Identification of the yeast gene encoding the tRNA m1G methyltransferase responsible for modification at position 9

Methylation of tRNA at the N-1 position of guanosine to form m(1)G occurs widely in nature. It occurs at position 37 in tRNAs from all three kingdoms, and the methyltransferase that catalyzes this reaction is known from previous work of others to be critically important for cell growth in Escherichia coli and the yeast Saccharomyces cerevisiae. m(1)G is also widely found at position 9 in eukaryotic tRNAs, but the corresponding methyltransferase was unknown. We have used a biochemical genomics approach with a collection of purified yeast GST-ORF fusion proteins to show that m(1)G(9) formation of yeast tRNA(Gly) is associated with ORF YOL093w, named TRM10. Extracts lacking Trm10p have undetectable levels of m(1)G(9) methyltransferase activity but retain normal m(1)G(37) methyltransferase activity. Yeast Trm10p purified from E. coli quantitatively modifies the G(9) position of tRNA(Gly) in an S-adenosylmethionine-dependent fashion. Trm10p is responsible in vivo for most if not all m(1)G(9) modification of tRNAs, based on two results: tRNA(Gly) purified from a trm10-Delta/trm10-Delta strain is lacking detectable m(1)G; and a primer extension block occurring at m(1)G(9) is removed in trm10-Delta/trm10-Delta-derived tRNAs for all 9 m(1)G(9)-containing species that were testable by this method. There is no obvious growth defect of trm10-Delta/trm10-Delta strains. Trm10p bears no detectable resemblance to the yeast m(1)G(37) methyltransferase, Trm5p, or its orthologs. Trm10p homologs are found widely in eukaryotes and many archaea, with multiple homologs in several metazoans, including at least three in humans.     

A protein extension to shorten RNA: elongated elongation factor-Tu recognizes the D-arm of T-armless tRNAs in nematode mitochondria

Nematode mitochondria possess extremely truncated tRNAs. Of 22 tRNAs, 20 lack the entire T-arm. The T-arm is necessary for the binding of canonical tRNAs and EF (elongation factor)-Tu (thermo-unstable). The nematode mitochondrial translation system employs two different EF-Tu factors named EF-Tu1 and EF-Tu2. Our previous study showed that nematode Caenorhabditis elegans EF-Tu1 binds specifically to T-armless tRNA. C. elegans EF-Tu1 has a 57-amino acid C-terminal extension that is absent from canonical EF-Tu, and the T-arm-binding residues of canonical EF-Tu are not conserved. In this study, the recognition mechanism of T-armless tRNA by EF-Tu1 was investigated. Both modification interference assays and primer extension analysis of cross-linked ternary complexes revealed that EF-Tu1 interacts not only with the tRNA acceptor stem but also with the D-arm. This is the first example of an EF-Tu recognizing the D-arm of a tRNA. The binding activity of EF-Tu1 was impaired by deletion of only 14 residues from the C-terminus, indicating that the C-terminus of EF-Tu1 is required for its binding to T-armless tRNA. These results suggest that C. elegans EF-Tu1 recognizes the D-arm instead of the T-arm by a mechanism involving its C-terminal region. This study sheds light on the co-evolution of RNA and RNA-binding proteins in nematode mitochondria.     

A previously unidentified activity of yeast and mouse RNA:pseudouridine synthases 1 (Pus1p) on tRNAs

Mouse pseudouridine synthase 1 (mPus1p) was the first vertebrate RNA:pseudouridine synthase that was cloned and characterized biochemically. The mPus1p was previously found to catalyze Psi formation at positions 27, 28, 34, and 36 in in vitro produced yeast and human tRNAs. On the other hand, the homologous Saccharomyces cerevisiae scPus1p protein was shown to modify seven uridine residues in tRNAs (26, 27, 28, 34, 36, 65, and 67) and U44 in U2 snRNA. In this work, we expressed mPus1p in yeast cells lacking scPus1p and studied modification of U2 snRNA and several yeast tRNAs. Our data showed that, in these in vivo conditions, the mouse enzyme efficiently modifies yeast U2 snRNA at position 44 and tRNAs at positions 27, 28, 34, and 36. However, a tRNA:Psi26-synthase activity of mPus1p was not observed. Furthermore, we found that both scPus1p and mPus1p, in vivo and in vitro, have a previously unidentified activity at position 1 in cytoplasmic tRNAArg(ACG). This modification can take place in mature tRNA, as well as in pre-tRNAs with 5' and/or 3' extensions. Thus, we identified the protein carrying one of the last missing yeast tRNA:Psi synthase activities. In addition, our results reveal an additional activity of mPus1p at position 30 in tRNA that scPus1p does not possess.     

