COB Transcripts Research Articles

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.

Differential expression of maize mitochondrial genes as dependent on mitochondria redox state

We studied the effects of changes in the mitochondrial redox state, induced by respiration inhibitors, on the expression of mitochondrial genes in Zea mays. The highest levels of cox1, cox3, and cob transcripts were observed in 4 h and atp9 transcripts in 6 h after treatment with antimycin A (antA). Treatment with salicyl hydroxamic acid (SHAM) resulted in a decrease in the transcripts during the first 10 h of incubation with subsequent recovering of this gene transcript level by 24 h. Treatment with SHAM did not essentially affect the level of cox1 transcripts, whereas the levels of cox3 and cob transcripts somewhat increased during the first 4 h and remained stable thereafter. After combined treatment with antA and SHAM, the level of cox1 and cox3 transcripts dropped below the control level during the first 4 h and then increased by the end of the treatment. The content of cob transcripts increased markedly after 24 h of treatment. The rps13 transcript changed irregularly, and its changes did not coincide with changes of other transcripts studied. Thus, we demonstrated differential expression of mitochondrial genes at changes in the redox state of the mitochondrial electron transport chain (ETC). The cox1, cox3, and cob gene expression responded to these changes rather coordinately. The results obtained indicate the involvement of the ETC redox state and/or the level of reactive oxygen species in the control of mitochondrial gene expression.

Cbp1 Is Required for Translation of the Mitochondrial Cytochromeb mRNA of Saccharomyces cerevisiae

Expression of the yeast mitochondrial cytochrome b gene (COB) is controlled by at least 15 nuclear-encoded proteins. One of these proteins, Cbp1, is required for COB mRNA stability. Delta cbp1 null strains fail to accumulate mature COB mRNA and cannot respire. Since Delta cbp1 null strains lack mature COB transcripts, the hypothesis that Cbp1 also plays a role in translation of these mRNAs could not be tested previously. 5'-End trimming of precursor COB RNA and other mitochondrial transcripts is dependent on Pet127. pet127 mutants accumulate high levels of precursor COB mRNA and have no mature mRNA. pet127 mutants respire well; this phenotype implies that COB precursor RNA is translated efficiently. With the expectation that a Delta cbp1 Delta pet127 strain might accumulate substantial levels of COB RNA, the double null strain was constructed and analyzed to test the hypothesis that Cbp1 is required for translation of COB RNA. The Delta cbp1 Delta pet127 strain does accumulate levels of COB precursor mRNA that are approximately 60% of the level of COB mRNA in the wild-type strain. However, cytochrome b protein is not synthesized, and thus the Delta cbp1 Delta pet127 strain does not respire. These results suggest that Cbp1 is required for translation of COB RNAs.

The formation of respiratory chain complexes in mitochondria is under the proteolytic control of the m-AAA protease.

Yta10p (Afg3p) and Yta12p (Rcal1p), members of the conserved AAA family of ATPases, are subunits of the mitochondrial m-AAA protease, an inner membrane ATP-dependent metallopeptidase. Deletion of YTA10 or YTA12 impairs degradation of non-assembled inner membrane proteins and assembly of respiratory chain complexes. Mutations of the proteolytic sites in either YTA10 or YTA12 have been shown to inhibit proteolysis of membrane-integrated polypeptides but not the respiratory competence of the cells, suggesting additional activities of Yta10p and Yta12p. Here we demonstrate essential proteolytic functions of the m-AAA protease in the biogenesis of the respiratory chain. Cells harbouring proteolytically inactive forms of both Yta10p and Yta12p are respiratory deficient and exhibit a pleiotropic phenotype similar to Deltayta10 and Deltayta12 cells. They show deficiencies in expression of the intron-containing mitochondrial genes COX1 and COB. Splicing of COX1 and COB transcripts is impaired in mitochondria lacking m-AAA protease, whilst transcription and translation can proceed in the absence of Yta10p or Yta12p. The function of the m-AAA protease appears to be confined to introns encoding mRNA maturases. Our results reveal an overlapping substrate specificity of the subunits of the m-AAA protease and explain the impaired assembly of respiratory chain complexes by defects in expression of intron-containing genes in mitochondria lacking m-AAA protease.

The ATP-dependent PIM1 protease is required for the expression of intron-containing genes in mitochondria.

