Summary
The Saccharomyces cerevisiae nuclear gene NAM2 codes for mitochondrial leucyl-tRNA synthetase (mLRS). Herbert et al. (1988, EMBO J 7:473–483) proposed that this protein is involved in mitochondrial RNA splicing. Here we present the construction and analyses of nine mutations obtained by creating two-codon insertions within the NAM2 gene. Three of these prevent respiration while maintaining the mitochondrial genome. These three mutants: (1) display in vitro a mLRS activity ranging from 0%–50% that of the wild type: (2) allow in vivo the synthesis of several mitochondrially encoded proteins; (3) prevent the synthesis of the COXII protein but not of its mRNA; (4) abolish the splicing of the group I introns bI4 and aI4; and (5) affect significantly the excision of the group I introns bI2, bI3 and aI3. Importation of the bI4 maturase from the cytoplasm into mitochondria in a nam2 − mutant strain does not restore the excision of the introns bI4 and aI4 implying that the splicing deficiency does not result from the absence of the bI4 maturase. We conclude that the mLRS is a splicing factor essential for the excision of the group I introns bI4 and aI4 and probably important for the excision of other group I introns.
Similar content being viewed by others
References
Akins RA, Lambowitz AM (1987) A protein required for splicing group I introns in Neurospora mitochondria is mitochondrial tyrosyl tRNA synthetase or a derivative there of. Cell 50:331–345
Banroques J, Delahodde A, Jacq C (1986) A mitochondrial mRNA maturase gene transferred to the yeast nucleus can control mitochondrial mRNA splicing. Cell 46:837–844
Banroques J, Perea J, Jacq C (1987) Efficient splicing of two yeast mitochondrial introns controlled by a nuclear-encoded maturase. EMBO J 6:1085–1091
Barany F (1985) Two-codon insertion mutagenesis of plasmid genes by using single-stranded hexameric oligonucleotides. Proc Natl Acad Sci USA 82:4202–4206
Budd M, Campbell JL (1987) Temperature-sensitive mutations in the yeast DNA polymerase I gene. Proc Natl Acad Sci USA 84:2838–2842
Burke JM (1988) Molecular genetics of group I introns: RNA structures and protein factors required for splicing — a review. Gene 73:273–293
Carignani G, Groudinsky O, Frezza D, Schiavon E, Bergantino E, Slonimski PP (1983) An mRNA maturase is encoded by the first intron of the mitochondrial gene for the subunit I of cytochrome oxidase in S. cerevisiae. Cell 35:733–742
Cech TR (1988) Conserved sequences and structures of group I introns: building an active site for RNA catalysis — a review. Gene 73:259–271
Claisse M, Slonimski PP, Johnson J, Mahler HR (1980) 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. Mol Gen Genet 177:375–387
Colleaux L, d'Auriol L, Galibert F, Dujon B (1988) Recognition and cleavage of the intron-encoded omega transposase. Proc Natl Acad Sci USA 85:6022–6026
Collins RA, Lambowitz AM (1985) RNA splicing in Neurospora mitochondria. Defective splicing of mitochondrial mRNA precursors in the nuclear mutant cyt18-1. J Mol Biol 184:413–428
Conde J, Fink GR (1976) A mutant of S. cerevisiae defective for nuclear fusion. Proc Natl Acad Sci USA 73:3651–3655
Coruzzi G, Bonitz SG, Thalenfeld BF, Tzagoloff A (1981) Assembly of the mitochondrial membrane system. Analysis of the nucleotide sequence and transcripts in the oxi1 region of yeast mitochondrial DNA. J Biol Chem 256:12780–12787
Delahodde A, Goguel V, Becam AM, Creusot F, Perea J, Banroques J, Jacq C (1989) Site-specific DNA endonuclease and RNA maturase activites of two homologous intron-encoded proteins from yeast mitochondria. Cell 56:431–441
DeLaSalle H, Jacq C, Slonimski PP (1982) Critical sequences within mitochondrial introns: pleiotropic mRNA maturase and cis-acting signals of the box intron controlling reductase and oxidase. Cell 28:721–732
Dhawale S, Hanson DK, Alexander NJ, Perlman PS, Mahler HR (1981) Regulatory interactions between mitochondrial genes: interactions between two mosaic genes. Proc Natl Acad Sci USA 78:1778–1782
Dujardin G, Pajot P, Groudinsky O, Slonimski PP (1980) Long range control circuits within mitochondria and between nucleus and mitochondria. I. Methodology and phenomenology of suppressors. Mol Gen Genet 179:469–482
Dujardin G, Labouesse M, Netter P, Slonimski PP (1983) Genetic and biochemical studies on the nuclear suppressor NAM2: Extraneous activation of a latent pleiotropic maturase. In: Schweyen RJ, Wolf K, Kaudewitz F (eds) Mitochondria 1983. W de Gruyter, Berlin, pp 233–250
Herbert CJ, Labouesse M, Dujardin G, Slonimski PP (1988a) The NAM2 proteins from the S. cerevisiae and S. douglasii are mitochondrial leucyl-tRNA synthetases, and are involved in mRNA splicing. EMBO J 7:473