A subcomplex of human mitochondrial RNase P is a bifunctional methyltransferase—extensive moonlighting in mitochondrial tRNA biogenesis

Transfer RNAs (tRNAs) reach their mature functional form through several steps of processing and modification. Some nucleotide modifications affect the proper folding of tRNAs, and they are crucial in case of the non-canonically structured animal mitochondrial tRNAs, as exemplified by the apparently ubiquitous methylation of purines at position 9. Here, we show that a subcomplex of human mitochondrial RNase P, the endonuclease removing tRNA 5′ extensions, is the methyltransferase responsible for m¹G9 and m¹A9 formation. The ability of the mitochondrial tRNA:m¹R9 methyltransferase to modify both purines is uncommon among nucleic acid modification enzymes. In contrast to all the related methyltransferases, the human mitochondrial enzyme, moreover, requires a short-chain dehydrogenase as a partner protein. Human mitochondrial RNase P, thus, constitutes a multifunctional complex, whose subunits moonlight in cascade: a fatty and amino acid degradation enzyme in tRNA methylation and the methyltransferase, in turn, in tRNA 5′ end processing.     

Identities and phylogenetic comparisons of posttranscriptional modifications in 16 S ribosomal RNA from Haloferax volcanii

Small subunit (16 S) rRNA from the archaeon Haloferax volcanii, for which sites of modification were previously reported, was examined using mass spectrometry. A census of all modified residues was taken by liquid chromatography/electrospray ionization-mass spectrometry analysis of a total nucleoside digest of the rRNA. Following rRNA hydrolysis by RNase T(1), accurate molecular mass values of oligonucleotide products were measured using liquid chromatography/electrospray ionization-mass spectrometry and compared with values predicted from the corresponding gene sequence. Three modified nucleosides, distributed over four conserved sites in the decoding region of the molecule, were characterized: 3-(3-amino-3-carboxypropyl)uridine-966, N(6)-methyladenosine-1501, and N(6),N(6)-dimethyladenosine-1518 and -1519 (all Escherichia coli numbering). Nucleoside 3-(3-amino-3-carboxypropyl)uridine, previously unknown in rRNA, occurs at a highly conserved site of modification in all three evolutionary domains but for which no structural assignment in archaea has been previously reported. Nucleoside N(6)-methyladenosine, not previously placed in archaeal rRNAs, frequently occurs at the analogous location in eukaryotic small subunit rRNA but not in bacteria. H. volcanii small subunit rRNA appears to reflect the phenotypically low modification level in the Crenarchaeota kingdom and is the only cytoplasmic small subunit rRNA shown to lack pseudouridine.     

N2-methylation of guanosine at position 10 in tRNA is catalyzed by a THUMP domain-containing, S-adenosylmethionine-dependent methyltransferase, conserved in Archaea and Eukaryota

In sequenced genomes, genes belonging to the cluster of orthologous group COG1041 are exclusively, and almost ubiquitously, found in Eukaryota and Archaea but never in Bacteria. The corresponding gene products exhibit a characteristic Rossmann fold, S-adenosylmethionine-dependent methyltransferase domain in the C terminus and a predicted RNA-binding THUMP (thiouridine synthases, RNA methyltransferases, and pseudouridine synthases) domain in the N terminus. Recombinant PAB1283 protein from the archaeon Pyrococcus abyssi GE5, a member of COG1041, was purified and shown to behave as a monomeric 39-kDa entity. This protein (EC 2.1.1.32), now renamed (Pab)Trm-G10, which is extremely thermostable, forms a 1:1 complex with tRNA and catalyzes the adenosylmethionine-dependent methylation of the exocyclic amino group (N(2)) of guanosine located at position 10. Depending on the experimental conditions used, as well as the tRNA substrate tested, the enzymatic reaction leads to the formation of either N(2)-monomethyl (m(2)G) or N(2)-dimethylguanosine (m(2)(2)G). Interestingly, (Pab)Trm-G10 exhibits different domain organization and different catalytic site architecture from another, earlier characterized, tRNA-dimethyltransferase from Pyrococcus furiosus ((Pfu)Trm-G26, also known as (Pfu)Trm1, a member of COG1867) that catalyzes an identical two-step dimethylation of guanosine but at position 26 in tRNAs and is also conserved among all sequenced Eukaryota and Archaea. The co-occurrence of these two guanosine dimethyltransferases in both Archaea and Eukaryota but not in Bacteria is a hallmark of distinct tRNAs maturation strategies between these domains of life.     

