Recognition of guanosine by dissimilar tRNA methyltransferases

Guanosines are important for biological activities through their specific functional groups that are recognized for RNA or protein interactions. One example is recognition of N(1) of G37 in tRNA by S-adenosyl-methionine (AdoMet)-dependent tRNA methyltransferases to synthesize m(1)G37-tRNA, which is essential for translational fidelity in all biological domains. Synthesis of m(1)G37-tRNA is catalyzed by TrmD in bacteria and by Trm5 in eukarya and archaea, using unrelated and dissimilar structural folds. This raises the question of how dissimilar proteins recognize the same guanosine. Here we probe the mechanism of discrimination among functional groups of guanosine by TrmD and Trm5. Guanosine analogs were systematically introduced into tRNA through a combination of chemical and enzymatic synthesis. Single turnover kinetic assays and thermodynamic analysis of the effect of each analog on m(1)G37-tRNA synthesis reveal that TrmD and Trm5 discriminate functional groups differently. While both recognize N(1) and O(6) of G37, TrmD places a much stronger emphasis on these functional groups than Trm5. While the exocyclic 2-amino group of G37 is important for TrmD, it is dispensable for Trm5. In addition, while an adjacent G36 is obligatory for TrmD, it is nonessential for Trm5. These results depict a more rigid requirement of guanosine functional groups for TrmD than for Trm5. However, the sensitivity of both enzymes to analog substitutions, together with an experimental revelation of their low cellular concentrations relative to tRNA substrates, suggests a model in which these enzymes rapidly screen tRNA by direct recognition of G37 in order to monitor the global state of m(1)G37-tRNA.     

NSUN6 is a human RNA methyltransferase that catalyzes formation of m5C72 in specific tRNAs

Many cellular RNAs require modification of specific residues for their biogenesis, structure, and function. 5-methylcytosine (m⁵C) is a common chemical modification in DNA and RNA but in contrast to the DNA modifying enzymes, only little is known about the methyltransferases that establish m⁵C modifications in RNA. The putative RNA methyltransferase NSUN6 belongs to the family of Nol1/Nop2/SUN domain (NSUN) proteins, but so far its cellular function has remained unknown. To reveal the target spectrum of human NSUN6, we applied UV crosslinking and analysis of cDNA (CRAC) as well as chemical crosslinking with 5-azacytidine. We found that human NSUN6 is associated with tRNAs and acts as a tRNA methyltransferase. Furthermore, we uncovered tRNA^Cys and tRNA^Thr as RNA substrates of NSUN6 and identified the cytosine C72 at the 3′ end of the tRNA acceptor stem as the target nucleoside. Interestingly, target recognition in vitro depends on the presence of the 3′-CCA tail. Together with the finding that NSUN6 localizes to the cytoplasm and largely colocalizes with marker proteins for the Golgi apparatus and pericentriolar matrix, our data suggest that NSUN6 modifies tRNAs in a late step in their biogenesis.     

Two proteins that form a complex are required for 7-methylguanosine modification of yeast tRNA

7-methylguanosine (m7G) modification of tRNA occurs widely in eukaryotes and bacteria, is nearly always found at position 46, and is one of the few modifications that confers a positive charge to the base. Screening of a Saccharomyces cerevisiae genomic library of purified GST-ORF fusion proteins reveals two previously uncharacterized proteins that copurify with m7G methyltransferase activity on pre-tRNA(Phe). ORF YDL201w encodes Trm8, a protein that is highly conserved in prokaryotes and eukaryotes and that contains an S-adenosylmethionine binding domain. ORF YDR165w encodes Trm82, a less highly conserved protein containing putative WD40 repeats, which are often implicated in macromolecular interactions. Neither protein has significant sequence similarity to yeast Abd1, which catalyzes m7G modification of the 5' cap of mRNA, other than the methyltransferase motif shared by Trm8 and Abd1. Several lines of evidence indicate that both Trm8 and Trm82 proteins are required for tRNA m7G-methyltransferase activity: Extracts derived from strains lacking either gene have undetectable m7G methyltransferase activity, RNA from strains lacking either gene have much reduced m7G, and coexpression of both proteins is required to overproduce activity. Aniline cleavage mapping shows that Trm8/Trm82 proteins modify pre-tRNAPhe at G46, the site that is modified in vivo. Trm8 and Trm82 proteins form a complex, as affinity purification of Trm8 protein causes copurification of Trm82 protein in approximate equimolar yield. This functional two-protein family appears to be retained in eukaryotes, as expression of both corresponding human proteins, METTL1 and WDR4, is required for m7G-methyltransferase activity.     

