High-throughput interrogation of programmed ribosomal frameshifting in human cells

Martin Mikl; Yitzhak Pilpel; Eran Segal

Comparing per position the median PRF signal for variants in which this position was changed reveals 243 a sensitivity profile around the PRF site ( Fig 2B, Fig S5C) , recapitulating these context specific 244 characteristics. Across all -1 PRF events tested positive in our assay, not only mutations at the 245 slippery site (marked in grey) and downstream of it, but also mutations immediately upstream of the 246 slippery site negatively affect PRF efficiency. Other -1 PRF events reported in the literature, but not 247 yielding a signal above background in our assay, tend to show substantially increased signal upon 248 A. Clustered heat map of median percent wild-type frameshifting of variants in which the indicated region (upstream, slippery site (including the preceding codon) and downstream) differs from the native one (n=37-325). B. The median percent wild-type PRF signal of variants in which the indicated position is changed is plotted for the entire length of the variable region; gray box: slippery site. C. Percent wild-type -1 PRF signal for variants with a point mutation only in the indicated position of the slippery site (n=6-22). D. Heatmap showing median percent GFP fluorescence conferred by replacing the native slippery site in -1 PRF sites tested positive in the assay (Fig 1C) with the indicated combination of slippery site elements. E. Distribution of percent wild-type GFP fluorescence of variants in which the slippery site was replaced by any possible combination of bases following the pattern XXXYYYZ (n=19-80). F. Distribution of normalized reads across GFP (+1 frame) expression bins for variants of the OAZ1 frameshifting site, in which either the first (right) or the second (left) codon is replaced; blue lines: wild-type; green and magenta lines: bin profile for the variant in which the native codon was replaced by the indicated one; grey lines: bin profiles for other codons at this position. G. Percent GFP fluorescence for variants in which the slippery site was replace with all possible combinations of the slippery site pattern found in west nile virus (XYYZZZZ) (n=16-56).