Argonaute Binding within 3'-Untranslated Regions Does Not Predict Gene Repression

Despite two decades of study, the full scope of RNAi in mammalian cells has remained obscure. Here we combine: 1) Systematic knockout of argonaute (AGO) variants; 2) RNA sequencing analysis of gene expression changes relative to wild-type cells; and 3) Crosslinking Immunoprecipitation Sequencing (CLIP-seq) using anti-AGO2 antibody to identify potential microRNA (miRNA) binding sites. We find that knocking out AGO1, AGO2, and AGO3 are necessary to achieve full impact on gene expression. CLIP-seq reveals several hundred significant AGO2 associations within the 3′-untranslated regions of cytoplasmic transcripts. The standard mechanism of miRNA action would suggest that these associations would repress gene expression. Contrary to this expectation, we observe that clusters are poorly correlated with gene repression in wild-type versus knockout cells. Many clusters are associated with increased gene expression in wild-type cells, including the strongest cluster within the MYC 3′-UTR. Our results suggest that assumptions about miRNA action should be re-examined and that claims related to the action of specific miRNAs should be better justified.