Purification and isoelectric heterogeneity of chicken tyrosinase

Alterations in rat liver transfer RNA (tRNA) methyltransferase activities have been observed after liver damage by various chemicals or by partial hepatectomy. The qualitative and quantitative nature of these activity changes and the time course for their induction have been studied. Since homologous tRNAs are essentially fully modified in vivo, E. coli tRNAs were used as in vitro substrates for the rat liver enzymes in these studies. Each of the liver-damaging agents tested rapidly caused increases in activities of the enzyme(s) catalyzing methyl group transfer to tRNAs that have an unmodified guanine at position 26 from the 5' end of the molecule. This group of tRNAs includes E. coli tRNANfmet, tRNAAla1, tRNALeu1, or Leu2, and tRNASer3 (Group 1). In each case N2-methylguanine and N2,N2-dimethylguanine represented 90% or more of the products of these in vitro methylations. The product and substrate specificity observed are characteristic of N2-guanine methyltransferase II (S-adenosyl-L-methionine : tRNA (guanine-2)-methyltransferase, EC 2.1.1.32). In crude and partially purified preparations derived from livers of both control and treated animals this enzyme activity was not diminished significantly by exposure to 50 degrees C for min. The same liver-damaging agents induced little or no change in the activities of enzymes that catalyze methyl group transfer to various other E. coli tRNAs that do not have guanine at position 26 (Group 2). The results of mixing experiments appear to rule out the likelihood that the observed enzyme activity changes are due to stimulatory or inhibitory materials present in the enzyme preparations from control or treated animals. Thus, our experiments indicate that liver damage by each of several different methods, including surgery or administration of chemicals that are strong carcinogens, hepatotoxins, or cancer-promoting substances, all produce changes in liver tRNA methyltransferase activity that represent a selective increase in activity of N2-guanine tRNA methyltransferase II. It is proposed that the specificity of this change is not fortuitous, but is the manifestation of an as yet unidentified regulatory process.     

Purification, structure, and properties of Escherichia coli tRNA pseudouridine synthase I

The RNA modification enzyme, tRNA pseudouridine synthase I has been isolated in 95% purity from an Escherichia coli strain harboring a multicopy plasmid with a 2.3-kilobase pair insert from the hisT operon. Its molecular size, amino acid composition, and amino-terminal sequence correspond to those predicted by the structure and expression of the hisT gene. Enzyme activity, as measured by a 3H release assay, is unaffected by pretreatment of tRNA pseudouridine synthase I with micrococcal nuclease and is optimized by the addition of a monovalent cation and thiol reductant. The activity is inhibited by all tRNA species tested, including substrates, modified tRNAs, nonsubstrates, or tRNAs containing 5-fluorouridine. Binding of tRNA pseudouridine synthase I occurs with both substrate and nonsubstrate tRNAs and does not require a monovalent cation. Our findings are consistent with a multistep mechanism whereby tRNA pseudouridine synthase I first binds nonspecifically and then forms transient covalent adducts with tRNA substrates. In the absence of other proteins, purified tRNA pseudouridine synthase I forms psi at all three modification sites known to be affected in hisT mutants. The 36.4-kDa polypeptide product of the gene adjacent to hisT, whose translation is linked to that of tRNA pseudouridine synthase I, is not a functional subunit for tRNA pseudouridine synthase I activity, nor is it a separate synthase acting at one of the three loci.     

