Mitochondrial RNase Research Articles

Rpm2, the protein subunit of mitochondrial RNase P in Saccharomyces cerevisiae, also has a role in the translation of mitochondrially encoded subunits of cytochrome c oxidase.

RPM2 is a Saccharomyces cerevisiae nuclear gene that encodes the protein subunit of mitochondrial RNase P and has an unknown function essential for fermentative growth. Cells lacking mitochondrial RNase P cannot respire and accumulate lesions in their mitochondrial DNA. The effects of a new RPM2 allele, rpm2-100, reveal a novel function of RPM2 in mitochondrial biogenesis. Cells with rpm2-100 as their only source of Rpm2p have correctly processed mitochondrial tRNAs but are still respiratory deficient. Mitochondrial mRNA and rRNA levels are reduced in rpm2-100 cells compared to wild type. The general reduction in mRNA is not reflected in a similar reduction in mitochondrial protein synthesis. Incorporation of labeled precursors into mitochondrially encoded Atp6, Atp8, Atp9, and Cytb protein was enhanced in the mutant relative to wild type, while incorporation into Cox1p, Cox2p, Cox3p, and Var1p was reduced. Pulse-chase analysis of mitochondrial translation revealed decreased rates of translation of COX1, COX2, and COX3 mRNAs. This decrease leads to low steady-state levels of Cox1p, Cox2p, and Cox3p, loss of visible spectra of aa(3) cytochromes, and low cytochrome c oxidase activity in mutant mitochondria. Thus, RPM2 has a previously unrecognized role in mitochondrial biogenesis, in addition to its role as a subunit of mitochondrial RNase P. Moreover, there is a synthetic lethal interaction between the disruption of this novel respiratory function and the loss of wild-type mtDNA. This synthetic interaction explains why a complete deletion of RPM2 is lethal.

Imported RNA in mammalian mitochondria

The ‘division of genetic labour’ between nuclear and mitochondrial DNA (mtDNA) in mitochondrial protein synthesis in animal cells is controversial. As far as is known, all mitochondrial tRNAs, plus the two major rRNAs, are encoded by mtDNA, whereas all of the proteins involved in mitochondrial gene expression are nuclear encoded. In plants, fungi and protists, there are many examples of imported (nuclear-encoded) tRNAs, but compelling evidence for RNAimport into mammalian mitochondria has been lacking – until now.One recently documented example is the 5S rRNA, a proportion of which is found inside mitochondria 1xEvidence for the presence of 5S rRNA in mammalian mitochondria. Magalhaes, P.J et al. Mol. Biol. Cell. 1998; 9: 2375Crossref | PubMedSee all References1, although its function there is unknown. Other putatively imported RNAs have long been suggested to have roles in mitochondrial RNA processing. A particularly curious example concerns RNase P, the ribozyme involved in tRNA 5′ end maturation, which is apparently conserved from bacteria to man. No distinct mitochondrial RNase P has been characterized, and no gene for the catalytically essential RNA component of the enzyme can be found in mammalian mtDNA, although it is clearly present in yeast mtDNA. This leaves us with two alternatives: either mitochondrial pre-tRNA 5′ processing uses the same enzyme as in the nucleus, or a distinct enzyme inside mitochondria is entirely protein based. Preliminary reports in the literature unfortunately seemed to favour both options!The controversy has probably been laid to rest by Puranam and Attardi 2xThe RNase P associated with HeLa cell mitochondria contains an essential RNA component identical in sequence to that of the nuclear RNase P. Puranam, R.S and Attardi, G. Mol. Cell. Biol. 2001; 21: 548–561Crossref | PubMed | Scopus (82)See all References2, who report the characterization of an RNaseP in highly purified mitochondria that appears to be identical to the nuclear enzyme. Crucially, the enzyme is able in vitro to process correctly a mitochondrial pre-tRNA substrate, for Ser-tRNAUCN. The RNA component of the nuclear and mitochondrial enzymes is the same 340-nt H1 RNA species, encoded by a nuclear gene. Only a fraction of a percent of the total cellular H1 RNA content is recovered with the mitochondria, but it can be removed only by treatments that destroy organelle integrity. These findings raise the obvious questions of how H1 RNA (or intact RNase P holoenzyme, in a 17S particle also containing protein) enters mitochondria. The same authors also found tiny amounts of other small nuclear RNAs inside mitochondria, notably the RNA component of MRP, a ribozyme involved in nuclear pre-rRNA processing that was also originally proposed as a mitochondrial endoribonuclease.Clearly, if the biochemical pathways of mitochondrial RNA import in mammalian cells can be elucidated, a novel route will be established, whereby mitochondrial RNA can be manipulated, even if mtDNA transformation itself remains technically unfeasible. This could well offer a long-term prospect of gene therapy for correcting mitochondrial disorders involving tRNA gene mutations. At the very least, it would allow us to investigate more directly the molecular and physiological consequences of the presence inside mitochondria of mutant tRNAs.

