N7 Methylation Research Articles

Structural and functional studies of the Thermus thermophilus 16S rRNA methyltransferase RsmG

The RsmG methyltransferase is responsible for N(7) methylation of G527 of 16S rRNA in bacteria. Here, we report the identification of the Thermus thermophilus rsmG gene, the isolation of rsmG mutants, and the solution of RsmG X-ray crystal structures at up to 1.5 A resolution. Like their counterparts in other species, T. thermophilus rsmG mutants are weakly resistant to the aminoglycoside antibiotic streptomycin. Growth competition experiments indicate a physiological cost to loss of RsmG activity, consistent with the conservation of the modification site in the decoding region of the ribosome. In contrast to Escherichia coli RsmG, which has been reported to recognize only intact 30S subunits, T. thermophilus RsmG shows no in vitro methylation activity against native 30S subunits, only low activity with 30S subunits at low magnesium concentration, and maximum activity with deproteinized 16S rRNA. Cofactor-bound crystal structures of RsmG reveal a positively charged surface area remote from the active site that binds an adenosine monophosphate molecule. We conclude that an early assembly intermediate is the most likely candidate for the biological substrate of RsmG.

Flaviviral methyltransferase/RNA interaction: Structural basis for enzyme inhibition

Flaviviruses are the causative agents of severe diseases such as Dengue or Yellow fever. The replicative machinery used by the virus is based on few enzymes including a methyltransferase, located in the N-terminal domain of the NS5 protein. Flaviviral methyltransferases are involved in the last two steps of the mRNA capping process, transferring a methyl group from S-adenosyl- l-methionine onto the N7 position of the cap guanine (guanine-N7 methyltransferase) and the ribose 2′O position of the first nucleotide following the cap guanine (nucleoside-2′O methyltransferase). The RNA capping process is crucial for mRNA stability, protein synthesis and virus replication. Such an essential function makes methyltransferases attractive targets for the design of antiviral drugs. In this context, starting from the crystal structure of Wesselsbron flavivirus methyltransferase, we elaborated a mechanistic model describing protein/RNA interaction during N7 methyl transfer. Next we used an in silico docking procedure to identify commercially available compounds that would display high affinity for the methyltransferase active site. The best candidates selected were tested in vitro to assay their effective inhibition on 2′O and N7 methyltransferase activities on Wesselsbron and Dengue virus (Dv) methyltransferases. The results of such combined computational and experimental screening approach led to the identification of a high-potency inhibitor.

Coronavirus Nonstructural Protein 16 Is a Cap-0 Binding Enzyme Possessing (Nucleoside-2′ O )-Methyltransferase Activity

The coronavirus family of positive-strand RNA viruses includes important pathogens of livestock, companion animals, and humans, including the severe acute respiratory syndrome coronavirus that was responsible for a worldwide outbreak in 2003. The unusually complex coronavirus replicase/transcriptase is comprised of 15 or 16 virus-specific subunits that are autoproteolytically derived from two large polyproteins. In line with bioinformatics predictions, we now show that feline coronavirus (FCoV) nonstructural protein 16 (nsp16) possesses an S-adenosyl-L-methionine (AdoMet)-dependent RNA (nucleoside-2'O)-methyltransferase (2'O-MTase) activity that is capable of cap-1 formation. Purified recombinant FCoV nsp16 selectively binds to short capped RNAs. Remarkably, an N7-methyl guanosine cap ((7Me)GpppAC(3-6)) is a prerequisite for binding. High-performance liquid chromatography analysis demonstrated that nsp16 mediates methyl transfer from AdoMet to the 2'O position of the first transcribed nucleotide, thus converting (7Me)GpppAC(3-6) into (7Me)GpppA(2')(O)(Me)C(3-6). The characterization of 11 nsp16 mutants supported the previous identification of residues K45, D129, K169, and E202 as the putative K-D-K-E catalytic tetrad of the enzyme. Furthermore, residues Y29 and F173 of FCoV nsp16, which may be the functional counterparts of aromatic residues involved in substrate recognition by the vaccinia virus MTase VP39, were found to be essential for both substrate binding and 2'O-MTase activity. Finally, the weak inhibition profile of different AdoMet analogues indicates that nsp16 has evolved an atypical AdoMet binding site. Our results suggest that coronavirus mRNA carries a cap-1, onto which 2'O methylation follows an order of events in which 2'O-methyl transfer must be preceded by guanine N7 methylation, with the latter step being performed by a yet-unknown N7-specific MTase.

