Fig 1: Mitochondria-associated mRNAs restore ATP through cytosolic translation in astrocytes. (A) Immunofluorescence showing the colocalization of MitoTracker+ mitochondria with the ribosomal protein RPS6, consistent with ribosome association at the outer mitochondrial membrane. Colocalization was quantified by Pearson’s correlation coefficient (Rr = 0.85143). (B,C) Western blot analysis demonstrating the presence of ribosomal proteins (RPS6, RPL26) in isolated mitochondrial fractions and their depletion following trypsin treatment. n = 5–6 from two independent experiments; *** p < 0.001, two-sided Student’s t-test. Data are shown as mean ± SD. (D) Quantification showing that trypsin treatment substantially reduced mitochondria-associated mRNAs without altering intrinsic mitochondrial ATP content. Although mRNA levels were markedly decreased, a small residual fraction remained detectable, likely reflecting protected or internal RNA species not accessible to proteolytic digestion. ATP: n = 24, mRNA: n = 9 from three independent experiments; **** p < 0.0001, two-sided Student’s t-test. Data are shown as mean ± SD. (E) ATP restoration by MitoCoat was significantly attenuated following the trypsin removal of surface-associated ribosomes and mRNAs. n = 48 from three independent experiments; *** p < 0.001, **** p < 0.0001, one-way ANOVA followed by Tukey’s test. Data are shown as mean ± SD. (F) Delivered mitochondria-associated mRNAs localized in proximity to mitochondria; this localization was reduced by CHX treatment. Colocalization was quantified by Pearson’s correlation coefficient (Rr = 0.37743). (G,H) Western blot analysis showing that H2O2 injury reduced glycolytic and oxidative phosphorylation proteins, while mRNA treatment partially restored these markers. ATP5A and PDH were significantly increased in a cytosolic translation-dependent manner. n = 3 from three independent experiments; * p < 0.05, ** p < 0.01, one-way ANOVA followed by Tukey’s test. Data are shown as mean ± SD. (I) Pharmacological inhibition of cytosolic translation with CHX (50 μg/mL) reduced ATP recovery, whereas the inhibition of mitochondrial translation with CAP (10 μg/mL) had no effect. n = 98 from seven independent experiments; * p < 0.05, **** p < 0.0001, one-way ANOVA followed by Tukey’s test. Data are shown as mean ± SD. (J) Working model illustrating that mitochondria-associated mRNAs support re-establishing metabolic coupling between glycolysis and mitochondrial oxidative phosphorylation and promoting ATP production in H2O2-injured cells.