The Crithidia fasciculata RNH1 gene encodes both nuclear and mitochondrial isoforms of RNase H.

The Crithidia fasciculata RNH1 gene encodes an RNase H, an enzyme that specifically degrades the RNA strand of RNA-DNA hybrids. The RNH1 gene is contained within an open reading frame (ORF) predicted to encode a protein of 53.7 kDa. Previous work has shown that RNH1 expresses two proteins: a 38 kDa protein and a 45 kDa protein which is enriched in kinetoplast extracts. Epitope tagging of the C-terminus of the RNH1 gene results in localization of the protein to both the kinetoplast and the nucleus. Translation of the ORF beginning at the second in-frame methionine codon predicts a protein of 38 kDa. Insertion of two tandem stop codons between the first ATG codon and the second in-frame ATG codon of the ORF results in expression of only the 38 kDa protein and the protein localizes specifically to the nucleus. Mutation of the second methionine codon to a valine codon prevents expression of the 38 kDa protein and results in exclusive production of the 45 kDa protein and localization of the protein only in the kinetoplast. These results suggest that the kinetoplast enzyme results from processing of the full-length 53.7 kDa protein. The nuclear enzyme appears to result from translation initiation at the second in-frame ATG codon. This is the first example in trypanosomatids of the production of nuclear and mitochondrial isoforms of a protein from a single gene and is the only eukaryotic gene in the RNase HI gene family shown to encode a mitochondrial RNase H.

Eukaryotic ribonucleases HI and HII generate characteristic hydrolytic patterns on DNA-RNA hybrids: further evidence that mitochondrial RNase H is an RNase HII.

RNase H activities from HeLa cells (either of cytoplasmic or mitochondrial origin), and from mitochondria of beef heart and Xenopus ovaries, have been tested with RNA-DNA substrates of defined length (20 bp) and sequence. Substrates were either blunt-ended, or presented DNA or RNA overhangs. The hydrolysis profiles obtained at early times of the digestion showed a good correlation between the class of RNase H, either type I or II assigned according to biochemical parameters, whatever the organism. Consequently, the pattern of primary cuts can be considered as a signature of the predominant RNase H activity. For a given sequence, hydrolysis profiles obtained are similar, if not identical, for either blunt-ended substrates or those presenting overhangs. However, profiles showed variations depending on the sequence used. Of the three sequences tested, one appears very discriminatory, class I RNases H generating a unique primary cut 3 nt from the 3' end of the RNA strand, whereas class II RNases H generated two simultaneous primary cuts at 6 and at 8 nt from the 5' end of the RNA strand. Hydrolysis profiles further confirm the assignation of the mitochondrial RNase H activity from HeLa cells, beef heart and Xenopus oocytes to the class II.

