TRM6/61 connects PKCα with translational control through tRNAi(Met) stabilization: impact on tumorigenesis.

Accumulating evidence suggests that changes of the protein synthesis machinery alter translation of specific mRNAs and participate in malignant transformation. Here we show that protein kinase C α (PKCα) interacts with TRM61, the catalytic subunit of the TRM6/61 tRNA methyltransferase. The TRM6/61 complex is known to methylate the adenosine 58 of the initiator methionine tRNA (tRNAi(Met)), a nuclear post-transcriptional modification associated with the stabilization of this crucial component of the translation-initiation process. Depletion of TRM6/61 reduced proliferation and increased death of C6 glioma cells, effects that can be partially rescued by overexpression of tRNAi(Met). In contrast, elevated TRM6/61 expression regulated the translation of a subset of mRNAs encoding proteins involved in the tumorigenic process and increased the ability of C6 cells to form colonies in soft agar or spheres when grown in suspension. In TRM6/61/tRNAi(Met)-overexpressing cells, PKCα overexpression decreased tRNAi(Met) expression and both colony- and sphere-forming potentials. A concomitant increase in TRM6/TRM61 mRNA and tRNAi(Met) expression with decreased expression of PKCα mRNA was detected in highly aggressive glioblastoma multiforme as compared with Grade II/III glioblastomas, highlighting the clinical relevance of our findings. Altogether, we suggest that PKCα tightly controls TRM6/61 activity to prevent translation deregulation that would favor neoplastic development.

Trm9-Catalyzed tRNA Modifications Regulate Global Protein Expression by Codon-Biased Translation.

Post-transcriptional modifications of transfer RNAs (tRNAs) have long been recognized to play crucial roles in regulating the rate and fidelity of translation. However, the extent to which they determine global protein production remains poorly understood. Here we use quantitative proteomics to show a direct link between wobble uridine 5-methoxycarbonylmethyl (mcm5) and 5-methoxy-carbonyl-methyl-2-thio (mcm5s2) modifications catalyzed by tRNA methyltransferase 9 (Trm9) in tRNAArg(UCU) and tRNAGlu(UUC) and selective translation of proteins from genes enriched with their cognate codons. Controlling for bias in protein expression and alternations in mRNA expression, we find that loss of Trm9 selectively impairs expression of proteins from genes enriched with AGA and GAA codons under both normal and stress conditions. Moreover, we show that AGA and GAA codons occur with high frequency in clusters along the transcripts, which may play a role in modulating translation. Consistent with these results, proteins subject to enhanced ribosome pausing in yeast lacking mcm5U and mcm5s2U are more likely to be down-regulated and contain a larger number of AGA/GAA clusters. Together, these results suggest that Trm9-catalyzed tRNA modifications play a significant role in regulating protein expression within the cell.

Biosynthesis of wyosine derivatives in tRNA(Phe) of Archaea: role of a remarkable bifunctional tRNA(Phe):m1G/imG2 methyltransferase.

The presence of tricyclic wyosine derivatives 3'-adjacent to anticodon is a hallmark of tRNA(Phe) in eukaryotes and archaea. In yeast, formation of wybutosine (yW) results from five enzymes acting in a strict sequential order. In archaea, the intermediate compound imG-14 (4-demethylwyosine) is a target of three different enzymes, leading to the formation of distinct wyosine derivatives (yW-86, imG, and imG2). We focus here on a peculiar methyltransferase (aTrm5a) that catalyzes two distinct reactions: N(1)-methylation of guanosine and C(7)-methylation of imG-14, whose function is to allow the production of isowyosine (imG2), an intermediate of the 7-methylwyosine (mimG) biosynthetic pathway. Based on the formation of mesomeric forms of imG-14, a rationale for such dual enzymatic activities is proposed. This bifunctional tRNA:m(1)G/imG2 methyltransferase, acting on two chemically distinct guanosine derivatives located at the same position of tRNA(Phe), is unique to certain archaea and has no homologs in eukaryotes. This enzyme here referred to as Taw22, probably played an important role in the emergence of the multistep biosynthetic pathway of wyosine derivatives in archaea and eukaryotes.