The ATP-dependent PIM1 protease, a Lon-like protease localized in the mitochondrial matrix, is required for mitochondrial genome integrity in yeast. Cells lacking PIM1 accumulate lesions in the mitochondrial DNA (mtDNA) and therefore lose respiratory competence. The identification of a multicopy suppressor, which stabilizes mtDNA in the absence of PIM1, enabled us to characterize novel functions of PIM1 protease during mitochondrial biogenesis. The synthesis of mitochondrially encoded cytochrome c oxidase subunit I (CoxI) and cytochrome b (Cob) is impaired in pim1 mutants containing mtDNA. PIM1-mediated proteolysis is required for the translation of mature COXI mRNA. Moreover, deficiencies in the splicing of COXI and COB transcripts, which appear to be restricted to introns encoding mRNA maturases, were observed in cells lacking the PIM1 gene. Transcripts of COXI and COB genes harboring multiple introns are degraded in the absence of PIM1. These results establish multiple, essential functions of the ATP-dependent PIM1 protease during mitochondrial gene expression.

In vivo analysis of sequences required for translation of cytochrome b transcripts in yeast mitochondria.

Respiratory chain proteins encoded by the yeast mitochondrial genome are synthesized within the organelle. Mitochondrial mRNAs lack a 5' cap structure and contain long AU-rich 5' untranslated regions (UTRs) with many potential translational start sites and no apparent Shine-Dalgarno-like complementarity to the 15S mitochondrial rRNA. However, translation initiation requires specific interactions between the 5' UTRs of the mRNAs, mRNA-specific activators, and the ribosomes. In an initial step toward identifying potential binding sites for the mRNA-specific translational activators and the ribosomes, we have analyzed the effects of deletions in the 5' UTR of the mitochondrial COB gene on translation of COB transcripts in vivo. The deletions define two regions of the COB 5' UTR that are important for translation and indicate that sequence just 5' of the AUG is involved in selection of the correct start codon. Taken together, the data implicate specific regions of the 5' UTR of COB mRNA as possible targets for the mitochondrial translational machinery.

Expression of Mitochondrial Genes in the Germinating Conidia of Neurospora crassa

The germinating asexual spores (conidia) of Neurospora crassa were employed to study steps in the accumulation of transcripts of groups of mitochondrial genes, including those for peptide subunits of cytochrome c oxidase (CO), ATPase (ATP), and apocytochrome b (COB). Physically clustered groups of genes were expressed as cohorts: transcripts of the ATP8-ATP6-mtATP9-CO2 genes were almost undetectable in the dormant spores, and they accumulated rapidly as a group immediately after spore activation. Transcripts of COB and the adjacent CO1 were abundant in the dormant spores, and the dormant and germinating spores contained size forms of the COB transcripts that were not evident in vegetative cells. Polyribosomes were prepared from mitochondrial lysates, and the polyribosomal RNA was probed to identify the mRNAs of specific genes; in several instances polycistronic mRNAs were present in the polyribosomes as were the smaller end products of the inferred transcript processing pathways. The expression of the physically dispersed genes for subunit peptides of cytochrome c oxidase appears to be regulated to the level of translation; these transcripts are accumulated in the total mitochondrial RNA with sharply different kinetics, but they appeared in the polyribosomes uniformly, their appearance correlating with the uniform synthesis of the subunit peptides. Transcripts for a previously reported non-functional mitochondrial gene, homologous to the functional nuclear gene for ATPase subunit 9, were found in the germinating spores, but were not detected in vegetative cells. These mtATP9 transcripts were also present in the polyribosomes and were apparently translated into a protein in vivo whose synthesis was insensitive to cycloheximide and detectable with an anti-ATP9 subunit antibody. Transcripts for two nuclear genes for mitochondrially localized proteins, ATP9 and CO5, were accumulated in unison and especially rapidly during spore germination

RNA editing of apocytochrome b (cob) transcripts in mitochondria from two genera of plants.

Editing of the complete coding region of cob transcripts from two genera of plants has been studied by cDNA sequence analysis. Eighteen and nine C residues are edited into U in the mitochondrial transcripts from wheat and potato respectively. Both systems share eight common editing sites; ten codons edited in wheat are "pre-edited" at the genomic level in potato, and one codon edited in potato is "pre-edited" in wheat. Most amino-acid modifications lead to hydrophobic residues and increase the homology between the COB polypeptides and the corresponding protein of other species. In two out of the nine potato cDNA clones, an additional C-to-T modification, which also leads to a change in the encoded amino acid, was identified. Heterogeneity observed at the carboxy-terminus of the COB open reading frame in Triticum aestivum and Triticum timopheevi is not corrected by editing.

