PT  - JOURNAL ARTICLE
AU  - Charlotte A. Cialek
AU  - Tatsuya Morisaki
AU  - Ning Zhao
AU  - Taiowa A. Montgomery
AU  - Timothy J. Stasevich
TI  - Imaging translational control by Argonaute with single-molecule resolution in live cells
AID  - 10.1101/2021.04.30.442135
DP  - 2021 Jan 01
TA  - bioRxiv
PG  - 2021.04.30.442135
4099  - http://biorxiv.org/content/early/2021/04/30/2021.04.30.442135.short
4100  - http://biorxiv.org/content/early/2021/04/30/2021.04.30.442135.full
AB  - A major challenge to our understanding of translational control has been deconvolving the individual impact specific regulatory factors have on the complex dynamics of mRNA translation. MicroRNAs (miRNAs), for example, guide Argonaute and associated proteins to target mRNAs, where they direct gene silencing in multiple ways that are not well understood. To better deconvolve these dynamics, we have developed technology to directly visualize and quantify the impact of human Argonaute2 (Ago2) on the translation and subcellular localization of individual reporter mRNAs in living cells. We show that our combined translation and Ago2 tethering system reflects endogenous miRNA-mediated gene silencing. Using the system, we find that Ago2 association leads to progressive silencing of translation at individual mRNA. The timescale of silencing was similar to that of translation, consistent with a role for Ago2 in blocking translation initiation, leading to ribosome runoff. At early time points, we observed occasional brief bursts of translational activity at Ago2-tethered mRNAs undergoing silencing, suggesting that translational repression may initially be reversible. At later time points, Ago2-tethered mRNAs cluster and coalesce with endogenous P-bodies, where a translationally silent state is maintained. These results provide a framework for exploring miRNA-mediated gene regulation in live cells at the single-molecule level. Furthermore, our tethering-based, single-molecule reporter system will likely have wide-ranging application in studying general RNA-protein interactions.Competing Interest StatementThe authors have declared no competing interest.