Nucleotide sequence and intron structure of the apocytochrome b gene of Neurospora crassa mitochondria

The sequence of the apocytochrome b (cob) gene of Neurospora crassa has been determined. The structural gene is interrupted by two intervening sequences of approximately 1260 bp each. The polypeptide encoded by the exons shows extensive homology with the cob proteins of Aspergillus nidulans and Saccharomyces cerevisiae (79% and 60%, respectively). The two introns are, however, located at sites different from those of introns in the cob genes of A. nidulans and S. cerevisiae (which contain highly homologous introns at the same site within the gene). The introns share several short regions of sequence homology (10-12 bp long) with each other and with other fungal mitochondrial introns. Moreover, the second intron contains a 50 nucleotide long sequence that is highly homologous with sequences within every ribosomal intron of fungal mitochondria sequenced to date. The conserved sequences may allow the formation of a core secondary structure, which is nearly identical in many mitochondrial introns. The conserved secondary structure may be required for intron splicing. The second intron contains an open reading frame, continuous with the preceding exon, of approximately 290 codons. Two stretches of 10 amino acid residues, conserved in many introns, are present in the open reading frame.     

Mutations within an intron and its flanking sites: Patterns of novel polypeptides generated by mutants in one segment of the cob-box region of yeast mitochondrial DNA

Summary We have undertaken a systematic examination of the polypeptides accumulating in thirteen (out of 23) mutants in the intron cluster box7 and its flanking clusters box2 and box9 of the cob-box (cytochrome b) region of the mitochondrial genome of Saccharomyces cerevisiae. We have subjected these polypeptides to fingerprint analysis, both sequential and in parallel, with two proteases in order to disclose sequence homologies and differences between the different novel polypeptides themselves, and between them and the wild type product of the gene, i.e. apocytochrome b. One of our aims has been to establish the existence of possible correlations between the nature of the novel polypeptides and the fine structure genetic map of that segment of the mitochondrial genome.Our results show that all box7 mutants accumulate the following set of polypeptides not seen in wild type cells: a) a characteristic set of large polypeptides consisting of three species: p56, p42 and p35 or p34.5; b) a polypeptide p23; c) a much shorter fragment (of which the apparent molecular weight varies from 12.5 to 13, according to the mutation) with the exception of two mutants; d) in addition, the majority accumulate in varying amounts a polypeptide p30 closely related to but not identical with apocytochrome b.Moreover only two box7 mutants accumulate a polypeptide in the range of mobilities corresponding to 25–27 Kd (referred to as class p26) while such a polypeptide is seen in all box9 and box2 mutants examined with one exception in box2.Only one mutant in box2 resembles box7 mutants with respect to criterion a), and no box2 or box9 mutants resemble box7 mutants with respect to criterion c); criteria b) and d) appear to apply equally well to mutants in all three clusters.Fingerprint analysis, carried out with polypeptides p56, p42, p35, p34.5, p30, p26, p23, discloses that a) The polypeptides belonging to the same class of mobility exhibit very similar if not identical sequences in various cases. b) These polypeptide classes, except p56, p42 and p26, share considerable sequence homologies with wild type apocytochrome b, perhaps encompassing 50% or more of the wild type sequences. b) Polypeptides belonging to the classes p42 and p26 exhibit less extensive but nevertheless significant homologies with the wild type sequence. c) Sequences in polypeptides belonging to class p56 are virtually indistinguishable from ones in cytochrome oxidase subunit I.The inferences from these findings are 1) one gene can produce a multiplicity of polypeptide products that share a common sequence at the promoter-proximal (N-terminal) portion, but diverge beyond these regions of homology. 2) Both the multiplicity of products in single mutants, and the protein structure found, argue against the divergent segments to be due to frameshift terminations, and instead suggest that the novel products are consequences of mRNA processing defects (excision and/or ligation) at and near intron regions. 3) Mutations at edges and the center of an intron can generate similar polypeptide patterns, i.e. produce analoguous mRNA processing defects. 4) Mutations in exons, at their boundary with introns, can produce polypeptide patterns indistinguishable from those at the neighbouring intron; they diverge and eventually become typically exonlike in mutants localized at increasing distances from the boundary. 5) Taken together these findings argue that pre-mRNA processing extends beyond the boundaries of the intron proper and that certain exonsequences participate in excision and ligation. 6) Accumulated pre-mRNAs, resulting from defects in splicing can be translated. 7) Product p56 is formally analogous to p23, as a faulty but highly conserved partial product of the wild type protein, the former of Cox I (oxi3 gene), the latter of the cob-box gene proper. Therefore both genes may utilize identical RNA processing elements which are affected by box7 mutations. 8) A small amount of product similar to p56 may accumulate even in some wild types but not in others. This observations suggests that in certain nuclear backgrounds RNA processing may be more error-prone than in others.Publication No. XXXX from the Department of Chemistry, Indiana UniversitySupported by Research Grant GM 12228 from the National Institute of General Medical Sciences, National Institutes of Health; recipient of Research Career Award K06 05060 from the same Institute.Supported by Délégation Générale à la Recherche Scientifique et Technique grant n⁰ 78-70341     