Separate molecules of West Nile virus methyltransferase can independently catalyze the N7 and 2′- O methylations of viral RNA cap

West Nile virus methyltransferase catalyzes N7 and 2′- O methylations of the viral RNA cap (GpppA-RNA → m 7GpppAm-RNA). The two methylation events are independent, as evidenced by efficient N7 methylation of GpppA-RNA → m 7GpppA-RNA and GpppAm-RNA → m 7GpppAm-RNA, and by the 2′- O methylation of GpppA-RNA → GpppAm-RNA and m 7GpppA-RNA → m 7GpppAm-RNA. However, the 2′- O methylation activity prefers substrate m 7GpppA-RNA to GpppA-RNA, thereby determining the dominant methylation pathway as GpppA-RNA → m 7GpppA-RNA → m 7GpppAm-RNA. Mutant enzymes with different methylation defects can trans complement one another in vitro. Furthermore, sequential treatment of GpppA-RNA with distinct methyltransferase mutants generates fully methylated m 7GpppAm-RNA, demonstrating that separate molecules of the enzyme can independently catalyze the two cap methylations in vitro.

Genetic Interactions among the West Nile Virus Methyltransferase, the RNA-Dependent RNA Polymerase, and the 5′ Stem-Loop of Genomic RNA

Flavivirus methyltransferase catalyzes both guanine N7 and ribose 2'-OH methylations of the viral RNA cap (GpppA-RNA-->m(7)GpppAm-RNA). The methyltransferase is physically linked to an RNA-dependent RNA polymerase (RdRp) in the flaviviral NS5 protein. Here, we report genetic interactions of West Nile virus (WNV) methyltransferase with the RdRp and the 5'-terminal stem-loop of viral genomic RNA. Genome-length RNAs, containing amino acid substitutions of D146 (a residue essential for both cap methylations) in the methyltransferase, were transfected into BHK-21 cells. Among the four mutant RNAs (D146L, D146P, D146R, and D146S), only D146S RNA generated viruses in transfected cells. Sequencing of the recovered viruses revealed that, besides the D146S change in the methyltransferase, two classes of compensatory mutations had reproducibly emerged. Class 1 mutations were located in the 5'-terminal stem-loop of the genomic RNA (a G35U substitution or U38 insertion). Class 2 mutations resided in NS5 (K61Q in methyltransferase and W751R in RdRp). Mutagenesis analysis, using a genome-length RNA and a replicon of WNV, demonstrated that the D146S substitution alone was lethal for viral replication; however, the compensatory mutations rescued replication, with the highest rescuing efficiency occurring when both classes of mutations were present. Biochemical analysis showed that a low level of N7 methylation of the D146S methyltransferase is essential for the recovery of adaptive viruses. The methyltransferase K61Q mutation facilitates viral replication through improved N7 methylation activity. The RdRp W751R mutation improves viral replication through an enhanced polymerase activity. Our results have clearly established genetic interactions among flaviviral methyltransferase, RdRp, and the 5' stem-loop of the genomic RNA.

The TbMTr1 Spliced Leader RNA Cap 1 2 ′-O-Ribose Methyltransferase from Trypanosoma brucei Acts with Substrate Specificity

In metazoa cap 1 (m(7)GpppNmp-RNA) is linked to higher levels of translation; however, the enzyme responsible remains unidentified. We have validated the first eukaryotic encoded cap 1 2'-O-ribose methyltransferase, TbMTr1, a member of a conserved family that modifies the first transcribed nucleotide of spliced leader and U1 small nuclear RNAs in the kinetoplastid protozoan Trypanosoma brucei. In addition to cap 0 (m(7)GpppNp-RNA), mRNA in these parasites has ribose methylations on the first four nucleotides with base methylations on the first and fourth (m(7)Gpppm(6,6)AmpAmpCmpm(3)Ump-SL RNA) conveyed via trans-splicing of a universal spliced leader. The function of this cap 4 is unclear. Spliced leader is the majority RNA polymerase II transcript; the RNA polymerase III-transcribed U1 small nuclear RNA has the same first four nucleotides as spliced leader, but it receives an m(2,2,7)G cap with hypermethylation at position one only (m(2,2,7)Gpppm(6,6)AmpApCpUp-U1 snRNA). Here we examine the biochemical properties of recombinant TbMTr1. Active over a pH range of 6.0 to 9.5, TbMTr1 is sensitive to Mg(2+). Positions Lys(95)-Asp(204)-Lys(259)-Glu(285) constitute the conserved catalytic core. A guanosine cap on RNA independent of its N(7) methylation status is required for substrate recognition, but an m(2,2,7G)-cap is not recognized. TbMTr1 favors the spliced leader 5' sequence, as reflected by a preference for A at position 1 and modulation of activity for substrates with base changes at positions 2 and 3. With similarities to human cap 1 methyltransferase activity, TbMTr1 is an excellent model for higher eukaryotic cap 1 methyltransferases and the consequences of cap 1 modification.

