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...