The influence of miRNAs on variation of gene expression during T-cell development

Abstract

MicroRNAs (miRNAs) are small non-coding RNAs which regulate gene expression post-transcriptionally, by binding to specific mRNAs. It has been suggested that miRNAs have a role in "canalising" development and reducing variability in gene expression. Using conditional deletions of the miRNA biogenesis enzyme Dicer in mice, I investigated the effect of miRNA depletion on gene expression during T-cell development. I used flow cytometry to obtain the distribution of proteins on a single cell basis. Proteins encoded by miRNA-regulated transcripts showed a Dicer-dependent increase in both mean protein expression and cell-cell variation. In particular, the genes Sca-1 and Cd44 show increased expression and cell-cell variation in Dicer-deleted double positive thymocytes, and Cd69 showed increased expression and cell-cell variation in Dicer-deleted thymocytes activated by stimulation of the T-cell receptor. Using fluorescent reporter constructs, the effect of the 3’ UTR of each mRNA on reporter expression was investigated to find miRNA binding sites. I identified binding sites for the miR-181, miR-130 and miR-20 miRNA families in the Cd69 3’ untranslated region (UTR). To further investigate how these miRNAs might regulate the expression of Cd69, I investigated how expression of miRNAs changed on T-cell activation: observing that the miR-181 family is downregulated after activation in thymocytes, and the miR-20 family is upregulated after activation in both thymocytes and mature peripheral T-cells. I used both miRNA inhibitors and deletion of specific miRNA families to confirm that the miR-181 and miR-20 families both regulate expression of Cd69 during thymocyte activation. Finally, I looked at theoretical models of how miRNAs might regulate biological noise, and found that the feedforward loop motif can reduce noise compared to an unregulated gene under conditions of moderate miRNA repression. These results show that depletion of miRNAs can result in increased cell-cell variation in developmentally regulated thymocyte genes.

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