High-throughput interrogation of programmed ribosomal frameshifting in human cells

Martin Mikl; Yitzhak Pilpel; Eran Segal

using Vienna RNAfold and pKiss, respectively, show good correlation ( Fig S6B) . We repeated the 353 . CC-BY-NC-ND 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder.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 above analysis and observed the same pattern as with RNAfold, namely widespread association 354 between secondary structure (low MFE) and higher PRF signal for small deviations from the native 355 sequence ( between PRF events whose downstream sequence was shown to fold into a hairpin structure (e.g. While mutations in the slippery site and the downstream region typically have a negative effect on 387 -1 PRF signal (e.g. PEG10, Fig S5D) . This suggests that inhibitory signals upstream of the 389 frameshifting site as found for SARS (Su et al., 2005) could be a more widespread property of PRF 390 sites, probably creating a balance to ensure that frameshift promoting signals like a rigid downstream 391 A. Schematic of sequence manipulations upstream and immediately downstream of the slippery site. B. Boxplots of % wild-type frameshifting rates for variants of "positive" -1 PRF sites in which the indicated number of nucleotides were deleted (negative numbers) or inserted (positive numbers, taken from two different constant sequences) after the slippery site (n=4-54). C. % GFP fluorescence for variants of "negative" -1 PRF sites with the sequence upstream of the slippery site being randomly recoded or replaced with sequences predicted to have the indicated secondary structure; the shaded area denotes the range of background fluorescence. D. % wild-type frameshifting rates (+1 for OAZ, blue, and -1 for HIV, green) of variants in which the native upstream region has been replaced by constant sequences up to the indicated position relative to the PRF site. E. Boxplot of percent of wild-type frameshifting rates for variants in which the -2 or -1 amino acid relative to the slippery site is replaced with an amino acid from the indicated groups, for codons which maintain the slippery site pattern XXXYYYZ (bottom) or not (top); n=10-115. F. Pearson correlation coefficient (blue) and associated p-values (green) between tAI at the indicated position of the original reading frame and % GFP fluorescence. G. Clustered heat map showing all possible combinations of 3 synthetic slippery sites, 34 synthetic downstream variants and up to 5 synthetic upstream regions (minimal value 1.3%). We expanded our search for properties affecting PRF efficiency and examined the effect of the codons 409 preceding the slippery site. We found that the presence of a charged amino acid immediately 410 upstream of the slippery site significantly reduced frameshifting signal, even when the sequence of 411 the slippery site was unchanged (Fig 4E, p<0 .0015, Wilcoxon signed-rank test). We examined also 412 the influence of decoding efficiency as measured by the tRNA adaptation index (tAI) on PRF and 413 detected an increasingly negative correlation between tAI and PRF signal in the 0 frame codons 414 leading up to the site of ribosome slippage (Fig 4F) . This observation might indicate that progressive 415 slowing down of the ribosome by limited tRNA availability might contribute to stalling at the slippery 416 site and frameshifting. 417