The yeast rapid tRNA decay pathway competes with elongation factor 1A for substrate tRNAs and acts on tRNAs lacking one or more of several modifications

The structural and functional integrity of tRNA is crucial for translation. In the yeast Saccharomyces cerevisiae, certain aberrant pre-tRNA species are subject to nuclear surveillance, leading to 3' exonucleolytic degradation, and certain mature tRNA species are subject to rapid tRNA decay (RTD) if they are appropriately hypomodified or bear specific destabilizing mutations, leading to 5'-3' exonucleolytic degradation by Rat1 and Xrn1. Thus, trm8-Δ trm4-Δ strains are temperature sensitive due to lack of m(7)G(46) and m(5)C and the consequent RTD of tRNA(Val(AAC)), and tan1-Δ trm44-Δ strains are temperature sensitive due to lack of ac(4)C(12) and Um(44) and the consequent RTD of tRNA(Ser(CGA)) and tRNA(Ser(UGA)). It is unknown how the RTD pathway interacts with translation and other cellular processes, and how generally this pathway acts on hypomodified tRNAs. We provide evidence here that elongation factor 1A (EF-1A) competes with the RTD pathway for substrate tRNAs, since its overexpression suppresses the tRNA degradation and the growth defect of strains subject to RTD, whereas reduced levels of EF-1A have the opposite effect. We also provide evidence that RTD acts on a variety of tRNAs lacking one or more different modifications, since trm1-Δ trm4-Δ mutants are subject to RTD of tRNA(Ser(CGA)) and tRNA(Ser(UGA)) due to lack of m(2,2)G(26) and m(5)C, and since trm8-Δ, tan1-Δ, and trm1-Δ single mutants are each subject to RTD. These results demonstrate that RTD interacts with the translation machinery and acts widely on hypomodified tRNAs.     

Point and deletion mutations eliminate one or both methyl group transfers catalysed by the yeast TRM1 encoded tRNA (m22G26)dimethyltransferase.

Guanosine at position 26 in eukaryotic tRNAs is usually modified to N2 , N2 -dimethylguanosine (m22G26). In Saccharomyces cerevisiae , this reaction is catalysed by the TRM1 encoded tRNA (m22G26)dimethyltransferase. As a prerequisite for future studies, the yeast TRM1 gene was expressed in Escherichia coli and the His-tagged Trm1 protein (rTrm1p) was extensively purified. rTrm1p catalysed both the mono- and dimethylation of G26 in vivo in Escherichia coli tRNA and in vitro in yeast trm1 mutant tRNA. The TRM1 gene from two independent wild-type yeast strains differed at 14 base positions causing two amino acid exchanges . Exchange of the original Ser467 for Leu caused a complete loss of enzyme activity in vitro against trm1 yeast tRNA. Comparatively short N- or C-terminal deletions from the 570 amino acid long Trm1 polypeptide decreased or eliminated the enzyme activity, as did some point mutations within these regions. This indicated that the protein is not a two domain peptide with the enzyme activity localised to one of the domains, but rather that both ends of the polypeptide seem to interact to influence the conformation of those parts that make up the RNA-binding site and/or the active site of the enzyme.     

Yeast mitochondrial initiator tRNA is methylated at guanosine 37 by the Trm5-encoded tRNA (guanine-N1-)-methyltransferase

