Differential protein- and RNA-interactions of Roquin specify alternative modes of post-transcriptional gene regulation

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Appropriate immune responses require CD4+ T cells to make profound cell fate decisions, which are placed under post-transcriptional control. A key player in these decisions is the RNA-binding protein (RBP) Roquin. While the prototypic interaction of Roquin with its consensus decay element and subsequent deadenylation and decapping of the target mRNA is well understood, many observations imply a much higher complexity of Roquin-mediated gene regulation. It not only involves interactions with additional cofactors and different post-transcriptional effectors causing different modes of post-transcriptional regulation but also interactions with different recognition elements in mRNAs and composite binding sites. In the first funding period, we studied the post-transcriptional regulation of Icos mRNA by the RNA-binding protein Roquin-1. We could show that Roquin-1 directly interacts with its cofactor Nufip2 and thereby increases Icos mRNA repression. Additional co-regulators identified by us and colleagues include the endonuclease Regnase-1 and the deadenylation factor Cnot1. In the second funding period we will therefore characterize and quantify the interaction of Roquin with Nufip2, Regnase-1, and Cnot1, map their interaction surfaces in case of direct interactions, and perform structural analyses of their co-complexes by X-ray crystallography, micro electron diffraction, and NMR. We will use this information to generate mutations that specifically abolish a particular interaction, verify their loss of binding, and characterize in T cells their global effects on gene regulation. In the first funding period, we performed a saturating mutagenesis screen with over 10,000 distinct mutations in the 2.6 kb long 3‘-UTR of Icos mRNA and used next-generation sequencing to quantify the effects of mutations on Icos mRNA regulation. In the second funding period we will perform an in-depth analysis of the results of this mutagenesis screen. Cis-acting elements identified in the screen will be reassessed in T cells using the CRISPR/Cas9 technology for reproducibility of their regulatory function and to understand their specific modes of regulation. Furthermore, we aim to identify proteins binding close to these cis-elements by using a RNA-tethered proximity ligation approach. RNA-binding proteins and co-factors identified this way, will be assessed for their binding to the corresponding cis-acting element, as well as to Roquin, Regnase-1 and Nufip2. The importance and specific functions of the most relevant factors will be assessed in T cells by knock-out, followed by target gene analysis. In summary, by elucidating how Roquin-1 cooperates with different trans-acting co-factors to select specific modes of translational inhibition we aim at identifying principles of combinatorial post-transcriptional gene regulation of Roquin-dependent target mRNAs.