A tRNA-specific function for tRNA methyltransferase Trm10 is associated with a new tRNA quality control mechanism in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, a single homolog of the tRNA methyltransferase Trm10 performs m1G9 modification on 13 different tRNAs. Here we provide evidence that the m1G9 modification catalyzed by S. cerevisiae Trm10 plays a biologically important role for one of these tRNA substrates, tRNATrp Overexpression of tRNATrp (and not any of 38 other elongator tRNAs) rescues growth hypersensitivity of the trm10Δ strain in the presence of the antitumor drug 5-fluorouracil (5FU). Mature tRNATrp is depleted in trm10Δ cells, and its levels are further decreased upon growth in 5FU, while another Trm10 substrate (tRNAGly) is not affected under these conditions. Thus, m1G9 in S. cerevisiae is another example of a tRNA modification that is present on multiple tRNAs but is only essential for the biological function of one of those species. In addition to the effects of m1G9 on mature tRNATrp, precursor tRNATrp species accumulate in the same strains, an effect that is due to at least two distinct mechanisms. The levels of mature tRNATrp are rescued in the trm10Δmet22Δ strain, consistent with the known role of Met22 in tRNA quality control, where deletion of met22 causes inhibition of 5'-3' exonucleases that catalyze tRNA decay. However, none of the known Met22-associated exonucleases appear to be responsible for the decay of hypomodified tRNATrp, based on the inability of mutants of each enzyme to rescue the growth of the trm10Δ strain in the presence of 5FU. Thus, the surveillance of tRNATrp appears to constitute a distinct tRNA quality control pathway in S. cerevisiae.

Thg1 family 3'-5' RNA polymerases as tools for targeted RNA synthesis.

Members of the 3'-5' RNA polymerase family, comprised of tRNAHis guanylyltransferase (Thg1) and Thg1-like proteins (TLPs), catalyze templated synthesis of RNA in the reverse direction to all other known 5'-3' RNA and DNA polymerases. The discovery of enzymes capable of this reaction raised the possibility of exploiting 3'-5' polymerases for posttranscriptional incorporation of nucleotides to the 5'-end of nucleic acids without ligation, and instead by templated polymerase addition. To date, studies of these enzymes have focused on nucleotide addition to highly structured RNAs, such as tRNA and other noncoding RNAs. Consequently, general principles of RNA substrate recognition and nucleotide preferences that might enable broader application of 3'-5' polymerases have not been elucidated. Here, we investigated the feasibility of using Thg1 or TLPs for multiple nucleotide incorporation to the 5'-end of a short duplex RNA substrate, using a templating RNA oligonucleotide provided in trans to guide 5'-end addition of specific sequences. Using optimized assay conditions, we demonstrated a remarkable capacity of certain TLPs to accommodate short RNA substrate-template duplexes of varying lengths with significantly high affinity, resulting in the ability to incorporate a desired nucleotide sequence of up to eight bases to 5'-ends of the model RNA substrates in a template-dependent manner. This work has further advanced our goals to develop this atypical enzyme family as a versatile nucleic acid 5'-end labeling tool.

tRNA m1G9 modification depends on substrate-specific RNA conformational changes induced by the methyltransferase Trm10.

The methyltransferase Trm10 modifies a subset of tRNAs on the base N1 position of the ninth nucleotide in the tRNA core. Trm10 is conserved throughout Eukarya and Archaea, and mutations in the human gene (TRMT10A) have been linked to neurological disorders such as microcephaly and intellectual disability, as well as defects in glucose metabolism. Of the 26 tRNAs in yeast with guanosine at position 9, only 13 are substrates for Trm10. However, no common sequence or other posttranscriptional modifications have been identified among these substrates, suggesting the presence of some other tRNA feature(s) that allow Trm10 to distinguish substrate from nonsubstrate tRNAs. Here, we show that substrate recognition by Saccharomyces cerevisiae Trm10 is dependent on both intrinsic tRNA flexibility and the ability of the enzyme to induce specific tRNA conformational changes upon binding. Using the sensitive RNA structure-probing method SHAPE, conformational changes upon binding to Trm10 in tRNA substrates, but not nonsubstrates, were identified and mapped onto a model of Trm10-bound tRNA. These changes may play an important role in substrate recognition by allowing Trm10 to gain access to the target nucleotide. Our results highlight a novel mechanism of substrate recognition by a conserved tRNA modifying enzyme. Further, these studies reveal a strategy for substrate recognition that may be broadly employed by tRNA-modifying enzymes which must distinguish between structurally similar tRNA species.

