Extended Data Fig. 7: uORF1 protects Atf4 mRNA from degradation by nonsense-mediated decay, while uORF2 regulates ATF4 protein synthesis in c-ISR.
From: Plasticity of the mammalian integrated stress response
(a) RT-qPCR analysis of Atf4 mRNA levels in WT or ΔuORF1 mouse ES Cells expressing control or Eif2b5 shRNAs and treated with vehicle or Tg (400 nM) for specified times. Data were normalized to Gapdh mRNA values. p-values were determined using two-tailed Student’s t-test. Data are presented as mean ± SEM (n = 3 independent experiments). (b, d-f, j) Parental WT or ΔuORF1 mouse ES Cells (b), or WT or ΔuORF1 mouse ES Cells expressing control or UPF1 shRNAs (d-f), or WT or ΔuORF1 mouse ES Cells treated with Tg (400 nM, 9 h) (j) were exposed to actinomycin D (10 μg/ml) for the indicated durations, to determine the half-life (t1/2) of the denoted mRNAs. mRNA levels were quantified by RT-qPCR analysis. Values were normalized to Gapdh mRNA and expressed as percentages of the levels before the addition of actinomycin D. t1/2 of mRNAs was calculated using exponential decay function. P-values; n.s., not significant evaluated by the two-tailed Student’s t-test (n = 3 independent experiments). (c,k) Western blot analysis of the indicated proteins in cell extracts isolated from WT or ΔuORF1 mouse ES Cells expressing control or Upf1 shRNAs (c) or WT and ΔuORF2 NIH3T3 cells treated with Tg (400 nM, 9 h) (k). Representative blots are shown (n = 3 independent experiments). The cartoon in (k) indicates CRSPR-Cas9 mutation in the Atf4 gene introduced in the initiation codon of uORF2. (g-i) RT-qPCR analysis of the indicated mRNAs in mouse ES Cells expressing control (shCon) or Upf1 (shUpf1) shRNAs. P-values, two-tailed Student’s t-test. Data are presented as mean ± SEM (n = 3 independent experiments). (l) Model for regulation of Atf4 mRNA translation and mRNA decay of the Atf4 mRNA via the functions of uORF1: (i) In the absence of stress, eIF4E-mediated translation of uORF1 in part inhibits translation of uORF2 and stabilizes Atf4 mRNA. (ii) When eIF2B activity is decreased in the absence of stress, translation of the Atf4 main ORF increases. Decreased uORF2 translation initiation and translation of the main Atf4 ORF, protects the Atf4 mRNA from NMD. (iii) Under chronic ER stress, Atf4 main ORF translation is independent of eIF4E and dependent on eIF3d6. The ribosomes (40S in red and 60S in yellow) are shown translating the mRNA as an 80S or as recycling/scanning units. The engagement of NMD via the translation of uORF2 is depicted as the mechanism of regulation of Atf4 mRNA abundance (indicated by the black lines). The degradation symbol has been sized as per the expected outcome of Atf4 mRNA decay under each condition.
