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.     

Efficient splicing of two yeast mitochondrial introns controlled by a nuclear-encoded maturase.

bI4 maturase encoded by the fourth intron of the yeast mitochondrial cytochrome b gene, controls the splicing of both the fourth intron of the cytochrome b gene and the fourth intron of the gene encoding subunit I of cytochrome oxidase. It has been shown previously that a cytoplasmically translated hybrid protein composed of the pre-sequence of subunit 9 of Neurospora ATPase fused to a part of the bI4 maturase can be guided to mitochondria where it could compensate maturase deficiencies. This in vivo complementation of maturase mutants can be easily estimated by restoration of respiration. This work examines the efficiency of different bI4 maturase constructions to restore respiration in different yeast maturase-deficient strains. It is shown that the N-terminal end of the bI4 maturase plays a crucial role in the maturase activity. Moreover, the 12 N-terminal amino acids of the mitochondrial outer membrane protein constitute the most efficient mitochondrial targeting sequence in this system. Surprisingly enough, it was found that the cytoplasmically translated bI4 maturase containing the 254 C-terminal amino acid coded by the intron open reading frame can complement maturase mutations without any added mitochondrial-targeting sequence.     

A mitochondrial RNA maturase gene transferred to the yeast nucleus can control mitochondrial mRNA splicing

bI4 maturase, encoded by the fourth intron of the yeast mitochondrial cytochrome b gene, controls the splicing of both the fourth intron of the cytochrome b gene and the fourth intron of the gene encoding subunit I of cytochrome oxidase. By fusing the encoding presequence of subunit 9 of the Neurospora ATPase to a restriction fragment containing the bI4 maturase coding sequence, we have constructed a hybrid gene that can be translated on yeast cytosolic ribosomes. The resulting protein is imported into mitochondria, which was revealed by its ability to restore to respiratory competence a yeast mutant defective in the bI4 maturase. Moreover, a protein reacting with antimaturase antibodies was detected in the mitochondria of the transformed cells; this imported maturase functioned similarly to the endogenous maturase.     

Construction of novel cytochrome b genes in yeast mitochondria by subtraction or addition of introns

The mitochondrial cob-box gene coding for apocytochrome b in yeast has five introns and six exons or two introns and three exons depending on the wild-type strain considered. Some intron mutations in this gene affect not only its expression but also that of another mitochondrial gene: oxi3. To understand better the function of introns in gene expression, we have constructed a series of new strains that differ only by the presence or absence of one of the five wild-type introns in the cytochrome b gene, the rest of the mitochondrial and nuclear genome remaining unchanged. All constructions result from in vivo recombination events between rho- donor and rho+ recipient mtDNA. The following genes have been constructed: [see text]. Interestingly, all the genes lead to the synthesis of cytochrome b, while only the genes having the intron bI4 allow the expression of oxi3. A nuclear gene, when mutated, can compensate for the absence of the intron bI4.     

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.     

Novel hybrid maturases in unstable pseudorevertants of maturaseless mutants of yeast mitochondrial DNA.

Unstable pseudorevertants of mitochondrial mutants of Saccharomyces cerevisiae lacking the maturase function encoded by the fourth intron of the cytochrome b gene (bI4) were isolated. They were found to be heteroplasmic cells owing their regained ability to respire (and grow on glycerol medium) to the presence of a rearranged (rho-) mtDNA that contains an in-frame fusion of the reading frames of the group I introns bI4 and intron 4 alpha of the coxl gene encoding subunit I of cytochrome c oxidase (aI4 alpha). The products of those gene fusions suppress the bI4 maturase deficiency still present in those heteroplasmic cells. Similar heteroplasmic pseudorevertants of a group II maturaseless mutant of the first intron of the coxI gene were characterized; they result from partial deletion of the coxI gene that fuses the reading frames of introns 1 and 2. These heteroplasms provide independent support for the existence of RNA maturases encoded by group I and group II introns. Also, since the petite/mit- heteroplasms arise spontaneously at very high frequencies they provide a system that can be used to obtain mutants unable to form or maintain heteroplasmic cells.     

Connections between RNA splicing and DNA intron mobility in yeast mitochondria: RNA maturase and DNA endonuclease switching experiments.