Expression of the split gene cob in yeast: Evidence for a precursor of a “maturase” protein translated from intron 4 and preceding exons

Intron 4 (14) of the split gene cob in mitochondrial DNA contains a long open reading frame in phase with the preceding exon. Mutations in this intron block the excision of the 14 sequence from the cob precursor RNA and, at the same time, generate a series of new polypeptides, parts of which apparently result from translation of 14 sequences. We sequenced six mutations clustered in the upstream part of the open reading frame, about 340 bp from the exon-intron boundary (box9 cluster). Four are base pair exchanges in the same triplet of this region; these form the polypeptides typical for 14 plus a trans-acting product encoded by 14, as shown by complementation studies. The other two mutations—a -2 bp deletion at the same site, causing frameshift with a chain-terminating codon within a few triplets, and a base pair exchange at a nearby site-affect both the formation of 14 typical translation products and the trans-acting function. These results on box9 mutants combined with results on box7 mutants suggest that an 14-encoded “maturase” protein (apparent molecular weight, 27,000) is cleaved off a precursor protein (apparent molecular weight, 55,000) encoded by exon sequences B1 to B4 and the intron open reading frame. We further discuss the role of the box9 nucleotide sequence in the maturation of cob-specific RNA.     

The identification of apocytochrome b as a mitochondrial gene product and immunological evidence for altered apocytochrome b in yeast strains having mutations in the COB region of mitochondrial DNA.

The yeast mitochondrial translation product of Mr 30 000 is identical with apocytochrome b. After labelling in vivo with [35S]sulphate in the presence of cycloheximide, the radioactivity in this product present in solubilized submitochondrial particles, was completely recovered in pure cytochrome bc1 complex as a single polypeptide. We show that this translation product is identical with apocytochrome b using peptide mapping by limited proteolysis according to Cleveland et al. [J. Biol. Chem. 250 (1977) 8236-8242] and by immunoprecipitation with a specific antiserum against apocytochrome b. New mitochondrial translation products in 36 strains of Saccharomyces cerevisiae having mutations in the COB region of the mitochondrial DNA, are precipitated by this antiserum. This is consistent with the assumption that many of the cob mutations are localized in the structural gene for apolcytochrome b on mitochondrial DNA. Mutations in two intervening sequences can give rise to products related to apocytochrome b that are considerably longer than normal apocytochrome b. We discuss the hypothesis that in these mutants splicing of the messenger RNA does not occur correctly and that, as a consequence of this, ribosomes read through in an intervening sequence.     

Mutations in putative intervening sequences of the mitochondrial cytochrome b gene of yeast produce abnormal cytochrome b polypeptides.

The apoprotein of yeast cytochrome b is translated on mitochondrial ribosomes and coded for by a split gene which is located in the COB-BOX region on mitochondrial DNA. With the aid of an antibody against cytochrome b, we identified the cytochrome b-cross-reacting polypeptides of respiration-deficient mutants mapping either in coding or intervening sequences of the cytochrome b gene. Most mutations in the coding regions caused the accumulation of a single apocytochrome b fragment whose apparent molecular weight (12,000 to 26,600) depended on the map position of the mutation. In contrast, mutations in putative intervening sequences often led to multiple new polypeptides immunologically related to apocytochrome b. Some of these abnormal polypeptides were considerably larger than wild type apocytochrome b. This suggests that mutations in intervening sequences can thus generate aberrant polypeptide products.     

