tRNA Methylase Research Articles

Elongator stabilizes microtubules to control central spindle asymmetry and polarized trafficking of cell fate determinants.

Asymmetric cell division gives rise to two daughter cells that inherit different determinants, thereby acquiring different fates. Polarized trafficking of endosomes containing fate determinants recently emerged as an evolutionarily conserved feature of asymmetric cell division to enhance the robustness of asymmetric cell fate determination in flies, fish and mammals. In particular, polarized sorting of signalling endosomes by an asymmetric central spindle contributes to asymmetric cell division in Drosophila melanogaster. However, how central spindle asymmetry arises remains elusive. Here we identify a moonlighting function of the Elongator complex-an established protein acetylase and tRNA methylase involved in the fidelity of protein translation-as a key factor for central spindle asymmetry. Elongator controls spindle asymmetry by stabilizing microtubules differentially on the anterior side of the central spindle. Accordingly, lowering the activity of Elongator on the anterior side using nanobodies mistargets endosomes to the wrong cell. Molecularly, Elongator regulates microtubule dynamics independently of its acetylation and methylation enzymatic activities. Instead, Elongator directly binds to microtubules and increases their polymerization speed while decreasing their catastrophe frequency. Our data establish a non-canonical role of Elongator at the core of cytoskeleton polarity and asymmetric signalling.

Put the Pedal to the METTL1: Adding Internal m7G Increases mRNA Translation Efficiency and Augments miRNA Processing.

Complementary papers by Zhang, Liu, and colleagues (Zhang etal., 2019) and Pandolfini, Barbieri, and colleagues (Pandolfini etal., 2019) develop new sequencing techniques that reveal that METTL1 N7-methylates internal guanosines in mRNAs and miRNAs to increase translation efficiency and miRNA processing, respectively.

DNA adenine methyltransferase (Dam) controls the expression of the cytotoxic enterotoxin (act) gene of Aeromonas hydrophila via tRNA modifying enzyme-glucose-inhibited division protein (GidA)

Aeromonas hydrophila is both a human and animal pathogen, and the cytotoxic enterotoxin (Act) is a crucial virulence factor of this bacterium because of its associated hemolytic, cytotoxic, and enterotoxic activities. Previously, to define the role of some regulatory genes in modulating Act production, we showed that deletion of a glucose-inhibited division gene (gidA) encoding tRNA methylase reduced Act levels, while overproduction of DNA adenine methyltransferase (Dam) led to a concomitant increase in Act-associated biological activities of a diarrheal isolate SSU of A. hydrophila. Importantly, there are multiple GATC binding sites for Dam within an upstream sequence of the gidA gene and one such target site in the act gene upstream region. We showed the dam gene to be essential for the viability of A. hydrophila SSU, and, therefore, to better understand the interaction of the encoding genes, Dam and GidA, in act gene regulation, we constructed a gidA in-frame deletion mutant of Escherichia coli GM28 (dam+) and GM33 (∆dam) strains. We then tested the expressional activity of the act and gidA genes by using a promoterless pGlow-TOPO vector containing a reporter green fluorescent protein (GFP). Our data indicated that in GidA+ strains of E. coli, constitutive methylation of the GATC site(s) by Dam negatively regulated act and gidA gene expression as measured by GFP production. However, in the ∆gidA strains, irrespective of the presence or absence of constitutively active Dam, we did not observe any alteration in the expression of the act gene signifying the role of GidA in positively regulating Act production. To determine the exact mechanism of how Dam and GidA influence Act, a real-time quantitative PCR (RT-qPCR) assay was performed. The analysis indicated an increase in gidA and act gene expression in the A. hydrophila Dam-overproducing strain, and these data matched with Act production in the E. coli GM28 strain. Thus, the extent of DNA methylation caused by constitutive versus overproduction of Dam, as well as possible conformation of DNA influence the expression of act and gidA genes in A. hydrophila SSU. Our results indicate that the act gene is under the control of both Dam and GidA modification methylases, and Dam regulates Act production via GidA.