In Vivo Analysis of Sequences Necessary for CBP1-Dependent Accumulation of Cytochrome b Transcripts in Yeast Mitochondria

In Saccharomyces cerevisiae, cytochrome b, an essential component of the respiratory chain, is encoded by the mitochondrial gene cob. The cob transcription unit includes the tRNA(Glu) gene from positions -1170 to -1099 relative to the cob ATG at +1. The initial tRNA(Glu)-cob transcript undergoes several processing events, including removal of tRNA(Glu) and production of the mature 5' end of cob mRNA at nucleotide -954. The nuclear gene product CBP1 is specifically required for the accumulation of cob mRNA. In cbp1 mutant strains, cob transcripts are not detectable by Northern (RNA) blot analysis, but the steady-state level of tRNA(Glu) is similar to that of wild type. The results of a previous study led to the conclusion that a 400-nucleotide region just downstream of tRNA(Glu) is sufficient for CBP1 function. In the present study, the microprojectile bombardment method of mitochondrial transformation was used to introduce deletions within this region of cob. The analysis of cob transcripts in strains carrying the mitochondrial deletion genomes indicates that a 63-nucleotide sequence that encompasses the cleavage site at -954 is sufficient both for CBP1 function and for correct positioning of the cleavage. Furthermore, the data indicate that CBP1 prevents the degradation of unprocessed cob transcripts produced by endonucleolytic cleavage at the 3' end of tRNA(Glu).

In vivo analysis of sequences necessary for CBP1-dependent accumulation of cytochrome b transcripts in yeast mitochondria.

In Saccharomyces cerevisiae, cytochrome b, an essential component of the respiratory chain, is encoded by the mitochondrial gene cob. The cob transcription unit includes the tRNA(Glu) gene from positions -1170 to -1099 relative to the cob ATG at +1. The initial tRNA(Glu)-cob transcript undergoes several processing events, including removal of tRNA(Glu) and production of the mature 5' end of cob mRNA at nucleotide -954. The nuclear gene product CBP1 is specifically required for the accumulation of cob mRNA. In cbp1 mutant strains, cob transcripts are not detectable by Northern (RNA) blot analysis, but the steady-state level of tRNA(Glu) is similar to that of wild type. The results of a previous study led to the conclusion that a 400-nucleotide region just downstream of tRNA(Glu) is sufficient for CBP1 function. In the present study, the microprojectile bombardment method of mitochondrial transformation was used to introduce deletions within this region of cob. The analysis of cob transcripts in strains carrying the mitochondrial deletion genomes indicates that a 63-nucleotide sequence that encompasses the cleavage site at -954 is sufficient both for CBP1 function and for correct positioning of the cleavage. Furthermore, the data indicate that CBP1 prevents the degradation of unprocessed cob transcripts produced by endonucleolytic cleavage at the 3' end of tRNA(Glu).

Structure and transcription of the gene coding for subunit 3 of cytochrome oxidase in wheat mitochondria

The wheat mitochondrial (mt) cox3 has been localized and sequenced. The gene exists as a single copy in the wheat mt master chromosome and is transcribed into a single 1.2 kb RNA, whose extremities have been mapped. Comparison of the wheat and Oenothera cox3 sequences gives ambiguous indications concerning the amino acid coded by the codon CGG. Upstream and downstream of the wheat cox3 gene, two short sequences of 43 bp and 69 bp respectively are present, which are almost identical to sequences present in the flanking regions of other plant mitochondrial genes. These common sequences seem to have played a role in the rearrangements which caused sequence divergence of the plant mt genomes during evolution. Furthermore, mapping of wheat and maize cox3 and cob transcripts suggests that some of these common sequences can play a role in the regulation of transcription or processing.

Nuclear functions required for cytochrome c oxidase biogenesis in Saccharomyces cerevisiae: multiple trans-acting nuclear genes exert specific effects on expression of each of the cytochrome c oxidase subunits encoded on mitochondrial DNA.

