Cob Gene Research Articles

Internal structure of a mitochondrial intron of Aspergillus nidulans.

The intron of the mitochondrial apocytochrome b gene, cobA, of Aspergillus nidulans has been subjected to sequence analysis. It contains an open reading frame of 957 base pairs contiguous with the preceding exon. Regions of the translated open reading frames of cobA and the third intron of the cob gene in yeast show high amino acid homology. Comparison of the cobA intron with this and other yeast introns indicates that cobA codes for a maturase protein that splices out the intron encoding it and possibly other mitochondrial introns. Two very similar decamer peptides are found in the protein sequences of the cobA intron, four mitochondrial yeast introns, and the yeast mitochondrial sequence reading frame 1 (RF-1) and may be diagnostic of one class of maturase-coding introns. Four short DNA sequences, two of which are in the region defined by box9 and box2 mutations in the cob gene of yeast, are conserved in cobA and certain yeast introns. Comparison with three yeast introns strongly suggests that the first 200 base pairs of the open reading frame of the cobA intron do not code for any amino acids present in the putative maturase protein but are required for splicing or the control of splicing, or both.

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 others 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.

Mitochondrial transcripts in glucose-repressed cells of Saccharomyces cerevisiae.

We have compared mitochondrial transcripts from yeast Saccharomyces cerevisiae strain D273-10B grown in the presence of 2% galactose (non-repressed cells) or 15% glucose (glucose-repressed cells). The ethidium-bromide-stained electrophoretic pattern of mitochondrial RNAs from glucose-repressed cells shows a clear decrease of tRNAs. In addition, some RNA bands appear to be specific for a single growth condition. To identify these RNA species we have performed hybridization experiments with 32P-labelled mitochondrial DNA from petite mutant cells. The mitochondrial repeat units of the mutants retained only one of the following genes: oxi1, oxi2, oxi3, oli2, cob and oli1. Unchanged amounts of oxi2 and oli2 transcripts and reduced concentrations of oli1 and oxi1 putative mRNAs are present in glucose-repressed cells. In the same growth condition we observe a decreased processing of a precursor RNA species from the split cob gene and reduced amounts of transcripts corresponding to the first, second and fifth intron of the split oxi3 gene. The oxi3 first and second introns, whose transcripts are the most variable, include long open reading frames in their nucleotide sequence, but at present it is not known whether the corresponding RNA species have a functional role. Our results show that their concentrations are related to the growth condition.

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.

Identification of splicing signals in introns of yeast mitochondrial split genes: mutational alterations in intron bI1 and secondary structures in related introns.

Four mitochondrial mutations are known to block excision of intron I1 of the cob gene in S.cerevisiae. The nucleotide sequence alteration of one of them, M4873, has been determined. It is a deletion of 1 bp in a run of five G's at a distance of 30 to 34 bp upstream to the 3' splice point. Reversion is found to occur by restoration of the run of five G's either by insertion of 1 G (wild type reversion) or by transition A leads to G next to this run of G's (pseudo-wild type reversion). The effect of mutation and reversion on RNA splicing indicates that the run of five G's is of critical importance for intron I1 excision, possibly in participating in the formation of a splice signal with a helical structure. This presumption is confirmed by the observation that this sequence is part of a larger sequence of some 80 bp next to the 3' splice point which is conserved to some extend in the four mitochondrial introns (bI1, aI1, aI2, aI5) that survive after excision as circular RNAs. Most striking is the conservation of this sequence at the level of secondary structure.

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

Electrophoretic separation of mitochondrial RNA followed by hybridization with restriction fragments of mtDNA has been used to identify transcripts of the split gen COB which codes for apocytochrome b.In wild type a major transcript of 18S is detected besides a 10S RNA and a series of transcripts with electrophoretic mobilities higher than 18S. Mutations in coding sequences do not significantly alter this transcript pattern. In contrast, mutations in intervening sequences give rise to different patterns: The 18S RNA, the putative messenger for apocytochrome b, is lacking; depending on the intervening sequence affected by mutation, one or the other of the larger transcripts (23S, 24S, 32S, 34S) is accumulated instead. In most mutants a 10S RNA species is present; it has not been detected, however, in case of a small cluster of mutations which lead to the accumulation of the largest transcript (34S) observed, which most likely contains all coding and intervening sequences of the split gene COB.These results suggest a pathway of splicing.