High-throughput interrogation of programmed ribosomal frameshifting in human cells

Martin Mikl; Yitzhak Pilpel; Eran Segal

To get an estimate of the size of the relevant downstream region in different PRF events, we replaced 298 native regions with a constant sequence, leaving stretches of different length after the slippery site 299 unchanged (Fig 3A, top; Methods). While in the case of SARS almost the entire downstream region 300 included in our reporter construct (120 nt) was crucial for frameshifting, in the HIV and SIVmac239 301 PRF sites only a comparatively small region (<24 nt) was required to achieve PRF signal >50% of 302 wild type (Fig 3B) . The shortness of the necessary downstream sequence in these cases was striking, To identify specific positions crucial for PRF we performed scanning mutagenesis of regions ranging 307 from 7 to 36 nt downstream of the slippery site (Fig 3A, bottom) . This revealed large differences in 308 the fraction of relevant downstream positions (Fig 3C) , ranging from 6% of single point mutations 309 reducing -1 PRF efficiency by more than half in the case of SIVmac239 to 53% in the case of SARS 310 (46% for +1 PRF at the OAZ1 site). The HIV gag-pol PRF site (among others) shows remarkable 311 resilience to point mutations ( Fig 3C, Fig S6A) suggesting that a reduced stem is still able to drive PRF at wild-type levels, and only forcing a 316 complete change in the structure affects frameshifting. 317 . CC-BY-NC-ND 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/469692 doi: bioRxiv preprint Figure 3 . Downstream RNA structure affects PRF efficiency in a context-dependent manner. A. Schematic of downstream sequence manipulations. B. Percent wild-type frameshifting rates of variants in which the native downstream region has been replaced by constant sequences, leaving the indicated number of nucleotides after the slippery site unchanged; n=1-3 per data point. C. Fraction of point mutations in the 40 nt downstream of the slippery site resulting in <50% of wild-type GFP fluorescence (green) and background fluorescence (<1.3% GFP fluorescence, blue), for -1 (top) and +1 (bottom) PRF events. D. The difference in mean % wild-type frameshifting between variants in which the indicated position is predicted to be paired vs unpaired along the variable region; gray box: slippery site. E. Schematic of downstream structure variations. F. Percent -1 GFP fluorescence of variants in which the downstream region is replaced by a synthetic sequence predicted to fold into the respective native secondary structure (using Vienna RNA (only stem-loop; light blue) or pKiss (including pseudoknots; dark blue; Methods)); the shaded area denotes the range of background fluorescence. G. % -1 GFP fluorescence of variants in which the downstream region is replaced by a synthetic sequence predicted to fold into the HIV (blue) or SRV1 (green) secondary structure (or variations thereof; Methods); the shaded area denotes the range of background fluorescence. H. % GFP fluorescence of variants in which the downstream region carried a single point mutation (left) or was replaced with sequences predicted to fold into variations of PRF inducing structures, plotted against the corresponding wild-type values; p-values (Mann-Whitney-U) for comparisons between groups of low (<4% GFP fluorescence) and high wild-type PRF (>4% GFP fluorescence). IJ. Clustered heatmap of Pearson correlation coefficients between percent -1 GFP fluorescence and minim