High-throughput interrogation of programmed ribosomal frameshifting in human cells

Martin Mikl; Yitzhak Pilpel; Eran Segal

Comparing the HIV gag-pol site with other PRF events shows the remarkable robustness and 593 exceptional tolerance for various non-native downstream stimulatory signals. More than in other 594 cases, maintaining the wild-type slippery site UUUUUUA is crucial for frameshifting, and tends to be 595 absolutely invariable across otherwise diverse natural isolates (Biswas et al., 2004) . The quantitative 596 nature of our assay allows us nevertheless to accurately quantify the effect of sequence variation 597 away from the slippery site, revealing sub-type specific differences. Furthermore, we devised a 598 machine learning model that is able to predict the natural variation in HIV frameshifting rates for novel 599 variants (Pearson r=0.60). Precise stoichiometry of frameshifted and non-frameshifted product is 600 crucial for HIV virulence, and the ability to predict frameshifting rates of naturally occurring variants of 601 (which also predicts pseudoknots) to determine the native secondary structure of each of the 15 PRF 671 events. We then created for each PRF event two sets of ten sequences each (using antaRNA) that 672 are predicted to fold into the downstream secondary structure as determined using ViennaRNA and 673 pKiss, respectively. Furthermore we designed variants of the predicted HIV, SRV1 and nsp2F 674 (PRRSV) downstream secondary structure, e.g. by extending or reducing the length of hairpins, and 675 completely synthetic secondary structures and again used antaRNA to create sets of corresponding 676 sequences that we used to replace the native downstream structure in all 15 PRF events. The parameters for the first PCR were 95 o C for 5 min, 18 cycles of 94 o C for 30s, 65 o C for 30s, and 802 72 o C for 60s, each, and one cycle of 72 o C for 5min. In the second PCR step, each reaction contained 803 2.5 μl of the first PCR product, 25 μl of Kapa Hifi ready mix X2 (KAPA Biosystems), 2.5 μl 10 μM 5′ 804 primer, and 2.5 μl 10 μM 3′ primer. The PCR program was 95 o C for 5 min, 24 cycles of 94 o C for 30s 805 and 72 o C for 30s, each, and one cycle of 72 o C for 5min. Specific primers corresponding to the constant 806 region of the plasmid were used. The 5′ primer contained a unique upstream 8-nt bin barcode 807 sequence, and three different barcodes were used for each bin. The 3′ primer was common to all bins. 808