Formation of m 2 G6 in Methanocaldococcus jannaschii tRNA catalyzed by the novel methyltransferase Trm14

The modified nucleosides N2-methylguanosine and -dimethylguanosine in transfer RNA occur at five positions in the D and anticodon arms, and at positions G6 and G7 in the acceptor stem. Trm1 and Trm11 enzymes are known to be responsible for several of the D/anticodon arm modifications, but methylases catalyzing post-transcriptional m2G synthesis in the acceptor stem are uncharacterized. Here, we report that the MJ0438 gene from Methanocaldococcus jannaschii encodes a novel S-adenosylmethionine-dependent methyltransferase, now identified as Trm14, which generates m2G at position 6 in tRNACys. The 381 amino acid Trm14 protein possesses a canonical RNA recognition THUMP domain at the amino terminus, followed by a γ-class Rossmann fold amino-methyltransferase catalytic domain featuring the signature NPPY active site motif. Trm14 is associated with cluster of orthologous groups (COG) 0116, and most closely resembles the m2G10 tRNA methylase Trm11. Phylogenetic analysis reveals a canonical archaeal/bacterial evolutionary separation with 20–30% sequence identities between the two branches, but it is likely that the detailed functions of COG 0116 enzymes differ between the archaeal and bacterial domains. In the archaeal branch, the protein is found exclusively in thermophiles. More distantly related Trm14 homologs were also identified in eukaryotes known to possess the m2G6 tRNA modification.

Identification of human tRNA:m5C methyltransferase catalysing intron-dependent m5C formation in the first position of the anticodon of the ${\hbox{ pre-tRNA }}_{\left(\hbox{ CAA }\right)}^{\hbox{ Leu }}$

We identified a human orthologue of tRNA:m5C methyltransferase from Saccharomyces cerevisiae, which has been previously shown to catalyse the specific modification of C34 in the intron-containing yeast . Using transcripts of intron-less and intron-containing human genes as substrates, we have shown that m5C34 is introduced only in the intron-containing tRNA precursors when the substrates were incubated in the HeLa extract. m5C34 formation depends on the nucleotide sequence surrounding the wobble cytidine and on the structure of the prolongated anticodon stem. Expression of the human Trm4 (hTrm4) cDNA in yeast partially complements the lack of the endogenous Trm4p enzyme. The yeast extract prepared from the strain deprived of the endogenous TRM4 gene and transformed with hTrm4 cDNA exhibits the same activity and substrate specificity toward human pre-tRNALeu transcripts as the HeLa extract. The hTrm4 MTase has a much narrower specificity against the yeast substrates than its yeast orthologue: human enzyme is not able to form m5C at positions 48 and 49 of human and yeast tRNA precursors. To our knowledge, this is the first report showing intron-dependent methylation of human and identification of human gene encoding tRNA methylase responsible for this reaction.

A new Drosophila gene wh ( wuho) with WD40 repeats is essential for spermatogenesis and has maximal expression in hub cells

Through mutagenesis by P-element transposition, we identified a series of mutants with deletions in topoisomerase 3β gene ( top3β) and an adjacent, previously uncharacterized gene CG15897, here named wuho ( wh). Whereas top3β truncation does not affect viability or fertility, wh null mutants display male sterile and female semi-sterile phenotypes. Furthermore, wh mutants can be fully rescued by wh transgenes, but not by top3β transgenes, suggesting that the fertility phenotypes are caused by wh deletion. The alignment of WH protein sequence with other eukaryotic putative homologues shows they are evolutionarily conserved proteins with 5 WD40 repeats in the middle portion of the protein, and a bipartite nuclear localization signal at the carboxyl terminus. Yeast homologue with 5 WD40 repeats, Trm82, is the non-catalytic subunit of a tRNA methylase. Immunostaining shows that WH has the highest expression in hub cells, a niche for germline stem cells of testis. However, WH is not required for the maintenance of hub cells or the germline stem cells. In wh mutant males, spermatogenesis is arrested at the elongating stage of the developing spermatids, resulting in an absence of mature sperms in the seminal vesicles. The decreased fertility in wh mutant females is mostly due to defects in oogenesis. There are abnormal egg chambers present in the mutant females, in which the cystocytes fail to arrest their cell division at the fourth mitotic cycle, resulting in more than 16 cells in a single egg chamber. Additionally, these abnormal cystocytes do not undergo multiple rounds of endoreplication as the nurse cells do in a normal egg chamber. Therefore, the cytological analyses demonstrate that wh has a critical function in cellular differentiation for germline cells during gametogenesis.

