Connecting E3 ligase and mRNA decay functions of Roquin proteins

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Roquin proteins prevent the development of autoimmune disease and control spontaneous T cell activation and inappropriate T cell differentiation. Roquin-1 and its paralog Roquin-2 share a highly similar domain structure in the N-terminal region, including a newly characterized RNA-binding domain called ROQ, and a RING finger. While a number of publications have established that Roquin with its ROQ domain directly binds to cis-elements in target mRNAs to induce degradation, the importance of the putative E3 ligase activity of Roquin-1 is currently unknown. In this proposal we will therefore set out to identify Roquin-1 and -2 E3 ligase substrates and cofactors and their activity in T cells. To do so we will apply a newly established approach, called BioID, that allows to biotinylate and purify proteins that had been in close proximity to Roquin in cells. Analyzing the resulting mass-spectrometry data, we will additionally determine whether peptides from already known Roquin target genes are enriched and compare this information to ribosome-profiling and mRNA next generation sequencing data. This bioinformatic analysis shall find out whether established targets of Roquin are inhibited by co-translational ubiquitination of nascent peptides. It will further clarify whether functions of the ROQ domain and the RING finger feed into convergent or independent regulatory processes. Finally, a major focus of this proposal will be to validate and comprehensively investigate the interactions of Roquin with its E3 ligase substrates or co-factors that either resulted from our BioID analysis or that came up in the functional whole genome CRISPR/Cas9 screen from our preliminary data. We further aim to determine the cellular impact of substrates or cofactors of Roquin in loss-of function and gain-of-function experiments in T cells to evaluate them as possible new targets for immune modulation.