Characterization of Human Mitochondrial RNase P: Novel Aspects in tRNA Processing

Human mitochondrial RNase P does not distinguish itself from other RNase P enzymes by most of its basic properties. 5′ phosphates on tRNA products, strict dependence on a divalent cation, independence of ATP or other cofactors, and sensitivity to puromycin are generally characteristic for RNase P. Slow sedimentation of human mitochondrial RNase P in glycerol gradients suggests a molecular weight considerably lower than that of bacterial or nuclear RNase P. In contrast to fungi, all putative components of mammalian mitochondrial RNase P are encoded by the nucleus. Intriguingly, no indication of the involvement of a trans-acting RNA was found in mammalian mitochondrial tRNA processing. Mitochondrial RNase P is resistant to rigorous treatments with nucleases and exhibits a protein-like density in Cs2SO4gradients. Moreover, an analysis of copurifying RNAs revealed no putative RNase P RNA candidates. These data suggest that mammalian mitochondrial RNase P, unlike its nuclear counterpart or its bacterial relatives, is not a ribonucleoprotein but a protein enzyme.

Ribonuclease P: unity and diversity in a tRNA processing ribozyme.

Ribonuclease P (RNase P) is the endoribonuclease that generates the mature 5'-ends of tRNA by removal of the 5'-leader elements of precursor-tRNAs. This enzyme has been characterized from representatives of all three domains of life (Archaea, Bacteria, and Eucarya) (1) as well as from mitochondria and chloroplasts. The cellular and mitochondrial RNase Ps are ribonucleoproteins, whereas the most extensively studied chloroplast RNase P (from spinach) is composed solely of protein. Remarkably, the RNA subunit of bacterial RNase P is catalytically active in vitro in the absence of the protein subunit (2). Although RNA-only activity has not been demonstrated for the archael, eucaryal, or mitochondrial RNAs, comparative sequence analysis has established that these RNAs are homologous (of common ancestry) to bacterial RNA. RNase P holoenzymes vary greatly in organizational complexity across the phylogenetic domains, primarily because of differences in the RNase P protein subunits: Mitochondrial, archaeal, and eucaryal holoenzymes contain larger, and perhaps more numerous, protein subunits than do the bacterial holoenzymes. However, that the nonbacterial RNase P RNAs retain significant structural similarity to their catalytically active bacterial counterparts indicates that the RNA remains the catalytic center of the enzyme.

Are tRNAs imported into the mitochondria of kinetoplastid protozoa as 5′-extended precursors?

All mitochondrial tRNAs in kinetoplastid protozoa are encoded in nuclear DNA and transported into the mitochondrion (Simpson et al., Nucl Acids Res 1989;17:5427–5445; Hancock and Hajduk, J Biol Chem 1990;265:19208–19215). It has been proposed that tRNAs in these cells are imported into the mitochondrion as 5′-extended precursors which are processed by a mitochondrial RNase P-like activity (Hancock et al., J Biol Chem 1992;267:23963–23971). We have examined this hypothesis by cloning and sequencing primer extension products of mitochondrial tRNAs from Leishmania tarentolae and Trypanosoma brucei, and have found that these are derived from circularized mature tRNA molecules. We suggest that these molecules are produced by the endogenous RNA ligase activity (Bakalara et al., J Biol Chem 1989;264:18679–18686) either in vivo or during mitochondrial isolation. We did not obtain any evidence for the existence of high molecular weight precursors of mitochondrial tRNAs. This negative result is consistent with previous in vivo transfection studies with both L. tarentolae (Lima and Simpson, RNA 1996;2:429–440) and T. brucei (Hauser and Schneider, EMBO J 1995;14:4212–4220; Schneider et al., Mol Cell Biol 1994;14:2317–2322), in which mitochondrial targeting of plasmid-expressed tRNAs was independent of the presence of 5′-flanking sequences. We conclude that the hypothesis for 5′-extended tRNA precursors in kinetoplastid mitochondrial importation remains to be verified.

Kluyveromyces lactis SEF1 and its Saccharomyces cerevisiae homologue bypass the unknown essential function, but not the mitochondrial RNase P function, of the S. cerevisiae RPM2 gene.

