Multi-color single molecule imaging uncovers extensive heterogeneity in mRNA decoding

Abstract

mRNA translation is a key step in decoding genetic information. Genetic decoding is surprisingly heterogeneous, as multiple distinct polypeptides can be synthesized from a single mRNA sequence. To study translational heterogeneity, we developed the MoonTag, a new fluorescence labeling system to visualize translation of single mRNAs. When combined with the orthogonal SunTag system, the MoonTag enables dual readouts of translation, greatly expanding the possibilities to interrogate complex translational heterogeneity. By placing MoonTag and SunTag sequences in different translation reading frames, each driven by distinct translation start sites, start site selection of individual ribosomes can be visualized in real-time. We find that start site selection is largely stochastic, but that the probability of using a particular start site differs among mRNA molecules, and can be dynamically regulated over time.Together, this study provides key insights into translation start site selection heterogeneity, and provides a powerful toolbox to visualize complex translation dynamics.

Following the messenger: Recent innovations in live cell single molecule fluorescence imaging

“…The SUperNova Tag (SunTag) utilizes a genetically encoded scFV fused to a superfolder green fluorescent protein (sfGFP) and allows a single molecule readout of translation processes in single cells (Tanenbaum et al, ; Wilson et al, ). The system relies on four modules: (a) up to 56 repeats of the 19 amino acid SunTag peptide epitope, which needs to be integrated into ORF sequence; (b) a ScFV‐sfGFP fusion targeting the epitope; (c) an mRNA modification suitable for labeling (such as the MS2 or PP7 RNA hairpins); and (d) a combined system for mRNA labeling with a distinct color and for membrane tethering (e.g., PCP‐mCherry‐CAAX expression module) (Boersma et al, ; Pichon et al, ; Tanenbaum et al, ).…”

“…In addition, the tethering to a relatively immobile structure such as the endoplasmic reticulum or the outer cell membrane improves the signal‐to‐noise during imaging and, combined with the equilibrium exchange with overexpressed, unbleached FPs, allows individual mRNA molecules to be tracked on long timescales (tens of minutes). The drawback of tethering is that it bears the risk of interfering with the translation process (Boersma et al, ; Tanenbaum et al, ).…”

“…As a step towards increasing the number of translated peptides that can simultaneously be studied, a second ScFV‐epitope pair called the MoonTag system was reported by Boersma et al (). Using this system, heterogeneity of initiation frequency during mRNA translation at different start sites could be tracked.…”

Computational design and interpretation of single-RNA translation experiments

“…elongation rates caused by codon-optimized sequences [10], the spatiotemporal 66 translation of specific genes in specific cellular compartments [7,8], ribosomal 67 frameshifting with bursty dynamics [14], and non-canonical forms of translation [15]. 68 As these experimental techniques rapidly evolve, they induce a growing need for 69 precise and flexible computational tools to interpret the resulting data and to design the 70 next wave of single-RNA translation experiments.…”

“…Furthermore, all of the 611 computational analyses described above are easily adapted to allow for analysis of 612 simultaneous multi-frame translation dynamics (e.g., when translation occurs on 613 overlapping open reading frames as is the case during frame-shifted translation), as we 614 implemented and described in [14]. Similarly, the code is easily extended to analyze 615 translation of genes that contain more than one set of fluorescence tags in multiple 616 colors, as has been explored experimentally in [15].…”