In vitro evaluation of the PARP inhibitor ABT-888 in combination with temozolomide for the treatment of pediatric leukemia

9528 Background: The alkylating agent temozolomide exerts its primary cytotoxic activity through the addition to O6 methyl adducts to guanine residues in DNA, and temozolomide resistance results either from increased expression of methyl-guanine DNA methyltransferase (MGMT), which removes O6 adducts, or from mutations in the mismatch repair system (MRS), which lead to microsatellite instability (MSI). Temozolomide also creates N3 and N7 methyl adducts that are efficiently removed by the base excision repair (BER) system. PARP inhibitors block BER and may potentiate the cytotoxic effects of temozolomide. Methods: The cytotoxicity of temozolomide in combination with the PARP inhibitor ABT-888 was evaluated in vitro in leukemia cell lines and primary leukemia cells using the MTT assay. PARP activity was measured using a commercially available PARP assay. Results: ABT-888 effectively enhanced temozolomide cytotoxicity in cell lines with elevated MGMT (Jurkat and HSB2 T-cell ALL cell lines) or MRS deficiencies (Jurkat and Molt-4 T cell ALL). The temozolomide IC50 decreased from 450 μM to 35 μM at an ABT-888 concentration of 5 μM in the Jurkat T-cell ALL cell line and from 340 μM to 7.5 μM in the HSB2 T-cell line. ABT-888 also enhanced temozolomide cytotoxicity in non-T-cell leukemia subtypes, decreasing the IC50 in the JM1 pre-B ALL cell line from 51 μM to 7.7 μM and decreasing the IC50 from 316 μM to 22 μM in tumor cells obtained from a patient with pre-B ALL. PARP activity was also examined. In contrast, the sensitivity of U937 AML cells to temozolomide showed no effect of the addition of a PARP inhibitor. These results are consistent with the findings that these cells have low or undetectable MGMT and no MSI to suggest MRS mutations. Conclusion: These results suggest that ABT-888 may enhance the cytotoxic activity of temozolomide in leukemia patients whose tumors are resistant to temozolomide because of elevated MGMT expression and mismatch repair defects. No significant financial relationships to disclose.

Pairing strength and proton characters of the N7,N9-dimethylated GC and AT base pairs: a density functional theory investigation

The pairing strength, proton properties associated with proton transfer and deprotonation as well as relevant structural perturbations of 7,9-dimethyl GC ([mGC]+) and 7,9-dimethyl AT ([mAT]+) base pairs have been investigated. Energies related to proton transfer and deprotonation have also been predicted. It is found that the guanine/adenine N7 methylation improves the stability of GC/AT base pair. The proton transfer between the guanine N1 and the cytosine N3 is observed in [mGC]+ with an out-of-plane transition state. However, no proton-transfer reaction occurs for [mAT]+. For the deprotonation of [mGC]+ and [mAT]+, guanine C8 and N1 and adenine C8 are the most favorable sites. Deprotonation from the pyrimidine N1 site generates the most stable deprotonated base pairs, and the dissociation energies surprisingly amount to ∼100.00 and ∼65.49 kcal mol−1, respectively, much higher than those of their neutral base pairs. Deprotonation from the other sites of pyrimidine exhibits the most significant structural changes and gives the most interesting deprotonated base pairs. This process is accompanied by a barrier-free proton transfer (BFPT) from guanine N1 to cytosine N3 or adenine N6 to thymine N3 or O4 site. In this way, two rare imino tautomers of cytosine (trans-C* or cis-C*) are easily generated by removing a proton from the N4 site of cytosine in [mGC]+. In addition, the reason for BFPT from guanine N1 to cytosine N3 has been explored.

