Connecting E3 ligase and mRNA decay functions of Roquin proteins

Roquin蛋白预防自身免疫性疾病的发展，控制自发T细胞活化和不适当的T细胞分化。Roquin-1及其相似的Roquin-2在n端区域具有高度相似的结构域结构，包括新发现的rna结合结构域ROQ和RING finger。虽然许多出版物已经确定Roquin及其ROQ结构域直接与目标mrna中的顺式元件结合以诱导降解，但Roquin-1的假定E3连接酶活性的重要性目前尚不清楚。因此，在本提案中，我们将着手鉴定Roquin-1和-2 E3连接酶底物和辅助因子及其在T细胞中的活性。为此，我们将采用一种名为BioID的新方法，该方法允许对细胞中与Roquin非常接近的蛋白质进行生物素化和纯化。分析所得质谱数据，我们还将确定已知Roquin靶基因的肽是否富集，并将此信息与核糖体分析和下一代mRNA测序数据进行比较。这个生物信息学分析将发现Roquin的既定靶点是否被新生肽的共翻译泛素化所抑制。它将进一步阐明ROQ结构域和无名指的功能是否参与聚合或独立的调控过程。最后，本提案的一个主要重点将是验证和全面研究Roquin与其E3连接酶底物或辅助因子的相互作用，这些相互作用要么来自我们的BioID分析，要么来自我们的初步数据的功能性全基因组CRISPR/Cas9筛选。我们进一步旨在确定Roquin底物或辅助因子在T细胞功能丧失和功能获得实验中的细胞影响，以评估它们作为免疫调节可能的新靶点。

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.