Outbreak of Salmonella enterica serotype Infantis producing ArmA 16S RNA methylase and CTX-M-15 extended-spectrum ß-lactamase in a neonatology ward in Constantine, Algeria.

Plasmid-mediated 16S rRNA methylases such as ArmA, which confer high levels of resistance to aminoglycosides, are increasingly reported in Enterobacteriaceae. This study investigated the molecular mechanism of ß-lactam and aminoglycoside resistance in extended-spectrum ß-lactamase (ESBL)-producing Salmonella enterica serotype Infantis isolated at the 53-bed neonatology ward of University Hospital Benabib in Constantine, Algeria. From September 2008 to January 2009, 200 S. enterica isolates were obtained from 138 patients (age range 8-80 months) hospitalised in the neonatology ward. Most isolates were from stool cultures, but also from two blood cultures and one gastric fluid. The isolates were multidrug-resistant and produced TEM-1 and CTX-M-15 enzymes as well as the 16S RNA methylase ArmA. The armA, bla(CTX-M-15) and bla(TEM-1) genes were located on the same 140-kb self-transferable plasmid belonging to the IncL/M incompatibility group. All of the S. Infantis isolates belonged to a single clone. Increased infection control measures and thorough biodecontamination of the rooms led to control of the outbreak but did not eradicate the epidemic strain. This study further illustrates the global emergence of ArmA methylase and its frequent association with bla(CTX-M) genes. Spread of 16S RNA methylase determinants at the same level as bla(CTX-M) genes in Enterobacteriaceae may seriously compromise the efficacy of aminoglycosides for treating Gram-negative infections.

Expression and purification of untagged and histidine-tagged folate-dependent tRNA:m5U54 methyltransferase from Bacillus subtilis.

Folate-dependent tRNA m(5)U methyltransferase TrmFO is a flavoprotein that catalyzes the C(5)-methylation of uridine at position 54 in the TPsiC loop of tRNA in several bacteria. Here we report the cloning and optimization of expression in Escherichia coli BL21 (DE3) of untagged, N-terminus, C-terminus (His)(6)-tagged TrmFO from Bacillus subtilis. Tagged and untagged TrmFO were purified to homogeneity by metal affinity or ion exchange and heparin affinity, respectively, followed by size-exclusion chromatography. The tag did not significantly alter the expression level, flavin content, activity and secondary structure of the protein.

Synthesis of a hetero subunit RNA modification enzyme by the wheat germ cell-free translation system.

Cell-free translation systems are a powerful tool for the production of many kinds of proteins. However, there are some barriers to improve the system in order to make it a more convenient approach. These include the fact that the production of proteins made up of hetero subunits is difficult. In this chapter, we describe the synthesis of yeast tRNA (m(7)G46) methyltransferase as a model protein. This enzyme catalyzes transfer of a methyl group from S-adenosyl-L-methionine to guanine at position 46 in tRNA and generates N(7)-methylguanine. Yeast tRNA (m(7)G46) methyltransferase is composed of two protein subunits, Trm8 and Trm82. To obtain the active Trm8-Trm82 complex, co-translation of both subunits is necessary. Preparation of mRNAs, in vitro synthesis and purification of the complex are explained in this chapter.

The expression of TRMT2A, a novel cell cycle regulated protein, identifies a subset of breast cancer patients with HER2 over-expression that are at an increased risk of recurrence.

