mRNP assembly at the 3' splice site studied by functional genomics and structural biology

RNA结合蛋白（rbp）通过结合顺式调控RNA元件（cis元件），如短RNA序列或结构化RNA元件，来调控各种mRNA加工步骤。RBP功能是由大信使核糖核蛋白颗粒（mRNPs）中蛋白质和RNA的相互作用形成的，mRNPs控制着每个转录物的命运和功能。这些rbp通常含有多个RNA结合域（rbd），这些RNA结合域介导组合RNA或蛋白质相互作用，以动态组装和重塑mRNPs。在这个项目中，我们的目标是通过在多个分辨率水平上剖析3 '剪接位点识别的分子机制来揭示mRNP编码的新原理。基于该SPP第一个资助期获得的结果，我们将大规模体内和体外RBP结合图与动力学和高分辨率结构信息相结合，以解决剪接调节中基本剪接因子U2AF的多层调节。该项目的目标是解剖3 '剪接位点的mRNP组装，重点关注U2AF结合作为剪接体招募过程中的先驱事件。在第一个资助期内，我们建立了一种独特的方法，将体内和体外转录范围的RBP结合图与功能分析和综合结构生物学相结合。这使我们能够展示U2AF65如何识别RNA序列，以及这是如何被分子内相互作用和新发现的反式作用因子调节的。我们将在这些发现的基础上解决三个目标：1)U2AF65的已知相互作用伙伴U2AF35和SF1如何调节3 '剪接位点识别？2) FUBP1/U2AF mRNPs如何调节U2AF活性。FUBP1是否作为剪接的一般调节因子，哪些3 '剪接位点特别易受影响？3) hnRNP A1的甘氨酸富区（GRR）如何促进hnRNP A1/U2AF mRNPs的形成。GRR和由该区域介导的液液相分离是否在剪接调节中发挥了校对和hnRNP A1去除错误组装的mRNPs的作用？根据这一策略，我们将破译3 '剪接位点识别的新分子机制-从组合RBP结合的机制原理到活细胞中mRNP组装的功能后果。因此，我们的方法有望对剪接调节及其他方面的结构/功能关系提供新的见解。该项目采用的互补方法的独特组合也将支持与SPP1935的其他研究人员的互动。

RNA-binding proteins (RBPs) regulate various mRNA processing steps by binding to cis-regulatory RNA elements (cis elements), such as short RNA sequences or structured RNA elements. RBP function is strongly shaped by the interplay of proteins and RNA in large messenger ribonucleoprotein particles (mRNPs) that control the fate and function of each transcript. These RBPs typically harbour multiple RNA binding domains (RBDs) that mediate combinatorial RNA or protein-protein interactions to dynamically assemble and remodel mRNPs.In this project, we aim to unravel novel principles of the mRNP code by dissecting the molecular mechanisms of 3’ splice site recognition at multiple levels of resolution. Building on the results obtained during the first funding period of this SPP, we integrate large-scale in vivo and in vitro RBP binding maps with kinetic and high-resolution structural information to address multiple layers of regulation of the essential splicing factor U2AF in splicing regulation. The goal of this project is to dissect mRNP assemblies at the 3’ splice site, focusing on U2AF binding as a pioneering event during spliceosome recruitment. During the first funding period, we established a unique approach combining transcript-wide in vivo and in vitro RBP binding maps with functional assays and integrative structural biology. This enabled us to show how U2AF65 recognizes RNA sequences and how this is modulated by intramolecular interactions and newly discovered trans-acting factors. We will build on these findings to address the three aims: 1) How do U2AF65’s known interaction partners U2AF35 and SF1 modulate 3’ splice site recognition? 2) How do FUBP1/U2AF mRNPs modulate U2AF activity. Does FUBP1 act as a general regulator of splicing, and which 3’ splice sites are particularly susceptible? 3) How does the glycine-rich region (GRR) of hnRNP A1 contribute to the formation of hnRNP A1/U2AF mRNPs. Does GRR and liquid-liquid phase separation mediated by this region play a role in proof-reading and the removal of erroneously assembled mRNPs by hnRNP A1 in splicing regulation?Following this strategy, we will decipher new molecular mechanisms of 3’ splice-site recognition - from the mechanistic principles of combinatorial RBP binding to the functional consequences of mRNP assembly in living cells. Our approach thereby promises new insights into structure/function relationships in splicing regulation and beyond. The unique combination of complementary methods employed in this project will also support interactions with other researchers in the SPP1935.