Regulation of translation initiation under stress conditions

应激条件下翻译起始的调节

• 批准号: RGPIN-2016-03938
• 负责人: Hirasawa, Kensuke(Ken)
• 金额: $ 2.26万
• 依托单位: Memorial University of Newfoundland
• 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=684045
• 关键词: Regulation; translation; initiation; under; stress

Two major steps of gene expression are transcription and translation. In the last 20 years, gene expression research has mainly focused on transcriptional regulation as microarray analysis became established. However, recent studies using genomic and proteomic profiling have revealed that changes in mRNA levels only partially correspond to those in protein levels. This suggests that translational regulation also plays critical roles in the regulation of gene expression. Translational regulation may be particularly important when cells respond to a rapid environmental change caused by cellular stresses. ***During the previous NSERC funding period, we investigated translation initiated by a unique RNA element in the 5'UTR of mRNA called the internal ribosomal entry site (IRES) under normal and stress conditions using a bicistronic reporter system. We determined that cellular stresses, such as amino acid starvation and mild hypothermia, increase the translation efficiency initiated by certain IRES elements. While the bicistronic reporter system has been very useful in these studies, it is not suitable for investigating a large number of IRES elements. Moreover, emerging evidence indicates that cis-acting elements other than IRES elements in the 5' and 3' UTR of mRNA can also regulate translation under stress conditions. Therefore, to fully understand how cellular stresses induce changes in translatomes, a global analysis is needed. ***To this end, we propose to investigate cellular stress-induced changes in the translatome using polysome profiling combined with RNA sequencing. The goal is to identify proteins involved in cell survival and death that are translationally regulated by cis-elements on the 5' and 3'UTR of mRNA under cellular stresses. ******Objective 1: Identification of proteins translationally regulated under stress conditions. mRNA preparations will be fractionated based on the number of bound ribosomes by conducting polysome profiling. mRNAs isolated from heavy polysome fractions (actively translated) of cells under normal and stress conditions will be compared by RNA sequencing. ***Objective 2: Validation of polysome profiling/RNA sequencing data. The results obtained in Objective 1 will be confirmed by western blot analysis, quantitative RT-PCR, de novo protein synthesis assay and 5' or 3'UTR reporter analysis. ***Objective 3: Investigation of functions of the validated proteins under stress conditions. Many of the proteins translationally upregulated during cellular stress are most likely very important for regulating cell survival and death. The functions of the proteins validated in Objective 2 will be determined by loss- and gain-of-function experiments.******To our knowledge, there are very few laboratories conducting a genome-wide scale analysis on translatome of cells under stress conditions. Thus, the proposed research program represents a new frontier. **

基因表达的两个主要步骤是转录和翻译。在过去的20年里，基因表达研究主要集中在转录调控的微阵列分析成为建立。然而，最近使用基因组和蛋白质组分析的研究表明，mRNA水平的变化仅部分对应于蛋白质水平的变化。这表明翻译调控在基因表达调控中也起着关键作用。当细胞对由细胞应激引起的快速环境变化做出反应时，翻译调节可能特别重要。* 在之前的NSERC资助期间，我们使用双顺反子报告系统研究了在正常和应激条件下由mRNA的5 'UTR中称为内部核糖体进入位点（IRES）的独特RNA元件启动的翻译。我们确定，细胞应激，如氨基酸饥饿和轻度低温，增加翻译效率启动某些IRES元素。虽然双顺反子报告系统在这些研究中非常有用，但它不适合研究大量的IRES元件。此外，新出现的证据表明，在mRNA的5'和3' UTR中除了IRES元件之外的顺式作用元件也可以在胁迫条件下调节翻译。因此，为了充分理解细胞应激如何诱导translatomes的变化，需要进行全局分析。* 为此，我们建议使用多核糖体分析结合RNA测序来研究细胞应激诱导的翻译组变化。目标是鉴定在细胞应激下由mRNA的5'和3' UTR上的顺式元件介导调节的参与细胞存活和死亡的蛋白质。 ** 目标1：鉴定在应激条件下受到抑制调节的蛋白质。通过进行多核糖体分析，基于结合的核糖体数量对mRNA制备物进行分级。将通过RNA测序比较在正常和应激条件下从细胞的重多核糖体部分（主动翻译）分离的mRNA。* 目标2：验证多核糖体分析/RNA测序数据。目标1中获得的结果将通过蛋白质印迹分析、定量RT-PCR、从头蛋白合成测定和5'或3' UTR报告基因分析来确认。 * 目的3：研究经验证的蛋白质在强制降解条件下的功能。许多蛋白质在细胞应激过程中被过度上调，很可能对调节细胞存活和死亡非常重要。目标2中验证的蛋白质的功能将通过功能丧失和获得实验来确定。**据我们所知，很少有实验室在应激条件下对细胞的翻译组进行全基因组规模的分析。因此，拟议的研究计划代表了一个新的前沿。**