Expression of the "split gene" cob in yeast mtDNA. Nuclear mutations specifically block the excision of different introns from its primary transcript

Five nuclear mutants falling into five different complementation groups are shown to block the maturation of long form mitochondrial cob RNA at five different processing steps. At the same time they prevent complete processing of the oxi 3 RNA, thus exhibiting the same phenotype as mitochondrial box mutants (cyt b- and oxi 3-). The different nuclear factors in question have varying ranges of specificity for the removal of introns from cob RNA, from only one to at the most three introns. Two mutated nuclear elements are shown to be specific for the processing of introns present only in the long form cob gene. One such mutation shows, as expected, no deleterious effect on the processing of the short form cob RNA exchanged into the mutant via cytoduction. The role of nuclear coded factors in the possible translation or activity of introncoded products ("maturases") is discussed for two mutants. Striking parallels are found between diverse polypeptide products, presumably translated from accumulated cob RNA intermediates, in pet- and mit- mutants blocked in the excision of the same intron.     

Molecular basis of the 'box effect', A maturase deficiency leading to the absence of splicing of two introns located in two split genes of yeast mitochondrial DNA

In the mitochondrial DNA of Saccharomyces cerevisiae, the genes cob-box and oxi3, coding for apocytochrome b and cytochrome oxidase subunit I respectively, are split. Several mutations located in the introns of the cob-box gene prevent the synthesis of cytochrome b and cytochrome oxidase subunit I (this is known as the 'box effect').-We have elucidated the molecular basis of this phenomenon: these mutants are unable to excise the fourth intron of oxi3 from the cytochrome oxidase subunit I pre-mRNA; the absence of a functional bI4 mRNA maturase, a trans-acting factor encoded by the fourth intron of the cob-box gene explains this phenomenon. This maturase was already known to control the excision of the bI4 intron; consequently we have demonstrated that it is necessary for the processing of two introns located in two different genes. Mutations altering this maturase can be corrected, but only partially, by extragenic suppressors located in the mitochondrial (mim2) or in the nuclear (NAM2) genome. The gene product of these two suppressors should, therefore, control (directly or indirectly) the excision of the two introns as the bI4 mRNA maturase normally does.     

Regulatory interactions between mitochondrial genes. I. Genetic and biochemical characterization of some mutant types affecting apocytochrome b and cytochrome oxidase.

The region of the mtDNA containing the structural gene for apocytochrome b is called the cob or box region. There is evidence that the same region is also involved in the regulation of cytochrome oxidase. We have isolated eight mit- mutants in this region and have ordered them using petite deletion mapping. Four of these mutants appear to map outside the boxII region on the oli2-proximal end. Analysis of restriction endonuclease fragments of the mtDNA from peptides used in the deletion mapping suggests a minimum size of 3.1 x 10(3) base pairs for the whole cob region. Although none of our mutants contained any cytochrome b or cytochrome b-linked activities, polypeptides apparently related to apocytochrome b were present in some but not all mutants. Additional regulatory effects (both positive and negative) on cytochrome oxidase by virtue of control of its subunit I were also observed. In addition to these phenotypic traits, some of the mutants accumulated novel, mitochondrially translated polypeptides not seen in wild type.     

Processing of yeast mitochondrial RNA: involvement of intramolecular hybrids in splicing of cob intron 4 RNA by mutation and reversion

Revertants have been obtained from six mutants of the box9 cluster, which are supposed to be defective in RNA splicing as a result of alterations in a splice signal sequence. This sequence is in the 5' part of intron 4 of the cob gene, 330 to 340 bp downstream from the 5' splice site. Sequencing reveals that reversion to splicing competence is achieved by restoration of the wild-type box9 sequence; by creation of novel box9 sequences; and by introduction of a second site or suppressor mutation (sup-) compensating for the effect of the primary box9- mutation. The sup- mutation alters a sequence in intron 4,293 bp upstream from the box9- primary mutation. The box9 sequence and this upstream sequence can base pair to form an intramolecular hybrid in intron RNA in which box9- and sup- are compensatory base pair exchanges (G----A and C----U, respectively). Thus intramolecular hybrid structures of intron RNA are essential for RNA splicing.     

