The RNA-binding Protein HuR Controls Thymocyte Development, Selection and Migration

The progressive development of T-cells in the thymus is a highly regulated process that aims to arm the body with T-cells against pathogens whilst maintaining tolerance to self. Although the role of gene transcription in this process has been extensively studied, little is known about the significance of associated post-transcriptional mechanisms. Post-transcriptional control is imposed by the dynamic interactions of RNA-binding proteins (RBPs) with RNA molecules which destine mRNA molecules towards translation or destruction. In this study we focus on the functions of an RBP called HuR. We made use of novel transgenic systems in which HuR is deleted in thymic T-cells to test whether HuR is involved in their developmental maturation. We show that HuR controls the generation and provision of T-cells competent for the proper recognition of antigens in the body and the elimination of harmfull cells. We further show that HuR controls T-cell development through its involvement in basic cellular processes like cell cycle regulation, cellular activation, death and migration which were associated to HuR’s efficacy in regulating the expression or function of key signaling components. Our studies provide important insight on the post-transcriptional regulation of fundamental cellular processes and identify HuR as an important modulator of adaptive immunity and autoimmunity. Total thymocytes were isolated from female 6-8 week old LckCre+ Elavl1+, LckCre+Elavl1fl/fl and Elavlfl/fl thymi following desegregation and pooled from n=6 littermate mice/group, were cultured in complete RPMI-1640 medium the presence or absence of PMA (10 ng/ml) and ionomycin (500ng/ml). Cells were collected at 0, 4 and 12 hrs post stimulation and washed in ice cold PBS via centrifugation, prior to lysis with Trizol (Invitrogen). Total RNA was then treated with DNase I to minimize contamination from genomic DNA. Further purification was carried out using the RNeasy columns (Qiagen). The experiment was performed two more times to complete a set of 3 biological replicates. In all cases, Ex vivo RNA samples were labeled with Cy3 and reference samples were labeled with Cy5. NaOH was used to destroy residual RNA.