TRNAGlu wobble uridine methylation by Trm9 identifies Elongator's key role for zymocin‐induced cell death in yeast

Zymocin-induced cell death in Saccharomyces cerevisiae requires the toxin-target (TOT) effector Elongator, a protein complex with functions in transcription, exocytosis and tRNA modification. In line with the latter, trm9Delta cells lacking a tRNA methylase specific for wobble uridine (U(34)) residues survive zymocin and in excess, the Trm9 substrate tRNA(Glu) copies zymocin protection of Elongator mutants. Phenotypes typical of a tot3/elp3Delta Elongator mutant are absent from trm9Delta cells but copied in a tot3Deltatrm9Delta double mutant suggesting that Elongator acts upstream of Trm9. Consistent with Elongator-dependent tRNA modification being more important to mRNA decoding than Trm9, SUP4 and SOE1TRNA suppressors are highly sensitive to loss of Elongator and tRNA U(34) hypomodification. As Trm9 overexpression counteracts the effect of high-copy tRNA(Glu), zymocin suppression by high-copy tRNA(Glu) may reflect tRNA hypomethylation of trm9Delta cells. Thus, Trm9 methylation may enable recognition of tRNA by zymocin, a notion supported by a dramatic reduction of tRNA(Glu) levels in zymocin-treated cells and by cytotoxic zymocin residues conserved between bacterial nucleases and a tRNA modifying GTPase. In sum, Trm9 is a bona fideTOT pathway component whose methylation may be hijacked by zymocin to target tRNA function and eventually, mRNA translation.

The tRNA methylase METTL1 is phosphorylated and inactivated by PKB and RSK in vitro and in cells.

A substrate for protein kinase B (PKB)alpha in HeLa cell extracts was identified as methyltransferase-like protein-1 (METTL1), the orthologue of trm8, which catalyses the 7-methylguanosine modification of tRNA in Saccharomyces cerevisiae. PKB and ribosomal S6 kinase (RSK) both phosphorylated METTL1 at Ser27 in vitro. Ser27 became phosphorylated when HEK293 cells were stimulated with insulin-like growth factor-1 (IGF-1) and this was prevented by inhibition of phosphatidyinositol 3-kinase. The IGF-1-induced Ser27 phosphorylation did not occur in 3-phosphoinositide-dependent protein kinase-1 (PDK1)-deficient embryonic stem cells, but occurred normally in PDK1[L155E] cells, indicating that the effect of IGF-1 is mediated by PKB. METTL1 also became phosphorylated at Ser27 in response to phorbol-12-myristate 13-acetate and this was prevented by PD 184352 or pharmacological inhibition of RSK. Phosphorylation of METTL1 by PKB or RSK inactivated METTL1 in vitro, as did mutation of Ser27 to Asp or Glu. Expression of METTL1[S27D] or METTL1[S27E] did not rescue the growth phenotype of yeast lacking trm8. In contrast, expression of METTL1 or METTL1[S27A] partially rescued growth. These results demonstrate that METTL1 is inactivated by PKB and RSK in cells, and the potential implications of this finding are discussed.

Theoretical mechanisms for synthesis of carcinogen-induced embryonic proteins: XXIV repeated embryonic genes in liver

Many embryonic genes may be sets of repeated genes. Processes of differentiation where the quantity of gene activity changes during embryogenesis may not reflect the transcription rate per se but instead be a result of the state of expression of constitutive repeated genes. Such repeated genes (in fact or theory) is dealt with here for embryonic, adult and neoplastic liver. Explanations are presented for 1) the variation in ATP: L-methionine S-adenosyltransferase activity in spontaneous hepatomas and embryonic liver; 2) differences tRNA L-methionine S-adenosyltransferase activity in spontaneous hepatomas and embryonic liver; 3) differences in tRNA methylase activity in embryonic liver, hepatomas of varying levels of differentiation, and ethionine treated liver; 4) relationships between rRNA genes and nucleolar organizers; and 5) the possibilities involving c-onc genes (proto-oncogenes) for situations of ‘over-expression’ in neoplastic and embryonic liver.

