Half of annotated human microRNAs are expressed at levels of questionable biological significance

MicroRNAs (miRNAs) are widely studied for their role in post-transcriptional gene regulation, often using exogenous overexpression to infer function. However, such approaches may not reflect physiological activity, as they rely on unnaturally high expression levels. To address this, we determined the minimal threshold required for detectable endogenous miRNA function using sensitive reporters. Comparison with small RNA sequencing data from thousands of cell lines and tissues revealed that over half of all miRNAs never reach functionally relevant levels, with more recently evolved miRNAs generally exhibiting lower expression. This challenges the validity of numerous studies reporting roles for lowly expressed miRNAs, suggesting many findings reflect artefacts of overexpression rather than true biological activity. Our work underscores the importance of evaluating miRNA function in a native context and supports the view that the functional human “microRNAome” is substantially smaller than estimates based solely on sequencing data. Overall design: RNA-seq profiling of small RNAs expressed in wildtype HEK-293T, HeLa, MDA-MB-231 and C32 cells