Exploiting variability of single cells to uncover the in vivo hierarchy of miRNA targets

MiRNAs are post-transcriptional repressors of gene expression that may additionally reduce the cell-to-cell variability in protein expression, induce correlations between target expression levels and provide a layer through which targets can influence each other's expression as 'competing RNAs' (ceRNAs). Here we combined single cell sequencing of human embryonic kidney cells in which the expression of two distinct miRNAs was induced over a wide range, with mathematical modeling, to estimate Michaelis-Menten (KM)-type constants for hundreds of evolutionarily conserved miRNA targets. These parameters, which we inferred here for the first time in the context of the entire network of endogenous miRNA targets, vary over ~2 orders of magnitude. They reveal an in vivo hierarchy of miRNA targets, defined by the concentration of miRNA-Argonaute complexes at which the targets are most sensitively down-regulated. The data further reveals miRNA-induced correlations in target expression at the single cell level, as well as the response of target noise to the miRNA concentration. The approach is generalizable to other miRNAs and post-transcriptional regulators and provides a deeper understanding of gene expression dynamics.