RPM2 is a Saccharomyces cerevisiae nuclear gene required for normal cell growth yet the only known function of Rpm2p is as a protein subunit of yeast mitochondrial RNase P, an enzyme responsible for the 5' maturation of mitochondrial tRNAs. Since mitochondrial protein synthesis in S. cerevisiae is not essential for viability, RPM2 must provide another function in addition to its known role as a mitochondrial tRNA processing enzyme. During a search for RPM2 homologues from Kluyveromyces lactis, we recovered a K. lactis gene that compensates for the essential function but not the RNase P function of RPM2. We have named this gene SEF1 (Suppressor of the Essential Function), DNA sequence analysis of SEF1 reveals it contains a Zn(2)-Cys(6) binuclear cluster motif found in a growing number of yeast transcription factors. The SEF1 homologue of S. cerevisiae also compensates for the essential function of RPM2. The two proteins share 49% identity and 72% amino acid sequence similarity.

5' end maturation and RNA editing have to precede tRNA 3' processing in plant mitochondria.

We report the characterization and partial purification of potato mitochondrial RNase Z, an endonuclease that generates mature tRNA 3' ends. The enzyme consists of one (or more) protein(s) without RNA subunits. Products of the processing reaction are tRNA molecules with 3' terminal hydroxyl groups and 3' trailers with 5' terminal phosphates. The main processing sites are located immediately 3' to the discriminator and one nucleotide further downstream. This endonucleolytic processing at and close to the tRNA 3' end in potato mitochondria suggests a higher similarity to the eukaryotic than to the prokaryotic tRNA 3' processing pathway. Partial purification and separation of RNase Z from the 5' processing activity RNase P allowed us to determine biochemical characteristics of the enzyme. The activity is stable over broad pH and temperature ranges, with peak activity at pH 8 and 30 degrees C. Optimal concentrations for MgCl2 and KCl are 5 mM and 30 mM, respectively. The potato mitochondrial RNase Z accepts only tRNA precursors with mature 5' ends. The precursor for tRNAPhe requires RNA editing for efficient processing by RNase Z.

Unusual Usage of Noncomplementary Dinucleotide Primers by the Yeast Mitochondrial RNA Polymerase

The mitochondrial RNase P RNA gene in yeastSaccharomyces cerevisiaeis transcribed from a variant mitochondrial promoter (SP). The sequence of this SP promoter [TATAAGAAG (+2)] differs from the conserved mitochondrial promoter sequence [TATAAGTAA (+2)] by −1T → A and +2A → G nucleotide substitutions. To determine the effect of these nucleotide alterations in mitochondrial promoter function, anin vitrotranscription analysis was carried out. In the presence of high concentrations of rNTPs (i.e., 125 μm), transcription initiation on the wild-type or variant promoter occurred at the conventional 3′ adenine nucleotide. However, at low rNTP concentrations (i.e., 5 μm) and in the presence of a complementary dinucleotide primer corresponding to positions −1 + 1, the mitochondrial RNA polymerase started transcription one nucleotide upstream of the conventional start site. Surprisingly, in the presence of some noncomplementary dinucleotides (i.e., GpA or CpA), which do not have perfect Watson–Crick base pairing with the initiator sequence, transcriptional initiation also occurred with the SP promoter but not with the conserved promoter sequence. This finding is the first example of utilization of noncomplementary dinucleotide primer by an RNA polymerase. Further analysis of mitochondrial promoter function by site-directed mutagenesis determined that the guanine nucleotide at position +2 is mainly responsible for this unusual function of the SP promoter.

Anin VitroTranscription Assay for Yeast Mitochondria Using Organellar Lysates

The nuclear geneNUC1encodes the major mitochondrial (mt) ribonuclease in the yeastSaccharomyces cerevisiae.We describe anin vitromt transcription assay system based on lysates of purified mitochondria from a petite (ρ−, mt deletion mutant) yeast strain in whichNUC1has been insertionally inactivated. Controlin vitrorun-on transcription assays using intact mitochondria demonstrate that the rate of incorporation of labeled precursor into mt RNA is identical in organelles from thenuc1ρ−mutant and its otherwise isochromosomalNUC1parent strain. Brij-35 lysates of mitochondria from thenuc1strain incorporate precursor into mt RNA at nearly the same rate as do intact organelles from that strain, while similar mt lysates fromNUC1cells show no such incorporation. Other control studies show that mt lysates from thenuc1strain retain functional mt cAMP-dependent protein kinase and other critical activities. When the cloned template DNA encoding the yeast mt 21S rRNA gene, which is not retained in thenuc1ρ−strain, is added to mt lysates from that strain, transcripts are produced from the template under standard assay conditions.

