Prioritizing Annotated miRNAs: Only a Small Percentage are Candidates for Biological Regulation

The potential for miRNAs to regulate gene expression remains controversial. DROSHA initiates the biogenesis of miRNAs while Argonaute (AGO) and TNRC6 proteins form complexes with miRNAs that recognize RNA. Here we investigate the fate of miRNAs in the absence of critical RNAi protein factors. Knockout of DROSHA expression reduced levels of some miRNAs, but not others. Knocking out AGO proteins, which directly contact the mature miRNA, decreased expression of miRNAs. Quantitative analysis indicates compensation to maintain the overall pool of AGO after knockout of AGO variants. Evaluation of miRNA binding to AGO proteins revealed that association between AGO and miRNAs was similar for AGO1 - 4. Contrary to the assumptions underlying many peer-reviewed reports, not all annotated miRNAs have equal potential as biological regulators. Cellular abundance, DROSHA dependence, and physical association with AGO must be considered when forming hypotheses related to their function. Our data prioritize sixty miRNAs – under two percent of the overall annotated miRNA repertoire – as being most likely to function as robust gene regulators. Our approach will facilitate identifying biologically active miRNAs.