Role of RNA-binding proteins in regulation of gene expression

背景资料：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。PTR的广泛性通过典型的真核生物基因组编码几百个RBP的事实而突出，每个RBP结合几百个转录物。RBP识别RNA中的三种顺式元件：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，我们已经证明其在体外识别ssRNA）的数百种体内mRNA靶标。计算分析的目标预测这些RBP结合的基序。这代表了预测赋予与dsRBP结合的特异性的二级结构的第一个定义和先前未发现的RBP家族TRIM-NHL蛋白的鉴定。目标：分析STAU dsRBP和果蝇的三种TRIM-NHL同源物BRAT、WECH和MEI-P26的结合特异性、机制和PTR中的功能。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开放阅读框融合的靶向3 '非翻译区（UTR）的报告基因进行体内分析。此外，我们将野生型或突变体结合基序的阵列插入到与荧光素酶或GFP融合的幼稚3 'UTR中用于体内研究。我们将通过RBP的免疫沉淀，然后进行RT-qPCR，评估基序与四种RBP结合的必要性和充分性。*目标二：为了分析四种RBP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）和通过突变（在胚胎中;通过RT-qPCR测定和通过RNAseq全面测定）去除RBP对内源性靶标和转基因报告基因的影响。目标3：为了鉴定RBP转录后控制其靶mRNA的机制，我们将免疫沉淀每个RBP，然后通过质谱法鉴定伴侣蛋白，其身份将提示机制。CCR 4/N 0 T-去腺苷酶复合物的组分与BRAT共纯化，表明该RBP触发去腺苷化以控制mRNA翻译和/或稳定性。将进行实验以评估内源性靶标和报告RNA的聚（A）尾长度，以及去腺苷酸化在BRAT靶标mRNA翻译和稳定性中的作用。*意义：RBPs的结合结构域和靶位点在后生动物中是保守的。因此，我们在苍蝇中对其原型的研究将导致对这些重要的RBP家族的机制和功能的全面了解。

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.**