Structural insight into the functional mechanism of Nep1/Emg1 N1-specific pseudouridine methyltransferase in ribosome biogenesis

Nucleolar Essential Protein 1 (Nep1) is required for small subunit (SSU) ribosomal RNA (rRNA) maturation and is mutated in Bowen-Conradi Syndrome. Although yeast (Saccharomyces cerevisiae) Nep1 interacts with a consensus sequence found in three regions of SSU rRNA, the molecular details of the interaction are unknown. Nep1 is a SPOUT RNA methyltransferase, and can catalyze methylation at the N1 of pseudouridine. Nep1 is also involved in assembly of Rps19, an SSU ribosomal protein. Mutations in Nep1 that result in decreased methyl donor binding do not result in lethality, suggesting that enzymatic activity may not be required for function, and RNA binding may play a more important role. To study these interactions, the crystal structures of the scNep1 dimer and its complexes with RNA were determined. The results demonstrate that Nep1 recognizes its RNA site via base-specific interactions and stabilizes a stem-loop in the bound RNA. Furthermore, the RNA structure observed contradicts the predicted structures of the Nep1-binding sites within mature rRNA, suggesting that the Nep1 changes rRNA structure upon binding. Finally, a uridine base is bound in the active site of Nep1, positioned for a methyltransfer at the C5 position, supporting its role as an N1-specific pseudouridine methyltransferase.     

Identification and characterization of the tRNA:Psi 31-synthase (Pus6p) of Saccharomyces cerevisiae.

To characterize the substrate specificity of the putative RNA:pseudouridine (Psi)-synthase encoded by the Saccharomyces cerevisiae open reading frame (ORF) YGR169c, the corresponding gene was deleted in yeast, and the consequences of the deletion on tRNA and small nuclear RNA modification were tested. The resulting DeltaYGR169c strain showed no detectable growth phenotype, and the only difference in Psi formation in stable cellular RNAs was the absence of Psi at position 31 in cytoplasmic and mitochondrial tRNAs. Complementation of the DeltaYGR169c strain by a plasmid bearing the wild-type YGR169c ORF restored Psi(31) formation in tRNA, whereas a point mutation of the enzyme active site (Asp(168)-->Ala) abolished tRNA:Psi(31)-synthase activity. Moreover, recombinant His(6)-tagged Ygr169 protein produced in Escherichia coli was capable of forming Psi(31) in vitro using tRNAs extracted from the DeltaYGR169c yeast cells as substrates. These results demonstrate that the protein encoded by the S. cerevisiae ORF YGR169c is the Psi-synthase responsible for modification of cytoplasmic and mitochondrial tRNAs at position 31. Because this is the sixth RNA:Psi-synthase characterized thus far in yeast, we propose to rename the corresponding gene PUS6 and the expressed protein Pus6p. Finally, the cellular localization of the green fluorescent protein-tagged Pus6p was studied by functional tests and direct fluorescence microscopy.     

Identity elements for N2-dimethylation of guanosine-26 in yeast tRNAs.

N2,N2-dimethylguanosine (m2(2)G) is a characteristic nucleoside that is found in the bend between the dihydro-uridine (D) stem and the anticodon (AC) stem in over 80% of the eukaryotic tRNA species having guanosine at position 26 (G26). However, since a few eukaryotic tRNAs have an unmodified G in that position, G26 is a necessary but not a sufficient condition for dimethylation. In yeast tRNA(Asp) G26 is unmodified. We have successively changed the near surroundings of G26 in this tRNA until G26 became modified to m2(2)G by a tRNA(m2(2)G26)methyltransferase in Xenopus laevis oocytes. In this way we have identified the two D-stem basepairs C11-G24, G10-C25 immediately preceding G26 as major identity elements for the dimethylating enzyme modifying G26. Furthermore, increasing the extra loop in tRNA(Asp) from four to the more usual five bases influenced the global structure of the tRNA such that the m2(2)G26 formation was drastically decreased even if the near region of G26 had the two consensus basepairs. We conclude that not only are the two consensus base pairs in the D-stem a prerequisite for G26 modification, but also is any part of the tRNA molecule that influence the 3D-structure important for the recognition between nuclear coded tRNAs and the tRNA(m2(2)G26)methyltransferase.     