High-molecular-weight forms of aminoacyl-tRNA synthetases and tRNA modification enzymes in Escherichia coli

The presence of high-molecular-weight complexes of aminoacyl-tRNA synthetases in Escherichia coli has been reported (C. L. Harris, J. Bacteriol. 169:2718-2723, 1987). In the current study, Bio-Gel A-5M gel chromatography of 105,000 x g supernatant preparations from E. coli Q13 indicated high molecular weights for both tRNA methylase (300,000) and tRNA sulfurtransferase (450,000). These tRNA modification enzymes did not appear to exist in the same multienzymic complex. On the other hand, 4-thiouridine sulfurtransferase eluted with aminoacyl-tRNA synthetase activity on Bio-Gel A-5M, and both of these activities were cosedimented after further centrifugation of cell supernatants at 160,000 x g for 18 h. Despite this evidence for association of the sulfurtransferase with the synthetase complex, isoleucyl-tRNA synthetase and tRNA sulfurtransferase were totally resolved from each other by DEAE-Sephacel chromatography. Subsequent gel chromatography showed little change in their elution positions on agarose. Hence, either nonspecific aggregation occurred here, or the modification enzymes studied are not members of the aminoacyl-tRNA synthetase complex in E. coli. These findings do suggest that some bacterial tRNA modification enzymes are present in multiprotein complexes of high molecular weight.

Theoretical mechanisms for synthesis of carcinogen-induced embryonic proteins: XXI oncogenes interpreted as embryonic genes

If oncogenes are interpreted as embryonic genes then the mechanisms for induction of these embryonic genes can be generalized and theoretical considerations can be derived as follows. Agents that induce activity by derepressing embryonic genes can be placed into major groups: i) translocation inducers, ii) non-mutating carcinogenic agents, iii) intercalating DNA groove distorters, and iv) mutating agents. Translocation inducers work via long terminal repeat insertions, antibody promoter region associations, or V-type position effects. Non-mutational agents such as ethionine cause hyporibosylation of nucleosome core histones or hypomethylation of promoter regions of conformation inducer proteins. All of these agents cause deheterochromatizations of facultative heterochromatin. These processes (or by a classical mutation with mutating agents), cause DNA replication to acquire a new abnormal methylationpattern that is held constant by maintenance DNA methylases. The resultant active series of repressed reduntant type embryonic genes, such as subsets of rDNA genes and tRNA methylase genes, including oncogenes, e.g., protein phosphokinases among other required genes, and spurious irrelevant embryonic genes to the process of carcinogenesis. From the above theory is derived the concept that normal sets of redundant genes, e.g., rDNA are normally activated by planar intercalating agents (steroids) that would simply be the limiting subset of the mechanism used to create an anomalous state when extended to the set of embryonically repressed genes that become activated.

Transfer RNA methylases and carcinogenesis

An increased tRNA methylase activity (100 %) accompanied by a 40 % decrease in the regulatory glycine methyltransferase activity was demonstrated in livers of mice fed on the carcinogenic (thioacetamide) diet, long before the onset of malignant transformation. Shortterm treatment with thioacetamide and phenobarbital independently, also brought about a significant increase in the rat liver tRNA methylase activity. A significant increase in the tRNA methylase activity was observed in the mammary glands of pregnant as well as lactating mice as against the negligible enzyme activity in the normal mammary glands of C3H and CBA mice, whereas a large increase in the tRNA methylase activity was evident in the spontaneously induced mammary tumours in these strains. Hepatic tRNA methylase activity was shown to remain unaffected in rats during various physiological stress conditions. It is suggested that elevation in the tRNA methylase activity may be one of the prerequisites during malignant transformation. A considerable increase in the tRNA methylase activity in host tissues of the tumour-bearing mice was also demonstrated

Activity patterns of aminoacyl-tRNA synthetases, tRNA methylases, arginyltransferase and tubulin: tyrosine ligase during development and ageing of Caenorhabditis elegans.

