A yeast nuclear gene, MRS1, involved in mitochondrial RNA splicing: nucleotide sequence and mutational analysis of two overlapping open reading frames on opposite strands.

We have cloned a 1.6-kb fragment of yeast nuclear DNA, which complements pet- mutant MK3 (mrs1). This mutant was shown to be defective in mitochondrial RNA splicing: the excision of intron 3 from the mitochondrial COB pre-RNA is blocked. The DNA sequence of the nuclear DNA fragment revealed two open reading frames (ORF1 with 1092 bp; ORF2 with 735 bp) on opposite strands, which overlap by 656 bp. As shown by in vitro mutagenesis, ORF1, but not ORF2, is responsible for complementation of the splice defect. Hence, ORF1 represents the nuclear MRS1 gene. Disruption of the gene (both ORFs) in the chromosomal DNA of the respiratory competent yeast strain DBY747 (long form COB gene) leads to a stable pet- phenotype and to the accumulation of the same mitochondrial RNA precursors as in strain MK3. The amino acid sequence of the putative ORF1 product does not exhibit any homology with other known proteins, except for a small region of homology with the gene product of another nuclear yeast gene involved in mitochondrial RNA splicing, CBP2. The function of the MRS1 (ORF1) gene in mitochondrial RNA splicing and the significance of the overlapping ORFs in this gene are discussed.     

p68 RNA helicase: identification of a nucleolar form and cloning of related genes containing a conserved intron in yeasts.

The human p68 protein is an RNA-dependent ATPase and RNA helicase which was first identified because of its immunological cross-reaction with a viral RNA helicase, simian virus 40 large T antigen. It belongs to a recently discovered family of proteins (DEAD box proteins) that share extensive regions of amino acid sequence homology, are ubiquitous in living organisms, and are involved in many aspects of RNA metabolism, including splicing, translation, and ribosome assembly. We have shown by immunofluorescent microscopy that mammalian p68, which is excluded from the nucleoli during interphase, translocates to prenucleolar bodies during telophase. We have cloned 55% identical genes from both Schizosaccharomyces pombe and Saccharomyces cerevisiae and shown that they are essential in both yeasts. The human and yeast genes contain a large intron whose position has been precisely conserved. In S. cerevisiae, the intron is unusual both because of its size and because of its location near the 3' end of the gene. We discuss possible functional roles for such an unusual intron in an RNA helicase gene.     

The DEAD-box RNA helicase ZmRH48 is required for the splicing of multiple mitochondrial introns,mitochondrial complex biosynthesis,and seed development in maize

RNA helicases participate in nearly all aspects of RNA metabolism by rearranging RNAs or RNA–protein complexes in an adenosine triphosphatedependent manner. Due to the large RNA helicase families in plants, the precise roles of many RNA helicases in plant physiology and development remain to be clarified. Here, we show that mutations in maize(Zea mays) DEAD-box RNA helicase48(Zm RH48) impair the splicing of mitochondrial introns, mitochondrial complex biosynthesis,and seed development. Loss of Z...     

Characterization of the I-Spom I endonuclease from fission yeast: insights into the evolution of a group I intron-encoded homing endonuclease

The first group I intron in the cox1 gene (cox1I1b ) of the mitochondrial genome of the fission yeast Schizosaccharomyces pombe is a mobile DNA element. The mobility is dependent on an endonuclease protein that is encoded by an intronic open reading frame (ORF). The intron-encoded endonuclease is a typical member of the LAGLIDADG protein family of endonucleases with two consensus motifs. In addition to this, analysis of several intron mutants revealed that this protein is required for intron splicing. However, this protein is one of the few group I intron-encoded proteins that functions in RNA splicing simultaneously with its DNA endonuclease activity. We report here on the biochemical characterization of the endonuclease activity of this protein artificially expressed in Escherichia coli. Although the intronic ORF is expressed as a fusion protein with the upstream exon in vivo, the experiments showed that a truncated translation product consisting of the C-terminal 304 codons of the cox1I1b ORF restricted to loop 8 of the intron RNA secondary structure is sufficient for the specific endonuclease activity in vitro. Based on the results, we speculate on the evolution of site-specific homing endonucleases encoded by group I introns in eukaryotes.     

The nuclear gene MRS2 is essential for the excision of group II introns from yeast mitochondrial transcripts in vivo.

RNA splicing defects in mitochondrial intron mutants can be suppressed by a high dosage of several proteins encoded by nuclear genes. In this study we report on the isolation, nucleotide sequence, and possible functions of the nuclear MRS2 gene. When present on high copy number plasmids, the MRS2 gene acts as a suppressor of various mitochondrial intron mutations, suggesting that the MRS2 protein functions as a splicing factor. This notion is supported by the observations that disruption of the single chromosomal copy of the MRS2 gene causes (i) a pet- phenotype and (ii) a block in mitochondrial RNA splicing of all four mitochondrial group II introns, some of which are efficiently self-splicing in vitro. In contrast, the five group I introns monitored here are excised from pre-mRNA in a MRS2-disrupted background although at reduced rates. So far the MRS2 gene product is unique in that it is essential for splicing of all four group II introns, but relatively unimportant for splicing of group I introns. In strains devoid of any mitochondrial introns the MRS2 gene disruption still causes a pet- phenotype and cytochrome deficiency, although the standard pattern of mitochondrial translation products is produced. Therefore, apart from RNA splicing, the absence of the MRS2 protein may disturb the assembly of mitochondrial membrane complexes.     