N2,N2-dimethylguanosine-specific tRNA methyltransferase contains both nuclear and mitochondrial targeting signals in Saccharomyces cerevisiae

The TRM1 gene of Saccharomyces cerevisiae encodes a tRNA modification enzyme, N2,N2-dimethylguanosine-specific tRNA methyltransferase, which modifies both mitochondrial and cytoplasmic tRNAs. The enzyme is targeted to mitochondria for the modification of mitochondrial tRNAs. Cellular fractionation and indirect immunofluorescence studies reported here demonstrate that this enzyme is also localized to the nucleus. Further, immunofluorescence experiments using strains that overproduce the enzyme show a staining at the periphery of the nucleus suggesting that the enzyme is found in a subnuclear destination near or at the nuclear membrane. There is no obvious cytoplasmic staining in these overproducing strains. Fusion protein technology was used to begin to localize sequences involved in the nuclear targeting of this enzyme. Indirect immunofluorescence studies indicate that sequences between the first 70 and 213 NH2-terminal amino acids of the methyltransferase are sufficient to target Escherichia coli beta-galactosidase to nuclei.     

Identification of pseudouridine methyltransferase in Escherichia coli

In ribosomal RNA, modified nucleosides are found in functionally important regions, but their function is obscure. Stem–loop 69 of Escherichia coli 23S rRNA contains three modified nucleosides: pseudouridines at positions 1911 and 1917, and N3 methyl-pseudouridine (m³Ψ) at position 1915. The gene for pseudouridine methyltransferase was previously not known. We identified E. coli protein YbeA as the methyltransferase methylating Ψ1915 in 23S rRNA. The E. coli ybeA gene deletion strain lacks the N3 methylation at position 1915 of 23S rRNA as revealed by primer extension and nucleoside analysis by HPLC. Methylation at position 1915 is restored in the ybeA deletion strain when recombinant YbeA protein is expressed from a plasmid. In addition, we show that purified YbeA protein is able to methylate pseudouridine in vitro using 70S ribosomes but not 50S subunits from the ybeA deletion strain as substrate. Pseudouridine is the preferred substrate as revealed by the inability of YbeA to methylate uridine at position 1915. This shows that YbeA is acting at the final stage during ribosome assembly, probably during translation initiation. Hereby, we propose to rename the YbeA protein to RlmH according to uniform nomenclature of RNA methyltransferases. RlmH belongs to the SPOUT superfamily of methyltransferases. RlmH was found to be well conserved in bacteria, and the gene is present in plant and in several archaeal genomes. RlmH is the first pseudouridine specific methyltransferase identified so far and is likely to be the only one existing in bacteria, as m³Ψ1915 is the only methylated pseudouridine in bacteria described to date.     

Pseudouridine formation in archaeal RNAs: The case of Haloferax volcanii

Pseudouridine (Ψ), the isomer of uridine, is commonly found at various positions of noncoding RNAs of all organisms. Ψ residues are formed by a number of single- or multisite specific Ψ synthases, which generally act as stand-alone proteins. In addition, in Eukarya and Archaea, specific ribonucleoprotein complexes, each containing a distinct box H/ACA guide RNA and four core proteins, can produce Ψ at many sites of different cellular RNAs. Cbf5 is the core Ψ synthase in these complexes. Using Haloferax volcanii as an archaeal model organism, we show that, contrary to eukaryotes, the Cbf5 homolog (HVO_2493) is not essential in this archaeon. The Cbf5-deleted strain of H. volcanii completely lacks Ψ at positions 1940, 1942, 2605, and 2591 (Escherichia coli positions 1915, 1917, 2572, and 2586) of its 23S rRNA, and contains reduced steady-state levels of some box H/ACA RNAs. Archaeal Cbf5 is known to have tRNA Ψ55 synthase activity in vitro but we could not confirm this activity in vivo in H. volcanii. Conversely, the Pus10 (previously PsuX) homolog (HVO_1979), which can produce tRNA Ψ55, as well as Ψ54 in vitro, is shown here to be essential in H. volcanii, whereas the corresponding tRNA Ψ55 synthases, Pus4 and TruB, are not essential in yeast and E. coli, respectively. Finally, we demonstrate that HVO_1852, the TruA/Pus3 homolog, is responsible for the pseudouridylation of position 39 in H. volcanii tRNAs and that the corresponding gene is not essential.