Computational design and interpretation of live-cell, single-RNA translation experiments

Abstract

Advances in fluorescence microscopy have introduced new assays to quantify live-cell translation dynamics at single-RNA resolution. We introduce a detailed, yet efficient sequence-based stochastic model that generates realistic synthetic data for several such assays, including Fluorescence Correlation Spectroscopy (FCS), ribosome Run-Off Assays (ROA) after Harringtonine application, and Fluorescence Recovery After Photobleaching (FRAP). We simulate these experiments under multiple imaging conditions and for thousands of human genes, and we evaluate through simulations which experiments are most likely to provide accurate estimates of elongation kinetics. Finding that FCS analyses are optimal for both short and long length genes, we integrate our model with experimental FCS data to capture the nascent protein statistics and temporal dynamics for three human genes: KDM5B, {beta}-actin, and H2B. Finally, we introduce a new open-source software package, RNA Sequence to NAscent Protein Simulator (O_SCPCAPRC_SCPCAPSNAPO_SCPCAPSIMC_SCPCAP), to easily simulate the single-molecule translation dynamics of any gene sequence for any of these assays and for different assumptions regarding synonymous codon usage, tRNA level modifications, or ribosome pauses. O_SCPCAPRC_SCPCAPSNAPO_SCPCAPSIMC_SCPCAP is implemented in Python and is available at: https://github.com/MunskyGroup/rSNAPsim.git.\n\nAuthor summaryTranslation is an essential step in which ribosomes decipher mRNA sequences to manufacture proteins. Recent advances in time-lapse fluorescence microscopy allow live-cell quantification of translation dynamics at the resolution of single mRNA molecules. Here, we develop a flexible computational framework to reproduce and interpret such experiments. We use this framework to explore how well different single-mRNA translation experiment designs would perform to estimate key translation parameters. We then integrate experimental data from the most flexible design with our stochastic model framework to reproduce the statistics and temporal dynamics of nascent protein elongation for three different human genes. Our validated computational method is packaged with a simple graphical user interface that (1) starts with mRNA sequences, (2) generates discrete, codon-dependent translation models, (3) provides visualization of ribosome movement as trajectories or kymographs, and (4) allows the user to estimate how optical single-mRNA translation experiments would be affected by different genetic alterations (e.g., codon substitutions) or environmental perturbations (e.g., tRNA titrations or drug treatments).