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: 2020
• 资助国家: 加拿大
• 起止时间: 2020-01-01 至 2021-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=711148
• 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：胚胎发育的忠实执行依赖于生物体适应压力的能力。在果蝇胚胎的早期阶段，核沉降过程能够选择性地消除在囊胚转变期间复制缺陷或过度 DNA 损伤的细胞核。长期以来，人们对控制核尘埃的分子机制仍知之甚少。在最近的一项研究中，我们的小组定义了一种新机制，通过该机制，由 Chk2 激酶介导的 DNA 损伤信号传导导致转录 mRNA 选择性保留在沉降核中，从而阻断编码关键蛋白的转录本的翻译。我们的工作确定了茎环结合蛋白 (SLBP)，一种组蛋白 mRNA 核输出所需的 RNA 结合蛋白 (RBP)，作为 Chk2 的直接靶标。虽然 SLBP 是解释组蛋白 mRNA 保留在沉降核中的关键靶标，但其他几种 mRNA 通过 SLBP 独立机制保留。因此，我们假设 Chk2 信号传导选择性抑制通常与 mRNA 核输出有关的其他 RBP 的功能，并且这些事件对于协调核沉降过程至关重要。我们将追求以下具体目标： 目标 1：识别调节核保留 mRNA 和核沉降的 RBP。我们将采用 RNA 捕获测定结合质谱法来鉴定与核保留 mRNA 特异性相互作用的 RBP。然后，我们将进行体内功能丧失研究，以评估哪些 RBP 在正常或应激条件下影响核沉降物。 目标 2：定义候选 RBP 如何与 Chk2 相关地发挥作用。为了确定候选 RBP 和 Chk2 之间的调控联系，我们将进行遗传上位研究，以对比 chk2 和/或 RBP 的表型胚胎突变体。我们通过对野生型和 chk2 突变体胚胎进行体外激酶测定和体内磷酸蛋白质组学研究来测试选定的 RBP 是否是 Chk2 靶标。 目标 3：确定候选 RBP 在正常或应激条件下的 RNA 结合特性。为了确定候选 RBP 的结合特性，我们将使用正常或应激条件下生长的胚胎提取物进行 RBP 免疫沉淀与 RNA 深度测序 (RIP-seq) 相结合。我们还将使用 RNA 深度测序来评估候选 RBP 中突变的转录组影响。 意义：虽然 DNA 损伤反应信号通路已被深入研究，但它们对转录后水平转录组调控机制的影响仍知之甚少。该项目将建立一个长期研究计划，旨在确定胚胎发育过程中维持转录组完整性的过程。