Extended Data Fig. 9: Nutrient starvation induces SCARF via eIF5B degradation.
From: Start codon-associated ribosomal frameshifting mediates nutrient stress adaptation
(a) Histogram showing the changes of ribosome density upon amino acid starvation. The mRNAs with top 25% increase (Inc) and decrease (Dec) are colored coded in red and blue, respectively. The right panels show the CDS IFR of both mRNA groups. P value was calculated by a permutation test. (b) Bar graphs show SCARF reporter assays in cells before and after amino acid starvation. Error bars, mean ± s.e.m.; two-tailed t-test, n = 3, *** P = 2.9 × 10−6 and P = 1.0 × 10−5 for comparison of Fluc levels in frame 0 or frame 1 of control cells; P = 2.3 × 10−5 and P = 1.5 × 10−4 for Fluc levels in cells with starvation. P = 5.6 × 10−6 and P = 6.5 × 10−5 for comparison of HiBit activities in frame 0 of cells with and without starvation. (c) The top panel shows the turnover of eIF5B in HEK293 cells before and after amino acid starvation. The bottom panel shows the turnover of eIF5B in starved HEK293 cells in the presence of 5 µM MG132. (d) Representative flow cytometry of HEK293-Kb cells transfected with SCARF reporters before and after amino acid starvation, in the absence or presence of 5 µM MG132. Bar plots show the relative 25D1 mean fluorescence intensity (MFI) of SCARF reporters. Error bars, mean ± s.e.m.; two-tailed t-test, n = 3, *** P = 1.2 × 10−5 and P = 8.6 × 10−4 for comparisons of 25D intensities in Frame 1; P = 7.5 × 10−4 and P = 1.3 × 10−3 for comparisons of 25D intensities in Frame 2. (e) Western blots of exogenous eIF5B in transfected HEK293 cells. (f) Representative flow cytometry of HEK293-Kb cells transfected with SCARF reporters before and after amino acid starvation, in the absence or presence of exogenous eIF5B. Bar plots show the relative 25D1 mean fluorescence intensity (MFI) of SCARF reporters. Error bars, mean ± s.e.m.; two-tailed t-test, n = 3, *** P = 5.8 × 10−4 for comparison of control vs starvation; P = 1.2 × 10−3 for comparison of exogenous eIF5B vs vector under starvation.