Diversity in Biological Function and Mechanism of the tRNA Methyltransferase Trm10.

ConspectusTransfer ribonucleic acid (tRNA) is the most highly modified RNA species in the cell, and loss of tRNA modifications can lead to growth defects in yeast as well as metabolic, neurological, and mitochondrial disorders in humans. Significant progress has been made toward identifying the enzymes that are responsible for installing diverse modifications in tRNA, revealing a landscape of fascinating biological and mechanistic diversity that remains to be fully explored. Most early discoveries of tRNA modification enzymes were in model systems, where many enzymes were not strictly required for viability, an observation somewhat at odds with the extreme conservation of many of the same enzymes throughout multiple domains of life. Moreover, many tRNA modification enzymes act on more than one type of tRNA substrate, which is not necessarily surprising given the similar overall secondary and tertiary structures of tRNA, yet biochemical characterization has revealed interesting patterns of substrate specificity that can be challenging to rationalize on a molecular level. Questions about how many enzymes efficiently select a precise set of target tRNAs from among a structurally similar pool of molecules persist.The tRNA methyltransferase Trm10 provides an exciting paradigm to study the biological and mechanistic questions surrounding tRNA modifications. Even though the enzyme was originally characterized in Saccharomyces cerevisiae where its deletion causes no detectable phenotype under standard lab conditions, several more recently identified phenotypes provide insight into the requirement for this modification in the overall quality control of the tRNA pool. Studies of Trm10 in yeast also revealed another characteristic feature that has turned out to be a conserved feature of enzymes throughout the Trm10 family tree. We were initially surprised to see that purified S. cerevisiae Trm10 was capable of modifying tRNA substrates that were not detectably modified by the enzyme in vivo in yeast. This pattern has continued to emerge as we and others have studied Trm10 orthologs from Archaea and Eukarya, with enzymes exhibiting in vitro substrate specificities that can differ significantly from in vivo patterns of modification. While this feature complicates efforts to predict substrate specificities of Trm10 enzymes in the absence of appropriate genetic systems, it also provides an exciting opportunity for studying how enzyme activities can be regulated to achieve dynamic patterns of biological tRNA modification, which have been shown to be increasingly important for stress responses and human disease. Finally, the intriguing diversity in target nucleotide modification that has been revealed among Trm10 orthologs is distinctive among known tRNA modifying enzymes and necessitates unusual and likely novel catalytic strategies for methylation that are being revealed by biochemical and structural studies directed toward various family members. These efforts will no doubt yield more surprising discoveries in terms of tRNA modification enzymology.

Tied up in knots: Untangling substrate recognition by the SPOUT methyltransferases.

The SpoU-TrmD (SPOUT) methyltransferase superfamily was designated when structural similarity was identified between the transfer RNA-modifying enzymes TrmH (SpoU) and TrmD. SPOUT methyltransferases are found in all domains of life and predominantly modify transfer RNA or ribosomal RNA substrates, though one instance of an enzyme with a protein substrate has been reported. Modifications placed by SPOUT methyltransferases play diverse roles in regulating cellular processes such as ensuring translational fidelity, altering RNA stability, and conferring bacterial resistance to antibiotics. This large collection of S-adenosyl-L-methionine-dependent methyltransferases is defined by a unique α/ß fold with a deep trefoil knot in their catalytic (SPOUT) domain. Herein, we describe current knowledge of SPOUT enzyme structure, domain architecture, and key elements of catalytic function, including S-adenosyl-L-methionine co-substrate binding, beginning with a new sequence alignment that divides the SPOUT methyltransferase superfamily into four major clades. Finally, a major focus of this review will be on our growing understanding of how these diverse enzymes accomplish the molecular feat of specific substrate recognition and modification, as highlighted by recent advances in our knowledge of protein-RNA complex structures and the discovery of the dependence of one SPOUT methyltransferase on metal ion binding for catalysis. Considering the broad biological roles of RNA modifications, developing a deeper understanding of the process of substrate recognition by the SPOUT enzymes will be critical for defining many facets of fundamental RNA biology with implications for human disease.

