Computational Design and Interpretation of Single-RNA Translation Experiments

Abstract

Translation is an essential step in which ribosomes decipher mRNA sequences to manufacture proteins. Recent advances in super-resolution fluorescence microscopy allow live-cell quantification of ribosome kinetics at single-molecule resolution. Here, we integrate single-molecule data and stochastic models to investigate canonical and non-canonical translation processes. Recent findings by our group using these modern methodologies allowed us to observe, for the first time, ribosomal frameshifting at single-molecule resolution. Our results corroborate that frameshifting is a bursty process, where the RNA stochastically switch between non-frameshifting and frameshifting states in which either 0% or 100% of ribosomes produce frameshifted proteins. We predicted that ribosomal traffic jams contribute to the persistence of the frameshifting state for long periods of time (Lyon, K., et al., 2019. Molecular cell). Additionally, we simulate single-molecule experiments under multiple imaging conditions and for thousands of human genes, and we evaluate which experiments are most likely to provide accurate estimates of elongation kinetics. In this study, we provide an interpretation for the well-established experimental procedures, such as Fluorescence Correlation Spectroscopy (FCS), ribosome Run-Off Assays (ROA) after Harringtonine application, and Fluorescence Recovery After Photobleaching (FRAP) (Aguilera, L.U., et al., 2019, Plos Computational Biology). Our results show previously unknown mechanisms taking place during translation at single-molecule resolution in living cells.