Live-cell single RNA imaging reveals bursts of translational frameshifting

Kenneth Lyon; Luis U. Aguilera; Tatsuya Morisaki; Brian Munsky; Timothy J. Stasevich

The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. . https: //doi.org/10.1101/478040 doi: bioRxiv preprint After RNA particles were identified and tracked using the custom Mathematica code described above, an average centered image of the first five frames from each track was created for RNA (JF646), 0 ORF (FLAG-Cy3 in the +FSS multi-frame tag), and -1 ORF (scFv-sfGFP in the +FSS multi-frame tag). The trims were then hand checked to remove any trims with artifacts, e.g., smears or non-diffraction limited spots. Next, a custom Mathematica code was used to detect particles in the 0 ORF or -1 ORF trim channels, sorting the spots into RNA only, 0 frame translation sites (0 TS only), 0 and -1 TS, and -1 only TS. For all cases, RNA always had to be present. Finally, frameshifting translation sites (the 0 and -1 TS or the -1 only TS) were validated by eye, to further remove artifacts.