Fellow’s research: Identification of a novel molecular inhibitor of the microRNA pathway
09 Sep 2019
Dr Sandeep M Eswarappa, Intermediate Fellow
Indian Institute of Science, Bengaluru
Our recently published study shows that translational readthrough—continuation of protein translation beyond the stop codon—in argonaute-encoding AGO1 messenger RNA (mRNA) generates a novel type of the protein, which has an ability to inhibit the function of microRNAs.This protein plays an important role in the function of microRNAs, which are a class of small non-coding RNAs involved in regulation of protein synthesis.
Genetic message encoded in an mRNA is decoded to synthesize proteins by a process called translation. Ribosomes, the central machinery in this process, read the mRNA sequence in three nucleotide units termed as codons. There are specific codons in the mRNA, which signal the ribosomes to start and stop the process of translation. However, under certain conditions, ribosomes ignore the stop signal and continue the process of translation until they encounter the next stop codon. This process is termed as translational readthrough.
We used fluorescence- and luminescence-based reporter assays to demonstrate translational readthrough in AGO1 mRNA. Data obtained from ribosome profiling and mass spectrometry provided additional evidence of translational readthrough in AGO1. The readthrough product (i.e. the longer, functionally similar form of the protein Ago1), Ago1x, was detected using a specific antibody both in cell lines and in mouse.
This readthrough process is directed by a sequence in AGO1 mRNA downstream of its canonical stop codon. This sequence has a binding site for the microRNA let-7a, and readthrough is promoted by this microRNA.
microRNAs bind to their target mRNAs leading to gene silencing; however, Ago1x can load microRNAs on target mRNAs without causing post-transcriptional gene silencing. Because of these properties, Ago1x can serve as a competitive inhibitor of the microRNA 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, which is mediated by the translational readthrough product of AGO1.
Although, microRNAs are well known to inhibit translation initiation, through our work, we have shown their influence on translation termination as well; this was not known until now. Our study has identified a molecular inhibitor of the miRNA pathway, which regulates global translation in mammalian cells
Let-7a-regulated translational readthrough of mammalian AGO1 generates a microRNA pathway inhibitor. Anumeha Singh, Lekha E Manjunath, Pradipta Kundu, Sarthak Sahoo, Arpan Das, Harikumar R Suma, Paul L Fox, Sandeep M Eswarappa. The EMBO Journal. August 2019
Banner Image Credits: MicroRNAs as the micro managers of gene expression