Role of RNA-binding proteins in regulation of gene expression

RNA结合蛋白在基因表达调控中的作用

• 批准号: RGPIN-2015-06246
• 负责人: Lipshitz, Howard
• 金额: $ 3.28万
• 依托单位: University of Toronto
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2018
• 资助国家: 加拿大
• 起止时间: 2018-01-01 至 2019-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=657711
• 关键词: Role; RNA; binding; proteins; regulation

Background: Post-transcriptional regulation (PTR) of mRNA stability, localization and translation regulates the spatial and temporal expression of proteins in all cell types and organisms. The widespread nature of PTR is highlighted by the fact that typical eukaryotic genomes encode several hundred RBPs, each of which binds to several hundred transcripts. RBPs recognize three classes of cis-elements in RNA: double-stranded (ds), single-stranded (ss), or hairpin.***Preliminary Results: We have identified hundreds of in vivo mRNA targets of Drosophila Staufen (STAU, a dsRBP) and Brain tumor (BRAT, a TRIM-NHL protein that we have shown recognizes ssRNA in vitro). Computational analysis of the targets predicted motifs bound by these RBPs. This represents the first definition of secondary structures predicted to confer specificity of binding to a dsRBP and identification of a previously undiscovered family of RBPs, the TRIM-NHL proteins.***Goal: To analyze the binding specificity, mechanisms and functions in PTR of the STAU dsRBP and Drosophila's three TRIM-NHL homologs, BRAT, WECH and MEI-P26.***Aim 1: To define and verify the binding specificity of the four RBPs we will use RNAcompete for in vitro-binding studies and reporters carrying target 3'-untranslated regions (UTRs) fused to luciferase or GFP open reading frames for in vivo analyses. In addition we will insert arrays of wild-type or mutant binding motifs into naïve 3'UTRs fused to luciferase or GFP for in vivo studies. We will assess the necessity and sufficiency of the motifs for binding to the four RBPs by immunoprecipitation of the RBP followed by RT-qPCR.***Aim 2: To analyze the roles of the four RBPs, we will define the post-transcriptional fate of the reporter mRNAs described in Aim 1 by assessing their stability, translational status and subcellular localization in embryos and/or S2 tissue culture cells. We will also assay the effects on endogenous targets and transgenic reporters, of removal of the RBPs by RNAi-induced 'knock down' (in S2 cells; assayed by RT-qPCR) and by mutation (in embryos; assayed by RT-qPCR and globally by RNAseq).***Aim 3: To identify the mechanisms by which the RBPs post-transcriptionally control their target mRNAs we will immunoprecipitate each RBP followed by mass spectrometry to identify partner proteins, the identity of which will suggest mechanism. Components of the CCR4/NOT-deadenylase complex co-purify with BRAT, suggesting that this RBP triggers deadenylation to control mRNA translation and/or stability. Experiments will be conducted to assess poly(A) tail length of endogenous targets and reporter RNAs, and the role of deadenylation in BRAT target mRNA translation and stability.***Significance: The binding domains and target sites of RBPs are conserved in metazoans. Thus our studies of their prototypes in flies will to lead to general insights into mechanisms and functions of these important families of RBPs.**

背景：mRNA 稳定性、定位和翻译的转录后调节 (PTR) 调节所有细胞类型和生物体中蛋白质的空间和时间表达。典型的真核基因组编码数百个 RBP，每个 RBP 与数百个转录本结合，这一事实凸显了 PTR 的广泛性。 RBP 可识别 RNA 中的三类顺式元件：双链 (ds)、单链 (ss) 或发夹。***初步结果：我们已经鉴定了果蝇 Staufen（STAU，一种 dsRBP）和脑肿瘤（BRAT，一种 TRIM-NHL 蛋白，我们已证明可在体外识别 ssRNA）的数百个体内 mRNA 靶标。对目标的计算分析预测了这些 RBP 结合的基序。这代表了预测赋予 dsRBP 结合特异性的二级结构的第一个定义，并鉴定了先前未发现的 RBP 家族 TRIM-NHL 蛋白。***目标：分析 STAU dsRBP 和果蝇的三种 TRIM-NHL 同源物 BRAT、WECH 和 MEI-P26 在 PTR 中的结合特异性、机制和功能。***目标 1：为了定义和验证四种 RBP 的结合特异性，我们将使用 RNAcompete 进行体外结合研究，并使用携带与荧光素酶或 GFP 开放阅读框融合的目标 3'-非翻译区 (UTR) 的报告基因进行体内分析。此外，我们会将野生型或突变体结合基序阵列插入与荧光素酶或 GFP 融合的天然 3'UTR 中，以进行体内研究。我们将通过 RBP 免疫沉淀和 RT-qPCR 来评估与四个 RBP 结合的基序的必要性和充分性。***目标 2：为了分析四个 RBP 的作用，我们将通过评估其在胚胎和/或 S2 组织培养细胞中的稳定性、翻译状态和亚细胞定位来定义目标 1 中描述的报告基因 mRNA 的转录后命运。我们还将测定通过 RNAi 诱导的“敲低”（在 S2 细胞中；通过 RT-qPCR 测定）和突变（在胚胎中；通过 RT-qPCR 测定和整体通过 RNAseq 测定）去除 RBP 对内源靶标和转基因报告基因的影响。***目标 3：为了确定 RBP 转录后控制其靶 mRNA 的机制，我们将 免疫沉淀每个 RBP，然后通过质谱分析来鉴定伴侣蛋白，其身份将提示其机制。 CCR4/NOT-脱腺苷酸酶复合物的成分与 BRAT 共纯化，表明该 RBP 触发脱腺苷化以控制 mRNA 翻译和/或稳定性。将进行实验以评估内源靶标和报告 RNA 的聚腺苷酸尾长度，以及脱腺苷化在 BRAT 靶标 mRNA 翻译和稳定性中的作用。***意义：RBP 的结合域和靶位点在后生动物中是保守的。因此，我们对其在果蝇中的原型的研究将有助于对这些重要 RBP 家族的机制和功能有一般性的了解。**