The intron-encoded proteins bI4 RNA maturase and aI4 DNA endonuclease can be faithfully expressed in yeast cytoplasm from engineered forms of their mitochondrial coding sequences. In this work we studied the relationships between these two activities associated with two homologous intron-encoded proteins: the bI4 RNA maturase encoded in the fourth intron of the cytochrome b gene and the aI4 DNA endonuclease (I-SceII) encoded in the fourth intron of the gene coding for the subunit I of cytochrome oxidase. Taking advantage of both the high recombinogenic properties of yeast and the similarities between the two genes, we constructed in vivo a family of hybrid genes carrying parts of both RNA maturase and DNA endonuclease coding sequences. The presence of a sequence coding for a mitochondrial targeting peptide upstream from these hybrid genes allowed us to study the properties of their translation products within the mitochondria in vivo. We thus could analyze the ability of the recombinant proteins to complement RNA maturase deficiencies in different strains. Many combinations of the two parental intronic sequences were found in the recombinants. Their structural and functional analysis revealed the following features. (i) The N-terminal half of the bI4 RNA maturase could be replaced in total by its equivalent from the aI4 DNA endonuclease without affecting the RNA maturase activity. In contrast, replacing the C-terminal half of the bI4 RNA maturase with its equivalent from the aI4 DNA endonuclease led to a very weak RNA maturase activity, indicating that this region is more differentiated and linked to the maturase activity. (ii) None of the hybrid proteins carrying an RNA maturase activity kept the DNA endonuclease activity, suggesting that the latter requires the integrity of the aI4 protein. These observations are interesting because the aI4 DNA endonuclease is known to promote the propagation, at the DNA level, of the aI4 intron, whereas the bI4 RNA maturase, which is required for the splicing of its coding intron, also controls the splicing process of the aI4 intron. We propose a scenario for the evolution of these intronic proteins that relies on a switch from DNA endonuclease to RNA maturase activity.     

Antibodies against a fused 'lacZ-yeast mitochondrial intron' gene product allow identification of the mRNA maturase encoded by the fourth intron of the yeast cob-box gene.

Several missense or nonsense mutations have been localized in the fourth intron open reading frame (ORF) of the yeast mitochondrial cytochrome b gene. These results and the phenotypes of mutants strongly suggested that a mRNA maturase, controlling the expression of both cytochrome b and cytochrome oxidase subunit I (COXI) genes, is encoded in this ORF. To investigate more directly the biosynthesis of mRNA maturase we raised antibodies against a part of the putative ORF translation product. For that purpose we inserted a fragment of the ORF sequence, in phase, into the C-terminal EcoRI site of lacZ gene. The hybrid gene was then expressed in Escherichia coli under the control of either the wild-type lac promoter or the thermoregulated lambda system PR/cI857. The hybrid protein was partially purified and antibodies were raised against it. These antibodies recognized a mitochondrially coded protein, p27, in intron mutants, whereas no such protein was detected in the wild-type cell. These results demonstrate that the p27 protein, previously shown to be associated with the mRNA maturase activity, is actually translated from the intron ORF. The autoregulated mRNA maturase synthesis model is discussed in relation to these results.     

The yeast mitochondrial leucyl-tRNA synthetase is a splicing factor for the excision of several group I introns

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.     

In vitro mutagenesis of the mitochondrial leucyl tRNA synthetase ofSaccharomyces cerevisiae shows that the suppressor activity of the mutant proteins is related to the splicing function of the wild-type protein

TheNAM2 gene ofSaccharomyces cerevisiae encodes the mitochondrial leucyl tRNA synthetase (mLRS), which is necessary for the excision of the fourth intron of the mitochondrialcytb gene (bI4) and the fourth intron of the mitochondrialcoxI gene (aI4), as well as for mitochondrial protein synthesis. Some dominant mutant alleles of the gene are able to suppress mutations that inactivate the bI4 maturase, which is essential for the excision of the introns aI4 and bI4. Here we report mutagenesis studies which focus on the splicing and suppressor functions of the protein. Small deletions in the C-terminal region of the protein preferentially reduce the splicing, but not the synthetase activity; and all the C-terminal deletions tested abolish the suppressor activity. Mutations which increase the volume of the residue at position 240 in the wild-type mLRS without introducing a charge, lead to a suppressor activity. The mutant 238C, which is located in the suppressor region, has a reduced synthetase activity and no detectable splicing activity. These data show that the splicing and suppressor functions are linked and that the suppressor activity of the mutant alleles results from a modification of the wild-type splicing activity.     

