Nuclear omnipotent suppressors of premature termination codons in mitochondrial genes affect the 37S mitoribosomal subunit

Summary nam3 and R705, yeast nuclear omnipotent suppressors of mitochondrial mit ^– mutations, reverse the superimposed spectrum of trans-recessive splicing defects by affecting the protein composition of the small mitoribosomal subunit. Analysis of the suppressor's interaction suggests that suppression results from mutations in the mitoribosomal polypeptides. These data indicate an obligatory connection between mitoribosome function and splicing of introns bI2, bI4 and aI1 in yeast mitochondria.     

A multitude of suppressors of group II intron-splicing defects in yeast

Disruption of the nuclear MRS2 gene (mrs2-1 mutation) causes a strong pet^- phenotype in strains with mitochondrial group II introns, and a leaky pet^- phenotype in strains without group II introns. MRS3 and MRS4, the genes for two mitochondrial-solute carrier proteins, can suppress both phenotypes when present in high-copy-number plasmids. In order to search for further multicopy suppressors of the mrs2-1 mutant phenotype, an yeast genomic DNA library, MW90, was constructed in YEp351 from a strain deleted for the MRS2, MRS3 and MRS4 genes. Ten different Sau3A DNA fragments that act as multicopy suppressors of the mrs2-1 respiratory-deficient phenotype were isolated from this library. Some of the newly isolated genes suppress the pet^- phenotypes of mrs2-1 cells in strains with and without mitochondrial group II introns. Other genes, however, are suppressors only for the mitochondrial intron-less strains. This supports the notion that the MRS2 gene product is bifunctional i.e., it is essential for the splicing of group II introns and is also involved in processes of mitochondrial biogenesis other than RNA splicing.     

The trans-spliced intron 1 in the psaA gene of the Chlamydomonas chloroplast: a comparative analysis

In the secondary structure model that has been proposed for the trans-spliced intron 1 in the Chlamydomonas reinhardtii psaA gene, a third RNA species (tscA RNA) interacts with the 5 and 3 intron parts flanking the exons to reconstitute a composite structure with several features of group-II introns. To test the validity of this model, we undertook the sequencing and modelling of equivalent introns in the psaA gene from other unicellular green algae belonging to the highly diversified genus Chlamydomonas. Our comparative analysis supports the model reported for the C. reinhardtii psaA intron 1, and also indicates that the 5 end of the tscA RNA and the region downstream from the psaA exon 1 cannot be folded into a structure typical of domain I as described for most group-II introns. It is possible that a fourth RNA species, yet to be discovered, provides the parts of domain I which are apparently missing.     

The group IIB intron from the green alga Scenedesmus obliquus mitochondrion: molecular characterization of the in vitro splicing products

Summary In the presence of high molar salt concentrations, the mitochondrial group IIB intron (rI1) from the green alga Scendesmus obliquus is capable of splicing in vitro. After establishing the optimal conditions for RNA processing the in vitro splicing products were unequivocally identified in self-splicing experiments by Northern hybridization analysis employing 3end-labelled RNAs or exon-and/or intron-specific probes. Finally, two trans-esterification products were identified by sequencing of the spliced RNA. From our data we conclude that the processing of group II introns from both algal and yeast mitochondria is preceded by identical consecutive trans-esterification steps. The predicted secondary and tertiary structure of intron rI1 of S. obliquus contains all the motifs necessary for optimal self-splicing and which are characteristic of other group IIB introns from different species.     

Euglena gracilis chloroplast DNA: analysis of a 1.6 kb intron of the psb C gene containing an open reading frame of 458 codons

Summary We retrieved a 1.6 kbp intron separating two exons of the psb C gene which codes for the 44 kDa reaction center protein of photosystem II. This intron is 3 to 4 times the size of all previously sequenced Euglena gracilis chloroplast introns. It contains an open reading frame of 458 codons potentially coding for a basic protein of 54 kDa of yet unknown function. The intron boundaries follow consensus sequences established for chloroplast introns related to class II and nuclear pre-mRNA introns. Its 3-terminal segment has structural features similar to class II mitochondrial introns with an invariant base A as possible branch point for lariat formation.     

