The RNA-Binding Proteins Regnase-1, Regnase-4 and PDAP1 Modulate Human T Helper Lymphocyte Cytokine Production and Proliferation

Abstract

T helper (TH) lymphocytes are essential cells of the immune system that produce cytokines which tailor the immune response to be most efficient against a specific pathogen. Gene expression of TH lymphocytes must be carefully and rapidly regulated, since they have to mount fast and specific responses upon encountering an antigen, while at the same time limiting excessive inflammation and tissue damage. Post-transcriptional regulation plays a key role in the function of TH lymphocytes by modulating the stability and degradation of mature mRNAs, including those encoding for cytokines and other immune-relevant genes. This level of regulation is tightly controlled by a complex network of RNA-binding proteins (RBPs) in cooperation with microRNAs (miRNAs). RBPs often have multiple functions, paralogues and dynamic expressions which is why their function remains partially elusive, while for other RBPs the functions are completely unknown. In the work presented in this thesis, I aimed to characterize the role of RBPs in modulating the function of TH lymphocytes. Specifically, in the first part of the study, I investigated the redundancy of two RBP paralogues (Regnase-1 and Regnase-4), which have some known common and different features, as seen on the mouse knockout (KO) phenotypes. Regnases are RBPs with intrinsic ribonuclease activity that directly degrade mRNAs of immune-related genes. By using CRISPR-Cas9, I identified that Regnase-4 does not have unique mRNA targets, however, the deletion of both Regnases lead to a combined derepression of multiple known Regnase targets. Interestingly, despite high homology, Regnase-1 and Regnase-4 have differing expression dynamics upon T cell activation. Therefore, the observed functional differences between Regnase-1 and Regnase-4 can be attributed to their different expression levels and gene dosage. In the second part of the study, I described how we identified and functionally characterized another RBP, namely PDAP1. We identified that RFX transcription factors modulate the expression of miR-150 which directly negatively regulates the expression of PDAP1. RNA-immunoprecipitation and sequencing (RIP-seq) of PDAP1 revealed directly bound mRNA targets that are crucial for T-cell activation, differentiation, and proliferation. Analysis of PDAP1 KO clones suggested that PDAP1 increases the stability of at least a subset of direct mRNA targets. The abrupt downregulation of miR-150 upon T cell activation releases PDAP1 from the negative brake enabling it to promote proliferation of TH lymphocytes. Overall, this study contributed to our understanding of the function of RBP paralogues and a novel RBP in modulating T cell function and proliferation