Fidelity of base-pair recognition by a 3'-5' polymerase: mechanism of the Saccharomyces cerevisiae tRNAHis guanylyltransferase.

The tRNAHis guanylyltransferase (Thg1) was originally discovered in Saccharomyces cerevisiae where it catalyzes 3'-5' addition of a single nontemplated guanosine (G-1) to the 5' end of tRNAHis In addition to this activity, S. cerevisiae Thg1 (SceThg1) also catalyzes 3'-5' polymerization of Watson-Crick (WC) base pairs, utilizing nucleotides in the 3'-end of a tRNA as the template for addition. Subsequent investigation revealed an entire class of enzymes related to Thg1, called Thg1-like proteins (TLPs). TLPs are found in all three domains of life and preferentially catalyze 3'-5' polymerase activity, utilizing this unusual activity to repair tRNA, among other functions. Although both Thg1 and TLPs utilize the same chemical mechanism, the molecular basis for differences between WC-dependent (catalyzed by Thg1 and TLPs) and non-WC-dependent (catalyzed exclusively by Thg1) reactions has not been fully elucidated. Here we investigate the mechanism of base-pair recognition by 3'-5' polymerases using transient kinetic assays, and identify Thg1-specific residues that play a role in base-pair discrimination. We reveal that, regardless of the identity of the opposing nucleotide in the RNA "template," addition of a non-WC G-1 residue is driven by a unique kinetic preference for GTP. However, a secondary preference for forming WC base pairs is evident for all possible templating residues. Similar to canonical 5'-3' polymerases, nucleotide addition by SceThg1 is driven by the maximal rate rather than by NTP substrate affinity. Together, these data provide new insights into the mechanism of base-pair recognition by 3'-5' polymerases.

Distinct substrate specificities of the human tRNA methyltransferases TRMT10A and TRMT10B.

The tRNA m1R9 methyltransferase (Trm10) family is conserved throughout Eukarya and Archaea. Despite the presence of a single Trm10 gene in Archaea and most single-celled eukaryotes, metazoans encode up to three homologs of Trm10. Several disease states correlate with a deficiency in the human homolog TRMT10A, despite the presence of another cytoplasmic enzyme, TRMT10B. Here we investigate these phenomena and demonstrate that human TRMT10A (hTRMT10A) and human TRMT10B (hTRMT10B) are not biochemically redundant. In vitro activity assays with purified hTRMT10A and hTRMT10B reveal a robust activity for hTRMT10B as a tRNAAsp-specific m1A9 methyltransferase and suggest that it is the relevant enzyme responsible for this newly discovered m1A9 modification in humans. Moreover, a comparison of the two cytosolic enzymes with multiple tRNA substrates exposes the enzymes' distinct substrate specificities, and suggests that hTRMT10B exhibits a restricted selectivity hitherto unseen in the Trm10 enzyme family. Single-turnover kinetics and tRNA binding assays highlight further differences between the two enzymes and eliminate overall tRNA affinity as a primary determinant of substrate specificity for either enzyme. These results increase our understanding of the important biology of human tRNA modification systems, which can aid in understanding the molecular basis for diseases in which their aberrant function is increasingly implicated.

Chemical footprinting and kinetic assays reveal dual functions for highly conserved eukaryotic tRNAHis guanylyltransferase residues.

