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。在早期果蝇胚胎中，核沉降过程能够在中囊胚过渡期间选择性地消除具有复制缺陷或过度DNA损伤的细胞核。长期以来，人们对控制核沉降物的分子机制知之甚少。在最近的一项研究中，我们的团队定义了一种新的机制，通过该机制，Chk2激酶介导的dna损伤信号传导导致放射性尘埃核中转录mrna的选择性保留，阻断编码关键蛋白的转录物的翻译。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的直接靶点。虽然SLBP是解释沉降核中组蛋白mRNA核保留的关键靶点，但其他一些mRNA的保留是通过与SLBP无关的机制进行的。因此，我们假设Chk2信号选择性地抑制其他rbp的功能，这些rbp通常与mRNA核输出有关，并且这些事件在协调核沉降过程中至关重要。具体目标是：