Bioinformatic analyses of the tRNA: (guanine 26, N2,N2)-dimethyltransferase (Trm1) family

Functional analyses of the tRNA:(guanine 26, N2,N2)-dimethyltransferase (Trm1) have been hampered by a lack of structural information about the enzyme and by low sequence similarity to better studied methyltransferases. Here we used computational methods to detect novel homologs of Trm1, infer the evolutionary relationships of the family, and predict the structure of the Trm1 methyltransferase. The N-terminal region of the protein is predicted to form an S-adenosylmethionine-binding domain, which harbors the active site. The C-terminal region is rich in predicted alpha-helices and, in analogy to other nucleic acid methyltransferases, may constitute the target recognition domain of the enzyme. Interposing these two domains, most Trm1 homologs possess a highly variable inserted sequence that is delimited by a Cys4 cluster, likely forming a Zn-finger structure. The residues of Trm1 predicted to participate in cofactor binding, target recognition, and catalysis, were mapped onto a preliminary structural model, providing a platform for designing new experiments to better understand the molecular functions of this protein family. In addition, identification of novel, atypical Trm1 homologs suggests candidates for cloning and biochemical characterization.     

New archaeal methyltransferases forming 1-methyladenosine or 1-methyladenosine and 1-methylguanosine at position 9 of tRNA

Two archaeal tRNA methyltransferases belonging to the SPOUT superfamily and displaying unexpected activities are identified. These enzymes are orthologous to the yeast Trm10p methyltransferase, which catalyses the formation of 1-methylguanosine at position 9 of tRNA. In contrast, the Trm10p orthologue from the crenarchaeon Sulfolobus acidocaldarius forms 1-methyladenosine at the same position. Even more surprisingly, the Trm10p orthologue from the euryarchaeon Thermococcus kodakaraensis methylates the N¹-atom of either adenosine or guanosine at position 9 in different tRNAs. This is to our knowledge the first example of a tRNA methyltransferase with a broadened nucleoside recognition capability. The evolution of tRNA methyltransferases methylating the N¹ atom of a purine residue is discussed.     

Assembly of protein-RNA complexes using natural RNA and mutant forms of an RNA cytosine methyltransferase

This work reveals that mutant forms of RNA methyltransferases that form 5-methylcytosine (m5C) have characteristics that may make them useful for biomacromolecular assembly. The experiments utilized bacterially expressed Trm4p, a tRNA methyltransferase cloned from Saccharomyces cerevisiae. Like DNA m5C methyltransferases, Trm4p mediates methylation using a covalent intermediate, which would allow Trm4p to be trapped as a stable protein-RNA complex when the substrate RNA contains a modified cytosine base such as 5-fluorocytosine. However, mutant forms of Trm4p are identified that fail to release RNA resulting in the formation of denaturant stable methyltransferase-RNA complexes that contain only natural nucleotides. The ability to form stable complexes with natural RNA gives these mutant forms of Trm4p greater potential versatility for biomacromolecule construction applications than the wild-type Trm4p enzyme or DNA methyltransferases for which the trapping of the covalent intermediate requires the presence of a nucleotide analogue at the site of modification.     

Crystal Structure of Methanocaldococcus jannaschii Trm4 Complexed with Sinefungin

tRNA:m⁵C methyltransferase Trm4 generates the modified nucleotide 5-methylcytidine in archaeal and eukaryotic tRNA molecules, using S-adenosyl-l-methionine (AdoMet) as methyl donor. Most archaea and eukaryotes possess several Trm4 homologs, including those related to diseases, while the archaeon Methanocaldococcus jannaschii has only one gene encoding a Trm4 homolog, MJ0026. The recombinant MJ0026 protein catalyzed AdoMet-dependent methyltransferase activity on tRNA in vitro and was shown to be the M. jannaschii Trm4. We determined the crystal structures of the substrate-free M. jannaschii Trm4 and its complex with sinefungin at 1.27 Å and 2.3 Å resolutions, respectively. This AdoMet analog is bound in a negatively charged pocket near helix α8. This helix can adopt two different conformations, thereby controlling the entry of AdoMet into the active site. Adjacent to the sinefungin-bound pocket, highly conserved residues form a large, positively charged surface, which seems to be suitable for tRNA binding. The structure explains the roles of several conserved residues that were reportedly involved in the enzymatic activity or stability of Trm4p from the yeast Saccharomyces cerevisiae. We also discuss previous genetic and biochemical data on human NSUN2/hTrm4/Misu and archaeal PAB1947 methyltransferase, based on the structure of M. jannaschii Trm4.     