tRNAHis guanylyltransferase (Thg1) adds a single guanine to the -1 position of tRNAHis as part of its maturation. This seemingly modest addition of one nucleotide to tRNAHis ensures translational fidelity by providing a critical identity element for the histidyl aminoacyl tRNA synthetase (HisRS). Like HisRS, Thg1 utilizes the GUG anticodon for selective tRNAHis recognition, and Thg1-tRNA complex structures have revealed conserved residues that interact with anticodon nucleotides. Separately, kinetic analysis of alanine variants has demonstrated that many of these same residues are required for catalytic activity. A model in which loss of activity with the variants was attributed directly to loss of the critical anticodon interaction has been proposed to explain the combined biochemical and structural results. Here we used RNA chemical footprinting and binding assays to test this model and further probe the molecular basis for the requirement for two critical tRNA-interacting residues, His-152 and Lys-187, in the context of human Thg1 (hThg1). Surprisingly, we found that His-152 and Lys-187 alanine-substituted variants maintain a similar overall interaction with the anticodon region, arguing against the sufficiency of this interaction for driving catalysis. Instead, conservative mutagenesis revealed a new direct function for these residues in recognition of a non-Watson-Crick G-1:A73 bp, which had not been described previously. These results have important implications for the evolution of eukaryotic Thg1 from a family of ancestral promiscuous RNA repair enzymes to the highly selective enzymes needed for their essential function in tRNAHis maturation.

A Family Divided: Distinct Structural and Mechanistic Features of the SpoU-TrmD (SPOUT) Methyltransferase Superfamily.

The SPOUT family of enzymes makes up the second largest of seven structurally distinct groups of methyltransferases and is named after two evolutionarily related RNA methyltransferases, SpoU and TrmD. A deep trefoil knotted domain in the tertiary structures of member enzymes defines the SPOUT family. For many years, formation of a homodimeric quaternary structure was thought to be a strict requirement for all SPOUT enzymes, critical for substrate binding and formation of the active site. However, recent structural characterization of two SPOUT members, Trm10 and Sfm1, revealed that they function as monomers without the requirement of this critical dimerization. This unusual monomeric form implies that these enzymes must exhibit a nontraditional substrate binding mode and active site architecture and may represent a new division in the SPOUT family with distinct properties removed from the dimeric enzymes. Here we discuss the mechanistic features of SPOUT enzymes with an emphasis on the monomeric members and implications of this "novel" monomeric structure on cofactor and substrate binding.

Mechanistic features of the atypical tRNA m1G9 SPOUT methyltransferase, Trm10.

The tRNA m1G9 methyltransferase (Trm10) is a member of the SpoU-TrmD (SPOUT) superfamily of methyltransferases, and Trm10 homologs are widely conserved throughout Eukarya and Archaea. Despite possessing the trefoil knot characteristic of SPOUT enzymes, Trm10 does not share the same quaternary structure or key sequences with other members of the SPOUT family, suggesting a novel mechanism of catalysis. To investigate the mechanism of m1G9 methylation by Trm10, we performed a biochemical and kinetic analysis of Trm10 and variants with alterations in highly conserved residues, using crystal structures solved in the absence of tRNA as a guide. Here we demonstrate that a previously proposed general base residue (D210 in Saccharomyces cerevisiae Trm10) is not likely to play this suggested role in the chemistry of methylation. Instead, pH-rate analysis suggests that D210 and other conserved carboxylate-containing residues at the active site collaborate to establish an active site environment that promotes a single ionization that is required for catalysis. Moreover, Trm10 does not depend on a catalytic metal ion, further distinguishing it from the other known SPOUT m1G methyltransferase, TrmD. These results provide evidence for a non-canonical tRNA methyltransferase mechanism that characterizes the Trm10 enzyme family.

A Temporal Order in 5'- and 3'- Processing of Eukaryotic tRNAHis.

For flawless translation of mRNA sequence into protein, tRNAs must undergo a series of essential maturation steps to be properly recognized and aminoacylated by aminoacyl-tRNA synthetase, and subsequently utilized by the ribosome. While all tRNAs carry a 3'-terminal CCA sequence that includes the site of aminoacylation, the additional 5'-G-1 position is a unique feature of most histidine tRNA species, serving as an identity element for the corresponding synthetase. In eukaryotes including yeast, both 3'-CCA and 5'-G-1 are added post-transcriptionally by tRNA nucleotidyltransferase and tRNAHis guanylyltransferase, respectively. Hence, it is possible that these two cytosolic enzymes compete for the same tRNA. Here, we investigate substrate preferences associated with CCA and G-1-addition to yeast cytosolic tRNAHis, which might result in a temporal order to these important processing events. We show that tRNA nucleotidyltransferase accepts tRNAHis transcripts independent of the presence of G-1; however, tRNAHis guanylyltransferase clearly prefers a substrate carrying a CCA terminus. Although many tRNA maturation steps can occur in a rather random order, our data demonstrate a likely pathway where CCA-addition precedes G-1 incorporation in S. cerevisiae. Evidently, the 3'-CCA triplet and a discriminator position A73 act as positive elements for G-1 incorporation, ensuring the fidelity of G-1 addition.