In vitro mutagenesis of the mitochondrial leucyl tRNA synthetase ofSaccharomyces cerevisiae shows that the suppressor activity of the mutant proteins is related to the splicing function of the wild-type protein

TheNAM2 gene ofSaccharomyces cerevisiae encodes the mitochondrial leucyl tRNA synthetase (mLRS), which is necessary for the excision of the fourth intron of the mitochondrialcytb gene (bI4) and the fourth intron of the mitochondrialcoxI gene (aI4), as well as for mitochondrial protein synthesis. Some dominant mutant alleles of the gene are able to suppress mutations that inactivate the bI4 maturase, which is essential for the excision of the introns aI4 and bI4. Here we report mutagenesis studies which focus on the splicing and suppressor functions of the protein. Small deletions in the C-terminal region of the protein preferentially reduce the splicing, but not the synthetase activity; and all the C-terminal deletions tested abolish the suppressor activity. Mutations which increase the volume of the residue at position 240 in the wild-type mLRS without introducing a charge, lead to a suppressor activity. The mutant 238C, which is located in the suppressor region, has a reduced synthetase activity and no detectable splicing activity. These data show that the splicing and suppressor functions are linked and that the suppressor activity of the mutant alleles results from a modification of the wild-type splicing activity.     

Two independent activities define Ccm1p as a moonlighting protein in Saccharomyces cerevisiae

Ccm1p is a nuclear-encoded PPR (pentatricopeptide repeat) protein that localizes into mitochondria of Saccharomyces cerevisiae. It was first defined as an essential factor to remove the bI4 [COB (cytochrome b) fourth intron)] and aI4 [COX1 (cytochrome c oxidase subunit 1) fourth intron] of pre-mRNAs, along with bI4 maturase, a protein encoded by part of bI4 and preceding exons that removes the intronic RNA sequence that codes for it. Later on, Ccm1p was described as key to maintain the steady-state levels of the mitoribosome small subunit RNA (15S rRNA). bI4 maturase is produced inside the mitochondria and therefore its activity depends on the functionality of mitochondrial translation. This report addresses the dilemma of whether Ccm1p supports bI4 maturase activity by keeping steady-state levels of 15S rRNA or separately and directly supports bI4 maturase activity per se. Experiments involving loss of Ccm1p, SMDC (sudden mitochondrial deprivation of Ccm1p) and mutations in one of the PPR (pentatricopeptide repeat) motifs revealed that the failure of bI4 maturase activity in CCM1 deletion mutants was not due to a malfunction of the translational machinery. Both functions were found to be independent, defining Ccm1p as a moonlighting protein. bI4 maturase activity was significantly more dependent on Ccm1p levels than the maintenance of 15S rRNA. The novel strategy of SMDC described here allowed the study of immediate short-term effects, before the mutant phenotype was definitively established. This approach can be also applied for further studies on 15S rRNA stability and mitoribosome assembly.     

The bI4 group I intron binds directly to both its protein splicing partners, a tRNA synthetase and maturase, to facilitate RNA splicing activity

The imported mitochondrial leucyl-tRNA synthetase (NAM2p) and a mitochondrial-expressed intron-encoded maturase protein are required for splicing the fourth intron (bI4) of the yeast cob gene, which expresses an electron transfer protein that is essential to respiration. However, the role of the tRNA synthetase, as well as the function of the bI4 maturase, remain unclear. As a first step towards elucidating the mechanistic role of these protein splicing factors in this group I intron splicing reaction, we tested the hypothesis that both leucyl-tRNA synthetase and bI4 maturase interact directly with the bI4 intron. We developed a yeast three-hybrid system and determined that both the tRNA synthetase and bI4 maturase can bind directly and independently via RNA-protein interactions to the large bI4 group I intron. We also showed, using modified two-hybrid and three-hybrid assays, that the bI4 intron bridges interactions between the two protein splicing partners. In the presence of either the bI4 maturase or the Leu-tRNA synthetase, bI4 intron transcribed recombinantly with flanking exons in the yeast nucleus exhibited splicing activity. These data combined with previous genetic results are consistent with a novel model for a ternary splicing complex (two protein: one RNA) in which both protein splicing partners bind directly to the bI4 intron and facilitate its self-splicing activity.     

A novel mitochondrial DEAD box protein (Mrh4) required for maintenance of mtDNA in Saccharomyces cerevisiae

In a screen of nuclear genes that assist splicing of mitochondrial localized group II introns in yeast we isolated low-copy number suppressors of splicing and respiratory-deficient point mutants of intron aI5gamma, the last intron of the gene encoding cytochrome c oxidase subunit I. One of the genes found contains the open reading frame (ORF) YGL064c that has previously been proposed to encode a putative RNA helicase of the DEAD box family. Deletion of the ORF gives rise to 100% cytoplasmic petites, indicating that the protein plays an essential role in the mitochondrial RNA metabolism. Overexpression of YGL064c-GFP fusions clearly revealed a mitochondrial localization of the protein. The gene encodes the fourth putative RNA helicase of Saccharomyces cerevisiae implicated in a mitochondrial function and was therefore termed MRH4 (for mitochondrial RNA helicase).     