Two distinct mechanisms for deletion in mitochondrial DNA of Schizosaccharomyces pombe mutator strains. Slipped mispairing mediated by direct repeats and erroneous intron splicing

Mutator strains of the fission yeast Schizosaccharomyces pombe produce mitochondrial respiratory deficient mutants at a high rate, and roughly 20% of these mutants carry deletions in the range of 50 to 1500 base-pairs. To elucidate the mechanism of deletion we have sequenced ten deletion mutants in the mosaic gene encoding apocytochrome b (cob) and three in the split gene coding for the first subunit of cytochrome c oxidase (cox1). Of 13 deletions, ten are correlated with the presence of direct repeats, which could promote deletions by slipped mispairing during DNA replication. In some of these mutants, the termini are located in possible DNA secondary structures. In three independently isolated mutants with identical deletions in the cob gene, the 5' deletion endpoint coincides with the 3' splice point of the intron, whereas the 3' endpoint of the deletion exhibits pronounced homology with the 5' splice point of the intron. This result suggests that these deletions might be initiated by erroneous RNA splicing.     

Regulatory interaction between mitochondrial genes. II. Detailed characterization of novel mutants mapping within one cluster in the cob2 region.

These studies describe the properties of three mit- mutants designated EM17, EM25, and PZ1, all mapping at two closely linked sites near one of the boundaries of the region of the mitochondrial genome concerned with the specification of cytochrome b. They all exhibit complex phenotypes affecting cytochrome b, cytochrome aa3, and additional polypeptides not found in the wild type. In the case of EM 17 this complexity can be ascribed to the presence of two mutations induced in the course of the initial mutagenic treatment: one, the cob2 mutation proper, is responsible for the loss of cytochrome b which is replaced by an altered, functionally inactive polypeptide, cytochrome b. This polypeptide can be further modified, or even eliminated, by the controlled introduction of another mutation in the cob1 segment of the cob region. The reduction in cytochrome oxidase subunit I, responsible for the effects on cytochrome aa3 and enzymatic activity in EM17, is due to a second (not mit-) mutation that has been located in the par1-proximal segment of the oxi3 region. This second mutation as well as the cob mutation can be overcome, and the respective aspect of wild type function restored to EM17, by recombination with rho- strains retaining the appropriate segment(s) of the wild type genome. The phenotype of the other two mutants is due to a single mutagenic event. This conclusion is confirmed by their ability to restore wild type functions by reversion. The mutation in EM25 appears to be due to a frameshift, which has led to premature chain termination, producing a polypeptide of Mr = 15,000 related to apocytochrome b. This change is accompanied by a decrease in the amount of subunit I of cytochrome oxidase. Revertants fall into three classes: on galactose two produce a polypeptide indistinguishable from apocytochrome b, but vary in its amount, while the third fails to increase apocytochrome b above mutant levels. Production of subunit I is increased but fails to reach wild type levels. Complete restoration of wild type functions can, however, be obtained by recombination of EM25 with rho- (cob2+) strains. Mutation PZ1 results in a complete absence of any polypeptide related to apocytochrome b and of cytochrome oxidase subunit I. These cells produce a novel polypeptide with a Mr = 45,000 not found in the wild type, and unrelated to all its normal polypeptides. Reversion or recombination with rho- (cob2+) strains results in virtually complete restoration of all wild type functions and the elimination of the novel polypeptide.     

Critical sequences within mitochondrial introns: cis-dominant mutations of the "cytochrome-b-like" intron of the oxidase gene

We have established the DNA sequence of two cis-dominant mutations located in the fourth intron, a14, of the yeast mitochondrial gene oxi3. These mutations prevent the synthesis of subunit I of cytochrome oxidase. Both mutations affect a very short DNA sequence located several hundred base pairs from the intron-exon junctions. An identical sequence is found in the cob-box gene; and this sequence is critical for the excision of the cytochrome b intron. Our interpretation is that this short sequence represents a common signal that must be recognized by the box7-encoded mRNA maturase, in conjunction with the mitochondrial ribosome, to splice out the introns in the two nonhomologous genes, cob-box and oxi3.     