Identification of distinct biological functions for four 3'-5' RNA polymerases.

The superfamily of 3'-5' polymerases synthesize RNA in the opposite direction to all other DNA/RNA polymerases, and its members include eukaryotic tRNA(His) guanylyltransferase (Thg1), as well as Thg1-like proteins (TLPs) of unknown function that are broadly distributed, with family members in all three domains of life. Dictyostelium discoideum encodes one Thg1 and three TLPs (DdiTLP2, DdiTLP3 and DdiTLP4). Here, we demonstrate that depletion of each of the genes results in a significant growth defect, and that each protein catalyzes a unique biological reaction, taking advantage of specialized biochemical properties. DdiTLP2 catalyzes a mitochondria-specific tRNA(His) maturation reaction, which is distinct from the tRNA(His) maturation reaction typically catalyzed by Thg1 enzymes on cytosolic tRNA. DdiTLP3 catalyzes tRNA repair during mitochondrial tRNA 5'-editing in vivo and in vitro, establishing template-dependent 3'-5' polymerase activity of TLPs as a bona fide biological activity for the first time since its unexpected discovery more than a decade ago. DdiTLP4 is cytosolic and, surprisingly, catalyzes robust 3'-5' polymerase activity on non-tRNA substrates, strongly implying further roles for TLP 3'-5' polymerases in eukaryotes.

Life without post-transcriptional addition of G-1: two alternatives for tRNAHis identity in Eukarya.

The identity of tRNA(His) is strongly associated with the presence of an additional 5'-guanosine residue (G-1) in all three domains of life. The critical nature of the G-1 residue is underscored by the fact that two entirely distinct mechanisms for its acquisition are observed, with cotranscriptional incorporation observed in Bacteria, while post-transcriptional addition of G-1 occurs in Eukarya. Here, through our investigation of eukaryotes that lack obvious homologs of the post-transcriptional G-1-addition enzyme Thg1, we identify alternative pathways to tRNA(His) identity that controvert these well-established rules. We demonstrate that Trypanosoma brucei, like Acanthamoeba castellanii, lacks the G-1 identity element on tRNA(His) and utilizes a noncanonical G-1-independent histidyl-tRNA synthetase (HisRS). Purified HisRS enzymes from A. castellanii and T. brucei exhibit a mechanism of tRNA(His) recognition that is distinct from canonical G-1-dependent synthetases. Moreover, noncanonical HisRS enzymes genetically complement the loss of THG1 in Saccharomyces cerevisiae, demonstrating the biological relevance of the G-1-independent aminoacylation activity. In contrast, in Caenorhabditis elegans, which is another Thg1-independent eukaryote, the G-1 residue is maintained, but here its acquisition is noncanonical. In this case, the G-1 is encoded and apparently retained after 5' end processing, which has so far only been observed in Bacteria and organelles. Collectively, these observations unearth a widespread and previously unappreciated diversity in eukaryotic tRNA(His) identity mechanisms.

A mutation in the THG1L gene in a family with cerebellar ataxia and developmental delay.