Herbert CJ, Dujardin G, Labouesse M, Slonimski PP (1988b) Divergence of the mitochondrial leucyl tRNA synthetase genes in two closely related yeasts Saccharomyces cerevisiae and Saccharomyces douglasii: A paradigm of incipient evolution. Mol Gen Genet 213:297–309
Hountondji C, Blanquet S, Lederer F (1985) Methionyl-tRNA synthetase from Escherichia coli: primary structure at the binding site for the 3′ end of tRNAf Met. Biochemistry 24:1175–1180
Ito H, Fukuda Y, Murata K, Kimura A (1983) Transformation of intact yeast cells treated with alkali cations. J Bacteriol 153:163–168
Jacquier A, Dujon B (1983) The intron of the mitochondrial 21SrRNA gene: distribution in different species and sequence comparison between Kluyveromyces thermotolerans and Saccharomyces cerevisiae. Mol Gen Genet 192:487–499
Jacquier A, Dujon B (1985) An intron-encoded protein is active in a gene conversion process that spreads an intron into a mitochondrial gene. Cell 41:383–394
Labouesse M, Slonimski PP (1983) Construction of novel cytochrome b genes in yeast mitochondria by subtraction or addition of introns. EMBO J 2:269–276
Labouesse M, Netter P, Schroeder R (1984) 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. Eur J Biochem 144:85–93
Labouesse M, Dujardin G, Slonimski PP 61985) The yeast nuclear gene NAM2 is essential for mitochondrial DNA integrity and can cure a mitochondrial RNA-maturase deficiency. Cell 41:133–143
Labouesse M, Herbert CJ, Dujardin G, Slonimski PP (1987) Three suppressor mutations which cure a mitochondrial RNA maturase deficiency occur at the same codon in the open reading frame of the nuclear NAM2 gene. EMBO J 6:713–721
Lamb MR, Anziano PQ, Glans KR, Hanson DK, Klapper HJ, Perlman PS, Mahler HR (1983) Functional domains in intronsRNA processing intermediates in cis and trans-acting mutants in the penultimate intron of the mitochondrial gene for cytochrome b. J Biol Chem 258:1991–1999
Lazowska J, Jacq C, Slonomski PP (1980) Sequence of introns and flanking exons in wild type and box3 mutants reveals an interlaced splicing protein coded by an intron. Cell 22:333–348
Lazowska J, Claisse M, Gargouri A, Kotylak Z, Spyridakis A, Slonimski PP 61989) The protein encoded by the third intron of cytochrome b gene in S. cerevisiae is an mRNA maturase: analysis of mitochondrial mutants, RNA transcripts, proteins and evolutionary perspectives. J Mol Biol 205:275–289
Majumder AL, Akins RA, Wilkinson JG, Kelley RL, Snook AJ, Lambowitz AM (1989) Involvement of tyrosyl-tRNA synthetase in splicing of group I introns in Neurospora crassa mitochondria: biochemical and immunochemical analyses of splicing activity. Mol Cell Biol 9:2089–2104
Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York
Michel F, Umesomo K, Ozeki H (1989) Comparative and functional anatomy of group II catalytic introns — a review. Gene 82:530
Netter P, Jacq C, Slonimski PP (1982) Critical sequences within mitochondrial introns: cis-dominant mutations of the “cytochrome-b-like” intron of the oxidase gene. Cell 28:733–738
Poutre CG, Fox TD (1987) PETIII, a Saccharomyces cerevisiae nuclear gene required for translation of the mitochondrial mRNA encoding cytochrome c oxidase subunit II. Genetics 115:637–647
Putney SD, Schimmel P (1981) An aminoacyl tRNA synthetase binds to a specific DNA sequence and regulates its gene transcription. Nature 291:632–635
Schimmel P (1987) Aminoacyl tRNA synthetases: general scheme of structure-function relationships in the polypeptides and recognition of transfer RNAs. Annu Rev Biochem 56:125–158
Séraphin B, Boulet A, Simon M, Faye G (1987) Construction of a yeast strain devoid of mitochondrial introns and its use to screen nuclear genes involved in mitochondrial splicing. Proc Natl Acad Sci USA 84:6810–6814
Springer M, Graffe M, Butler JS, Grunberg-Manago M (1986) Genetic definition of the translational operator of the threonine tRNA ligase gene in E. coli. Proc Natl Acad Sci USA 83:4384–4388
Wenzlau JM, Saldanha RJ, Butow RA, Perlman PS (1989) A latent intron-encoded maturase is also an endonuclease needed for intron mobility. Cell 56:421–430
Zinn AR, Butow RA (1985) Nonreciprocal exchange between alleles of the yeast mitochondrial 21SrRNA gene: kinetics and the involvement of a double-strand break. Cell 40:887–895
Author information
Authors and Affiliations
Additional information
Communicated by W. Gajewski
Rights and permissions
About this article
Cite this article
Labouesse, M. The yeast mitochondrial leucyl-tRNA synthetase is a splicing factor for the excision of several group I introns. Molec. Gen. Genet. 224, 209–221 (1990). https://doi.org/10.1007/BF00271554
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF00271554