BACKGROUND: Over-expression of HER2 in a subset of breast cancers (HER2+) is associated with high histological grade and aggressive clinical course. Despite these distinctive features, the differences in response of HER2+ patients to both adjuvant cytotoxic chemotherapy and targeted therapy (e.g. trastuzumab) suggests that unrecognized biologic and clinical diversity is confounding treatment strategies. Furthermore, the small but established risk of cardiac morbidity with trastuzumab therapy compels efforts towards the identification of biomarkers that might help stratify patients. METHODS: A single institution tissue array cohort assembled at the Clearview Cancer Institute of Huntsville (CCIH) was screened by immunohistochemistry staining using a large number of novel and commercially available antibodies to identify those with a univariate association with clinical outcome in HER2+ patients. Staining with antibody directed at TRMT2A was found to be strongly associated with outcome in HER2+ patients. This association with outcome was tested in two independent validation cohorts; an existing staining dataset derived from tissue assembled at the Cleveland Clinic Foundation (CCF), and in a new retrospective study performed by staining archived paraffin blocks available at the Roswell Park Cancer Institute (RPCI). RESULTS: TRMT2A staining showed a strong correlation with likelihood of recurrence at five years in 67 HER2+ patients from the CCIH discovery cohort (HR 7.0; 95% CI 2.4 to 20.1, p < 0.0004). This association with outcome was confirmed using 75 HER2+ patients from the CCF cohort (HR 3.6; 95% CI 1.3 to 10.2, p < 0.02) and 64 patients from the RPCI cohort (HR 3.4; 95% CI 1.3-8.9, p < 0.02). In bivariable analysis the association with outcome was independent of grade, tumor size, nodal status and the administration of conventional adjuvant chemotherapy in the CCIH and RPCI cohorts. CONCLUSIONS: Studies from three independent single institution cohorts support TRMT2A protein expression as a biomarker of increased risk of recurrence in HER2+ breast cancer patients. These results suggest that TRMT2A expression should be further studied in the clinical trial setting to explore its predictive power for response to adjuvant cytotoxic chemotherapy in combination with HER2 targeted therapy.

Production of yeast (m2G10) methyltransferase (Trm11 and Trm112 complex) in a wheat germ cell-free translation system.

Transfer RNA (guanine-N(2)-)-methyltransferase [tRNA (m(2)G10) methyltransferase] catalyzes a methyl-transfer from S-adenosyl-L-methionine to N(2)-atom of guanine at position 10 (G10) in tRNA and generates N(2)-methylguanine at position 10 (m(2)G10). Yeast enzyme contains two protein subunits (Trm11 and Trm112). Trm11 protein is expected to be a catalytic subunit and Trm112 contains a Zinc-finger. In yeast cells, Trm112 binds not only to Trm11 but also to other proteins such as Lys9, Trm9, and Mtq2. Therefore, the Trm112 protein may regulate population of several protein complexes. To address these issues, we started the study on synthesis of Trm112 related protein complexes. In this meeting, we report synthesis of active Trm11-Trm112 complex in a wheat germ cell-free translation system.

Hetero subunit interaction and RNA recognition of yeast tRNA (m7G46) methyltransferase synthesized in a wheat germ cell-free translation system.

Yeast tRNA (m(7)G46) methyltransferase contains two protein subunits (Trm8 and Trm82). The enzyme catalyzes a methyl-transfer from S-adenosyl-L-methionine to the N(7) atom of guanine at position 46 in tRNA. We deviced synthesis of active Trm8-Trm82 heterodimer in a wheat germ cell-free translation system. When Trm8 or Trm82 mRNA were used for a synthesis, Trm8 or Trm82 protein could be synthesized. Upon mixing the synthesized Trm8 and Trm82 proteins, no active Trm8-Trm82 heterodimer was produced. Active Trm8-Trm82 heterodimer was only synthesized under conditions, in which both Trm8 and Trm82 mRNAs were co-translated. To address the RNA recognition mechanism of the Trm8-Trm82 complex, we investigated methyl acceptance activities of eight truncated yeast tRNA(Phe) transcripts. In this meeting, we demonstrate that yeast Trm8-Trm82 has stricter recognition requirements for the tRNA molecule as compared to the bacterial enzyme, TrmB.

Identification and characterization of mouse TRMU gene encoding the mitochondrial 5-methylaminomethyl-2-thiouridylate-methyltransferase.

