Abstract
Mitochondrial translation appears to involve two stalled-ribosome rescue factors (srRFs). One srRF is an ICT1 protein from humans that rescues a “non-stop” type of mitochondrial ribosomes (mitoribosomes) stalled on mRNA lacking a stop codon, while the other, C12orf65, reportedly has functions that overlap with those of ICT1; however, its primary role remains unclear. We herein demonstrated that the Saccharomyces cerevisiae homolog of C12orf65, Pth3 (Rso55), preferentially rescued antibiotic-dependent stalled mitoribosomes, which appear to represent a “no-go” type of ribosomes stalled on intact mRNA. On media containing a non-fermentable carbon source, which requires mitochondrial gene expression, respiratory growth was impaired significantly more by the deletion of PTH3 than that of the ICT1 homolog PTH4 in the presence of antibiotics that inhibit mitochondrial translation, such as tetracyclines and macrolides. Additionally, the in organello labeling of mitochondrial translation products and quantification of mRNA levels by quantitative RT-PCR suggested that in the presence of tetracycline, the deletion of PTH3, but not PTH4, reduced the protein expression of all eight mtDNA-encoded genes at the post-transcriptional or translational level. These results indicate that Pth3 can function as a mitochondrial srRF specific for ribosomes stalled by antibiotics and plays a role in antibiotic resistance in fungi.
Highlights
Mitochondrial translation appears to involve two stalled-ribosome rescue factors
We demonstrated that the S. cerevisiae homolog of C12orf[65], Pth[3], preferentially rescued stalled mitoribosomes caused by various antibiotics that have different mechanisms of action even more efficiently than the homolog of ICT1, Pth[4]
It is possible that an excessive amount of either of stalled-ribosome rescue factor (srRF) causes its unexpected binding to normal translating mitoribosomes, preventing normal elongation or termination. These results revealed that Pth[3] plays a particular role in antibiotic resistance, suggesting the presence of specific mitoribosome stalling caused by the antibiotics that is only rescued by Pth[3]
Summary
Mitochondrial translation appears to involve two stalled-ribosome rescue factors (srRFs). The in organello labeling of mitochondrial translation products and quantification of mRNA levels by quantitative RT-PCR suggested that in the presence of tetracycline, the deletion of PTH3, but not PTH4, reduced the protein expression of all eight mtDNA-encoded genes at the post-transcriptional or translational level These results indicate that Pth[3] can function as a mitochondrial srRF specific for ribosomes stalled by antibiotics and plays a role in antibiotic resistance in fungi. ICT1 belongs to the RF-1 family, as represented by class I peptide release factors (RFs) from bacteria and mitochondria[10] This family is characterized by a catalytic structural domain containing a conserved Gly–Gly–Gln (GGQ) motif, termed the GGQ domain (Supplementary Fig. 1). When YaeJ is bound to the ribosome, a section of its C-terminal extension (termed the C-terminal tail) forms a helical structure, which lies in the mRNA entry channel, downstream of the A-site that is vacant in the 30S subunit
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