Pea chloroplast genes encoding a 4kDa polypeptide of photosystem I and a putative enzyme of C1 metabolism

Summary The nucleotide sequence of 3.2 kbp of pea chloroplast DNA located upstream from the petA gene for cytochrome f, and previously reported to contain the gene for a photosystem I polypeptide, has been determined. Three open reading frames of 587, 40 and 157 codons have been identified. Orf40 encodes a highly conserved, hydrophobic, membrane-spanning polypeptide, and is identified as the gene psaI for the 4 kDa subunit of photosystem I. Orf587 is an extended version of the gene zfpA previously identified as encoding a conserved putative zinc-finger protein. The product of orf587 shows extensive homology to an unidentified open reading frame cotranscribed with a gene for folate metabolism in Escherichia coli and local homology to a region of the subunit of rat mitochondrial propionyl-CoA carboxylase. It is suggested that the product of orf587 is an enzyme of C₁ metabolism and is unlikely to be a regulatory DNA-binding protein. Orf157 potentially encodes an unidentified basic protein, but the protein sequence is not conserved in other plants.     

Frequent site-specific mit− deletions at cryptic exon-intron junctions in the COX1 gene of yeast mtDNA

A class of large site-specific deletions (del-B) occurs with exceptionally-high frequencies of 10^-3 in the mitochondrial COX1 gene of Mn²⁺-treated yeast cells. This work shows that del-B deletions are associated with COX1 intron aI1. All five deletion mutants studied have their upstream end at the authentic 3 splice site of this intron. The deletion ends 8.2 kb downstream in intron aI5b. This downstream deletion-end constitutes a potentially-cryptic 5 splice site for intron aI1. The coincidences of the del-B deletion-ends with authentic and cryptic RNA splice sites suggest that the group-II intron aI1, and/or the RNA maturase encoded in it, plays an active role in this exceptionally-frequent, site-specific deletion process.     

Three nuclear genes suppress a yeast mitochondrial splice defect when present in high copy number

Summary A gene bank of a yeast wild type DNA in the high copy number vector YEp 13 was screened for recombinant plasmids which suppress the mitochondrial RNA splice defect exerted by mutant M1301, a –1 by deletion in the first intron of the mitochondrial COB gene (bIl). A total of 17 recombinant plasmids with similar suppressor activity were found. Restriction mapping and cross-hybridization of the inserts revealed that these 17 plasmids contain three different inserts, all lacking any extended sequence homology. Each of the inserts, when present in high copy number, has a similar suppressor activity: high in the presence of mutation M1301 in bll, a group II intron, and low but significant with the presence of few mutants in bI2 and bI3 of the COB gene, both of which are group I introns.     

Suppression of a mitochondrial point mutation in a tRNA gene can cast light on the mechanisms of 3′ end-processing

We used a genetic approach to study the nuclear factors involved in the biogenesis of mitochondrial tRNAs. A point mutation in the mitochondrial tRNA^Asp gene of Saccharomyces cerevisiae had previously been shown to result in a temperature-sensitive respiratory-deficient phenotype as a result of the absence of 3 end-processing of the tRNA^Asp. Analysis of mitochondrial revertants has shown that all revertants sequenced have a G-A compensatory change at position 53, which restores the hydrogen-bond with the mutated nucleotide. We then searched for nuclear suppressors to identify the nuclear gene(s) involved in mitochondrial tRNA 3 end-processing. One such suppressor mutation was further characterized: it restores tRNA^Asp maturation and growth at 36°C on glycerol medium in heterozygous diploids, but leads to a defective growth phenotype in haploids.     

Synthesis and function of the mitochondrial intron —encoded bI4 RNA maturase from Saccharomyces cerevisiae

Summary We have analyzed the expression and function of the intron-encoded bI4 maturase when frame-shift mutations in the upstream exon alter the translational process. By constructing secondary cis-acting mutations within the b14 intron, we observed (1) that the bI4 maturase is still translated in the presence of the upstream mutation, albeit in very low amounts, and (2) that the limited amounts of bI4 maturase made under these conditions is no longer able to promote the splicing process of the aI4 intron. These observations, which further strengthen the maturase model, strongly suggest that bI4 maturase acts sequentially on the bI4 intron and then on the aI4 intron.     

