Computational design and interpretation of single-RNA translation experiments.

Recent advances in time-lapse fluorescence microscopy allow live-cell quantification of ribosome kinetics at single-molecule resolution. Here, we integrate single-molecule experimental data and stochastic models to investigate canonical and non-canonical translation processes. In a first study, we developed a new fluorescent tag system that 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 state (Lyon, K., et al., 2019. Molecular Cell). In a second study, we developed novel stochastic models to estimate biophysical parameters, such as ribosomal elongation and initiation rates. Our methods were used to simulate single-molecule experiments under multiple imaging conditions and for thousands of human genes, and we evaluate which experiments provide accurate estimates of elongation kinetics. With this, we present an interpretation for the well-established experimental procedures, including Correlation Spectroscopy, ribosome Run-Off Assays, and FRAP (Aguilera, L., et al., 2019, Plos Comp Biology).