Quantifying Single mRNA Translation Kinetics in Living Cells

Abstract

One of the last hurdles to quantifying the full central dogma of molecular biology in living cells with single-molecule resolution has been the imaging of single messenger RNA (mRNA) translation. Here we describe how recent advances in protein tagging and imaging technologies are being used to precisely visualize and quantify the synthesis of nascent polypeptide chains from single mRNA in living cells. We focus on recent applications of repeat-epitope tags and describe how they enable quantification of single mRNA ribosomal densities, translation initiation and elongation rates, and translation site mobility and higher-order structure. Together with complementary live-cell assays to monitor translation using fast-maturing fluorophores and mRNA-binding protein knockoff, single-molecule studies are beginning to uncover striking and unexpected heterogeneity in gene expression at the level of translation.

Figure.1.

Quantifying messenger RNA (mRNA) translation at the single-molecule level. Many mechanistic details of mRNA translation can now be measured in living cells. (Top) The rates of ribosome initiation, nascent chain elongation, ribosome stalling, and translation termination can be measured, as can the density of ribosomes within polysomes. (Bottom) Polysome shape, higher-order structure, mobility, and localization can also be measured.

Figure 2.

Repeat-epitope and accessory tags for single-molecule imaging of mRNA translation. A schematic showing how repeat-epitope tags are used to visualize single translation sites in living cells. (A) Nascent peptides (small triangles) encoded by repeat epitopes (green mRNA) placed in the amino terminus of a protein of interest are co-translationally labeled by fluorescent antibody fragments (green spheres). (B) An mRNA tag can facilitate single mRNA translation imaging. Stem loops within the 3′ untranslated region (UTR) of mRNA are co-transcriptionally labeled by coat proteins (red spheres). In combination with a repeat-epitope tag, translation sites are marked in green and red. (C) Immobilization tags can restrict the movement of mRNA so that translation sites can be tracked with less frequent imaging. The cytoplasmic end of an endoplasmic reticulum signal-anchor membrane protein (CytERM) tag (blue) embeds nascent chains into the endoplasmic reticulum (ER) co-translationally, whereas a CAAX domain embeds transcripts with an mRNA tag into the plasma membrane via bound coat proteins (red spheres). (D) A degron (blue mRNA) encoded upstream of the repeat epitopes enhances the degradation of proteins, so nascent chains can be preferentially imaged (rather than mature proteins).

Figure 3.

Quantifying translation elongation rates with repeat-epitope tags. The translation elongation rate k_elong can be measured using repeat-epitope tags in multiple ways. Common assumptions are (1) every ribosome that finishes translation is on average replaced by another that starts translation; (2) ribosomes are assumed to always complete translation, so no partial proteins are produced; and (3) completed nascent chains quickly leave the translation sites. L_Tag is the length of the repeat-epitope tag and L_POI is the length of the tagged protein of interest. (Top, left) Addition of the translation initiation inhibitor harringtonine leads to ribosome runoff. During runoff, polysome fluorescence on average drops in four phases (I–IV). The rate of fluorescence decay depends on the elongation rate. (Bottom, left) The fluorescence signal during monosome translation runoff also depends on the elongation rate. (Top, right) Fluorescence recovery after photobleaching (FRAP) of polysomes leads to an average fluorescence recovery that is approximately the inverse of the average polysome runoff fluorescence decay. (Bottom, right) Fluorescence correlation spectroscopy (FCS) at the translation sites produces a decaying autocorrelation function (ACF) that depends on the elongation rate.