Evidence that tRNA modifying enzymes are important in vivo targets for 5-fluorouracil in yeast

We have screened a collection of haploid yeast knockout strains for increased sensitivity to 5-fluorouracil (5-FU). A total of 138 5-FU sensitive strains were found. Mutants affecting rRNA and tRNA maturation were particularly sensitive to 5-FU, with the tRNA methylation mutant trm10 being the most sensitive mutant. This is intriguing since trm10, like many other tRNA modification mutants, lacks a phenotype under normal conditions. However, double mutants for nonessential tRNA modification enzymes are frequently temperature sensitive, due to destabilization of hypomodified tRNAs. We therefore tested if the sensitivity of our mutants to 5-FU is affected by the temperature. We found that the cytotoxic effect of 5-FU is strongly enhanced at 38 degrees C for tRNA modification mutants. Furthermore, tRNA modification mutants show similar synthetic interactions for temperature sensitivity and sensitivity to 5-FU. A model is proposed for how 5-FU kills these mutants by reducing the number of tRNA modifications, thus destabilizing tRNA. Finally, we found that also wild-type cells are temperature sensitive at higher concentrations of 5-FU. This suggests that tRNA destabilization contributes to 5-FU cytotoxicity in wild-type cells and provides a possible explanation why hyperthermia can enhance the effect of 5-FU in cancer therapy.     

Transfer RNA methytransferases and their corresponding modifications in budding yeast and humans: activities, predications, and potential roles in human health

Throughout the kingdoms of life, transfer RNA (tRNA) undergoes over 100 enzyme-catalyzed, methyl-based modifications. Although a majority of the methylations are conserved from bacteria to mammals, the functions of a number of these modifications are unknown. Many of the proteins responsible for tRNA methylation, named tRNA methyltransferases (Trms), have been characterized in Saccharomyces cerevisiae. In contrast, only a few human Trms have been characterized. A BLAST search for human homologs of each S. cerevisiae Trm revealed a total of 34 human proteins matching our search criteria for an S. cerevisiae Trm homolog candidate. We have compiled a database cataloging basic information about each human and yeast Trm. Every S. cerevisiae Trm has at least one human homolog, while several Trms have multiple candidates. A search of cancer cell versus normal cell mRNA expression studies submitted to Oncomine found that 30 of the homolog genes display a significant change in mRNA expression levels in at least one data set. While 6 of the 34 human homolog candidates have confirmed tRNA methylation activity, the other candidates remain uncharacterized. We believe that our database will serve as a resource for investigating the role of human Trms in cellular stress signaling.     

Substrate tRNA recognition mechanism of a multisite-specific tRNA methyltransferase, Aquifex aeolicus Trm1, based on the X-ray crystal structure

Archaeal and eukaryotic tRNA (N(2),N(2)-guanine)-dimethyltransferase (Trm1) produces N(2),N(2)-dimethylguanine at position 26 in tRNA. In contrast, Trm1 from Aquifex aeolicus, a hyper-thermophilic eubacterium, modifies G27 as well as G26. Here, a gel mobility shift assay revealed that the T-arm in tRNA is the binding site of A. aeolicus Trm1. To address the multisite specificity, we performed an x-ray crystal structure study. The overall structure of A. aeolicus Trm1 is similar to that of archaeal Trm1, although there is a zinc-cysteine cluster in the C-terminal domain of A. aeolicus Trm1. The N-terminal domain is a typical catalytic domain of S-adenosyl-l-methionine-dependent methyltransferases. On the basis of the crystal structure and amino acid sequence alignment, we prepared 30 mutant Trm1 proteins. These mutant proteins clarified residues important for S-adenosyl-l-methionine binding and enabled us to propose a hypothetical reaction mechanism. Furthermore, the tRNA-binding site was also elucidated by methyl transfer assay and gel mobility shift assay. The electrostatic potential surface models of A. aeolicus and archaeal Trm1 proteins demonstrated that the distribution of positive charges differs between the two proteins. We constructed a tRNA-docking model, in which the T-arm structure was placed onto the large area of positive charge, which is the expected tRNA-binding site, of A. aeolicus Trm1. In this model, the target G26 base can be placed near the catalytic pocket; however, the nucleotide at position 27 gains closer access to the pocket. Thus, this docking model introduces a rational explanation of the multisite specificity of A. aeolicus Trm1.     

