Let-7a-regulated translational readthrough of mammalian AGO1 generates a microRNA pathway inhibitor

Translational readthrough generates proteins with extended C terminus which often possess distinct properties. Here, we have demonstrated translational readthrough of AGO1 using various reporter assays. Analysis of ribosome profiling data and mass-spectrometry data provided additional evidences for translational readthrough of AGO1. We could also detect endogenous readthrough product, Ago1x, using a specific antibody both in vitro and in vivo. This process is programmed by a cis sequence downstream to the canonical stop codon of AGO1 which could drive readthrough even in a heterologous context. The cis sequence has a let-7a miRNA binding site and the readthrough process is promoted by let-7a miRNA. Interestingly, Ago1x can load miRNAs on target mRNAs without causing post-transcriptional gene silencing due to its inability to interact with GW182. Because of these properties, Ago1x can serve as a competitive inhibitor of miRNA pathway. In support of this, we observed increased global translation in cells overexpressing Ago1x. Overall, our results reveal a negative feedback loop in the miRNA pathway mediated by the translational readthrough product of AGO1.