Working on this project:
The subject of this proposal is to determine the molecular background of cytokine-based diseases related to the stabilization of target mRNAs by the recently identified RNA-binding protein (RBP) Arid5a (AT-rich interactive domain (ARID)-containing protein 5A). Arid5a opposes the function of decaying RBPs, e.g. the nuclease Regnase, through either the competition for common structured RNA cis regulatory elements or the usage of adjacent Arid5a responsive elements, e.g. in the interleukin-6 (IL-6) mRNA 3’-untranslated region (UTR). A clear Arid5a target consensus has, however, remained elusive and there is currently no atomic data that unambiguously prove the interaction with any of the suggested RNA cis elements. In the course of this project, we aim at solving structures of all Arid5a RNA-binding moieties in complex with RNA, pre-eminently the predicted ARID domain, by nuclear magnetic resonance (NMR) spectroscopy and crystallography (together with B7). Biophysical analysis will reveal affinities, kinetics and stoichiometries of components within these protein-RNA complexes. We plan to define an Arid5a target RNA consensus using SELEX (Systematic Evolution of Ligands by EXponential enrichment) in collaboration with A2 and look for novel target mRNAs in vivo by iCLIP (individual-nucleotide resolution Cross-Linking and Immuno-Precipitation) with B13. We will then specifically examine structural determinants of mRNA regulatory hubs to unravel mechanisms of mRNA stabilization, in particular for the IL-6 3’-UTR, and including the counteracting Regnase in an integrated structural biology approach. One aspect will be the role of RNA flexibility for the availability of structured ciselements to Arid5a and Regnase. Studies will be expanded to a functional validation in reporter assays in collaboration with B14 and B15 that will support in vitro findings on a cellular level and complete the picture of Arid5a-co-regulated mRNA regulatory hubs in disease.