DNA Damage Signaling and Transcriptome Regulation in Drosophila Embryogenesis

果蝇胚胎发生中的 DNA 损伤信号转导和转录组调控

• 批准号: RGPIN-2017-04614
• 负责人: Lecuyer, Eric
• 金额: $ 2.91万
• 依托单位: Université de Montréal
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2019
• 资助国家: 加拿大
• 起止时间: 2019-01-01 至 2020-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=687656
• 关键词: DNA; Damage; Signaling; Transcriptome; Regulation

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.**

背景：忠实的胚胎发展执行取决于有机体适应压力的能力。在早期果蝇胚胎中，核辐射的过程可以选择性消除核中具有复制缺陷或过度DNA损伤的核。控制核辐射的分子机制长期以来一直在了解不足。在最近的一项研究中，我们的小组定义了一种新的机制，通过该机制，由CHK2激酶介导的DNA破坏信号传导导致在辐射核中选择性保留转录的mRNA，从而阻止编码密钥蛋白的转录本的翻译。我们的工作确定了组蛋白mRNA核导出所需的RNA结合蛋白（RBP）的茎环结合蛋白（SLBP）为直接靶靶CHK2。虽然SLBP是解释辐射核中组蛋白mRNA核保留的关键靶标，但其他几种mRNA是通过独立于SLBP的机制保留的。因此，我们假设CHK2信号有选择地抑制了通常与mRNA核出口有关的其他RBP的功能，并且这些事件对于协调核辐射过程至关重要。我们将追求以下具体目标：******目标1：确定调节核保留mRNA和核辐射的RBP。我们将采用RNA捕获测定法与质谱法相结合，以鉴定与核保留的mRNA特别相互作用的RBP。然后，我们将进行体内功能丧失研究，以评估哪些RBP在正常或应力条件下影响核后果。 *** AIM 2：定义候选RBP与CHK2的功能。为了定义候选RBPS和CHK2之间的调节联系，我们将进行遗传性遗传研究，以对比CHK2和/或RBP的表型胚胎突变体。我们通过在野生型和CHK2突变胚胎上进行体外激酶测定和体内磷酸化 - 磷酸化 - 磷酸化 - 磷酸化 - 磷酸化 - 磷酸化 - 磷酸化 - 磷酸化酶学研究是否是CHK2靶标。 ***目标3：在正常或应力条件下定义候选RBP的RNA结合特性。为了定义候选RBP的结合特性，我们将使用在正常或应力条件下生长的胚胎提取物进行RBP免疫沉淀与RNA深测序（RIP-SEQ）结合使用。我们还将使用RNA深度测序来评估候选RBP中突变的t骨影响。******重要性：虽然已经对DNA损伤响应信号传导途径进行了深入研究，但它们对作用于文字后水平的转录组调节机制的影响仍然很差。该项目将使建立一项长期研究计划，旨在定义在胚胎开发过程中维持trancriptome完整性的过程。**