An RNase P RNA subunit mutation affects ribosomal RNA processing.

RNase P is a ribonucleoprotein endoribonuclease responsible for the 5' maturation of precursor tRNAs in all organisms. While analyzing mutations in conserved positions of the yeast nuclear RNase P RNA subunit, significant accumulation of an aberrant RNA of approximately 193 nucleotides was observed. This abundant RNA was identified as a 3'extended form of the 5.8S rRNA. This strain also displays a slightly elevated level of other rRNA processing intermediates with 5-ends at processing site A2 in the internal transcribed spacer 1 (ITS1) region of the rRNA primary transcript. To test whether pre-rRNA in the region of ITS1/5.8S/ITS2 is a substrate for RNase P in vitro, nuclear RNase P was partially purified to remove contaminating nucleases. Cleavage assays were performed using an rRNA substrate transcribed in vitro which includes the 5.8S region and its surrounding processing sites in ITS1 and ITS2. Discrete cleavages of this rRNA substrate were coincident with the peak fractions of nuclear RNase P, but not with fractions corresponding to mitochondrial RNase P or ribonuclease MRP RNA. The cleavage activity is sensitive to treatment with micrococcal nuclease, also consistent with an activity attributable to RNase R The strong RNase P cleavage sites were mapped and their possible relationships to steps in the rRNA processing pathway are considered. These observations suggest an intimate relationship between the processes of tRNA and rRNA maturation in the eukaryotic nucleus.

The evolution of the RNase P- and RNase MRP-associated RNAs: Phylogenetic analysis and nucleotide substitution rate

We report a detailed evolutionary study of the RNase P- and RNase MRP- associated RNAs. The analyses were performed on all the available complete sequences of RNase MRP (vertebrates, yeast, plant), nuclear RNase P (vertebrates, yeast), and mitochondrial RNase P (yeast) RNAs. For the first time the phylogenetic distance between these sequences and the nucleotide substitution rates have been quantitatively measured.The analyses were performed by considering the optimal multiple alignments obtained mostly by maximizing similarity between primary sequences. RNase P RNA and MRP RNA display evolutionary dynamics following the molecular clock. Both have similar rates and evolve about one order of magnitude faster than the corresponding small rRNA sequences which have been, so far, the most common gene markers used for phylogeny. However, small rRNAs evolve too slowly to solve close phylogenetic relationships such as those between mammals. The quicker rate of RNase P and MRP RNA allowed us to assess phylogenetic relationships between mammals and other vertebrate species and yeast strains. The phylogenetic data obtained with yeasts perfectly agree with those obtained by functional assays, thus demonstrating the potential offered by this approach for laboratory experiments.

Yeast mitochondrial RNase P RNA synthesis is altered in an RNase P protein subunit mutant: insights into the biogenesis of a mitochondrial RNA-processing enzyme.

Rpm2p is a protein subunit of Saccharomyces cerevisiae yeast mitochondrial RNase P, an enzyme which removes 5' leader sequences from mitochondrial tRNA precursors. Precursor tRNAs accumulate in strains carrying a disrupted allele of RPM2. The resulting defect in mitochondrial protein synthesis causes petite mutants to form. We report here that alteration in the biogenesis of Rpm1r, the RNase P RNA subunit, is another consequence of disrupting RPM2. High-molecular-weight transcripts accumulate, and no mature Rpm1r is produced. Transcript mapping reveals that the smallest RNA accumulated is extended on both the 5' and 3' ends relative to mature Rpm1r. This intermediate and other longer transcripts which accumulate are also found as low-abundance RNAs in wild-type cells, allowing identification of processing events necessary for conversion of the primary transcript to final products. Our data demonstrate directly that Rpm1r is transcribed with its substrates, tRNA met f and tRNAPro, from a promoter located upstream of the tRNA met f gene and suggest that a portion also originates from a second promoter, located between the tRNA met f gene and RPM1. We tested the possibility that precursors accumulate because the RNase P deficiency prevents the removal of the downstream tRNAPro. Large RPM1 transcripts still accumulate in strains missing this tRNA. Thus, an inability to process cotranscribed tRNAs does not explain the precursor accumulation phenotype. Furthermore, strains with mutant RPM1 genes also accumulate precursor Rpm1r, suggesting that mutations in either gene can lead to similar biogenesis defects. Several models to explain precursor accumulation are presented.

