Supplementary Figure 7: RCK Member Dhh1 is a Post-transcriptional Repressor of Autophagy in Yeast.
From: A conserved mechanism of TOR-dependent RCK-mediated mRNA degradation regulates autophagy
(a,b) Dhh1 represses the expression of ATG genes in nutrient-replete conditions. Wild-type (WT; WLY176) and dhh1Δ cells (YAB269) were grown in rich medium (a), then shifted to nitrogen starvation for 1 h (b). Total RNA was extracted and analysed as in Fig. 1a. Error bars: standard deviation (SD). (a) n = 5 independent experiments except n = 3 for ATG9, 32,34; n = 4 for ATG2 and n = 6 for n = 5 for ATG1,7,8. (b) n = 3 independent experiments except n = 2 for ATG4 in WT cells. Student’s t-test, ∗p < 0.05,∗∗∗p < 0.001. (c–e) Mild overexpression of DHH1 modestly represses autophagy. WT (YAB281) and OE DHH1 cells (YAB282) were grown in YPGal medium (+N) until mid-log phase, then starved for nitrogen (−N) for 5 h (c,d) or 1 h (e). (c) Protein extracts were analysed by western blot. (d) Pho8Δ60 activity was normalized to that of WT cells (set to 100%). Error bars: SD, n = 4 independent experiments. Student’s t-test, ∗∗p < 0.01. (e) Total RNA was extracted and the mRNA levels were quantified by RT-qPCR. The mRNA level of ATG8 was normalized to that in WT cells in rich conditions (set to 1). Error bars: SD, n = 4 independent experiments. Student’ t-test, NS > 0.05. (f) Dcp2 represses autophagy. WT (YTS158) and dcp2-7Δ cells were grown in YPD at 24 °C until early log-phase, then shifted to 38.5 °C for 1 h and starved for 3 h at the same temperature. Pho8Δ60 activity was normalized to that of WT cells (set to 100%). Error bars: SD, n = 5 independent experiments. Student’s t-test, ∗∗∗p < 0.001. (g) Dcp2 represses the expression of Atg1 and Atg9. WT and dcp2-7Δ cells were grown in rich medium until early-log phase. Cells were shifted to 38.5 °C for 1 h then starved. Protein extracts were analysed by western blot. (h) WT (BY4742) and dcp2-7Δ cells were grown in rich medium until early log phase. Cells were then shifted to 38.5 °C for 1 h then starved for 1 h. The mRNA level of individual ATG genes was normalized to the mRNA level of the corresponding gene in WT cells (set to 1). Error bars: SD, n = 3 independent experiments.
