable the establishment of a long-term research program aimed at defining the processes involved in maintaining trancriptome integrity during embryonic development.**