Role of RNA-binding proteins in regulation of gene expression

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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.**