The TRM5 gene encodes a tRNA (guanine-N1-)-methyltransferase (Trm5p) that methylates guanosine at position 37 (m(1)G37) in cytoplasmic tRNAs in Saccharomyces cerevisiae. Here we show that Trm5p is also responsible for m(1)G37 methylation of mitochondrial tRNAs. The TRM5 open reading frame encodes 499 amino acids containing four potential initiator codons within the first 48 codons. Full-length Trm5p, purified as a fusion protein with maltose-binding protein, exhibited robust methyltransferase activity with tRNA isolated from a Delta trm5 mutant strain, as well as with a synthetic mitochondrial initiator tRNA (tRNA(Met)(f)). Primer extension demonstrated that the site of methylation was guanosine 37 in both mitochondrial tRNA(Met)(f) and tRNA(Phe). High pressure liquid chromatography analysis showed the methylated product to be m(1)G. Subcellular fractionation and immunoblotting of a strain expressing a green fluorescent protein-tagged version of the TRM5 gene revealed that the enzyme was localized to both cytoplasm and mitochondria. The slightly larger mitochondrial form was protected from protease digestion, indicating a matrix localization. Analysis of N-terminal truncation mutants revealed that a Trm5p active in the cytoplasm could be obtained with a construct lacking amino acids 1-33 (Delta1-33), whereas production of a Trm5p active in the mitochondria required these first 33 amino acids. Yeast expressing the Delta1-33 construct exhibited a significantly lower rate of oxygen consumption, indicating that efficiency or accuracy of mitochondrial protein synthesis is decreased in cells lacking m(1)G37 methylation of mitochondrial tRNAs. These data suggest that this tRNA modification plays an important role in reading frame maintenance in mitochondrial protein synthesis.     

tRNA m7G methyltransferase Trm8p/Trm82p: evidence linking activity to a growth phenotype and implicating Trm82p in maintaining levels of active Trm8p

We show that Saccharomyces cerevisiae strains lacking Trm8p/Trm82p tRNA m7G methyltransferase are temperature-sensitive in synthetic media containing glycerol. Bacterial TRM8 orthologs complement the growth defect of trm8-Delta, trm82-Delta, and trm8-Delta trm82-Delta double mutants, suggesting that bacteria employ a single subunit for Trm8p/Trm82p function. The growth phenotype of trm8 mutants correlates with lack of tRNA m7G methyltransferase activity in vitro and in vivo, based on analysis of 10 mutant alleles of trm8 and bacterial orthologs, and suggests that m7G modification is the cellular function important for growth. Initial examination of the roles of the yeast subunits shows that Trm8p has most of the functions required to effect m7G modification, and that a major role of Trm82p is to maintain cellular levels of Trm8p. Trm8p efficiently cross-links to pre-tRNAPhe in vitro in the presence or absence of Trm82p, in addition to its known residual tRNA m7G modification activity and its SAM-binding domain. Surprisingly, the levels of Trm8p, but not its mRNA, are severely reduced in a trm82-Delta strain. Although Trm8p can be produced in the absence of Trm82p by deliberate overproduction, the resulting protein is inactive, suggesting that a second role of Trm82p is to stabilize Trm8p in an active conformation.     

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.     

The 2'-O-methyltransferase responsible for modification of yeast tRNA at position 4

The methylation of the ribose 2'-OH of RNA occurs widely in nature and in all stable RNAs and occurs at five positions in yeast tRNA. 2'-O-methylation of tRNA at position 4 is interesting because it occurs in the acceptor stem (which is normally undermodified), it is the only 2'-O-methylation that occurs in the middle of a duplex region in tRNA, the modification is conserved in eukaryotes, and the features of the tRNA necessary for substrate recognition are poorly defined. We show here that Saccharomyces cerevisiae ORF YOL125w (TRM13) is necessary and sufficient for 2'-O-methylation at position 4 of yeast tRNA. Biochemical analysis of the S. cerevisiae proteome shows that Trm13 copurifies with 2'-O-methylation activity, using tRNAGlyGCC as a substrate, and extracts made from a trm13-Delta strain have undetectable levels of this activity. Trm13 is necessary for activity in vivo because tRNAs isolated from a trm13-Delta strain lack the corresponding 2'-O-methylated residue for each of the three known tRNAs with this modification. Trm13 is sufficient for 2'-O-methylation at position 4 in vitro since yeast Trm13 protein purified after expression in Escherichia coli has the same activity as that produced in yeast. Trm13 protein binds substrates tRNAHis and tRNAGlyGCC with KD values of 85+/-8 and 100+/-14 nM, respectively, and has a KM for tRNAHis of 10 nM, but binds nonsubstrate tRNAs very poorly (KD>1 microM). Trm13 is conserved in eukaryotes, but there is no sequence similarity between Trm13 and other known methyltransferases.     

