Quantification of microRNA regulation and its consequences at the single cell level

Abstract

MicroRNAs (miRNAs) are a class of small non-coding RNAs which play important roles in post transcriptional gene regulation. miRNAs regulate more than half of mammalian protein-coding genes. They have been found to participate in almost every cellular process and their dysregulation is associated with many diseases. miRNAs recognize their targets by base paring to miRNA response elements (MREs), which are predominantly located at 3' untranslated region (3'UTR) of mRNAs. This thesis focuses on a microRNA activity reporter system to investigate various aspects of miRNA regulation on its endogenous 3'UTR targets. Mutation of selected MREs on 3'UTRs (MutUTRs) was designed and validated as miRNA unregulated control. It does not require genetic modifications of cellular background and effectively abolishes the majority of miRNA regulation with minimum perturbation to the UTR sequences. MicroRNAs can induce target silencing via mRNA transcript degradation and translational inhibition. But the relative contributions from the two sources have been under debate. It is also unclear how miRNA regulation varies for different target expression. MicroRNA regulation at the transcriptional and translational levels was quantified at single cell resolution over a target expression range of more than 100 fold using our reporter system. The transcriptional regulation was found to be uniform throughout the range of measurement, whereas translational regulation decreases at high target expression. Our data also suggests that translational regulation increase initially at low target expression for certain targets. For all UTRs under study, miRNA regulation from the two sources were found to be on the same order. In addition to target repression, miRNAs also control target expression noise. MicroRNAs decrease protein expression noise for lowly expressed genes, but increase noise for highly expressed genes, and the noise regulation seems to happen at translational level. By linking reporter assays to transcriptome expression, our findings suggest that microRNAs confer precision to protein expression in vivo, and transcriptional regulation might dominate for endogenous targets. Finally we applied the reporter system as miRNA decoys to study miRNA-mediated- crosstalk. We also propose that the reporter systems could be used to study alternative polyadenylation, which is usually accompanied by consequential loss of MREs.