Ccm1p is a 15S rRNA primary transcript processing factor as elucidated by a novel in vivo system in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, the mitoribosomal RNA of the minor subunit, 15S rRNA, is transcribed as a bicistronic transcript along with tRNAW. 5' and 3' sequences flanking the mature transcript must be removed by cleavage at the respective junctions before incorporating it into the mitoribosome. An in vivo dose-response triphasic system was created to elucidate the role of Ccm1p in the processing of 15S rRNA: Ccm1p supply ("On"), deprivation ("Off"), and resupply ("Back on"). After 72 h under "Off" status, the cells started to exhibit a complete mutant phenotype as assessed by their lack of growth in glycerol medium, while keeping their mitochondrial DNA integrity (ρ+). Full functionality of mitochondria was reacquired upon "Back on." 15S rRNA levels and phenotype followed the Ccm1p intramitochondrial concentrations throughout the "On-Off-Back on" course. Under "Off" status, cells gradually accumulated unprocessed 5' and 3' junctions, which reached significant levels at 72-96 h, probably due to a saturation of the mitochondrial degradosome (mtEXO). The Ccm1p/mtEXO mutant (Δccm1/Δdss1) showed a copious accumulation of 15S rRNA primary transcript forms, which were cleaved upon Ccm1p resupply. The gene that codes for the RNA component of RNase P was conserved in wild-type and mutant strains. Our results indicate that Ccm1p is crucial in processing the 15S rRNA primary transcript and does not stabilize the already mature 15S rRNA. Consequently, failure of this function in Δccm1 cells results, as it happens to any other unprocessed primary transcripts, in total degradation of 15S rRNA by mtEXO, whose mechanism of action is discussed.

Ccm1p/Ygr150cp, a pentatricopeptide repeat protein, is essential to remove the fourth intron of both COB and COX1 pre-mRNAs in Saccharomyces cerevisiae.

YGR150C gene product (Ygr150cp) is one of the three mitochondrially located Saccharomyces cerevisiae proteins with pentatricopeptide repeat (PPR) motifs. Ygr150cp is essential for mitochondrial functionality but its molecular targets are still unknown. This study was undertaken to define the role of Ygr150cp in mitochondria biogenesis. Repression of Ygr150cp expression in complemented mutants prevented their use of glycerol or lactate, but allowed limited growth on ethanol-containing medium. RNA hybridization studies showed that Deltaygr150c meiotic segregants produced COB and COX1 transcripts but failed to process them into the mature forms. Detailed RT-PCR assays revealed that Deltaygr150c specifically failed to remove the fourth intron of both COB and COX1 pre-mRNAs while all other group I introns were excised. Expression of Ygr150cp mutants without any of the PPR motifs did not complement the growth phenotype. Accordingly, we designate YGR150C as CCM1 (COB and COX1 mRNA maturation). This report provides the first evidence of PPR protein involvement in the specific removal of group I introns in mitochondria of S. cerevisiae.

Two independent activities define Ccm1p as a moonlighting protein in Saccharomyces cerevisiae.

Ccm1p is a nuclear-encoded PPR (pentatricopeptide repeat) protein that localizes into mitochondria of Saccharomyces cerevisiae. It was first defined as an essential factor to remove the bI4 [COB (cytochrome b) fourth intron)] and aI4 [COX1 (cytochrome c oxidase subunit 1) fourth intron] of pre-mRNAs, along with bI4 maturase, a protein encoded by part of bI4 and preceding exons that removes the intronic RNA sequence that codes for it. Later on, Ccm1p was described as key to maintain the steady-state levels of the mitoribosome small subunit RNA (15S rRNA). bI4 maturase is produced inside the mitochondria and therefore its activity depends on the functionality of mitochondrial translation. This report addresses the dilemma of whether Ccm1p supports bI4 maturase activity by keeping steady-state levels of 15S rRNA or separately and directly supports bI4 maturase activity per se. Experiments involving loss of Ccm1p, SMDC (sudden mitochondrial deprivation of Ccm1p) and mutations in one of the PPR (pentatricopeptide repeat) motifs revealed that the failure of bI4 maturase activity in CCM1 deletion mutants was not due to a malfunction of the translational machinery. Both functions were found to be independent, defining Ccm1p as a moonlighting protein. bI4 maturase activity was significantly more dependent on Ccm1p levels than the maintenance of 15S rRNA. The novel strategy of SMDC described here allowed the study of immediate short-term effects, before the mutant phenotype was definitively established. This approach can be also applied for further studies on 15S rRNA stability and mitoribosome assembly.