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.