DNA Damage Signaling and Transcriptome Regulation in Drosophila Embryogenesis

背景：胚胎发育的忠实执行依赖于生物体适应压力的能力。在早期阶段的果蝇胚胎中，核沉降的过程使得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。控制核沉降的分子机制长期以来一直知之甚少。在最近的一项研究中，我们的研究小组定义了一种新的机制，通过这种机制，Chk 2激酶介导的DNA损伤信号传导导致转录的mRNA选择性保留在沉降核中，阻止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），组蛋白mRNA核输出所需的RNA结合蛋白（RBP），作为直接靶点Chk 2。虽然SLBP是解释组蛋白mRNA在沉降核中的核保留的关键目标，但其他几种mRNA通过SLBP独立机制保留。因此，我们假设Chk 2信号选择性地抑制其他RBPs的功能，通常涉及mRNA核出口，这些事件是至关重要的，在协调核沉降过程。我们将追求以下具体目标：** 目标1：确定调节核保留mRNA和核沉降物的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。* 目标2：定义候选RBP如何相对于Chk 2发挥作用。为了确定候选RBP和Chk 2之间的调控联系，我们将进行遗传上位性研究，以对比chk 2和/或RBP的表型胚胎突变体。我们通过对野生型和chk 2突变体胚胎进行体外激酶测定和体内磷酸化蛋白质组学研究来测试选择的RBP是否是Chk 2靶点。* 目标3：定义候选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，我们将使用在正常或应激条件下生长的胚胎提取物进行RBP免疫沉淀结合RNA深度测序（RIP-seq）。我们还将使用RNA深度测序来评估候选RBP中突变的转录组学影响。重要性：虽然DNA损伤反应信号通路已被深入研究，其对转录后水平的转录组调控机制的影响仍然很差的特点。该项目将建立一个长期的研究计划，旨在确定在胚胎发育过程中维持转录组完整性的过程。

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.**