The nucleotide modification in tRNA plays a pivotal role in the fidelity of translational process. The mutated mitochondrial tRNA (mt tRNA) associated with human diseases often exhibited a defect in nucleotide modification at wobble position of anticodons. Recently, the product of trmU, 5-methylaminomethyl-2-thiouridylate-methyltransferase, has been shown to be one component of enzyme complex for the biosynthesis of mnm5s2U in the wobble position of the bacterial tRNAs. Here we report the identification and characterization of mouse TRMU homolog. A 1532 bp TRMU cDNA has been isolated and the genomic organization of TRMU has been elucidated. The mouse TRMU gene containing 11 exons encodes a 417 residue protein with a strong homology to the TRMU-like proteins of bacteria and other homologs related to tRNA modification. The mouse TRMU is ubiquitously expressed in various tissues, but abundantly in tissues with high metabolic rates including heart, liver and brain. Furthermore, immunofluorescence analysis of NIH3T3 cells expressing TRMU-GFP fusion protein demonstrated that the mouse Trmu localizes in mitochondria. These observations suggest that the mouse TRMU is a structural and functional homolog of bacterial TrmU, thereby playing a role in the mt tRNA modification and protein synthesis.

Dietary fish oil reduces O6-methylguanine DNA adduct levels in rat colon in part by increasing apoptosis during tumor initiation.

There is epidemiological, clinical, and experimental evidence that dietary fish oil, containing n-3 polyunsaturated fatty acids, protects against colon tumor development. However, its effects on colonocytes in vivo remain poorly understood. Therefore, we investigated the ability of fish oil to modulate colonic methylation-induced DNA damage, repair, and deletion. Sprague Dawley rats were provided with complete diets containing either corn oil or fish oil (15% by weight). Animals were injected with azoxymethane, and the distal colon was removed 3, 6, 9, or 12 h later. Targeted apoptosis and DNA damage were assessed by cell position within the crypt using the terminal deoxynucleotidyl transferase-mediated nick end labeling assay and quantitative immunohistochemical analysis of O6-methylguanine adducts, respectively. Localization and expression of the alkyl group acceptor, O6-methylguanine-DNA-methyltransferase, was also determined. Lower levels of adducts were detected at 6, 9, and 12 h in fish oil- versus corn oil-fed animals (P < 0.05). In addition, fish oil supplementation had the greatest effect on apoptosis in the top one-third of the crypt, increasing the apoptotic index compared with corn oil-fed rats (P < 0.05). In the top one-third of the crypt, fish oil feeding caused an incremental stimulation of apoptosis as adduct level increased. In contrast, a negative correlation between apoptosis and adduct incidence occurred with corn oil feeding (P < 0.05). Diet had no main effect (all tertiles combined) on O6-methylguanine-DNA-methyltransferase expression over the time frame of the experiment. The enhancement of targeted apoptosis combined with the reduced formation of O6-methylguanine adducts may account, in part, for the observed protective effect of n-3 polyunsaturated fatty acids against experimentally induced colon cancer.

High-level expression and rapid purification of tRNA (m5U54)-methyltransferase.

We report an extremely high-level expression system for tRNA (m5U54)-methyltransferase (RUMT), and a purification strategy which routinely yields 20 to 50 mg of homogeneous RUMT per liter of Escherichia coli cells. The RUMT gene (trmA) was cloned into a pET vector and transformed into E. coli BL21 (DE3) cells. Following induction, this system produces active enzyme at a level approaching 50% of the total soluble protein. A purification scheme consisting of DEAE-cellulose chromatography to remove nucleic acids, followed by phosphocellulose chromatography, provides homogeneous enzyme. The entire procedure, from cell growth to purified enzyme, takes less than 2 days. This represents a significant improvement over the previously published expression/purification protocol for RUMT (Gu, X, and Santi, D.V., Protein Expression Purif. 2, 66-68, 1991), which typically nets 5- to 10-fold less enzyme per liter of cells and is substantially more labor intensive.

