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