Autosomal-recessive cerebellar atrophy is usually associated with inactivating mutations and early-onset presentation. The underlying molecular diagnosis suggests the involvement of neuronal survival pathways, but many mechanisms are still lacking and most patients elude genetic diagnosis. Using whole exome sequencing, we identified homozygous p.Val55Ala in the THG1L (tRNA-histidine guanylyltransferase 1 like) gene in three siblings who presented with cerebellar signs, developmental delay, dysarthria, and pyramidal signs and had cerebellar atrophy on brain MRI. THG1L protein was previously reported to participate in mitochondrial fusion via its interaction with MFN2. Abnormal mitochondrial fragmentation, including mitochondria accumulation around the nuclei and confinement of the mitochondrial network to the nuclear vicinity, was observed when patient fibroblasts were cultured in galactose containing medium. Culturing cells in galactose containing media promotes cellular respiration by oxidative phosphorylation and the action of the electron transport chain thus stimulating mitochondrial activity. The growth defect of the yeast thg1Δ strain was rescued by the expression of either yeast Thg1 or human THG1L; however, clear growth defect was observed following the expression of the human p.Val55Ala THG1L or the corresponding yeast mutant. A defect in the protein tRNAHis guanylyltransferase activity was excluded by the normal in vitro G-1 addition to either yeast tRNAHis or human mitochondrial tRNAHis in the presence of the THG1L mutation. We propose that homozygosity for the p.Val55Ala mutation in THG1L is the cause of the abnormal mitochondrial network in the patient fibroblasts, likely by interfering with THG1L activity towards MFN2. This may result in lack of mitochondria in the cerebellar Purkinje dendrites, with degeneration of Purkinje cell bodies and apoptosis of granule cells, as reported for MFN2 deficient mice.

Mitochondrial tRNA 5'-editing in Dictyostelium discoideum and Polysphondylium pallidum.

Mitochondrial tRNA (mt-tRNA) 5'-editing was first described more than 20 years ago; however, the first candidates for 5'-editing enzymes were only recently identified in a eukaryotic microbe (protist), the slime mold Dictyostelium discoideum. In this organism, eight of 18 mt-tRNAs are predicted to be edited based on the presence of genomically encoded mismatched nucleotides in their aminoacyl-acceptor stem sequences. Here, we demonstrate that mt-tRNA 5'-editing occurs at all predicted sites in D. discoideum as evidenced by changes in the sequences of isolated mt-tRNAs compared with the expected sequences encoded by the mitochondrial genome. We also identify two previously unpredicted editing events in which G-U base pairs are edited in the absence of any other genomically encoded mismatches. A comparison of 5'-editing in D. discoideum with 5'-editing in another slime mold, Polysphondylium pallidum, suggests organism-specific idiosyncrasies in the treatment of U-G/G-U pairs. In vitro activities of putative D. discoideum editing enzymes are consistent with the observed editing reactions and suggest an overall lack of tRNA substrate specificity exhibited by the repair component of the editing enzyme. Although the presence of terminal mismatches in mt-tRNA sequences is highly predictive of the occurrence of mt-tRNA 5'-editing, the variability in treatment of U-G/G-U base pairs observed here indicates that direct experimental evidence of 5'-editing must be obtained to understand the complete spectrum of mt-tRNA editing events in any species.

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.

Diversity in mechanism and function of tRNA methyltransferases.

tRNA molecules undergo extensive post-transcriptional processing to generate the mature functional tRNA species that are essential for translation in all organisms. These processing steps include the introduction of numerous specific chemical modifications to nucleotide bases and sugars; among these modifications, methylation reactions are by far the most abundant. The tRNA methyltransferases comprise a diverse enzyme superfamily, including members of multiple structural classes that appear to have arisen independently during evolution. Even among closely related family members, examples of unusual substrate specificity and chemistry have been observed. Here we review recent advances in tRNA methyltransferase mechanism and function with a particular emphasis on discoveries of alternative substrate specificities and chemistry associated with some methyltransferases. Although the molecular function for a specific tRNA methylation may not always be clear, mutations in tRNA methyltransferases have been increasingly associated with human disease. The impact of tRNA methylation on human biology is also discussed.

TRMT10A dysfunction is associated with abnormalities in glucose homeostasis, short stature and microcephaly.

