Cis- and trans-acting determinants of mRNA stability, structure and fate (as a continuation)

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Posttranscriptional regulation steers gene expression e.g., during development and immune response. mRNAs contain structured and unstructured cis-regulatory elements in their 3'-untranslated regions (UTR), recognized by trans-acting RNA-binding proteins (RBPs) with specified domains (RBDs) mediating the mRNA’s fate. We often lack details about the role of RNA sequence, structure and modification in regulatory protein-mRNA hubs (mRNPs). Also, the 3D-arrangement of mRNPs often escapes structural analysis, in total or relevant details. In the work prior to this proposal, we have tackled the role of RNA-regulatory hubs composed of either multiple linear or structured cis-regulatory elements for the fate of target mRNAs when read by the multi-domain RBPs IMP3 and Roquin. The two proteins each significantly affect specific mRNA surveillance: Roquin in immune regulation by recognition of mRNA stem-loops; IMP3, an oncogenic RBP relevant in cancer progression through protective engagement with multiple short linear mRNA cis elements. Extending the static picture of single cis-trans, i.e. RNA motif-RBD interactions, we have discovered cis-trans networks in specific mRNA recognition and processing for both systems. A focus was on the role of multiple-cis-element composition in sequence as compared to their 3D-arrangement in mRNPs and to derive general concepts of mRNA fate-decision. Our work, and novel insights from other labs, have provoked pestering new questions to address, related to the above. This includes the role of RNP stoichiometries mediated by protein-protein (PPI) and RNA-RNA interactions as well as, specifically, the role of RNA structure influenced by m6A-methylation for complex formation. As a consequent continuation of the prior work, this proposal will address both proteins and their target RNAs in the remaining and newly occurring aims. Based on the obtained knowledge in complex formation of the Roquin ROQ domain with the 3’UTR of T cell co-receptor Ox40, we will use X-ray crystallography and Cryo-EM to obtain high-resolution structures with multiple structured cis elements. Further, we will investigate the role of Roquin- and target RNA oligomers for RNP formation with the help of NMR and native mass spectrometry. Both together shall aid the determination of a high-resolution structure of the Roquin N-terminus with a combined target RNA motif via integrated structural biology. For the recognition of clustered ssRNA elements in oncogene-mRNAs by the multi-domain RBP IMP3 we will focus on the role of KH3-4-mediated PPI for RNA-affinity and -specificity using systematic RNP biochemistry and SAXS. This will engage with work on expanded IMP3 RBD-RBD and RBD-RNA interfaces in full-length contexts using mass spectrometry approaches. Finally, a project part on the basis of NMR and accessory biophysical approaches will address the differential m6A-RNA recognition by tandem RBDs of IMP3 in comparison to the well-studied m6A-reader IMP1.