Imaging translational control by Argonaute with single-molecule resolution in live cells

Abstract

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 sensor reflects endogenous miRNA-mediated gene silencing. Using the sensor, we find that Ago2 association leads to progressive silencing of translation at individual mRNA. Silencing was occasionally interrupted by brief bursts of translational activity and took 3–4 times longer than a single round of translation, consistent with a gradual increase in the inhibition of translation initiation. At later time points, Ago2-tethered mRNAs cluster and coalesce with 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 RNA-protein interactions.

Using Mechanistic Models and Machine Learning to Design Single-Color Multiplexed Nascent Chain Tracking Experiments

“…Temporally- or spatially-resolved activation or repression of translation provides for a potent mechanism by which cells could rapidly alter their protein content in response to cellular signals (Sonenberg and Hinnebusch, 2009; Fabian et al, 2010; Hershey et al, 2012; Zhao et al, 2019; Peer et al, 2019). Recent advances in Nascent Chain Tracking (NCT) experiments has made it possible to observe this regulation at the level of single mRNA molecules in living cells (Lyon et al, 2019; Moon et al, 2019; Koch et al, 2020; Cialek et al, 2022; Kobayashi and Singer, 2022). However, limitations on the number of distinct fluorophores prevents current NCT experiments from exploring more than one or two different mRNA species at a time.…”

“…NCT has been utilized to investigate many processes of interest such as mRNA frameshifting (Lyon et al, 2019), mRNA IRES-mediated translation (Koch et al, 2020), mRNA decay (Horvathova et al, 2017;Hoek et al, 2019), translation suppression during cellular stress (Moon et al, 2019), and mRNA spot to spot heterogeneity (Boersma et al, 2019). NCT has also proven beneficial to extract important biophysical parameters, including elongation rates, initiation rates and ribosomal densities (Morisaki et al, 2016;Wu et al, 2016;Yan et al, 2016;Pichon et al, 2016), and microRNA mediated decay (Kobayashi and Singer, 2022;Cialek et al, 2022). Notwithstanding NCT's current adoption and importance, application of NCT to understand how different cellular signals affect translation of different mRNA is currently prevented by strong limits on the fluorophore color palette.…”

Muscleblind-like proteins use modular domains to localize RNAs by riding kinesins and docking to membranes

“…RNA-MRI reveals RBP properties by directly tethering RBP domains to the MS2 coat protein and assembling them onto an MS2 reporter RNA, painting a "dynamic caricature" of the RBP. Our assay has similarities to the "TnT translation and tether" assay (Cialek et al, 2022), but uses Halo signal to directly image RNPs that contain the RBP of interest. Inducible components yield signal to noise properties required to generate high quality movies amenable to automated computational analyses and could be expanded to many additional RBPs.…”

Expression of miRNA-Targeted and Not-Targeted Reporter Genes Shows Mutual Influence and Intercellular Specificity

“…At present, the model is mainly conceptual, and more detailed experimental studies of the relevant RNA–RNA and protein–RNA interactions are needed. New methods which allow for the exploration of the multiple conformations and interactions of RNAs in living cells and which follow processes ongoing on single molecules [ 51 , 52 ] create new possible directions for further investigations.…”