The Saccharomyces cerevisiae Pus2 protein encoded by YGL063w ORF is a mitochondrial tRNA:Psi27/28-synthase

The RNA:pseudouridine (Psi)-synthase family is one of the most complex families of RNA modification enzymes. Ten genes encoding putative RNA:Psi-synthases have been identified in S. cerevisiae. Most of the encoded enzymes have been characterized experimentally. Only the putative RNA:Psi-synthase Pus2p (encoded by the YGL063w ORF) had no identified substrate. Here, we analyzed Psi residues in cytoplasmic and mitochondrial tRNAs extracted from S. cerevisiae strains, carrying disruptions in the PUS1 and/or PUS2 ORFs. Our results demonstrate that Pus2p is a mitochondrial-specific tRNA:Psi-synthase acting at positions 27 and 28 in tRNAs. The importance of the Asp56 residue in the conserved ARTD motif of the Pus2p catalytic site is demonstrated in vivo. Interestingly, in spite of the absence of a characteristic N-terminal targeting signal, our data strongly suggest an efficient and rapid targeting of Pus2p in yeast mitochondria. In contradiction with the commonly held idea that a unique nuclear gene encodes the enzyme required for both cytoplasmic and mitochondrial tRNA modifications, here we show the existence of an enzyme specifically dedicated to mitochondrial tRNA modification (Pus2p), the corresponding modification in cytoplasmic tRNAs being catalyzed by another protein (Pus1p).     

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.     

The mitochondrial tyrosyl-tRNA synthetase of Podospora anserina is a bifunctional enzyme active in protein synthesis and RNA splicing.

The Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (mt tyrRS), which is encoded by the nuclear gene cyt-18, functions not only in aminoacylation but also in the splicing of group I introns. Here, we isolated the cognate Podospora anserina mt tyrRS gene, designated yts1, by using the N. crassa cyt-18 gene as a hybridization probe. DNA sequencing of the P. anserina gene revealed an open reading frame (ORF) of 641 amino acids which has significant similarity to other tyrRSs. The yts1 ORF is interrupted by two introns, one near its N terminus at the same position as the single intron in the cyt-18 gene and the other downstream in a region corresponding to the nucleotide-binding fold. The P. anserina yts1+ gene transformed the N. crassa cyt-18-2 mutant at a high frequency and rescued both the splicing and protein synthesis defects. Furthermore, the YTS1 protein synthesized in Escherichia coli was capable of splicing the N. crassa mt large rRNA intron in vitro. Together, these results indicate that YTS1 is a bifunctional protein active in both splicing and protein synthesis. The P. anserina YTS1 and N. crassa CYT-18 proteins share three blocks of amino acids that are not conserved in bacterial or yeast mt tyrRSs which do not function in splicing. One of these blocks corresponds to the idiosyncratic N-terminal domain shown previously to be required for splicing activity of the CYT-18 protein. The other two are located in the putative tRNA-binding domain toward the C terminus of the protein and also appear to be required for splicing. Since the E. coli and yeast mt tyrRSs do not function in splicing, the adaptation of the Neurospora and Podospora spp. mt tyrRSs to function in splicing most likely occurred after the divergence of their common ancestor from yeast.     

The NAM8 gene in Saccharomyces cerevisiae encodes a protein with putative RNA binding motifs and acts as a suppressor of mitochondrial splicing deficiencies when overexpressed.

Summary We have characterized the nuclear geneNAM8 inSaccharomyces cerevisiae. It acts as a suppressor of mitochondrial splicing deficiencies when present on a multicopy plasmid. The suppressed mutations affect RNA folding and are located in both group I and group II introns. The gene is weakly transcribed in wildtype strains, its overexpression is a prerequisite for the suppressor action. Inactivation of theNAM8 gene does not affect cell viability, mitochondrial function or mitochondrial genome stability. TheNAM8 gene encodes a protein of 523 amino acids which includes two conserved (RNP) motifs common to RNA-binding proteins from widely different organisms. This homology with RNA-binding proteins, together with the intronic location of the suppressed mitochondrial mutations, suggests that the NAM8 protein could be a non-essential component of the mitochondrial splicing machinery and, when present in increased amounts, it could convert a deficient intron RNA folding pattern into a productive one.     

