Immune tolerance is key to the maintenance of the integrity of organisms against foreign invaders with respect to self-constituents. Deregulation of this mechanism promotes the occurrence of life-threatening autoimmune diseases that affect 5-10% of the general population. Previously, the product of the autoimmune regulator (Aire) gene was shown to play a key role in immune tolerance. Indeed, Aire induces medullary epithelial cells in the thymus (MECs) to synthesize and present a large repertoire of peripheral self-antigens (a “self-shadow”), leading to the clonal deletion of self-reactive maturing T cells and thereby protecting against autoimmune manifestations. Recently, important advances have enlightened the intriguing mode of action of Aire in showing that Aire recruits the transcriptional machinery at silenced genes and activates transcription elongation. In addition to activating transcription elongation, preliminary results indicate that Aire induces 3’UTR shortening of its sensitive transcripts in MECs, and that these cells show an accumulation of miRNAs. The overall goal of my project is to describe a post-transcriptional control of the Aire-driven expression in the thymus that leads to higher levels of Aire-dependent self-antigens, and to identify key molecular players involved in this mechanism. I propose as a first task to establish a widespread increase of miRNAs in MECs by quantifying the expression of a comprehensive panel of miRNAs. As miRNAs bind to 3’UTRs, and as 3’UTRs are subjected to dynamic regulation, we will assay the extent of 3’UTR length regulation in MECs by using Affymetrix whole-transcript microarrays and by analyzing the data at the single probe level (task 2). In order to characterize the escape of the Aire-induced genes from a repressive effect of miRNAs, we will assay the Aire-specific 3’UTR shortening in WT vs Aire-KO MECs by mRNA high-throughput sequencing (task 3). Subsequently, we will perform reporter assays in a MEC cell line in order to evaluate the increase of protein levels of short 3’UTR isoforms compared to long 3’UTR isoforms. We will also assay the impact of miRNAs on the post-transcriptional repression, by mutating the complementary sites of miRNAs in long 3’UTR reporters. The following objective (task 4) will be to identify the factors involved in the 3’UTR shortening triggered by Aire. We will first set up an in vitro model of Aire-triggered 3’UTR shortening, based on transient transfection of a MEC cell line with an Aire expression vector and a dual-luciferase reporter construct containing a prototypic 3’UTR whose transcript undergoes Aire-specific 3’UTR shortening as a read-out. Subsequently, an extended set of RNA-binding factors potentially involved in Aire’s mode of action will be screened by short-hairpin RNA (shRNA)-containing lentivirus infection of the in vitro model that we set up. Finally, to validate in vivo the effect of some RNA-binding proteins identified by the shRNA screen (the best candidates), we will generate knock-down mice using a high-speed lentigenic approach, which is based on oocyte infection with very-high titer shRNA-containing lentiviruses (task 5). Altogether the expected results should uncover an important layer of control of self-antigen expression in the thymus, at the post-transcriptional level. Completion of this project should shed a new light on our understanding of the processes that mediate the establishment of immune tolerance in the thymus, and provide new potential targets for therapeutic intervention in autoimmune diseases.