miRNA mediated repression depends on number of active binding sites and miRNA expression level
miRNAs are predominantly known to be modulators of the target gene expression. However, recent studies related to miRNA mediated target repression at the single-cell level indicate that they can even influence the target gene repression following a bi-stable switching mechanism. Under these circumstances, miRNA abundance and the number of MREs have a greater impact on the repression kinetics.53,54 Mukherji et al53 used dual-color fluorescent reporters with and without binding sites with imperfect complementarity to miR-20a in the 3’UTR of the fluorescent genes. They have seen that there exists an mRNA threshold level below which the miRNA highly represses the corresponding mRNA. Once the mRNA level increases above the threshold, then mRNAs become free of the miRNA mediated repression. Mukherji et al53 explained the dynamics of microRNA-mediated switch-like repression kinetics using a molecular titration model (Fig. 3 ).53 As the mRNA level increases, miRNA gets titrated due to the formation of miRNA:mRNA complex, and after a certain threshold, mRNA can escape the repressive effect. The model recreates the threshold behavior in miRNA-mediated repression observed in experiments. They further studied the fold repression by introducing a single binding site with perfect complementarity to miR-20a into the fluorescent gene. However, there was no mRNA threshold for gene expression in the presence of miR-20a. Instead, the fold repression gradually decreased with an increase in mRNA levels.
Next, they investigated the importance of various parameters such as miRNA level and rate of association between miRNA and mRNA associated with miRNA mediated target regulation. Increasing the association rate increased the sharpness of the threshold without affecting the threshold mRNA amount. However, altering the miRNA level influences both the sharpness of the threshold as well as the amount of mRNA accumulation required for escape from repression (Fig. 3a ). Experimentally, to increase the binding affinity, the number of miR-20a binding sites in the 3’UTR were increased and miRNA levels were modulated using miR-20a mimics. They observed that increasing the binding sites sharpened the switch from full repression to escape from mir-20a mediated repression (Fig. 3b ). However, the fold repression was higher at low protein levels for an increase in the binding sites, and there was no significant change at high protein levels. For four miR-20a binding sites in the 3’UTR, increasing the concentration of miRNA mimics sharpened the transition from repression to escape. The miRNA levels also modulated the target mRNA level required for the transition as predicted by the titration model.53