Fourteen nuclear complementation groups of mutants that specifically affect the three mitochondrially-encoded subunits of yeast cytochrome c oxidase have been characterized. Genes represented by these complementation groups are not required for mitochondrial transcription, transcript processing, or translation per se but are required for the expression of one of the three genes--COX1, COX2, or COX3--which encode the cytochrome c oxicase subunits I, II, or III, respectively. Five of these genes affect the biogenesis of cytochrome c oxidase subunit I, 3 affect the biogenesis of subunit II, 3 affect the biogenesis of subunit III and 3 affect the biogenesis of both cytochrome c oxidase subunit I and cytochrome b, the product of COB. Among the 5 complementation groups of mutants that affect the expression of COX1, 2 lack COX1 transcripts, 1 produces incompletely processed COX1 transcripts, and 2 contain normal levels of normal-sized COX1 transcripts. In contrast, all 3 complementation groups which affect the expression of COX2 and all 3 complementation groups which affect the expression of COX3 exhibit no, or little, detectable difference with respect to the wild type pattern of transcripts. The 3 complementation groups which affect the expression of both COX1 and COB all have aberrant COX1 and COB transcript patterns. These findings indicate that multiple trans-acting nuclear genes are required for specific expression of each COX gene encoded on mitochondrial DNA and suggest that their products act at different steps in the expression of these mitochondrial genes.

Cloning of a nuclear gene MRS1 involved in the excision of a single group I intron (bI3) from the mitochondrial COB transcript in S. cerevisiae.

The respiratory deficient yeast nuclear mutant MK3 is defective in the synthesis of the mature transcripts of the mitochondrial COB and OX13 genes, which code for apocytochrome b and subunit I of cytochrome c oxidase, resp. Introns 3 and 4 of the COB transcript (bI3 and bI4) and intron 4 (aI4) of the OXI3 transcript can not be excised (Pillar et al. 1983a, b). When combined with mitochondrial genomes lacking introns bI1, bI2 and bI3, or lacking intron bI3 alone the mutant is respiratory competent. Thus, the non-excision of bI4 and aI4 turns out to be an indirect effect of the mutation. From a wild type yeast genebank a plasmid has been isolated with a 3.3 kb DNA insert, which complements the mutant. Subcloning experiments assigned the functional gene to a 1.6 kb HaeIII-Sau3A fragment. Hybridization experiments showed, that it is (i) a single copy gene, (ii) also present in strain D273-10B, containing the "short form" mitochondrial genome (lacking the COB introns bI1-bI3), and (iii) located on chromosome IX. The nuclear gene defective in mutant MK3, was named MRS1 (Mitochondrial RNA Splicing). The involvement of this nuclear gene in the excision of a single group I mitochondrial intron (bI3) of the COB transcript is discussed.

Molecular cloning of the yeast nuclear genes CBS1 and CBS2.

The yeast nuclear genes CBS1 and CBS2 are both required for translation of the mitochondrial COB transcripts. Here we report on the identification of two unique chromosomal DNA-sequences of 2 kb and 2.3 kb from yeast wild type gene banks which functionally complement cbs1 and cbs2 mutants, respectively. Disruption of the homologous DNA-fragments by insertion of the URA3 gene generates respiratory deficient cells which fail to complement the original mutants. Cells with these gene disruptions are phenotypically identical to the original cbs1 and cbs2 mutants with respect to cytochrome spectra and mitochondrial translation products. The results exclude the possibility that suppressor genes have been cloned and confirm the conclusion that both genes, CBS1 and CBS2, specifically are involved in translation of mitochondrial COB RNA.

Two yeast nuclear genes, CBS1 and CBS2, are required for translation of mitochondrial transcripts bearing the 5'-untranslated COB leader.

Mutations in one of the yeast nuclear genes CBS1 or CBS2 both prevent the excision of the maturase-coding introns bI2, bI3 and bI4 from the mitochondrial COB precursor transcript. Mutant strain MK2 (cbs1-1) has recently been reported to be primarily defective in the translation of COB transcripts, as it can be suppressed by a fusion of the COB structural gene with the 5' untranslated leader of the mitochondrial OLI1 gene (G. Rödel, A. Körte and F. Kaudewitz, Curr Genet 9: 641-648). Here I report that the effect of mutation cbs2-1, too, is suppressed by this gene rearrangement. CBS2 is the second nuclear gene identified which is involved in the translation of mitochondrial transcripts bearing the 5' untranslated COB leader. Gene specific translation control appears to be a major mode of regulation of mitochondrial gene expression in Saccharomyces cerevisiae.

RNA processing in Neurospora crassa mitochondria: transfer RNAs punctuate a large precursor transcript.