Rapid tRNA decay can result from lack of nonessential modifications

The biological role of many nonessential tRNA modifications outside of the anticodon remains elusive despite their evolutionary conservation. We show here that m7G46 methyltransferase Trm8p/Trm82p acts as a hub of synthetic interactions with several tRNA modification enzymes, resulting in temperature-sensitive growth. Analysis of three double mutants indicates reduced levels of tRNA(Val(AAC)), consistent with a role of the corresponding modifications in maintenance of tRNA levels. Detailed examination of a trm8-delta trm4-delta double mutant demonstrates rapid degradation of preexisting tRNA(Val(AAC)) accompanied by its de-aminoacylation. Multiple copies of tRNA(Val(AAC)) suppress the trm8-delta trm4-delta growth defect, directly implicating this tRNA in the phenotype. These results define a rapid tRNA degradation (RTD) pathway that is independent of the TRF4/RRP6-dependent nuclear surveillance pathway. The degradation of an endogenous tRNA species at a rate typical of mRNA decay demonstrates a critical role of nonessential modifications for tRNA stability and cell survival.     

A domain of the actin binding protein Abp140 is the yeast methyltransferase responsible for 3-methylcytidine modification in the tRNA anti-codon loop

The 3-methylcytidine (m3C) modification is widely found in eukaryotic species of tRNA(Ser), tRNA(Thr), and tRNA(Arg); at residue 32 in the anti-codon loop; and at residue e2 in the variable stem of tRNA(Ser). Little is known about the function of this modification or about the specificity of the corresponding methyltransferase, since the gene has not been identified. We have used a primer extension assay to screen a battery of methyltransferase candidate knockout strains in the yeast Saccharomyces cerevisiae, and find that tRNA(Thr(IGU)) from abp140-Δ strains lacks m3C. Curiously, Abp140p is composed of a poorly conserved N-terminal ORF fused by a programed +1 frameshift in budding yeasts to a C-terminal ORF containing an S-adenosylmethionine (SAM) domain that is highly conserved among eukaryotes. We show that ABP140 is required for m3C modification of substrate tRNAs, since primer extension is similarly affected for all tRNA species expected to have m3C and since quantitative analysis shows explicitly that tRNA(Thr(IGU)) from an abp140-Δ strain lacks m3C. We also show that Abp140p (now named Trm140p) purified after expression in yeast or Escherichia coli has m3C methyltransferase activity, which is specific for tRNA(Thr(IGU)) and not tRNA(Phe) and occurs specifically at C??. We suggest that the C-terminal ORF of Trm140p is necessary and sufficient for activity in vivo and in vitro, based on analysis of constructs deleted for most or all of the N-terminal ORF. We also suggest that m3C has a role in translation, since trm140-Δ trm1-Δ strains (also lacking m2,2G??) are sensitive to low concentrations of cycloheximide.     

Identification of the TRM2 gene encoding the tRNA(m5U54)methyltransferase of Saccharomyces cerevisiae

The presence of 5-methyluridine (m5U) at position 54 is a ubiquitous feature of most bacterial and eukaryotic elongator tRNAs. In this study, we have identified and characterized the TRM2 gene that encodes the tRNA(m5U54)methyltransferase, responsible for the formation of this modified nucleoside in Saccharomyces cerevisiae. Transfer RNA isolated from TRM2-disrupted yeast strains does not contain the m5U54 nucleoside. Moreover, a glutathione S-transferase (GST) tagged recombinant, Trm2p, expressed in Escherichia coli displayed tRNA(m5U54)methyltransferase activity using as substrate tRNA isolated from a trm2 mutant strain, but not tRNA isolated from a TRM2 wild-type strain. In contrast to what is found for the tRNA(m5U54)methyltransferase encoding gene trmA+ in E. coli, the TRM2 gene is not essential for cell viability and a deletion strain shows no obvious phenotype. Surprisingly, we found that the TRM2 gene was previously identified as the RNC1/NUD1 gene, believed to encode the yNucR endo-exonuclease. The expression and activity of the yNucR endo-exonuclease is dependent on the RAD52 gene, and does not respond to increased gene dosage of the RNC1/NUD1 gene. In contrast, we find that the expression of a trm2-LacZ fusion and the activity of the tRNA(m5U54)methyltransferase is not regulated by the RAD52 gene and does respond on increased gene dosage of the TRM2 (RNC1/NUD1) gene. Furthermore, there was no nuclease activity associated with a GST-Trm2 recombinant protein. The purified yNucR endo-exonuclease has been reported to have an NH2-D-E-K-N-L motif, which is not found in the Trm2p. Therefore, we suggest that the yNucR endo-exonuclease is encoded by a gene other than TRM2.     