BACKGROUND: Trm10 is a tRNA m(1)G9 methyltransferase, which in yeast modifies 12 different tRNA species, yet is considered non-essential for viability under standard growth conditions. In humans, there are three Trm10 orthologs, one mitochondrial and two presumed cytoplasmic. A nonsense mutation in one of the cytoplasmic orthologs (TRMT10A) has recently been associated with microcephaly, intellectual disability, short stature and adolescent onset diabetes. METHODS AND RESULTS: The subjects were three patients who suffered from microcephaly, intellectual disability, short stature, delayed puberty, seizures and disturbed glucose metabolism, mainly hyperinsulinaemic hypoglycaemia. A homozygous Gly206Arg (G206R) mutation in the TRMT10A gene was identified using whole exome sequencing. The mutation segregated in the family and was absent from large control cohorts. Determination of the methylation activity of the expressed wild-type (WT) and variant TRMT10A enzymes with transcripts of (32)P -tRNA(Gly) GCC as a substrate revealed a striking defect (<0.1% of WT activity) for the variant enzyme. The binding affinity of the G206R variant enzyme to tRNA, determined by fluorescence anisotropy, was similar to that of the WT enzyme. CONCLUSIONS: The completely abolished m(1)G9 methyltransferase activity of the mutant enzyme is likely due to significant defects in its ability to bind the methyl donor S-adenosyl methionine. We propose that TRMT10A deficiency accounts for abnormalities in glucose homeostasis initially manifesting both ketotic and non-ketotic hypoglycaemic events with transition to diabetes in adolescence, perhaps as a consequence of accelerated ß cell apoptosis. The seizure disorder and intellectual disability are probably secondary to mutant gene expression in neuronal tissue.

Absence of a universal element for tRNAHis identity in Acanthamoeba castellanii.

The additional G(-1) nucleotide on tRNA(His) is a nearly universal feature that specifies tRNA(His) identity in all three domains of life. In eukaryotes, the G(-1) identity element is obtained by a post-transcriptional pathway, through the unusual 3'-5' polymerase activity of the highly conserved tRNA(His) guanylyltransferase (Thg1) enzyme, and no examples of eukaryotic histidyl-tRNAs that lack this essential element have been identified. Here we report that the eukaryote Acanthamoeba castellanii lacks the G(-1) identity element on its tRNA(His), consistent with the lack of a gene encoding a bona fide Thg1 ortholog in the A. castellanii genome. Moreover, the cytosolic histidyl-tRNA synthetase in A. castellanii exhibits an unusual tRNA substrate specificity, efficiently aminoacylating tRNA(His) regardless of the presence of G(-1). A. castellanii does contain two Thg1-related genes (encoding Thg1-like proteins, TLPs), but the biochemical properties we associate here with these proteins are consistent with a function for these TLPs in separate pathways unrelated to tRNA(His) metabolism, such as mitochondrial tRNA repair during 5'-editing.

Saccharomyces cerevisiae Thg1 uses 5'-pyrophosphate removal to control addition of nucleotides to tRNA(His.).

In eukaryotes, the tRNA(His) guanylyltransferase (Thg1) catalyzes 3'-5' addition of a single guanosine residue to the -1 position (G-1) of tRNA(His), across from a highly conserved adenosine at position 73 (A73). After addition of G-1, Thg1 removes pyrophosphate from the tRNA 5'-end, generating 5'-monophosphorylated G-1-containing tRNA. The presence of the 5'-monophosphorylated G-1 residue is important for recognition of tRNA(His) by its cognate histidyl-tRNA synthetase. In addition to the single-G-1 addition reaction, Thg1 polymerizes multiple G residues to the 5'-end of tRNA(His) variants. For 3'-5' polymerization, Thg1 uses the 3'-end of the tRNA(His) acceptor stem as a template. The mechanism of reverse polymerization is presumed to involve nucleophilic attack of the 3'-OH from each incoming NTP on the intact 5'-triphosphate created by the preceding nucleotide addition. The potential exists for competition between 5'-pyrophosphate removal and 3'-5' polymerase reactions that could define the outcome of Thg1-catalyzed addition, yet the interplay between these competing reactions has not been investigated for any Thg1 enzyme. Here we establish transient kinetic assays to characterize the pyrophosphate removal versus nucleotide addition activities of yeast Thg1 with a set of tRNA(His) substrates in which the identity of the N-1:N73 base pair was varied to mimic various products of the N-1 addition reaction catalyzed by Thg1. We demonstrate that retention of the 5'-triphosphate is correlated with efficient 3'-5' reverse polymerization. A kinetic partitioning mechanism that acts to prevent addition of nucleotides beyond the -1 position with wild-type tRNA(His) is proposed.