Expression of the mitochondrial RNase P RNA subunit-encoding gene from a variant promoter sequence in Saccharomyces cerevisiae

Ribonuclease P (RNase P) is a common tRNA processing enzyme that removes the 5′ leader sequence of precursor tRNAs. This activity is identified in yeast mitochondria as a separate enzyme from the nuclear RNase P. Like other RNase P enzymes, the mitochondrial (mt) RNase P is also a ribonucleoprotein composed of both RNA and protein subunits. The RNA subunit is encoded by a mt gene and the protein subunit is supplied by a nuclear gene. Earlier studies described one active promoter ( FP1) located 5′ to the mt tRNA fMet-RNase P RNA-tRNA Pro gene cluster, so that the mitochondrially encoded RNA subunit was thought to be co-transcribed with two of its substrate tRNAs. However, the results of in vitro transcription and primer extension experiments presented here demonstrate that the mt RNase P RNA subunit-encoding gene ( RPM1) is transcribed from a new promoter ( SP) which is located between the tRNA fMet and RPM1 genes. The sequence [5′-TATAAGAA(+1)] of the new promoter varies from the conserved promoter sequence [5′-TATAAGTA(+1)], but is one of the sequences that is active in the in vitro transcription assay to determine the consensus promoter sequence [5′-T A T/a A A/g/c G T/a/c N(+1)]. This result demonstrates that a naturally occurring variant promoter is used by RPM1. Identification of the novel SP promoter suggests that the synthesis of the mt RNase P RNA subunit might be uncoupled from the expression of upstream tRNA fMet gene, and that RPMI might be independently transcribed in Saccharomyces cerevisiae

9] Genetic and biochemical approaches for analysis of mitochondrial RNase P from Saccharomyces cerevisiae

Publisher Summary Yeast mitochondrial RNase P is the endonuclease, which is is composed of both RNA and protein subunits coded by the mitochondrial and nuclear genomes, respectively. This chapter describes the in vivo and in vitro approaches used to understand the nucleocytoplasmic interactions necessary for the biosynthesis of this dually derived complex of yeast mitochondria and to gain insight into RNase P structure and function. Many questions about this enzyme and its biosynthesis remain despite the identification and current characterization of Rpm1r and Rpm2p. First, none of the experiments eliminates the possible presence of a nuclear encoded RNA in this enzyme. Second, although Rpm2p clearly fills all the criteria expected of a protein subunit of the enzyme and is the most abundant protein in near homogeneous preparations, it is possible that other proteins play either essential or stimulatory roles. Successful production of sufficient Rpm2p for in vitro reconstitution studies should determine if Rpm1r and Rpm2p are the only components necessary to support full activity in vitro. If they are, studies on the biochemistry and biogenesis of a well-defined yeast mitochondrial RNase P can be initiated. Immunoprecipitation of complexes or cross-linking between the known RNA and proteins offer other approaches to identify additional components that can be required for in vitro reconstitution or be associated with the enzyme in vivo.

Purification and characterization of mitochondrial ribonuclease P from Aspergillus nidulans.