Conserved amino acids in each subunit of the heteroligomeric tRNA m1A58 Mtase from Saccharomyces cerevisiae contribute to tRNA binding

In Saccharomyces cerevisiae, a two-subunit methyltransferase (Mtase) encoded by the essential genes TRM6 and TRM61 is responsible for the formation of 1-methyladenosine, a modified nucleoside found at position 58 in tRNA that is critical for the stability of tRNA(Met)i The crystal structure of the homotetrameric m1A58 tRNA Mtase from Mycobacterium tuberculosis, TrmI, has been solved and was used as a template to build a model of the yeast m1A58 tRNA Mtase heterotetramer. We altered amino acids in TRM6 and TRM61 that were predicted to be important for the stability of the heteroligomer based on this model. Yeast strains expressing trm6 and trm61 mutants exhibited growth phenotypes indicative of reduced m1A formation. In addition, recombinant mutant enzymes had reduced in vitro Mtase activity. We demonstrate that the mutations introduced do not prevent heteroligomer formation and do not disrupt binding of the cofactor S-adenosyl-L-methionine. Instead, amino acid substitutions in either Trm6p or Trm61p destroy the ability of the yeast m1A58 tRNA Mtase to bind tRNA(Met)i, indicating that each subunit contributes to tRNA binding and suggesting a structural alteration of the substrate-binding pocket occurs when these mutations are present.     

Unexpected expansion of tRNA substrate recognition by the yeast m1G9 methyltransferase Trm10

N-1 Methylation of the nearly invariant purine residue found at position 9 of tRNA is a nucleotide modification found in multiple tRNA species throughout Eukarya and Archaea. First discovered in Saccharomyces cerevisiae, the tRNA methyltransferase Trm10 is a highly conserved protein both necessary and sufficient to catalyze all known instances of m¹G₉ modification in yeast. Although there are 19 unique tRNA species that contain a G at position 9 in yeast, and whose fully modified sequence is known, only 9 of these tRNA species are modified with m¹G₉ in wild-type cells. The elements that allow Trm10 to distinguish between structurally similar tRNA species are not known, and sequences that are shared between all substrate or all nonsubstrate tRNAs have not been identified. Here, we demonstrate that the in vitro methylation activity of yeast Trm10 is not sufficient to explain the observed pattern of modification in vivo, as additional tRNA species are substrates for Trm10 m¹G₉ methyltransferase activity. Similarly, overexpression of Trm10 in yeast yields m¹G₉ containing tRNA species that are ordinarily unmodified in vivo. Thus, yeast Trm10 has a significantly broader tRNA substrate specificity than is suggested by the observed pattern of modification in wild-type yeast. These results may shed light onto the suggested involvement of Trm10 in other pathways in other organisms, particularly in higher eukaryotes that contain up to three different genes with sequence similarity to the single TRM10 gene in yeast, and where these other enzymes have been implicated in pathways beyond tRNA processing.     

