The Pet309 pentatricopeptide repeat motifs mediate efficient binding to the mitochondrialCOX1transcript in yeast

Abstract

Mitochondrial synthesis of Cox1, the largest subunit of the cytochrome c oxidase complex, is controlled by Mss51 and Pet309, two mRNA-specific translational activators that act via the COX1 mRNA 5′-UTR through an unknown mechanism. Pet309 belongs to the pentatricopeptide repeat (PPR) protein family, which is involved in RNA metabolism in mitochondria and chloroplasts, and its sequence predicts at least 12 PPR motifs in the central portion of the protein. Deletion of these motifs selectively disrupted translation but not accumulation of the COX1 mRNA. We used RNA coimmunoprecipitation assays to show that Pet309 interacts with the COX1 mRNA in vivo and that this association is present before processing of the COX1 mRNA from the ATP8/6 polycistronic mRNA. This association was not affected by deletion of 8 of the PPR motifs but was undetectable after deletion of the entire 12-PPR region. However, interaction of the Pet309 protein lacking 12 PPR motifs with the COX1 mRNA was detected after overexpression of the mutated form of the protein, suggesting that deletion of this region decreased the binding affinity for the COX1 mRNA without abolishing it entirely. Moreover, binding of Pet309 to the COX1 mRNA was affected by deletion of Mss51. This work demonstrates an in vivo physical interaction between a yeast mitochondrial translational activator and its target mRNA and shows the cooperativity of the PPR domains of Pet309 in interaction with the COX1 mRNA.

Mitochondrial genetics revisited

“…Interestingly, the CCM1 protein also acts directly on intron splicing efficiency, independently of its action on the mitoribosome (Moreno et al, ). This example illustrates the importance of PPR containing proteins for the function of mitochondrial RNA molecules (Herbert, Golik, & Bonnefoy, ), predicting additional cases of mitonuclear incompatibilities (Zamudio‐Ochoa, Camacho‐Villasana, Garcia‐Guerrero, & Pérez‐Martinez, ).…”

Cbp3 and Cbp6 are dispensable for synthesis regulation of cytochrome b in yeast mitochondria

“…Mitochondria from BY4742 and D273-10b strains expressing the CBP3-HA or CBP3 gene were solubilized with dodecylmaltoside. The mitochondrial extract was immunoprecipitated using antibodies against the HA epitope, and RNA was isolated and analyzed by reverse transcription-PCR (30). For cDNA synthesis and PCR amplification, we used primers specific for COB and also for VAR1 (used as a negative control because translation of this mRNA is independent of Cbp3).…”

“…This technique was performed as previously described (30). Mitochondria (500 g) were lysed with 0.7% n-dodecyl ␤-Dmaltoside, 20 mM Tris-HCl, pH 7.4, 150 mM NaCl, RNaseOUT (Invitrogen), and Minicomplete protease inhibitors (Roche).…”

Human mitochondrial COX1 assembly into cytochrome c oxidase at a glance

“…In the case of COX1 mRNA, these activators are Mss51 and Pet309 (Barrientos et al, 2004;Pérez-Martinez et al, 2003;Tavares-Carreón et al, 2008;Zambrano et al, 2007;Zamudio-Ochoa et al, 2014). Pet309 belongs to the pentatricopeptide repeat (PPR) protein family, which is typically involved in mRNA metabolism.…”

“…Pet309 belongs to the pentatricopeptide repeat (PPR) protein family, which is typically involved in mRNA metabolism. Pet309 has been shown to bind COX1 mRNA and is required for its translation (Manthey and McEwen, 1995;TavaresCarreón et al, 2008;Zamudio-Ochoa et al, 2014). Mss51 is able to bind to COX1 mRNA, as well as to the newly synthesized Cox1 protein (Barrientos et al, 2004;Pérez-Martinez et al, 2003;Pérez-Martinez et al, 2009;Zambrano et al, 2007).…”