Translation Dynamics of Single mRNAs in Live Cells

Abstract

The translation of messenger RNA (mRNA) into proteins represents the culmination of gene expression. Recent technological advances have revolutionized our ability to investigate this process with unprecedented precision, enabling the study of translation at the single-molecule level in real time within live cells. In this review, we provide an overview of single-mRNA translation reporters. We focus on the core technology, as well as the rapid development of complementary probes, tags, and accessories that enable the visualization and quantification of a wide array of translation dynamics. We then highlight notable studies that have utilized these reporters in model systems to address key biological questions. The high spatiotemporal resolution of these studies is shedding light on previously unseen phenomena, uncovering the full heterogeneity and complexity of translational regulation.

Keywords: central dogma; fluorescence microscopy; gene expression; intrabodies; live-cell imaging; mRNA; messenger RNA; single-molecule tracking; translation.

Figure 1.. Imaging single-mRNA translation in living cells.

a) With a traditional GFP tag placed on the N-terminus of a protein of interest, the fluorophore matures too slowly to capture the translation process. b) Using an N-terminal repeat epitope tag, already fluorescent GFP-tagged intrabodies (Fab, scFv, nanobodies) can be recruited to the site of translation quickly. Fluorescence at the translation site is amplified above background because repeat epitopes can recruit more than one GFP-tagged intrabody and also because ribosomes can translate multiple nascent chains from a single mRNA. This creates bright fluorescent puncta that mark the translation sites for tracking in living cells. c) Sample translation sites in living U2OS cells. Addition of puromycin causes translation spots to quickly disappear. Scale bar, 10 µm.

Figure 2.. Intrabodies for single-mRNA translation imaging.

Structures of translation imaging intrabodies bound to their target peptides. Structures were predicted using Alphafold2 and color-coded by low to high confidence (red to blue). Target peptides are shown (prolines in ALFA-Tag not shown), along with binding affinity and live-cell FRAP recovery half-time, if known. Tag accessories (solubilization domain GB1 and tested fluorescent fusion tags) are also indicated, but not shown.

Figure 3.. Tag arrangements for imaging translation.

a) Left, epitopes are commonly arranged one after the other in a linear sequence for translation imaging, as in the 24×SunTag (left box). Right, linear epitopes can alternatively be embedded within structures to help spread them out and create binding hotspots, as in the 10×FLAG spaghetti monster tag (right box). b) Two types of epitopes can be arranged into reporters to compare the translation kinetics of (i) two transcripts, (ii) canonical and non-canonical (IRES) translation, (iii) translation from 0 and ±1 frames, or (iv) barcoded proteins with unique fluorescence fluctuations for multiplexed imaging.

Figure 4.. Modular accessories for improved single-mRNA translation imaging.

A variety of accessory modules can be added to the core translation imaging tag to, from left to right, induce translation, degrade protein faster, immobilize nascent chains in the ER membrane, or light up transcripts with an mRNA tag. The mRNA tag can also be used to assay mRNA degradation (using the TREAT assay), recruit translation regulatory factors, or immobilize mRNA to the plasma membrane.

Figure 5.. Measuring single mRNA translation dynamics:

a Some measurables of single-mRNA translation reporters: i. translation subcellular location (random vs. nuclear periphery), ii. mobility (motored vs. diffusive), iii. polysome density (from low to high), and iv. translation efficiency (fraction of mRNA with translation signals). b. Fluorescence signals from single-mRNA translation reporters naturally fluctuate up and down through time t as ribosomes initiate and terminate translation. c. The elongation time can be quantified in a variety of ways: i. fluorescence autocorrelation G(τ) of natural fluctuations, ii. treatment with the translation initiation inhibitor harringtonine (leads to steady loss in average fluorescence signal I(t) as ribosomes run off transcripts one by one without further initiation), iii. treatment with the elongation inhibitor cycloheximide (freezes translation, reducing fluctuations), and iv. fluorescence recovery after photobleaching (FRAP).

Figure 6.. Biological applications of single-mRNA translation reporters in live cells.

Many aspects of translation have now been studied using single-mRNA translation reporters, including: a) the structural organization of single-mRNA during translation, b) local translation in special sub-cellular locations such as the ER, c) the clustering of translation sites in translation factories, d) endogenous translation using genome editing techniques, e) viral translation using increasingly realistic viral translation reporters such as replicons, f) translation quality control which monitors for ribosomal traffic jams, g) translational silencing during stress and the relocalization of mRNA to stress granules (where translation may not always be off), h) nonsense mediated decay and siRNA-mediated translation silencing, and i) miRNA-mediated translational silencing, which may involve recruitment to P-bodies.