Trm11p and Trm112p are both required for the formation of 2-methylguanosine at position 10 in yeast tRNA

N(2)-Monomethylguanosine-10 (m(2)G10) and N(2),N(2)-dimethylguanosine-26 (m(2)(2)G26) are the only two guanosine modifications that have been detected in tRNA from nearly all archaea and eukaryotes but not in bacteria. In Saccharomyces cerevisiae, formation of m(2)(2)G26 is catalyzed by Trm1p, and we report here the identification of the enzymatic activity that catalyzes the formation of m(2)G10 in yeast tRNA. It is composed of at least two subunits that are associated in vivo: Trm11p (Yol124c), which is the catalytic subunit, and Trm112p (Ynr046w), a putative zinc-binding protein. While deletion of TRM11 has no detectable phenotype under laboratory conditions, deletion of TRM112 leads to a severe growth defect, suggesting that it has additional functions in the cell. Indeed, Trm112p is associated with at least four proteins: two tRNA methyltransferases (Trm9p and Trm11p), one putative protein methyltransferase (Mtc6p/Ydr140w), and one protein with a Rossmann fold dehydrogenase domain (Lys9p/Ynr050c). In addition, TRM11 interacts genetically with TRM1, thus suggesting that the absence of m(2)G10 and m(2)(2)G26 affects tRNA metabolism or functioning.     

The primary target of the killer toxin from Pichia acaciae is tRNA(Gln)

The Pichia acaciae killer toxin (PaT) arrests yeast cells in the S-phase of the cell cycle and induces DNA double-strand breaks (DSBs). Surprisingly, loss of the tRNA-methyltransferase Trm9 – along with the Elongator complex involved in synthesis of 5-methoxy-carbonyl-methyl (mcm⁵) modification in certain tRNAs – conferred resistance against PaT. Overexpression of mcm⁵-modified tRNAs identified tRNA^Gln_(UUG) as the intracellular target. Consistently, toxin-challenged cells displayed reduced levels of tRNA^Gln and in vitro the heterologously expressed active toxin subunit disrupts the integrity of tRNA^Gln_(UUG). Other than Kluyveromyces lactis zymocin, an endonuclease specific for tRNA^Glu_(UUC), affecting its target in a mcm⁵-dependent manner, PaT exerts activity also on tRNA^Gln lacking such modification. As sensitivity is restored in trm9 elp3 double mutants, target tRNA cleavage is selectively inhibited by incomplete wobble uridine modification, as seen in trm9 , but not in elp3 or trm9 elp3 cells. In addition to tRNA^Gln_(UUG), tRNA^Gln_(CUG) is also cleaved in vitro and overexpression of the corresponding gene increased resistance. Consistent with tRNA^Gln_(CUG) as an additional TRM9 -independent target, overexpression of PaT's tRNase subunit abolishes trm9 resistance. Most interestingly, a functional DSB repair pathway confers PaT but also zymocin resistance, suggesting DNA damage to occur generally concomitant with specific tRNA offence.     

The human tRNA(m(2)(2)G(26))dimethyltransferase: functional expression and characterization of a cloned hTRM1 gene

This paper presents the first example of a complete gene sequence coding for and expressing a biologically functional human tRNA methyltransferase: the hTRM1 gene product tRNA(m²₂G)dimethyltransferase. We isolated a human cDNA (1980 bp) made from placental mRNA coding for the full-length (659 amino acids) human TRM1 polypeptide. The sequence was fairly similar to Saccharomyces cerevisiae Trm1p, to Caenorhabditis elegans TRM1p and to open reading frames (ORFs) found in mouse and a plant (Arabidopsis thaliana) DNA. The human TRM1 gene was expressed at low temperature in Escherichia coli as a functional recombinant protein, able to catalyze the formation of dimethylguanosine in E.coli tRNA in vivo. It targeted solely position G₂₆ in T7 transcribed spliced and unspliced human tRNA^Tyr in vitro and in yeast trm1 mutant tRNA. Thus, the human TRM1 protein is a tRNA(m²₂G₂₆)dimethyltransferase. Compared with yeast Trm1p, hTRM1p has a C-terminal protrusion of ~90 amino acids which shows similarities to a mouse protein related to RNA splicing. A deletion of these 90 C-terminal amino acids left the modification activity in vitro intact. Among point mutations in the hTRM1 gene, only those located in conserved regions of hTRM1p completely eliminated modification activity.