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. 2023 Jul 10;42(16):e112446. doi: 10.15252/embj.2022112446

Figure 1. mRNA degradation and CCR4‐NOT complex components physically and functionally associate with mitochondria.

Figure 1

  1. NTL‐2 foci localize in close proximity to mitochondria in young adult animals; hypodermis is imaged (green: NTL‐2, red: TMRE (tetramethylrhodamine, ethyl ester, perchlorate), a mitochondrial membrane potential‐dependent dye) (n = 3 independent experiments).
  2. Localization of NTL‐2 foci relative to mitochondria in young adult animals; body wall muscle cells are imaged (green: NTL‐2, red: TOMM‐20, an outer mitochondrial membrane (OMM) protein marker of mitochondria) (n = 3 independent experiments).
  3. EDC‐3 foci localize in close proximity to mitochondria in young adult animals; body wall muscle cells are imaged (green: EDC‐3, red: TMRE) under control conditions (n = 3 independent experiments).
  4. Localization of EDC‐3 foci relative to mitochondria in young adult animals; body wall muscle cells are imaged (red: EDC‐3, green: mitochondrial matrix targeted by GFP) (n = 3 independent experiments).
  5. Quantification of NTL‐2 and EDC‐3 foci (shown in dots) with depicted distances from mitochondria is shown (n = 3 independent experiments) based on experiments presented in images (A–D).
  6. Immunoblot analysis of the cytoplasmic and mitochondria‐containing fractions obtained from whole animal extracts, showing that NTL‐2, CCF‐1, EDC‐3 and DCAP‐2 are localized in the cytoplasm and co‐precipitate with mitochondria (n = 3 independent experiments).
  7. Mitochondrial ROS production is elevated in animals subjected to either dcap‐2 or ntl‐2 RNAi, as evidenced by staining with Mitotracker Red CM‐H2X ROS (n = 3 independent experiments with at least 113 animals/experiment; ****P < 0.0001; one‐way analysis of variance (ANOVA)).
  8. Mitochondrial membrane potential (Δψ) is increased in animals subjected to either dcap‐2 or ntl‐2 RNAi as evidenced by TMRE staining (n = 3 independent experiments with at least 144 animals/experiment; ****P < 0.0001; one‐way analysis of variance (ANOVA)).
  9. EDC‐3 and NTL‐2 foci lose their specific localization close to mitochondria upon paraquat treatment of transgenic animals expressing mitochondria‐targeted GFP; top: EDC‐3 foci, bottom: NTL‐2 foci (n = 3 independent experiments).
  10. Measurement of the distances between EDC‐3 foci (shown in dots) and mitochondria under paraquat treatment as compared with their control counterparts (n = 3 independent experiments with at least 30 animals/experiment; ****P < 0.0001; two‐tailed unpaired t‐test).
  11. EDC‐3 foci are increased upon paraquat treatment (n = 3 Independent experiments with at least 30 animals/experiment; ****P < 0.0001; two‐tailed unpaired t‐test).
  12. EDC‐3 and NTL‐2 foci lose their specific localization in the vicinity of mitochondria upon genetic inhibition of mrps‐5; top: EDC‐3 foci, bottom: NTL‐2 foci (n = 3 independent experiments).
  13. Measurement of the distances between EDC‐3 foci (shown in dots) and mitochondria upon genetic inhibition of mrps‐5 as compared to control (n = 3 independent experiments with at least 30 animals/experiment; ****P < 0.0001; two‐tailed unpaired t‐test).
  14. Quantification of NTL‐2 protein bound on mitochondria under control conditions and upon genetic inhibition of mrps‐5 (western blot shown in Fig 5E, n = 2 independent experiments; *P < 0.05; one‐way analysis of variance (ANOVA) followed by Dunnett's test).

Data information: Images were acquired using an ×63 objective lens. Scale bars, 20 μm. Error bars denote SEM.