As a step in the characterization of development and ageing in the nematode Caenorhabditis elegans, the activities of different groups of enzymes that supposedly exert modulating functions in and after protein synthesis have been determined. From embryonic (E), the four juvenile larval stages (L1-L4) and the gravid adult (A,A+), the selection of defined developmental stages extends to two different preparations of aged nematodes (S10, S12). Some aminoacyl-tRNA synthetase activities remain nearly unchanged in all stages up to the adult, some increase continuously during the larval stages and the remaining activities show stage-specific alterations. Upon ageing all activities except the one for tryptophan decrease sharply, tRNA methylase activities increase from E to L4, decrease from L4 to adult and to aged nematodes with only qualitative alterations in substrate specificity. The activity of tubulin: tyrosine ligase exhibits a parallel pattern, while arginyltransferase activity has a plateau between L2 and L4. The results are consistent with the idea of a modulation of protein synthesis and other cellular processes by quantitative activity changes during development and ageing.

Effects of cortisol on tRNA methylase activities in rat mammary carcinoma.

Mammary carcinomas induced in rats by DMBA were divided into three types: I, hard proliferating tumors; II, tumors presenting from an early stage the first signs of cystic degeneration; III, lactating tumors. In all three types, cortisol reduced the protein content by 26%-30%. The already high tRNA methyltransferase activity in type I increased by 200% after cortisol treatment. Hormonal treatment of type II increased the previously reduced control methyltransferases by 37%. In the type III lactating tumors, the total tRNA methyltransferases were inhibited by 35% after cortisol treatment. The methyltransferases of types I and II were separated chromatographically into seven analogous peaks, while the enzymes from type III presented a modified pattern. In each case, cortisol treatment affected the activities of several methyltransferases simultaneously without obvious specificity.

Theoretical mechanisms for synthesis of carcinogen-induced embryonic proteins: XI a theoretical interpretation of the sequential methylation of yeast phenylalanine tRNA

Theoretical studies on the mechanisms of methylation of tRNA was reported in a previous paper dealing with the induction of genic activity of proposed “embryonic” tRNA methylases. The mechanism described only the first methylation of a tRNA that had a known structure, namely yeast phenylalanine tRNA. In this paper, a complete scheme of methylation for the molecule is reported in detail using molecular model building. The mechanism uses only one specific uridine residue with which S-adenosyl-L-methionine complexs for all required methylation sites.

S-adenosylhomocysteine hydrolases as the primary target enzymes in androgen regulation of methylation complexes

tRNA methylation complexes consisting of S-adenosylmethionine (AdoMet) synthetase, tRNA methylases, and S-adenosylhomocysteine (AdoHcy) hydrolase have been prepared from rat Novikoff hepatoma cells. The existence of the ternary enzyme complex is supported by dissociation and reconstitution of the ternany tRNA methylation complexes. In rat prostate and testis, two isozymes each for AdoMet synthetase and AdoHcy hydrolase are detected. The K m (methionine) values for the two AdoMet synthetases are 3.1 and 23.7 μ m and the K m (adenosine) values for the two AdoHcy hydrolases are 0.33 and 1.8 μ m. Correspondingly, two groups of methylation complexes are detectable, sedimenting in a sucrose gradient as 7 S and 8 S. The 7 S complexes are composed of AdoMet synthetase and AdoHcy hydrolase with the higher K m values, and the 8 S complexes are composed of the respective isozymes with the lower K m values. tRNA methylation complexes belong to the 8 S group. In hormone-depleted rat prostates and testes following hypophysectomy, the specific activities of AdoMet synthetases, tRNA methylases, and AdoHcy hydrolases are decreased severely, but are restored promptly after administration of testosterone. Thus, methylation enzymes are responsive to the regulation by steroid hormone. AdoHcy hydrolases from hormone-depleted tissues are unstable, and ternary tRNA methylation complexes are easily dissociable into individual activities. The stability of AdoHcy hydrolases is markedly improved by testosterone, and the integrity of ternary tRNA methylation complexes is maintained in the presence of testosterone. These results suggest that AdoHcy hydrolases are the primary target enzymes in adrogen regulation of methylation complexes.

TRNA methylation A rapid and simple method for determination of total radioactivity and methylated base distribution in the same sample

A simple and rapid method is described for the analysis of in vitro methylated tRNA. The total radioactivity is first determined after trichloroacetic acid-precipitation and filtration. Then the tRNA is hydrolyzed while still on the filter, and the bases are separated by thin-layer chromatography. This method eliminates the need for duplicate sample preparation, and has proved especially useful when the amount of tRNA and/or tRNA methylase is limited.

