Elucidating translation control of Argonaute in live cells by developing a tetherable biosensor with single-mRNA resolution
Date
2021
Authors
Cialek, Charlotte A., author
Stasevich, Timothy, advisor
Montgomery, Taiowa, advisor
Hoerndli, Fred, committee member
Nishimura, Erin, committee member
Ross, Eric, committee member
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Abstract
Translation is an essential step for all living beings. It stands as the final hurdle of converting our genetic code into functional protein products. A culmination of specific factors heavily regulates what, when, and how much of a given peptide product is translated (Chapter 1). Though many technological advancements have expanded our understanding of translational control, they have often opened many new questions due to the high complexity of this process. Recently, a technique called Nascent Chain Tracking (NCT) was able to image translation with single molecule resolution in living cells (Chapter 2). NCT-based technologies have overcome some of the limitations of conventional in vivo and in vitro approaches to study translation. Though these NCT-based technologies accomplished detection of heterogeneity of translation dynamics, they were not capable of studying how specific factors mediate translational control. The process of translation can be controlled by small RNA silencing pathways that restrict or completely block protein production. Of these, microRNAs (miRNAs) direct translational repression and mRNA decay by guiding the enzyme Argonaute and its associated proteins to partially complementary sequences on target mRNAs (Chapter 1). The dynamics of miRNA-mediated gene silencing, and in particular the role of Argonaute on translation, remain difficult to interpret due to pathway's complexity, long (minutes-to-hours) timescale, and conflicting results from different studies. To address these problems, we developed technology to directly visualize and quantify the impact of human Argonaute2 (Ago2) on translation and subcellular localization of individual reporter mRNAs in living cells (Chapter 3). Translation and Tethering (TnT) is a tethering-based single molecule reporter that simultaneously monitors translation and Ago2-tethering status in live human cells. Since this technique is microscopy-based, its readout includes valuable subcellular localization and intensity information over timeframes ranging from seconds to hours, which describe when, where, and how much translation and tethering is occurring per single-mRNA. Finally, to simplify using this multi-construct, multi-probe system, we adapted a cell loading technique, called bead loading, to introduce TnT plasmids and proteins simultaneously into adherent cells (Chapter 4). Our TnT system reflects endogenous miRNA-mediated gene silencing when we compared it to natural miRNA-target site recruitment. Using the TnT system, we find that Ago2 association leads to progressive repression 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 and subsequent runoff of the ribosomes already engaged in translation elongation. At early timepoints, we observed occasional brief bursts of translational activity at Ago2-tethered mRNAs undergoing silencing, suggesting that translational repression may initially be reversible. At late timepoints, Ago2-tethered mRNA were redirected into P-bodies where they remained translationally silenced for 10+ hours, which was the duration of the experiment. These results provide a framework for exploring miRNA-mediated gene regulation in live cells at the single molecule level (Chapter 5). Furthermore, due to the adaptability of the TnT system, it will likely have wide-ranging application in studying RNA-protein interactions more generally.
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Subject
fluorescence
microRNA
translation control
gene expression
Argonaute
microscopy