Molecular titration by MicroRNAs and target mimic inhibitors

Abstract

MicroRNAs (miRNAs) are short, highly conserved non-coding RNA molecules that repress gene expression in a sequence-dependent manner. Each miRNA is predicted to target hundreds of genes, and a majority of protein-coding genes are computationally predicted to be miRNA targets. To test miRNA functions experimentally, we introduced the miRNA "sponge" method, which uses miRNA target mimics to sequester mature miRNAs and thereby create continuous miRNA loss of function in cell lines and transgenic organisms. Sponge RNAs contain complementary binding sites to a miRNA of interest, and are produced from transgenes within cells. As with most miRNA target genes, a sponge's binding sites are specific to the miRNA seed region, which allows them to block a whole family of related miRNAs. This transgenic approach has proven to be a powerful tool to generate miRNA phenotypes in a variety of experimental systems. Bulk measurements on populations of cells have indicated that, although pervasive, repression due to miRNAs is on average quite modest. To assay repression in single cells, we performed quantitative fluorescence microscopy and flow cytometry to monitor a target gene's protein expression in the presence and absence of regulation by miRNA. We found that repression among individual cells varies dramatically. miRNAs establish a threshold level of target mRNA below which protein production is highly repressed and above which expression responds ultra sensitively to target mRNA input until reaching high enough mRNA levels to almost escape repression. We constructed a mathematical model describing molecular titration of target mRNAs by miRNAs. The model predicted, and experiments confirmed, that the ultrasensitive regime could be shifted to higher target mRNA levels by increasing the miRNA concentration or the number of miRNA binding sites in the 3' untranslated region (UTR) of the target mRNA. Thus even a single species of miRNA can act both as a switch to effectively silence gene expression and as a fine-tuner of gene expression. This fits the emerging paradigm in which miRNAs help to confer robustness to biological processes by reinforcing transcriptional programs, attenuating leaky transcripts, and perhaps buffering random fluctuations in transcript copy number.