Figure 4.
Knockdown of ribosomal subunits blocks UPR
mt
activation and ATFS-1 translation upon mitochondrial stress. (A) RNAi of multiple ribosomal subunits attenuated UPRmt activation induced by cco-1 RNAi. RNAi targeting cco-1 occupies 40%, RNAi targeting ribosomal subunits occupies 60%. Scale bar, 0.3 mm. (B and C) Western blots (B) and qRT-PCR analysis of transcripts (n = 4 biologically independent samples; C) in atfs-1p::atfs-1::flag::gfp worms fed with control, rps-8, rps-10, rpl-27 or rpl-36 (60%) RNAi with or without cco-1 (40%) RNAi. (D–F) Western blots of atfs-1p::H1-wCherry worms fed with control or cco-1 (40%) RNAi, in combination with let-363 or rheb-1 (60%) RNAi (D), vha-1, vha-4, vha-16, or vha-19 (25%) RNAi (E), or rps-8, rps-10, rpl-27 or rpl-36 (60%) RNAi (F). (G) Representative polysome profiles of worms fed with control or cco-1 (50%) RNAi, with or without or vha-1 (25%) RNAi. The relative positions of the ribosome subunits (40 and 60 S), monosomes (80 S), and polysomes (P1-P6) are indicated. (H) qRT-PCR analysis of transcripts (n = 3 biologically independent samples) in the polysomal fractions (highly translated) of worms as indicated in G. Error bars denote SEM. Statistical analysis was performed by ANOVA followed by Tukey post-hoc test (*P < 0.05; **P < 0.01; ***P < 0.001). Source data are available for this figure: SourceData F4.