The yeast nuclear gene NAM2 is essential for mitochondrial DNA integrity and can cure a mitochondrial RNA-maturase deficiency

Dominant mutations in the yeast nuclear gene NAM2 cure the RNA splicing deficiency resulting from the inactivation of the bI4 maturase encoded by the fourth intron of the mitochondrial cytochrome b gene. This maturase is required to splice the fourth intron of this gene and to splice the fourth intron of the mitochondrial gene oxi3 encoding cytochrome oxidase subunit I. We have cloned the nuclear gene NAM2, which codes for two overlapping RNAs, 3.2 kb and 3.0 kb long, which are transcribed in the same direction but differ at their 5' ends. NAM2 compensating mutations probably result from point mutations in the structural gene. Integration of the cloned gene occurs at its homologous locus on the right arm of chromosome XII. Inactivation of the NAM2 gene either by transplacement with a deleted copy of the gene, or by disruption, is not lethal to the cell, but leads to the destruction of the mitochondrial genome with the production of 100% cytoplasmic petites.     

Pleiotropic mutations within two yeast mitochondrial cytochrome genes block mRNA processing.

The mRNAs from two yeast mitochondrial genes cob-box (cytochrome b) and oxi-3 (cytochrome oxidase 40,000 dalton subunit) are processed from large (7-10 kb) precursors. Certain mutations in each gene block the maturation of the RNAs from both genes at a variety of specific steps. The pleiotropic cytochrome b mutants seem to lack a functional trans-acting RNA required for the processing of both messengers. In contrast, the oxi-3 mutants may act by producing an activity that inhibits specific steps.     

Respiratory-deficient Mutants in Saccharomyces lactis

Six independent ultraviolet-induced respiratory-deficient mutants (petites) of Saccharomyces lactis were isolated and characterized. Two possessed a normal cytochrome spectrum, another displayed an increased level of all the cytochromes, and three suffered from a partial or complete loss of one or more of the cytochromes a, b, c, and c₁. All of the mutants were segregational petites; none was vegetative. Determination of linkage relationships between mutants was restricted because matings between mutants, homozygous or heterozygous, for loci affecting cytochrome content were blocked at various stages in the mating-sporulating sequence. At least three of the petites were genetically nonidentical. Three of the mutations appeared to occupy loci within the same linkage group; two of the three mutations that mapped within this region were cytochrome-deficient. Growth at high or low temperatures, under increased osmotic pressure or in media supplemented with various fatty acids or sterols, did not relieve the physiological defects in these mutants. Reasons for the differences in survival of segregational and vegetative petites within this species are examined.     

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     

Influence of RNA structural stability on the RNA chaperone activity of the Escherichia coli protein StpA

Proteins with RNA chaperone activity are able to promote folding of RNA molecules by loosening their structure. This RNA unfolding activity is beneficial when resolving misfolded RNA conformations, but could be detrimental to RNAs with low thermodynamic stability. In order to test this idea, we constructed various RNAs with different structural stabilities derived from the thymidylate synthase (td) group I intron and measured the effect of StpA, an Escherichia coli protein with RNA chaperone activity, on their splicing activity in vivo and in vitro. While StpA promotes splicing of the wild-type td intron and of mutants with wild-type-like stability, splicing of mutants with a lower structural stability is reduced in the presence of StpA. In contrast, splicing of an intron mutant, which is not destabilized but which displays a reduced population of correctly folded RNAs, is promoted by StpA. The sensitivity of an RNA towards StpA correlates with its structural stability. By lowering the temperature to 25°C, a temperature at which the structure of these mutants becomes more stable, StpA is again able to stimulate splicing. These observations clearly suggest that the structural stability of an RNA determines whether the RNA chaperone activity of StpA is beneficial to folding.     

The chloroplastic DEVH‐box RNA helicase INCREASED SIZE EXCLUSION LIMIT 2 involved in plasmodesmata regulation is required for group II intron splicing

INCREASED SIZE EXCLUSION LIMIT 2 (ISE2) encodes a putative DEVH‐box RNA helicase originally identified through a genetic screening for Arabidopsis mutants altered in plasmodesmata (PD) aperture. Depletion of ISE2 also affects chloroplasts activity, decreases accumulation of photosynthetic pigments and alters expression of photosynthetic genes. In this work, we show the chloroplast localization of ISE2 and decipher its role in plastidic RNA processing and, consequently, PD function. Group II intron‐containing RNAs from chloroplasts exhibit defective splicing in ise2 mutants and ISE2‐silenced plants, compromising plastid viability. Furthermore, RNA immunoprecipitation suggests that ISE2 binds in vivo to several splicing‐regulated RNAs. Finally, we show that the chloroplast clpr2 mutant (defective in a subunit of a plastidic Clp protease) also exhibits abnormal PD function during embryogenesis, supporting the idea that chloroplast RNA processing is required to regulate cell–cell communication in plants.