Translational readthrough of AGO1 mRNA and its regulation by let-7a microRNA

Abstract

During translation, the message encoded in mRNAs is decoded by ribosomes to synthesize proteins following strict canonical decoding rules. This process is tightly regulated and several mechanisms exist in cells that ensure accuracy of this process. However, under certain circumstances, translational recoding events that defy the canonical rules of translation might occur. Translational readthrough (or stop-codon readthrough) is one such event. Translational readthrough is the phenomenon of continuation of protein synthesis beyond a stop codon, till the next in-frame stop codon. This process gives rise to a C-terminally extended isoform of the protein. Translational readthrough has been well studied in viruses, fungi and invertebrates. However, our understanding of translational readthrough in vertebrates is very limited. Several mRNAs have been predicted to undergo translational readthrough in mammals. A genome wide bioinformatics screen revealed that AGO1 mRNA (encodes Argonaute 1) is a potential translational readthrough candidate. Argonaute 1 (Ago1) is a key protein in miRNA-mediated gene silencing pathway. In this thesis, we discuss a translational readthrough event in mammalian AGO1 mRNA, which generates a microRNA pathway inhibitor and the regulation of this phenomenon by miRNA let-7a. In the first part, we demonstrate translational readthrough of mammalian AGO1 mRNA by multiple reporter-based translational readthrough assays. We also provide supporting evidence from analysis of previously published ribosome profiling and mass spectrometry data. Endogenous readthrough product of AGO1, termed as Ago1x, was detected by a specific antibody both in vitro and in vivo. Our results demonstrate that the cis sequence present downstream of the canonical AGO1 stop codon is important for translational readthrough of AGO1. In the second part, we demonstrate that miRNA let-7a binding to the cis-sequence present downstream of the stop codon, enhances the translational readthrough of AGO1. We could also demonstrate that this cis sequence is sufficient to drive translational readthrough even in a heterologous context. In the third part, we investigated the function of the translational readthrough product of AGO1, termed Ago1x. Our results demonstrate that Ago1x, like Ago1, interacts with miRNAs and loads them onto the target mRNAs. However, due to its inability to interact with the protein GW182, Ago1x cannot cause post-transcriptional gene silencing. In the fourth part, we investigate the physiological significance of translational readthrough of AGO1. We observed increased global translation upon overexpression of Ago1x, suggesting that Ago1x acts as a competitive inhibitor of miRNA pathway. We also observed a decrease in translational readthrough of AGO1 during serum starvation, which is associated with attenuated global translation. In summary, our study reveals a novel isoform of Ago1 generated as a result of translational readthrough of AGO1 mRNA. This isoform acts as a miRNA pathway inhibitor. Our study also describes a hitherto unknown function of miRNAs in translational recoding at the stop codon.