Figure S1.
Reducing mitochondrial translation and fusion suppresses animals’ growth and exclusively provokes mitochondrial stress response.(A) Transcript level of eat-3 upon eat-3 RNAi or mrps-5;eat-3 double RNAi was measured by qPCR in day 1 adult N2 animals. RNAi of eat-3 alone or in conjunction with mrps-5 RNAi results in an around 66.3% reduction in the mRNA level. (B) Transcript level of fzo-1 upon fzo-1 RNAi or mrps-5;fzo-1 double RNAi was measured in day 1 adult N2 animals. Around 43.7% decrease in the mRNA level of fzo-1 was observed in worms treated with RNAi against fzo-1 alone or mrps-5; fzo-1 in combination. The expression levels of eat-3 or fzo-1 were normalized to reference genes y45f10d.4 and f35g12.2 and compared with the mean value of ev-treated controls. Mean ± SD of n = 3 biological replicates. ns, not significant; *, P < 0.5; ***, P < 0.001 by one-way ANOVA with Tukey’s multiple comparison tests. (C) Representative micrographs of worms showing that double RNAi of mrps-5;eat-3 exhibits the strongest suppression on age-dependent growth in body length. Scale bar in ev-treated day 3 condition is 200 µm and valid for all the images in C. The length of worms was normalized to the mean value of ev-treated day 3 controls. Mean ± SD of n = 17–24 images. Significance was calculated using one-way ANOVA with Tukey’s multiple comparison tests; ***, P < 0.001; ****, P < 0.0001; ns, not significant. (D) Fragmenting mitochondrial network through RNAi against eat-3 or fzo-1 triggers the UPRMT, visualized in hsp-6::GFP reporter strain on day 3 and day 7 of adulthood. Scale bar in ev-treated hsp-6::GFP animals represents 200 µm and is valid for all the images in D. (E) Quantification of hsp-6::GFP expression in D. The GFP fluorescence intensity was normalized to the mean value of ev-treated controls. Mean ± SD of n = 17 images. Significance was calculated using one-way ANOVA with Tukey’s multiple comparisons test; ****, P < 0.0001. (F) RNAi of mrps-5, eat-3, and fzo-1, individually or in combination, does not influence ER proteostasis, visualized in hsp-4::GFP reporter strain at day 7 of adulthood. Scale bar in ev-treated hsp-4::GFP animals represents 1 mm and is valid for all the images in F. (G) Measurement of hsp-4::GFP fluorescence intensity in F using a TECAN plate reader. Mean ± SD of n = 5 or 6 replicates. ns, not significant; ****, P < 0.0001 by one-way ANOVA with Tukey’s multiple comparisons test. (H) Basal and maximum OCR upon the RNAi knockdown of eat-3 and fzo-1. Basal OCR is not influenced by RNAi of eat-3 or fzo-1, whereas maximum OCR is strongly diminished. Mean ± SD of 11–16 replicates; Significance was calculated using one-way ANOVA with Tukey’s multiple comparisons test; ****, P < 0.0001; ns, not significant. (I) Raw averaged traces of oxygen consumption from wild type N2 worms treated with RNAi against eat-3 and fzo-1, respectively. Mean ± SEM (n = 16); FCCP and sodium azide were added at the indicated time.

Reducing mitochondrial translation and fusion suppresses animals’ growth and exclusively provokes mitochondrial stress response. (A) Transcript level of eat-3 upon eat-3 RNAi or mrps-5;eat-3 double RNAi was measured by qPCR in day 1 adult N2 animals. RNAi of eat-3 alone or in conjunction with mrps-5 RNAi results in an around 66.3% reduction in the mRNA level. (B) Transcript level of fzo-1 upon fzo-1 RNAi or mrps-5;fzo-1 double RNAi was measured in day 1 adult N2 animals. Around 43.7% decrease in the mRNA level of fzo-1 was observed in worms treated with RNAi against fzo-1 alone or mrps-5; fzo-1 in combination. The expression levels of eat-3 or fzo-1 were normalized to reference genes y45f10d.4 and f35g12.2 and compared with the mean value of ev-treated controls. Mean ± SD of n = 3 biological replicates. ns, not significant; *, P < 0.5; ***, P < 0.001 by one-way ANOVA with Tukey’s multiple comparison tests. (C) Representative micrographs of worms showing that double RNAi of mrps-5;eat-3 exhibits the strongest suppression on age-dependent growth in body length. Scale bar in ev-treated day 3 condition is 200 µm and valid for all the images in C. The length of worms was normalized to the mean value of ev-treated day 3 controls. Mean ± SD of n = 17–24 images. Significance was calculated using one-way ANOVA with Tukey’s multiple comparison tests; ***, P < 0.001; ****, P < 0.0001; ns, not significant. (D) Fragmenting mitochondrial network through RNAi against eat-3 or fzo-1 triggers the UPRMT, visualized in hsp-6::GFP reporter strain on day 3 and day 7 of adulthood. Scale bar in ev-treated hsp-6::GFP animals represents 200 µm and is valid for all the images in D. (E) Quantification of hsp-6::GFP expression in D. The GFP fluorescence intensity was normalized to the mean value of ev-treated controls. Mean ± SD of n = 17 images. Significance was calculated using one-way ANOVA with Tukey’s multiple comparisons test; ****, P < 0.0001. (F) RNAi of mrps-5, eat-3, and fzo-1, individually or in combination, does not influence ER proteostasis, visualized in hsp-4::GFP reporter strain at day 7 of adulthood. Scale bar in ev-treated hsp-4::GFP animals represents 1 mm and is valid for all the images in F. (G) Measurement of hsp-4::GFP fluorescence intensity in F using a TECAN plate reader. Mean ± SD of n = 5 or 6 replicates. ns, not significant; ****, P < 0.0001 by one-way ANOVA with Tukey’s multiple comparisons test. (H) Basal and maximum OCR upon the RNAi knockdown of eat-3 and fzo-1. Basal OCR is not influenced by RNAi of eat-3 or fzo-1, whereas maximum OCR is strongly diminished. Mean ± SD of 11–16 replicates; Significance was calculated using one-way ANOVA with Tukey’s multiple comparisons test; ****, P < 0.0001; ns, not significant. (I) Raw averaged traces of oxygen consumption from wild type N2 worms treated with RNAi against eat-3 and fzo-1, respectively. Mean ± SEM (n = 16); FCCP and sodium azide were added at the indicated time.

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