tRNA and protein methylase complexes mediate zymocin toxicity in yeast

Modification of Saccharomyces cerevisiae tRNA anticodons at the wobble uridine (U34) position is required for tRNA cleavage by the zymocin tRNase killer toxin from Kluyveromyces lactis . Hence, U34 modification defects including lack of the U34 tRNA methyltransferase Trm9 protect against tRNA cleavage and zymocin. Using zymocin as a tool, we have identified toxin-resistant mutations in TRM9 that are likely to affect the U34 methylation reaction. Most strikingly, C-terminal truncations in Trm9 abolish interaction with Trm112, a protein shown to individually purify with Lys9 and two more methylases, Trm11 and Mtq2. Downregulation of a GAL1-TRM112 allele protects against zymocin whereas LYS9 , TRM11 and MTQ2 are dosage suppressors of zymocin. Based on immune precipitation studies, the latter scenario correlates with competition for Trm112 and in excess, some of these Trm112 partners interfere with formation of the toxin-relevant Trm9·Trm112 complex. In contrast to trm11 Δ or lys9 Δ cells, trm112 Δ and mtq2 Δ null mutants are zymocin resistant. In line with the identified role that methylation of Sup45 by Mtq2 has for translation termination by the release factor dimer Sup45·Sup35, we observe that SUP45 overexpression and sup45 mutants suppress zymocin. Intriguingly, this suppression correlates with upregulated levels of tRNA species targeted by zymocin's tRNase activity.     

Cysteine of sequence motif VI is essential for nucleophilic catalysis by yeast tRNA m5C methyltransferase

Sequence comparison of several RNA m(5)C methyltransferases identifies two conserved cysteine residues that belong to signature motifs IV and VI of RNA and DNA methyltransferases. While the cysteine of motif IV is used as the nucleophilic catalyst by DNA m(5)C methyltransferases, this role is fulfilled by the cysteine of motif VI in Escherichia coli 16S rRNA m(5)C967 methyltransferase, but whether this conclusion applies to other RNA m(5)C methyltransferases remains to be verified. Yeast tRNA m(5)C methyltransferase Trm4p is a multisite-specific S-adenosyl-L-methionine-dependent enzyme that catalyzes the methylation of cytosine at C5 in several positions of tRNA. Here, we confirm that Cys310 of motif VI in Trm4p is essential for nucleophilic catalysis, presumably by forming a covalent link with carbon 6 of cytosine. Indeed, the enzyme is able to form a stable covalent adduct with the 5-fluorocytosine-containing RNA substrate analog, whereas the C310A mutant protein is inactive and unable to form the covalent complex.     

Detection and discovery of RNA modifications using microarrays

Using a microarray that tiles all known yeast non-coding RNAs, we compared RNA from wild-type cells with RNA from mutants encoding known and putative RNA modifying enzymes. We show that at least five types of RNA modification (dihydrouridine, m1G, m2(2)G, m1A and m6(2)A) catalyzed by 10 different enzymes (Trm1p, Trm5, Trm10p, Dus1p-Dus4p, Dim1p, Gcd10p and Gcd14p) can be detected by virtue of differential hybridization to oligonucleotides on the array that are complementary to the modified sites. Using this approach, we identified a previously undetected m1A modification in GlnCTG tRNA, the formation of which is catalyzed by the Gcd10/Gcd14 complex. complex.     

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.     

Frequent increased gene copy number and high protein expression of tRNA (cytosine-5-)-methyltransferase (NSUN2) in human cancers

NSUN2, also known as SAKI or MISU, is a methyltransferase which catalyses (cytosine-5-)-methylation of tRNA. The human NSUN2 gene is located on chromosome 5p15.31-33. We show that NSUN2 gene copy number is increased in oral and colorectal cancers. Protein expression levels of NSUN2 were determined by immunoblot using novel polyclonal antibodies raised against a synthetic peptide corresponding to the C-terminal region of the protein. In most normal tissues, NSUN2 expression levels were extremely low. On the other hand, oral and colorectal cancers typically expressed high levels of NSUN2. The level of NSUN2 was similar in interphase and mitotic cells, and immunohistochemical analysis demonstrated strong staining for NSUN2 in oral and colon cancer tissues when compared with normal tissues, providing a distinct diagnostic significance for NSUN2 in comparison with Ki-67, a widely used marker of actively proliferating cells. In addition, elevated protein expression of NSUN2 was confirmed by immunohistochemical analysis of various cancers including esophageal, stomach, liver, pancreas, uterine cervix, prostate, kidney, bladder, thyroid, and breast cancers. NSUN2 is regulated by Aurora-B, a newly developed molecular target for cancer therapy, leading us to propose that NSUN2 might become a valuable target for cancer therapy and a cancer diagnostic marker.