Mitochondrial mRNA and the small subunit rRNA in budding yeasts undergo 3'-end processing at conserved species-specific elements.

Respiration in eukaryotes depends on mitochondrial protein synthesis, which is performed by organelle-specific ribosomes translating organelle-encoded mRNAs. Although RNA maturation and stability are central events controlling mitochondrial gene expression, many of the molecular details in this pathway remain elusive. These include and -regulatory factors that generate and protect the 3' ends. Here, we mapped the 3' ends of mitochondrial mRNAs of yeasts classified into multiple families of the subphylum Saccharomycotina. We found that the processing of mitochondrial 15S rRNA and mRNAs involves species-specific sequence elements, which we term 3'-end RNA processing elements (3'-RPEs). In the 3'-RPE has long been recognized as a conserved dodecamer sequence, which recent studies have shown specifically interacts with the nuclear genome-encoded pentatricopeptide repeat protein Rmd9. We also demonstrate that, analogous to Rmd9 in , two Rmd9 orthologs from the family interact with their respective 3'-RPEs found in mRNAs and 15S rRNA. Thus, Rmd9-dependent processing of mitochondrial RNA precursors may be a common mechanism among the families of the Saccharomycotina subphylum. Surprisingly, we observed that 3'-RPEs often occur upstream of stop codons in complex I subunit mRNAs from yeasts of the CUG-Ser1 clade. We examined two of these mature mRNAs and found that their stop codons are indeed removed. Thus, translation of these stop-codon-less transcripts would require a noncanonical termination mechanism. Our findings highlight Rmd9 as a key evolutionarily conserved factor in both mitochondrial mRNA metabolism and mitoribosome biogenesis in a variety of yeasts.