Role of RNA-binding proteins in regulation of gene expression

背景：转录后调节(PTR)对mRNA的稳定性、定位和翻译进行调节，调节蛋白质在所有细胞类型和生物体中的时空表达。典型的真核生物基因组编码数百个限制性商业惯例，每个限制性商业惯例都与数百个转录本结合，这一事实突显了PTR的广泛性质。限制性商业惯例识别RNA中的三类顺式元件：双链(Ds)、单链(Ss)或发夹。*初步结果：我们已经在体内确定了数百个靶标：果蝇Staufen(Stau，一种dsRBP)和脑瘤(Brat，一种我们在体外识别ssRNA的Trim-NHL蛋白)。对这些限制性商业惯例结合的目标预测基序进行了计算分析。这代表了第一个预测赋予与dsRBP结合特异性的二级结构的定义，并鉴定了一个以前未发现的限制性商业惯例家族，TRIM-NHL蛋白。*目标：分析Stau dsRBP和果蝇的三个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：为了定义和验证四个RBPs的结合特异性，我们将使用RNA竞争性进行体外结合研究，并携带靶标3‘-非翻译区(UTRs)融合到荧光素酶或GFP开放阅读框架的记者进行体内分析。此外，我们将把野生型或突变型结合基序插入到与荧光素酶或GFP融合的朴素3‘UTRs中，用于活体研究。我们将通过RBP的免疫沉淀和RT-qPCR来评估与四个RBPs结合的基序的必要性和充分性。*目标2：为了分析四个RBPs的作用，我们将通过评估它们在胚胎和/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mRNAs的转录后命运。我们还将分析RNAi诱导的“击倒”(在S2细胞中；用RT-qPCR检测)和突变(在胚胎中；通过RT-qPCR检测和全局RNAseq检测)对内源性靶标和转基因记者的影响。*目标3：为了确定RBPs转录后控制其靶mRNAs的机制，我们将免疫沉淀每个RBP，然后用质谱仪鉴定伙伴蛋白，其鉴定将揭示机制。CCR4/非死烯基酶复合体的成分与Brat共纯化，表明该RBP触发死烯基化来控制mRNA的翻译和/或稳定性。实验将评估内源性靶标和报告RNA的聚(A)尾长，以及去烯化在Brat靶mRNA翻译和稳定性中的作用。*意义：在后生动物中，限制性商业惯例的结合域和靶点是保守的。因此，我们对它们在果蝇中的原型的研究将导致对这些重要限制性商业惯例家族的机制和功能的总体洞察。

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.**