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

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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.