Mitochondrial ribonuclease (RNase) P from Aspergillus nidulans was purified to near homogeneity using whole-cell extract as the starting material. A 4400-fold purification with a yield of 5.2% was achieved by ammonium sulfate fractionation, heat treatment, and five types of column chromatography, including tRNA-affinity column chromatography. This enzyme, which has a molecular mass of 232 kDa determined by glycerol gradient sedimentation analysis, appears to be composed of seven polypeptides and an RNA moiety. These seven polypeptides consistently copurified with the RNase P activity through two ion-exchange chromatography columns and in a glycerol gradient. As judged by nuclease sensitivity, the enzyme requires an RNA component for its activity. The 3'-end-labeled RNAs that copurified with the enzyme displayed identical sequences but had variable lengths for the 5' end, indicating that they originated from a common RNA molecule, the putative RNA component of RNase P. The purified enzyme cleaved mitochondrial precursor tRNAHis, resulting in an 8-bp acceptor stem. This implies that the purified RNase P is a mitochondrial enzyme and that an additional guanylate residue (at position -1) of tRNAHis in A. nidulans mitochondria is generated by a mode that is analogous to the generation of their counterparts in prokaryotes and chloroplasts.

The RNA component of mitochondrial ribonuclease P from Aspergillus nidulans.

Several RNA molecules that copurified with Aspergillus nidulans mitochondrial ribonuclease (RNase) P were identified [Lee, Y C., Lee, B. J., Hwang, D. S. & Kang, H. S. (1996) Eur J. Biochem. 235, 289-296], and their partial sequences were determined. Using an oligonucleotide probe, we cloned and mapped the gene encoding this putative RNA component of RNase P (RNase P-RNA), situated between URFA3 (unidentified reading frame A3) and cobA (apocytochrome b) genes in the mitochondrial genome of A. nidulans. The gene is extremely (A+T)-rich and contains two regions of sequence similarity conserved among the known mitochondrial RNase P-RNAs and the eubacterial RNase P-RNAs. The determination of 5' and 3' termini by primer extension and sequencing indicated that the length of the RNA transcript is 232 nucleotides. Northern-blot analysis revealed that its only subcellular location was the mitochondria. Two RNase P-RNA fragments of 110 nucleotides and 80 nucleotides, each containing one of the two conserved regions, could be recovered from the nuclease-treated enzyme without significant loss of activity. The sizes of these fragments appeared to be the minimum lengths required for the vitro activity of the enzyme.

RPM2, independently of its mitochondrial RNase P function, suppresses an ISP42 mutant defective in mitochondrial import and is essential for normal growth.

RPM2 is identified here as a high-copy suppressor of isp42-3, a temperature-sensitive mutant allele of the mitochondrial protein import channel component, Isp42p. RPM2 already has an established role as a protein component of yeast mitochondrial RNase P, a ribonucleoprotein enzyme required for the 5' processing of mitochondrial precursor tRNAs. A relationship between mitochondrial tRNA processing and protein import is not readily apparent, and, indeed, the two functions can be separated. Truncation mutants lacking detectable RNase P activity still suppress the isp42-3 growth defect. Moreover, RPM2 is required for normal fermentative yeast growth, even though mitochondrial RNase P activity is not. The portion of RPM2 required for normal growth and suppression of isp42-3 is the same. We conclude that RPM2 is a multifunctional gene. We find Rpm2p to be a soluble protein of the mitochondrial matrix and discuss models to explain its suppression of isp42-3.

Human Mitochondrial tRNA Processing

tRNA processing is a central event in mammalian mitochondrial gene expression. We have identified key enzymatic activities (ribonuclease P, precursor tRNA 3'-endonuclease, and ATP(CTP)-tRNA-specific nucleotidyltransferase) that are involved in HeLa cell mitochondrial tRNA maturation. Different mitochondrial tRNA precursors are cleaved precisely at the tRNA 5'- and 3'-ends in a homologous mitochondrial in vitro processing system. The cleavage at the 5'-end precedes that at the 3'-end, and the tRNAs are substrates for the specific CCA addition in the same in vitro system. Using a comparative enzymatic approach as well as biochemical and immunological techniques, we furthermore demonstrate that human cells contain two distinct enzymes that remove 5'-extensions from tRNA precursors, the previously characterized nuclear and the newly identified mitochondrial ribonuclease P. These two cellular isoenzymes have different substrate specificities that seem to be well adapted to their structurally disparate mitochondrial and nuclear tRNA substrates. This kind of approach may also help to understand the structural diversities and commonalities of tRNAs.