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.
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"By the time non-complemented nascent Δccm1 segregants reached t=0 (Figure 1A), the levels of the molecular targets were too low to be analysed . Repression of the GAL1 promoter by dextrose in pCCM1LC was enough to cause deficient growth in non-fermentable substrates , but a more stringent mitochondrial deprivation of Ccm1p was required to dissect events at the molecular level. Limitations upon GAL1-repression were previously reported [12,32]. "
[Show abstract][Hide abstract]ABSTRACT: Ccm1p is a nuclear-encoded pentatricopeptide repeat (PPR) protein that localizes into mitochondria of Saccharomyces cerevisiae. It was first defined as an essential factor to remove the fourth intron of COB and COX1 pre-mRNA, along with bI4 maturase, a protein encoded by part of the COB fourth intron 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, sudden mitochondrial deprivation of Ccm1p (SMDC), and mutations in one of the PPR 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.
"This includes the Mss116 RNA helicase , which is a general mitochondrial splicing factor that acts in facilitating the RNA folding reaction required for self-splicing [68, 69] and the Suv3 RNA helicase, which is required for the processing of intron aI5β by recycling the intron-splicing factor Mrs1 . At least two proteins are required for COX1 aI4 intron splicing, Ccm1  and Nam2  . Other proteins are involved, which specific functions in most cases remain to be fully understood such as the COX3 mRNA translational activator Pet54 [73, 74], Mrs1 , Mne1  and Mss18 , four proteins required for the processing of COX1 aI5β intron, and the recently identified Cox24, required for splicing of COX1 aI2 and aI3 introns . "
[Show abstract][Hide abstract]ABSTRACT: Eukaryotic cytochrome c oxidase (COX) is the terminal enzyme of the mitochondrial respiratory chain. COX is a multimeric enzyme formed by subunits of dual genetic origin which assembly is intricate and highly regulated. The COX catalytic core is formed by three mitochondrial DNA encoded subunits, Cox1, Cox2 and Cox3, conserved in the bacterial enzyme. Their biogenesis requires the action of messenger-specific and subunit-specific factors which facilitate the synthesis, membrane insertion, maturation or assembly of the core subunits. The study of yeast strains and human cell lines from patients carrying mutations in structural subunits and COX assembly factors has been invaluable to identify these ancillary factors. Here we review the current state of knowledge of the biogenesis and assembly of the eukaryotic COX catalytic core and discuss the degree of conservation of the players and mechanisms operating from yeast to human. This article is part of a Special Issue entitled: Biogenesis/Assembly of Respiratory Enzyme Complexes.
"It is therefore obvious that splicing defects are not a primary deficiency responsible for the deletion phenotype. The loss of functional mtDNA, observed in our study and reported previously (Ellis et al. 2004; Merz and Westermann 2009; Moreno et al. 2009) in strains carrying the null allele of YGR150C is, on the other hand, consistent with the disruption of mitochondrial translation, caused by degradation of the small subunit ribosomal RNA, being the initial dysfunction leading to the irreversible collapse of the entire mitochondrial gene expression system. Evidence gathered so far leads to a conclusion that the product of the DMR1 gene is an RNA binding protein , with the small subunit mitochondrial 15S rRNA being its target. "
[Show abstract][Hide abstract]ABSTRACT: Pentatricopeptide repeat (PPR) proteins form the largest known RNA-binding protein family and are found in all eukaryotes, being particularly abundant in higher plants. PPR proteins localize mostly in mitochondria and chloroplasts, where they modulate organellar genome expression on the post-transcriptional level. The Saccharomyces cerevisiae DMR1 (CCM1, YGR150C) encodes a PPR protein that localizes to mitochondria. Deletion of DMR1 results in a complete and irreversible loss of respiratory capacity and loss of wild-type mtDNA by conversion to rho(-)/rho(0) petites, regardless of the presence of introns in mtDNA. The phenotype of the dmr1Delta mitochondria is characterized by fragmentation of the small subunit mitochondrial rRNA (15S rRNA), that can be reversed by wild-type Dmr1p. Other mitochondrial transcripts, including the large subunit mitochondrial rRNA (21S rRNA), are not affected by the lack of Dmr1p. The purified Dmr1 protein specifically binds to different regions of 15S rRNA in vitro, consistent with the deletion phenotype. Dmr1p is therefore the first yeast PPR protein, which has an rRNA target and is probably involved in the biogenesis of mitochondrial ribosomes and translation.