Argonaute binding within 3'-untranslated regions poorly predicts gene repression.

David R Corey,Shinnichi Yokota,Yongjun Chu,Jing Liu,Krystal C Johnson,Audrius Kilikevicius

doi:10.1093/nar/gkaa478

David R Corey, Shinnichi Yokota + Show 4 more

Open Access

https://doi.org/10.1093/nar/gkaa478

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Abstract

Despite two decades of study, the full scope of RNAi in mammalian cells has remained obscure. Here we combine: (i) Knockout of argonaute (AGO) variants; (ii) RNA sequencing analysis of gene expression changes and (iii) Enhanced Crosslinking Immunoprecipitation Sequencing (eCLIP-seq) using anti-AGO2 antibody to identify potential microRNA (miRNA) binding sites. We find that knocking out AGO1, AGO2 and AGO3 together are necessary to achieve full impact on steady state levels of mRNA. eCLIP-seq located AGO2 protein associations within 3′-untranslated regions. The standard mechanism of miRNA action would suggest that these associations should repress gene expression. Contrary to this expectation, associations between AGO and RNA are poorly correlated with gene repression in wild-type versus knockout cells. Many clusters are associated with increased steady state levels of mRNA in wild-type versus knock out cells, including the strongest cluster within the MYC 3′-UTR. Our results suggest that assumptions about miRNA action should be re-examined.

Highlights

RNA interference (RNAi) is a powerful mechanism for controlling steady state levels of mRNA in mammalian cells [1,2] and is achieving success in the clinic [3,4]
We obtained CRISPR gene knockouts to investigate the role of argonaute protein (AGO) in controlling steady state levels of mRNA
Our goal was to test this hypothesis by combining AGO knockout cells, RNA sequencing (RNAseq), and anti-AGO2 enhanced crosslinking immunoprecipitation (eCLIP) (Figure 1), followed by analysis of the linkage between AGO2 binding within 3 UTRs and altered gene expression

Summary

Introduction

RNA interference (RNAi) is a powerful mechanism for controlling steady state levels of mRNA in mammalian cells [1,2] and is achieving success in the clinic [3,4]. Chromosomally-encoded hairpin RNAs are expressed and are processed into microRNAs (miRNAs) [5] These miRNAs are loaded into argonaute protein (AGO) to form a ribonucleoprotein in which the miRNA directs the complex to bind complementary RNA sequences. AGO2 is the best studied and is known as the catalytic engine for RNAi because it possesses an enzyme active site capable of cleaving target RNA transcripts [10,11]. This ability to cleave mRNA is critical for applying fully complementary synthetic RNAs to gene silencing and contributes to the robustness of RNAi as a method for controlling gene expression in the laboratory and the clinic. AGO4 had been observed to make a negligible contribution to RNAi in the cell lines tested [12]

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