The two genes for the P700 chlorophyll a apoproteins on the Euglena gracilis chloroplast genome contain multiple introns

Summary We have determined the complete nucleotide sequence of the two genes encoding the P₇₀₀ chlorophyll a apoproteins of the photosystem I reaction center of the Euglena gracilis chloroplast genome. The two genes are separated by 77 bp, are of the same polarity, and span a region which is greater than 9.0 kbp. The psaA gene (751 codons) is interrupted by three introns and the psaB gene (734 codons) by six introns. The introns range in size from 361 to 590 bp, whereas the exons range in size from 42 to 1,194 bp. The introns are extremely AT rich with a pronounced base bias of T > A > G > C in the RNA-like strand. Like other interrupted protein genes in the Euglena chloroplast genome, the psaA and psaB introns are similar to mitochondrial group II introns in having the splice junction consensus sequence, 5 GTGCGNTTCG ..... INTRON ..... TTAATTTTAT 3 and conserved secondary structural features. Except for the placement of the first intron, the intron-exon organization of these two highly homologous genes is not conserved. The other introns fall at or near putative surface domains of the predicted gene products. The psaA and psaB gene products are 74% homologous to one another and 93% and 95% homologous, respectively, to the psaA and psaB gene products of higher plant chloroplasts. The predicted secondary structure derived from the primary amino acid sequence has 11 potential membrane-spanning domains. Abbreviations and notations: Gene names follow the convention of Hallick and Bottomley (1983: psaA, psaB, genes for the P₇₀₀ apoprotein; psbE an psbF, genes foe the subunits of cytochrome b ₅₅₉; orfN, open reading frame of N condons     

The yeast protein Mrs6p,a homologue of the rabGDI and human choroideraemia proteins,affects cytoplasmic and mitochondrial functions

MRS6 is a newly-identified gene in the yeast Saccharomyces cerevisiae. Its product Mrs6p shows significant homology to the mammalian GDP dissociation inhibitor (GDI) of Rab/Ypt-type small G proteins and to the human choroideraemia protein (CHM), the component A of Rab-specific GGTase II. The interaction of Mrs6p with G proteins is indicated by our observation that the MRS6 gene suppresses the effect of a temperature-sensitive ypt1 mutation. Disruption of the MRS6 gene is lethal to haploid yeast cells. This is consistent with the notion that Mrs6p is interacting with Rab/Ypt-type small G proteins, which are known to have essential functions in vesicular transport. Unexpeciedly, the MRS6 gene product also affects mitochondrial functions as revealed by the facts that highcopy numbers of MRS6 (1) suppress the pet ^- phenotype of mrs2-1 mutant strains and (2) cause a weak pet ^- phenotype in wild-type strains. We conclude from these results that the MRS6 gene product has a vital function in connection with Rab/Ypt-type proteins in the cytoplasm and, in addition, affects mitochondrial functions.     

Mating-type suppression of the DNA-repair defect of the yeast rad6Δ mutation requires the activity of genes in the RAD52 epistasis group

The RAD6 gene of Saccharomyces cerevisiae is required for post-replication repair of UV-damaged DNA, UV mutagenesis, and sporulation. Here, we show that the radiation sensitivity of a MAT a rad6 strain can be suppressed by the MAT2 gene carried on a multicopy plasmid. The a1-2 suppression is specific to the RAD6 pathway, as mutations in genes required for nucleotide excision repair or for recombinational repair do not show such mating-type suppression. The a1-2 suppression of the rad6 mutation requires the activity of the RAD52 group of genes, suggesting that suppression occurs by channelling of post-replication gaps present in the rad6 mutant into the RAD52 recombinational repair pathway. The a1-2 repressor could mediate this suppression via an enhancement in the expression, or the activity, of recombination genes.