Determination of functional domains in intron bI1 of yeast mitochondrial RNA by studies of mitochondrial mutations and a nuclear suppressor.

The sequence of intron 1 in the cob gene in mtDNA (bI1) of the yeast strain 777-3A has been determined. Furthermore, we have performed a systematic search for complementary sequence stretches within this intron RNA, and within the RNA of intron 5 gamma of the oxi3 gene (aI5 gamma) which shares distinctive sequences with bI1. Possible secondary structure models derived from this analysis show nearly identical core structures for bI1 and aI5 gamma RNA with conserved sequence stretches in prominent positions. These core structures are similar to those previously reported for RNAs of introns having very limited sequence homology with bI1 and aI5 gamma. In two mutants which are defective in bI1 excision from cob pre-mRNA, nucleotide sequence alterations in bI1 have been determined. One mutation (G5049) apparently affects the stability of a hybrid stretch in the proposed secondary structure of bI1 RNA whereas the other one (M1301), a deletion of one A in a run of five As, affects a sequence which is conserved in bI1 and aI5 gamma and is involved in the formation of a distinct secondary structure. Out of seven revertants of M1301, three were found to have restored the wild-type bI1 sequence AAAAA, three others had the related sequence AAAAG which is functionally indistinguishable from wild-type, whereas one revertant had a nuclear mutation which suppresses the splicing defect exerted by the mitochondrial mutation M1301. This nuclear suppressor (SUP-101) is allele specific and dominant. The possible role of the sequence affected by M1301 in terms of a recognition site for a nuclear gene product will be discussed.     

Protein encoded by the third intron of cytochrome b gene in Saccharomyces cerevisiae is an mRNA maturase. Analysis of mitochondrial mutants, RNA transcripts proteins and evolutionary relationships

We have established the nucleotide sequence of the wild-type and that of a trans-acting mutant located in the third (bi3) intron of the Saccharomyces cerevisiae mitochondrial cytochrome b gene. The intron, 1691 base-pairs long, has an open reading frame 1045 base-pairs long, in phase with the preceding exon and the mutation replaces the evolutionarily conserved Gly codon of the second consensus motif by an Asp codon and blocks the formation of mature cytochrome b mRNA. Splicing intermediates of 5300 and 3900 bases with unexcised bi3 intron and a characteristic novel polypeptide (p50), the size of which corresponds to the chimeric protein encoded by upstream exons and the bi3 intronic open reading frame (ORF), accumulate in this and other bi3 splicing-deficient mutants. We conclude that the protein encoded by the bi3 ORF is a specific mRNA maturase involved in the splicing of the cytochrome b mRNA. The open reading frame of the third intron is remarkably similar to that of the unique intron of the cytochrome b gene (cob A) of Aspergillus nidulans. Both are located in exactly the same position and possibly derive from a recent common ancestor by a horizontal transfer. We have established the nucleotide sequence of an exonic mutant located in the B3 exon. This missense mutation changes the Phe codon 151 into a Cys codon and leads to the absence of functional cytochrome b but does not affect splicing. Finally, we have studied the splicing pathway leading to the synthesis of cytochrome b mRNA by analysing, in a comprehensive manner, the 22 splicing intermediates of several mutants located in bi3.     

Functional domains in introns: trans-acting and cis-acting regions of intron 4 of the cob gene

We have sequenced the mutational changes in eight mutants in the open reading frame of intron 4 of the cob gene on yeast mitochondrial DNA. Three have a cis-acting splicing defect, while the other inactivate a trans-recessive intron domain that specifies a trans-acting splicing factor. From phenotypic evidence, including analyses of the allele-specific extra proteins, we have identified a protein (P27) encoded wholly within the intron that appears to be the intron 4 splicing factor (maturase). The evidence suggests that P27 is a secondary translation product resulting from the proteolytic cleavage of a larger precursor encoded by exon and intron sequences, but an alternative model, in which P27 is a primary translation product, has not been ruled out.