Characterization of Non-Coding RNAs in Regulating Thymic Epithelial Cell Responses to Pathophysiological Stress

The thymus is uniquely sensitive to several forms of stress. Stress initiates a transient involution that can reduce overall thymic volume up to 90%. The thymus is predominantly composed of developing thymocytes and specialized epithelial cells. The type of stress predicates whether the thymocytes or epithelial cells initiate the involutionary response. MicroRNAs (miRs) are small non-coding RNAs ~18-22 nucleotides in length that maintain cellular homeostasis and regulate stress responses. Previous work in the laboratory identified several microRNAs involved in regulating thymocyte responses to stress. Thymocytes have been the main population studied in response to stress. However, it has become increasingly clear that the epithelial cells play a critical role in thymus involution and the subsequent recovery of thymopoiesis. The work presented in this thesis characterizes an epithelial specific miR, miR-205, and its surrounding long noncoding RNA, MIR205.001 in regulating TEC functions. A deficiency of miR-205 specifically in TECs renders these cells more susceptible to stress mediated thymic involution. This is revealed by a significant loss in developing thymocytes, altered migration, delayed recovery of single positive thymocyte selection, and proliferative defects in cortical TECs. Gene expression comparisons revealed miR-205 deficient TECs had reduced levels of the TEC master transcriptional regulator, Foxn1, as well as the expression of multiple chemokines. MiR-205 mimics introduced into miR-205 deficient fetal thymic organ cultures were able to restore the levels of Foxn1 and selected chemokines. This work demonstrates that miR-205 positively regulates Foxn1 and chemokine expression following stress. MiR-205 resides within a putative long noncoding RNA (lncRNA), MIR205.001. TECs deficient in this lncRNA are also sensitive to stress, but do not experience a delay in thymopoiesis nor cortical TEC proliferation defects. This suggests these noncoding RNAs have non-overlapping functions within the TECs. Mice deficient in MIR205.001 also have distinct phenotypes, displaying reduced mendelian ratios indicative of a lethality. The surviving animals display reduced body size, weight, and fat mass. Current experiments are addressing whether this is a metabolic defect or due to changes in feeding behavior.