Insights into the translational activation mechanisms of the COX1 mRNA in yeast mitochondria

Abstract

Mitochondrial translation is a crucial regulatory step in mitochondrial genome expression. In Saccharomyces cerevisiae, translational activators are believed to bind to the 5' UTRs of their target mRNAs to position the mitochondrial ribosome at the start codon. Pet309 and Mss51 are translational activators of COX1 mRNA, which encodes subunit one of cytochrome c oxidase. Pet309 physically interacts with COX1 mRNA, but no direct interaction of Mss51 with its target mRNA has been detected. Currently, the mechanisms underlying translational activation of COX1, or any other mitochondrial gene, remain poorly understood. To explore in depth the mechanism of COX1 mRNA translational activation, we studied the association of Pet309 and Mss51 with the mitochondrial ribosome. Both Pet309 and Mss51 interact with the mitoribosome regardless of the presence of COX1 mRNA or of each other. The association of Pet309 with the ribosome and with COX1 mRNA depends on its N-terminal domain. These findings indicate that Pet309 and Mss51 stably interact with the mitoribosome independently of active translation. By integrating our data with previously published research, we propose a new mechanism of COX1 mRNA translation activation.

Keywords: COX1 mRNA; Mitochondria; Mitoribosome; Mss51; Pet309; Translation.

Fig. 1.

Pet309 associates with the mitoribosome. (A) 500 µg of mitochondrial protein from the PET309-3xHA strain were lysed with 1% digitonin. Clarified lysate was centrifuged in a discontinuous 20–40% sucrose gradient. Seven fractions were collected and analyzed by SDS-PAGE and western blotting using the indicated antibodies. (B) Western blot analysis with indicated amounts of mitochondrial protein from the PET309-3xHA and PET309-3xHA ρ⁰ strains. (C) Same experiment as shown in A with lysed mitochondria from the ρ⁰ strain. For efficient immunodetection, a strain overexpressing PET309-3xHA^OE was used in this experiment. In all experiments citrate synthase (CS) was used as loading control or a protein independent form the respiratory chain function. bS1, small mitoribosomal subunit (Mrp51); uL23 large mitoribosomal subunit (Mrp21); T, total fraction equivalent to 7% from the loaded clarified lysate. For all experiments, we used n=3 biological replicates. Uncropped blots are shown in Fig. S5.

Fig. 2.

Pet309 constitutively associates with the mitoribosome. Mitochondria bearing Pet309–3xHA with the mutants (A) cox1Δ or mss51Δ were lysed and separated through a sucrose gradient as in Fig. 1. Fractions from the gradient were analyzed by western blotting. (C) Steady state levels of Pet309–3xHA in the indicated mutants were analyzed by western blotting. (D) Western blot analysis from a sucrose gradient separation of the mitochondrial lysate from Pet309–3xHA mitochondria bearing the double mutants cox1Δ, mss51Δ. Citrate synthase (CS) was used as loading control or a protein independent from the respiratory chain function. bS1, small subunit protein; uL23, large subunit protein; T, Total fraction, equivalent to 7% of the load. For all experiments, we used n=3 biological replicates. Uncropped blots are shown in Fig. S5.

Fig. 3.

The N-terminal PPR module of Pet309 mediates interaction with the mitoribosome. (A) Diagram indicating the three regions form Pet309 that were analyzed in this study. Pet309Δnt lacks residues A53 to Q311, Pet309Δ12ppr lacks residues N312 to N759 and Pet309Δct lacks residues L760 to V962. The triple hemagglutinin epitope (3xHA) was used to immunodetect Pet309 proteins with antibodies as indicated. Bottom panel, a model of Pet309 was downloaded from the AlphaFold database of structural modeling (Jumper et al., 2021). The three proposed modules that conform Pet309 are indicated by colors: N-terminus module, blue; central module bearing 12 PPRs, red; C-terminus module, yellow. The figure was created using Pymol. (B–D) Western blot analysis of the sucrose gradient separation from mitochondria carrying the pet309Δ12ppr-3xHA (B), pet309Δnt-3xHA (C) and pet309Δnt-3xHA (D) constructs. bS1, small subunit protein. uL23, large subunit protein. CS. Citrate synthase. T, Total fraction, equivalent to 7% of the load. For all experiments, we used n=3 biological replicates. Uncropped blots are shown in Fig. S6.

Fig. 4.

The N-terminal module of Pet309 is necessary for interaction with the COX1 mRNA. Mitochondria from PET309-3xHA (WT lanes), (A) pet309Δct-3xHA or (C) pet309Δnt-3xHA^OE and the untagged strain were lysed with digitonin. The lysates were subjected to immunoprecipitation with anti-HA antibodies coupled to agarose beads. 5% of the lysate and 25% of the immunoprecipitated (IP) were separated by SDS-PAGE and analyzed by western blotting using an anti-HA antibody (HA) to show the efficiency of immunoprecipitation. An anti-citrate synthase antibody (CS) was used as a negative control. Next, RNA was extracted from the total (T), immunoprecipitated (IP) and supernatant (S) fractions from (B) pet309Δct-3xHA or (D) pet309Δnt-3xHA^OE samples. After DNase treatment, cDNA was prepared using reverse transcriptase (RT) (+) and adding primers for COX1 and VAR1 genes (Table S2). Samples without RT (−) were included as a control for DNA contamination. Resulting cDNAs were used as a template for PCR amplification of COX1 and VAR1 genes and run in an agarose gel. For all experiments, we used n=3 biological replicates Uncropped blots are shown in Fig. S6.

Fig. 5.

The N-terminal module from Pet309 is involved in stabilization of the COX1 mRNA. The PET309-3xHA (WT lanes), pet309Δct-3xHA (A) pet309Δnt-3xHA (B) were cloned on centromeric plasmid for low copy number (Low) and 2 µ plasmid for high copy number (High) and transformed into a pet309Δ strain. An empty plasmid was also transformed and used as negative control. 10 µg of total RNA was analyzed by northern blotting. As a negative control, total RNA from pet309Δ strains bearing empty plasmids was also analyzed. The membrane was hybridized with ³²P-labeled probes complementary to COX1, COX2 and 15S rRNA probes. (B,D) Quantification of the COX1 signals from three independent experiments are represented in a bar graph (mean±s.d.). COX1 signals were normalized against 15S rRNA and the result from PET309-3xHA in low copy plasmid was taken as 100% (§). For all experiments we used n=3 biological replicates. *P<0.05 (two-way ANOVA with Bonferroni post-hoc test).

Fig. 6.

Mss51 constitutively interacts with the mitoribosome. Mitochondria from MSS51-3xHA cells carrying (A) WT mitochondrial DNA or (B) devoid of mtDNA (ρ°) were solubilized and separated by ultracentrifugation on a sucrose gradient. The resultant seven fractions from this gradient were analyzed by western blotting with the indicated antibodies. Similar experiments were carried out with mitochondria bearing (C) a deletion of the COX1 gene (cox1Δ), (D) the mutant PET309 gene (pet309Δ) and (E) the double mutant cox1Δ pet309Δ. For all experiments we used n=3 biological replicates. Uncropped blots are shown in Fig. S7. *Scratch on the original film, not a western blot signal.

Fig. 7.

Model for the mechanisms of Pet309 and Mss51 on translation of the COX1 mRNA. See text in Discussion section for details. Created in BioRender by Perez, X., 2025. https://BioRender.com/jqiwlc8. This figure was sublicensed under CC-BY 4.0 terms.