Working on this project:
Post-transcriptional gene regulation by miRNAs plays a fundamental role in many biological processes. The molecular mechanisms of miRNA function and target recognition are only partially understood. In particular, it remains unclear how an individual miRNA can mediate a robust biological effect despite generally modest repression of target mRNAs. Recent evidence suggests that target selection of miRNAs is highly context-dependent and can only partially be explained by established principles of miRNA binding to miRNA response elements (MREs) in target mRNA. We have identified and characterized miRNAs contributing to development and function of the adaptive immune system. We showed that one of these miRNAs, miR-181a, is critical for development and function of non-conventional T cells. Furthermore, in a pre-clinical model of Graft-versus-Host-Disease, we demonstrated that miR-181a is also involved in controlling T-cell function. Using T-cell development as a defined physiologic model, we propose to address the following questions: How do defined concentrations of a miRNA functionally repress a distinct set of target genes in the presence of an excess of MREs to maintain a physiologic state? Can a model of dose-dependent repression by miRNAs be generated based on previously established targeting principles? To answer these questions, we will employ classical and well-established inducible systems of dose-dependent gene expression as well as novel light-inducible tools developed in the previous funding period. We will combine functional readouts of T-cell development and function with global target identification assays and molecular characterization of MREs at single-nucleotide resolution. Together, the proposed experiments will shed light on the role of miRNA concentration and the stoichiometry of miRNA and its cognate MREs in miRNA-mediated gene regulation in physiologically relevant settings.