AtnMat2, a nuclear-encoded maturase required for splicing of group-II introns in Arabidopsis mitochondria

Mitochondria (mt) in plants house about 20 group-II introns, which lie within protein-coding genes required in both organellar genome expression and respiration activities. While in nonplant systems the splicing of group-II introns is mediated by proteins encoded within the introns themselves (known as “maturases”), only a single maturase ORF (matR) has retained in the mitochondrial genomes in plants; however, its putative role(s) in the splicing of organellar introns is yet to be established. Clues to other proteins are scarce, but these are likely encoded within the nucleus as there are no obvious candidates among the remaining ORFs within the mtDNA. Intriguingly, higher plants genomes contain four maturase-related genes, which exist in the nucleus as self-standing ORFs, out of the context of their evolutionary-related group-II introns “hosts.” These are all predicted to reside within mitochondria and may therefore act “in-trans” in the splicing of organellar-encoded introns. Here, we analyzed the intracellular locations of the four nuclear-encoded maturases in Arabidopsis and established the roles of one of these genes, At5g46920 (AtnMat2), in the splicing of several mitochondrial introns, including the single intron within cox2, nad1 intron2, and nad7 intron2.     

Yeast and human mitochondrial helicases

Mitochondria are semiautonomous organelles which contain their own genome. Both maintenance and expression of mitochondrial DNA require activity of RNA and DNA helicases. In Saccharomyces cerevisiae the nuclear genome encodes four DExH/D superfamily members (MSS116, SUV3, MRH4, IRC3) that act as helicases and/or RNA chaperones. Their activity is necessary for mitochondrial RNA splicing, degradation, translation and genome maintenance. In humans the ortholog of SUV3 (hSUV3, SUPV3L1) so far is the best described mitochondrial RNA helicase. The enzyme, together with the matrix-localized pool of PNPase (PNPT1), forms an RNA-degrading complex called the mitochondrial degradosome, which localizes to distinct structures (D-foci). Global regulation of mitochondrially encoded genes can be achieved by changing mitochondrial DNA copy number. This way the proteins involved in its replication, like the Twinkle helicase (c10orf2), can indirectly regulate gene expression. Here, we describe yeast and human mitochondrial helicases that are directly involved in mitochondrial RNA metabolism, and present other helicases that participate in mitochondrial DNA replication and maintenance. This article is part of a Special Issue entitled: The Biology of RNA helicases — Modulation for life.     

Molecular genetics of group I introns: RNA structures and protein factors required for splicing--a review

In vivo and in vitro genetic techniques have been widely used to investigate the structure-function relationships and requirements for splicing of group-I introns. Analyses of group-I introns from extremely diverse genetic systems, including fungal mitochondria, protozoan nuclei, and bacteriophages, have yielded results which are complementary and highly consistent. In vivo genetic studies of fungal mitochondrial systems have served to identify cis-acting sequences within mitochondrial introns, and trans-acting protein products of mitochondrial and nuclear genes which are important for splicing, and to show that some mitochondrial introns are mobile genetic elements. In vitro genetic studies of the self-splicing intron within the Tetrahymena thermophila nuclear large ribosomal RNA precursor (Tetrahymena LSU intron) have been used to examine essential and nonessential RNA sequences and structures in RNA-catalyzed splicing. In vivo and in vitro genetic analysis of the intron within the bacteriophage T4 td gene has permitted the detailed examination of mutant phenotypes by analyzing splicing in vivo and self-splicing in vitro. The genetic studies combined with phylogenetic analysis of intron structure based on comparative nucleotide sequence data [Cech 73 (1988) 259-271] and with biochemical data obtained from in vitro splicing experiments have resulted in significant advances in understanding the biology and chemistry of group-I introns.     

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.     

Mutations in Nuclear Gene Cyt-4 of Neurospora Crassa Result in Pleiotropic Defects in Processing and Splicing of Mitochondrial Rnas

The nuclear cyt-4 mutants of Neurospora crassa have been shown previously to be defective in splicing the group I intron in the mitochondrial large rRNA gene and in 3' end synthesis of the mitochondrial large rRNA. Here, Northern hybridization experiments show that the cyt-4-1 mutant has alterations in a number of mitochondrial RNA processing pathways, including those for cob, coI, coII and ATPase 6 mRNAs, as well as mitochondrial tRNAs. Defects in these pathways include inhibition of 5' and 3' end processing, accumulation of aberrant RNA species, and inhibition of splicing of both group I introns in the cob gene. The various defects in mitochondrial RNA synthesis in the cyt-4-1 mutant cannot be accounted for by deficiency of mitochondrial protein synthesis or energy metabolism, and they suggest that the cyt-4-1 mutant is defective in a component or components required for processing and/or turnover of a number of different mitochondrial RNAs. Defective splicing of the mitochondrial large rRNA intron in the cyt-4-1 mutant may be a secondary effect of failure to synthesize pre-rRNAs having the correct 3' end. However, a similar explanation cannot be invoked to account for defective splicing of the cob pre-mRNA introns, and the cyt-4-1 mutation may directly affect splicing of these introns.