The effect of sinefungin and synthetic analogues on RNA and DNA methyltransferases from Streptomyces.

Sinefungin is an antibiotic structurally related to S-adenosylmethionine. It has been described as an inhibitor of RNA transmethylation reactions in viruses and eukaryotic organisms, but not in bacteria. We show here that sinefungin strongly inhibits RNA methyltransferase activity, but not the biosynthesis of these enzymes in Streptomyces. All the methylated bases found in Streptomyces RNA (1-methyladenine, N6-methyladenine, N6,N6-dimethyladenine and 7-methylguanine) are inhibited by this antibiotic. Experiments with sinefungin analogues show that specific changes in the ornithine radical of the molecule still preserve its inhibitory capability. The substitution of the adenine radical by uridine causes the loss of the inhibitory effect. These results and our former studies on Streptomyces DNA methylation, suggest that nucleic acid modification is the main target of sinefungin in Streptomyces.

Expression of Sindbis virus nsP1 and methyltransferase activity in Escherichia coli.

We have constructed two plasmids, pSR5-42 and pSR5-Toto, which under lac control expressed the SVLM21 and the SVToto forms, respectively, of the Sindbis virus nonstructural protein, nsP1. The induced protein, which was the major protein made following induction with IPTG, had an apparent molecular weight of 60,000 and an amino terminal sequence in agreement with that expected for nsP1. Following induction with IPTG, cells carrying pSR5-42 (which contains the SVLM21 gene sequence) generated much higher RNA methyltransferase activity than cells carrying pSR5-Toto (which contains the SVToto gene sequence). This result is in agreement with what is observed when methyltransferase is measured in cells infected with SVLM21 and SVSTD (or SVToto), respectively. These results provide strong evidence that nsP1 has methyltransferase activity in the absence of any other viral nonstructural proteins.

Mutations that confer resistance to mycophenolic acid and ribavirin on Sindbis virus map to the nonstructural protein nsP1.

SVMPA, a mutant of Sindbis virus derived by serial passage on Aedes albopictus mosquito cells maintained after infection in the presence of mycophenolic acid (MPA), is resistant not only to MPA but also to ribavirin. Both of these compounds inhibit the synthesis of GMP and thereby reduce the level of GTP. We had suggested earlier that SVMPA had become resistant to MPA because it coded for an altered RNA guanylyltransferase enzyme with an increased affinity for GTP, enabling it to replicate in cells with reduced levels of GTP. We now report that the MPA-resistant phenotype of SVMPA has been mapped to the coding region for the nonstructural viral protein, nsP1. By replacing the nucleotide sequence between 88 and 1404 of the infectious clone of Sindbis virus (i.e., the Toto 1101 plasmid) with the corresponding sequence from SVMPA cDNA, we were able to generate recombinant Sindbis virus expressing the drug-resistant phenoptype. SVMPA has three base substitutions in the region between nucleotides 88 and 1404 which lead to predicted amino acid changes in the Sindbis virus nsP1 protein: the replacement of Gln at residue 21 by Lys, Ser at residue 23 by Asn, and Val at residue 302 by Met. These results, taken together with previous data from our laboratory associating the RNA methyltransferase with nsP1, (1) are consistent with the idea that an alteration of the RNA guanylyltransferase is responsible for the MPA-resistant phenotype and (2) support the idea that an important function of nsP1 relates to the modification of the 5' terminus of the Sindbis virus mRNAs.

High-level expression of Escherichia coli tRNA (m5U54)-methyltransferase.

A cloning and high-expression system for tRNA (m5U54)-methyltransferase (RUMT) is described. Polymerase chain reaction (PCR) was used to replicate the coding sequence and create flanking restriction sites for cloning. The PCR product was then inserted into expression vectors containing the tac and PL promoters. With the PL promoter, induced cells produced about 1.5% of their soluble protein as catalytically active RUMT. With the tac promoter, up to 8% of the total cell protein was active enzyme, and RUMT was purified to near homogeneity in three steps.