Synthesis of catechol estrogens by human uterus and leiomyoma

Homogenates of human endometrial, myometrial and leiomyoma tissues were incubated with (2,4,6,7- 3H)-estradiol and tritiated catechol estrogens were isolated and identified. Though 2-and 4-hydroxylations were about the same in endometrium, 4-hydroxylation was two to four fold higher than 2-hydroxylation in myometrium and leiomyoma. However, endometrium showed greater capacity to form both 2- and 4-hydroxyestrogens than the other two tissues. Both 2- and 4-hydroxylations were significantly less than in myometrium. In view of the reports indicating that inhibitors of catechol 0-methyl transferease (COMT) might act as anti-neoplastic agents due to their interferance with t-RNA methylases and since catechol estrogens inhibit COMT, the present results suggest that endogenous synthesis of catechol estrogens may play an important role in the pathophysiology of uterine leiomyoma.

The effect of the methionine antagonist l-2-amino-4-methoxy- trans-3-butenoic acid on the growth and metabolism of walker carcinosarcoma in vitro

l-2-Amino-4-methoxy- trans-3-butenoic acid (Ro07–7957) is a structural analogue ot methionine with a potent tumour growth inhibitory activity in vitro. This agent is transported by the methionine carrier system in Walker carcinoma and causes an initial dose-related depression of the acid-soluble pool of methionine. The depression of the incorporation of l-[methyl- 3H]methionine into acid-insoluble material in the presence of Ro07–7957 is greater than that of l-[2- 3H]methionine and l-[4,5( n)- 3H]lysine, suggesting an inhibition of the methylation of macromolecules as well as an inhibition of protein synthesis. There is no effect of the drug on the incorporation of [ 3H]thymidine into acid-insoluble material during the first 24 hr after treatment, while the incorporation of [5- 3H]uridine is stimulated 40 per cent. The ratio of incorporation of l-[2- 3H]methionine to l-[methyl- 14C]methionine into proteins increases with increasing drug concentration, suggesting an inhibition of protein methylation. This effect is more prevalent at 24 hr than after 8 hr of treatment. The specific activity of tRNA methylase using E. coli MRE 600 tRNA as substrate is elevated more than two-fold within 24 hr after treatment, as is also the intracellular level of S-adenosyl- l-methionine (SAM). The effects of this agent on macromolecular metabolism is in some respects similar to that observed with the carcinogen ethionine, and suggests the initial formation of an inhibitor of methylation, which is followed by a later attempt by the cells to maintain homeostasis by production of increased amounts of tRNA methylating enzymes.

Biochemical alterations in hepatic tissue of ethionine and N-nitrosodimethylamine treated mice, and in a transplantable tumor of rat.

Considerably lowere CO2-14C production from the degradative pathway of thymidine -14C utilization was observed in the hepatic tissues of mice (C3H) that recieved multiple intraperitoneal doses of ethionine (400 mg/kg on day 1 5, 9, 13, 17, 21 and 25). While the incorporation of thymidine -14C into DNA was unchanged throughout this period, the tRNA methylase activity of these mice receiving multiple doses of ethionine was significantly lowered at the final sacrifice. When a lower single dose of ethionine (80 mg/kg) was given to mice, the depression of CO2-14C produced from thymidine -14C utilization was less pronounced.Transplantable dimethylaminobenz(a)anthracene (DMBA) induced manunary tumor of rat showed both increased incorporation of thymidine -14C into DNA and t-RNA methylase activity. Also, transplantable Morris hepatoma, although not available for testing in this study, shares a similar pattern of thymidine utilization with transplantable mammary tumor and in general, all other transplantable tumors. In contrast to transplantable tumors N-nitrosodimethylamine (NDMA) induced hepatomas of mice showed no statistically significant alteration in their utilization of thymidine -14C.The known hepatotoxins, bromobenzene and carbon tetra-chloride, when administered to rats in a single dose all lowered the heptatic CO2-14C production from thymidine -14C utilization as compared to non-treated controls (p<0.001, p<0.01 respectively). The implication of these findings is discussed.