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

Anumeha Singh,Pradipta Kundu,Sandeep M Eswarappa,Paul L Fox,Sarthak Sahoo,Lekha E Manjunath,Harikumar R Suma,Arpan Das

doi:10.15252/embj.2018100727

Anumeha Singh, Pradipta Kundu + Show 6 more

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https://doi.org/10.15252/embj.2018100727

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Abstract

Translational readthrough generates proteins with extended C‐termini, which often possess distinct properties. Here, we have used various reporter assays to demonstrate translational readthrough of AGO1 mRNA. Analysis of ribosome profiling data and mass spectrometry data provided additional evidence for translational readthrough of AGO1. The endogenous readthrough product, Ago1x, could be detected by a specific antibody both in vitro and in vivo. This readthrough process is directed by a cis sequence downstream of the canonical AGO1 stop codon, which is sufficient to drive readthrough even in a heterologous context. This cis sequence has a let‐7a miRNA‐binding site, and readthrough 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.

Highlights

Translating ribosomes normally terminate at the first in-frame stop codon they encounter on the mRNA
These constructs were transfected in HEK293 cells, and the translational readthrough was measured as firefly luciferase (FLuc) activity normalized to the activity of co-transfected Renilla luciferase (RLuc)
Analysis of the previously published ribosome profiling data and the mass spectrometry data strengthened the evidence for AGO1 translational readthrough

Summary

Introduction

Translating ribosomes normally terminate at the first in-frame stop codon they encounter on the mRNA. In some transcripts, under certain conditions, translating ribosomes continue to translate beyond the stop codon until they encounter another in-frame stop codon. This process, termed as translational readthrough (or stop codon readthrough), generates longer isoforms with unique C-terminal extensions, which can have different functions or different localization (Eswarappa et al, 2014; Schueren et al, 2014; Hofhuis et al, 2016), contributing to proteome expansion. Functional translational readthrough is usually programmed by the downstream cis-acting RNA elements such that only select transcripts undergo this process. Readthrough can be regulated by trans-acting molecules that bind these RNA elements as shown in the case of VEGFA where an RNA-binding protein, hnRNPA2/B1, promotes readthrough (Eswarappa et al, 2014)

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