Long range control circuits within mitochondria and between nucleus and mitochondria

Summary In the preceding paper of this series (Dujardin et al. 1980a) we described general methods of selecting and genetically characterizing suppressor mutations that restore the respiratory capacity of mit ^- mitochondrial mutations. Two dominant nuclear (NAM1-1 and NAM2-1) and one mitochondrial (mim2-1) suppressors are more extensively studied in this paper. We have analysed the action spectrum of these suppressors on 433 mit ^- mutations located in various mitochondrial genes and found that they preferentially alleviate the effects of mutations located within intron open reading frames of the cob-box gene. We conclude that these suppressors permit the maturation of cytochrome b mRNA by restoring the synthesis of intron encoded protein(s) catalytically involved in splicing i.e. mRNA-maturase(s) (cf. Lazowska et al. 1980). NAM1-1 is allele specific and gene non-specific: it suppresses mutations located within different introns. NAM2-1 and mim2-1 are intron-specific: they suppress mutations all located in the same (box7) intron of the cobbox gene. Analyses of cytochrome absorption spectra and mitochondrial translation products of cells in which the suppressors are associated with various other mit ^- mutations show that the suppressors restore cytochrome b and/or cytochrome oxidase (cox 1) synthesis, as expected from their growth phenotype. This suppression is, however, only partial: some new polypeptides characteristic of the mit ^- mutations can be still detected in the presence of suppressor. Interestingly enough when box7 specific suppressors NAM2-1 and mim2-1 are associated with a complete cob-box deletion (leading to a total deficiency of cytochrome b and oxidase) partial restoration of cox I synthesis is observed while cytochrome b is still totally absent. These results show that in strains carrying NAM2-1 or mim2-1 the presence of cytochrome b gene is no longer required for the expression of the oxi3 gene pointing out to the possibility of a mutational switch-on of silent genes, whether mitochondrial, mim2-1, or nuclear, NAM2-1. This switch-on would permit the synthesis of an active maturase acting as a substitute for the box7 maturase in order to splice the cytochrome b and oxidase mRNAs.     

Antibodies against synthetic oligopeptides allow identification of the mRNA-maturase encoded by the second intron of the yeast cob-box gene.

Genetic and biochemical evidence has strongly suggested that several introns located in yeast mitochondrial genes specifying apocytochrome b or cytochrome oxidase encode trans-acting proteins (termed mRNA-maturases) responsible for splicing the cognate intron and maturation of the mRNA. We have chemically synthesized three oligopeptides, predicted from the DNA sequence of the open reading frame (ORF) present in the second intron of the cob-box gene, and raised antibodies against them. These antibodies have allowed us to identify a protein of 42 kd as the product translated from the ORF of the wild-type intron. In two splicing-deficient mutants this protein is replaced by shorter polypeptides whose lengths and antigenic properties are in full agreement with the positions of TAA codons established by the DNA sequence of the intron's ORF.     

Maturase and endonuclease functions depend on separate conserved domains of the bifunctional protein encoded by the group I intron aI4 alpha of yeast mitochondrial DNA.

Intron 4 alpha (aI4 alpha) of the yeast mitochondrial COXI gene is a mobile group I intron that contains a reading frame encoding both the homing endonuclease I-SceII and a latent maturase capable of splicing both aI4 alpha and the fourth intron of the cytochrome b (COB) gene (bI4). The aI4 alpha reading frame is a member of a large gene family recognized by the presence of related dodecapeptide sequence motifs called P1 and P2. In this study, missense mutations of P1 and P2 were placed in mitochondrial DNA by biolistic transformation. The effects of the mutations on intron mobility, endonuclease I-SceII activity and maturase function were tested. The mutations of P1 strongly affected mobility and endonuclease I-SceII activity, but had little or no effect on maturase function; mutations of P2 affected splicing but not mobility or endonuclease I-SceII activity. Surprisingly, the conditional (temperature-sensitive) mutations at P1 and P2 block one or the other function of the protein but not both. This study indicates that the two functions depend on separate domains of the intron-encoded protein.