Appropriate adaptive immune responses require T helper cells to make profound cell fate decisions, which involve post-transcriptional level of gene regulation. 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. It also involves interactions with different recognition elements in mRNAs and on 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 cofactors Nufip2 and the endonuclease Regnase-1. Here, we will characterize and quantify the interactions of Roquin with Nufip2 and Regnase-1, as well as with its downstream effector Cnot1. We will map their interaction surfaces in case of direct interactions and perform structural analyses of their co-complexes. 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. We will also identify proteins binding close to Roquin regulated 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-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 or mRNA decay we aim at identifying principles of combinatorial post-transcriptional gene regulation of Roquin- dependent target mRNAs.

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