Ribosomal protein methyltransferases in the yeast Saccharomyces cerevisiae: Roles in ribosome biogenesis and translation - PubMed

FIG. 4. Cells deficient in ribosomal protein methyltransferases have reduced translational fidelity

Translation elongation and termination accuracy were measured in cells lacking each of the ten ribosomal protein methyltransferases. A dual-luciferase reporter assay consisting of a Renilla luciferase gene fused C-terminally to a firefly luciferase gene separated by a linker region was used to measure stop codon readthrough, amino acid misincorporation, and programmed −1 ribosomal frameshifting (−1 PRF), as described previously [12,22,24]. (A) Percent readthrough was calculated by taking the firefly/Renilla luminescence ratio of the UAA-containing vector divided by the same ratio in the respective control. Error bars represent standard deviation. hpm1Δ p = 0.0077, rkm1Δ p < 0.0001, rkm2Δ p = 0.006, rkm3Δ p = 0.0001, rkm4Δ p = 0.0158, rkm5Δ p < 0.0001, ntm1Δ p = 0.0001, rmt1Δ p < 0.0001, rmt2Δ p < 0.0001, sfm1Δ p < 0.0001. Wild type was assayed 11 independent times, hpm1Δ 8 times and mutants were assayed three independent times. Data for the wild type and hpm1Δ cells were previously reported [13]. (B) Same as in (A) except a UAG stop codon was used. Wild type was assayed for a total of 12 biological replicates, hpm1Δ was assayed 7 times, and mutants 3–4 times. Data for wild type and hpm1Δ cells were previously reported [13]. hpm1Δ p = 0.0811, rkm1Δ p = 0.6114, rkm2Δ p = 0.626, rkm3Δ p = 0.0024, rkm4Δ p = 0.0003, rkm5Δ p = 0.0001, ntm1Δ p = 0.05, rmt1Δ p = 0.0017, rmt2Δ p < 0.0001, sfm1Δ p < 0.0001. (C) Percent amino acid misincorporation was measured as in (A). Wild type was assayed 10 independent times, hpm1Δ was assayed 8 times, and mutants were assayed 3–4 independent time. Data for wild type and hpm1Δ cells were previously reported [13]. hpm1Δ p = 0.0621, rkm1Δ p = 0.0054, rkm2Δ p = 0.7818, rkm3Δ p = 0.0051, rkm4Δ p < 0.0001, rkm5Δ p = 0.0043, ntm1Δ p = 0.9623, rmt1Δ p = 0.0032, rmt2Δ p = 0.0069, sfm1Δ p = 0.001. (D) Percent −1 PRF was calculated by taking the firefly/Renilla luminescence ratio of cells containing pJD376 (L-A virus gag-pol frameshift signal) divided by the same ratio of cells containing the no frameshift control (pJD375). Error bars represent standard deviation of at least two independent experiments. Strains were assayed 2–4 independent times as indicated by the number of data points. hpm1Δ p = 0.03, rkm1Δ p = 0.006, rkm3Δ p = 0.03, rkm5Δ p = 0.03, ntm1Δ p = 0.02, sfm1Δ p = 0.02. Frameshift vectors were generously provided by Jonathan Dinman at the University of Maryland, MD and described [24]. p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), p < 0.0001 (****).