The pattern of transcripts arising from a large contiguous portion of the Neurospora crassa mitochondrial genome and the processing of these transcripts have been investigated. Evidence is presented for the transcription of a single, at least 12.5 kb, precursor RNA that contains sequences corresponding to the apocytochrome b (cob), tRNACys, cytochrome oxidase subunit 1 (CO I), tRNAArg and unidentified reading frame (URF) 1 genes. The two tRNA sequences serve as punctuation signals for the cleavage of this large transcript. Processing at the tRNACys and tRNAArg sequences directly generates the proposed CO I mRNA, as well as the mature 3' end of the cob transcripts. Moreover, three other sites have been identified that are specifically involved in further 5' end processing events. One of the two 5' end processing sites of the cob transcript shares homology with the 5' end processing sites of the mitochondrial 19S and 25S rRNA precursors. PstI palindromes do not act as processing signals because six of these palindromic sequences are present in the cob and CO I mRNAs.

Evidence for ribosomes involved in splicing of yeast mitochondrial transcripts.

We have investigated the processing of transcripts of the split gene COB in yeast mitochondrial DNA from cells whose mitochondrial translation was blocked by chloramphenicol for several generations of cell growth. First analysis of transcripts by electrophoresis and RNA/DNA-hybridization clearly showed that cell growth in the presence of CAP leads to an inhibition of processing yielding an increasing amount of splicing intermediates of the COB transcript and decreasing amounts of the 18S mRNA coding for apocytochrome b. This observation is in accordance with the now widely favoured idea that mitochondrial proteins are involved in splicing of COB transcripts and that their reduction should hamper processing and - therefore - lead to an accumulation of pre-mRNAs. However, further information obtained by pulse-labeling of pre-mRNA in vivo in the presence of CAP for various times shows that even 30 minutes after addition of CAP a reduction of the processing rate is obtained. Based on these findings we conclude that maturation of mtRNAs is not only dependent on mitochondrial proteins, but also on a more direct interaction of the translation machinery and RNA processing whose nature is so far unknown.

Pathways of transcript splicing in yeast mitochondria. Mutations in intervening sequences of the split gene COB reveal a requirement for intervening sequence-encoded products.

We have studied the transcript processing of the split gene COB (or BOX) in yeast mtDNA, in both wild type and cob- mutants. Using various DNA fragments specific for coding or intervening sequences of this gene, we have determined the composition of splicing intermediates by DNA/RNA hybridization. The pattern of splicing intermediates detected in wild type reveals differing rates of the five splicings resulting in an apparent pathway of processing rather than an absolute order among the five cut and splice events. Effects of mutations in four of the five sequences have been studied. All of them interfere with transcript processing. Some block the excision of the sequence mutated only, but allow other splicing events to occur essentially as in the wild type. They suggest that in these mutants any order of splicings is possible, but that some are preferred. In contrast, other mutations located in four different sequences block several splicings simultaneously and thus suggest the existence of an obligatory order of events. In order to reconcile these findings we discuss the following hypotheses. (i) Some intervening sequences in COB specify products which are involved in transcript splicing; (ii) the biosynthesis of trace amounts of these products occurs on splicing intermediates. Their formation requires a certain order of splicing events to occur on a small number of COB transcripts. (iii) If expressed and functional, the intervening sequence-encoded products, together with other components, act on the bulk of COB transcripts, resulting in the steady state pattern of splicing intermediates observed in wild type.

Expression of the “split gene” COB in yeast mtDNA. Translation of intervening sequences in mutant strains.

This study deals with the effects that mutations in the COB region of yeast mtDNA have on the expression of mitochondrially made polypeptides. Based on the detection of two series of polypeptide chain-terminating mutations, we conclude that two proteins are specified by this region, apocytochrome b (Mr = 30,000) and a polypeptide of Mr = 42,000. One series of mutations generates new polypeptides ranging in size from 8,000 to 29,000 daltons; all of them are precipitated by serum direct against apocytochrome b. These mutations are located in five distinct segments of the COB region, the sequences alpha to epsilon coding for apocytochrome b. The second series of mutations, generating new polypeptides ranging in size from 17,000 to 41,000 daltons, is located within the first intervening sequence (alpha/beta) of the split gene for apocytochrome b. These mutations cause premature chain termination in the COOH-terminal part of a 42,000-dalton polypeptide. Its NH2-terminal part is likely to be specified by sequence alpha and thus to be homologous to that of apocytochrome b. We conclude that the 42,000-dalton polypeptide is translated on a processing intermediate of th COB transcript by reading through sequence alpha into sequence alpha/beta. We discuss the hypothesis that this polypeptide has a function in the expression of the COB region, possibly at the level of transcript processing.