Final Report Summary - RC3H1/2-SPECIFICITY (Specificity of Rc3h1/2 proteins in post-transcriptional control of immunity and autoimmune disease)
Our research in the grant 218666_Rc3h1/2-specificity was successful as it showed that the Roquin-1 and Roquin-2 encoding Rc3h1 and Rc3h2 genes serve redundant functions in T cells, but nevertheless each one of them is essential for the survival of mice. These findings provided a first explanation for the contradictory observations that autoimmunity develops in mice with the sanroque pointmutation in the Roquin-1 encoding gene Rc3h1, but is absent when this gene was deleted. Together, we demonstrated that Roquin-2 functionally replaces Roquin-1 in the prevention of autoimmunity, but cannot do so when Roquin-1 is present in its mutated form.
On the cellular level we were able to show that Roquin proteins exert essential control over spontaneous T cell activation and T helper differentiation into Th1, Tfh and Th17 subsets and we have uncovered that these proteins regulate targets within the pathways that control differentiation of CD4+ T cells into these subsets. Moreover, using tools that were generated for this project we uncovered post-translational regulation of Roquin proteins downstream of T cell receptor (TCR) signaling through proteolytic cleavage by the paracaspase MALT1.
Another strong focus in this project was on the identification and definition of Roquin binding sites. In close collaborations with structural biologists we defined how Roquin interacts through its ROQ domain with RNA to recognize secondary structures in the RNA like the constitutive decay elements (CDE). Employing "Systematic Evolution of Ligands by Exponential Enrichment (SELEX) we then identified a novel alternative decay element (ADE) and by Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation we have now defined a linear response element (LRE) as another novel Roquin binding site.
According to our work these binding sites differ very much in their affinity and different mRNA targets with composite binding sites can be recognized by Roquin in concert with other RNA-binding proteins like Regnase-1 thereby creating cellular targets of very different avidity. These insights have led us to propose a model in which increasing TCR signal strength gradually inactivates Roquin, thus causing differential target mRNA derepression that specifies cell fate decisions and effector functions of T cells.
On the cellular level we were able to show that Roquin proteins exert essential control over spontaneous T cell activation and T helper differentiation into Th1, Tfh and Th17 subsets and we have uncovered that these proteins regulate targets within the pathways that control differentiation of CD4+ T cells into these subsets. Moreover, using tools that were generated for this project we uncovered post-translational regulation of Roquin proteins downstream of T cell receptor (TCR) signaling through proteolytic cleavage by the paracaspase MALT1.
Another strong focus in this project was on the identification and definition of Roquin binding sites. In close collaborations with structural biologists we defined how Roquin interacts through its ROQ domain with RNA to recognize secondary structures in the RNA like the constitutive decay elements (CDE). Employing "Systematic Evolution of Ligands by Exponential Enrichment (SELEX) we then identified a novel alternative decay element (ADE) and by Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation we have now defined a linear response element (LRE) as another novel Roquin binding site.
According to our work these binding sites differ very much in their affinity and different mRNA targets with composite binding sites can be recognized by Roquin in concert with other RNA-binding proteins like Regnase-1 thereby creating cellular targets of very different avidity. These insights have led us to propose a model in which increasing TCR signal strength gradually inactivates Roquin, thus causing differential target mRNA derepression that specifies cell fate decisions and effector functions of T cells.