Identification of a new region in the vesicular stomatitis virus L polymerase protein which is essential for mRNA cap methylation

The vesicular stomatitis virus (VSV) L polymerase protein possesses two methyltransferase (MTase) activities, which catalyze the methylation of viral mRNA cap structures at the guanine- N7 and 2′- O-adenosine positions. To identify L sequences required for the MTase activities, we analyzed a host range ( hr) and temperature-sensitive ( ts) mutant of VSV, hr8, which was defective in mRNA cap methylation. Sequencing hr8 identified five amino acid substitutions, all residing in the L protein. Recombinant VSV were generated with each of the identified L mutations, and the presence of a single G1481R substitution in L, located between conserved domains V and VI, was sufficient to produce a dramatic reduction (about 90%) in overall mRNA methylation. Cap analysis showed residual guanine- N7 methylation and reduced 2′- O-adenosine methylation, identical to that of the original hr8 virus. When recombinant viruses were tested for virus growth under conditions that were permissive and nonpermissive for the hr8 mutant, the same single L mutation, G1481R, was solely responsible for both the hr and ts phenotypes. A spontaneous suppressor mutant of the rG1481R virus that restored both growth on nonpermissive cells and cap methylation was identified and mapped to a single change, L1450I, in L. Site-directed mutagenesis of the region between domains V and VI, amino acids 1419–1672 of L, followed by the rescue of recombinant viruses identified five additional virus mutants, K1468A, R1478A/D1479A, G1481A, G1481N, and G1672A, that were all hr and defective in mRNA cap methylation. Thus, in addition to the previously characterized domain VI [Grdzelishvili, V.Z., Smallwood, S., Tower, D., Hall, R.L., Hunt, D.M., Moyer, S.A., 2005. A single amino acid change in the L-polymerase protein of vesicular stomatitis virus completely abolishes viral mRNA cap methylation. J. Virol. 79, 7327–7337; Li, J., Fontaine-Rodriguez, E.C., Whelan, S.P., 2005. Amino acid residues within conserved domain VI of the vesicular stomatitis virus large polymerase protein essential for mRNA cap methyltransferase activity. J. Virol. 79, 13373–13384], a new region between L amino acids 1450–1481 was identified which is critical for mRNA cap methylation.

Specificity and Mechanism of RNA Cap Guanine-N2 Methyltransferase (Tgs1)

The 2,2,7-trimethylguanosine (TMG) cap structure is characteristic of certain eukaryotic small nuclear and small nucleolar RNAs. Prior studies have suggested that cap trimethylation might be contingent on cis-acting elements in the RNA substrate, protein components of a ribonucleoprotein complex, or intracellular localization of the RNA substrate. However, the enzymatic requirements for TMG cap formation remain obscure because TMG synthesis has not been reconstituted in vitro from defined components. Tgs1 is a conserved eukaryal protein that was initially identified as being required for RNA cap trimethylation in vivo in budding yeast. Here we show that purified recombinant fission yeast Tgs1 catalyzes methyl transfer from S-adenosylmethionine (AdoMet) to m7GTP and m7GDP. Tgs1 also methylates the cap analog m(7)GpppA but is unreactive with GTP, GDP, GpppA, m2,2,7GTP, m2,2,7GDP, ATP, CTP, UTP, and ITP. The products of methyl transfer to m7GTP and m7GDP formed under conditions of excess methyl acceptor are 2,7-dimethyl GTP and 2,7-dimethyl GDP, respectively. Under conditions of limiting methyl acceptor, the initial m2,7GDP product is converted to m2,2,7GDP in the presence of excess AdoMet. We conclude that Tgs1 is guanine-specific, that N7 methylation must precede N2 methylation, that Tgs1 acts via a distributive mechanism, and that the chemical steps of TMG synthesis do not require input from RNA or protein cofactors.

Structure and Mechanism of mRNA Cap (Guanine-N7) Methyltransferase

A suite of crystal structures is reported for a cellular mRNA cap (guanine-N7) methyltransferase in complex with AdoMet, AdoHcy, and the cap guanylate. Superposition of ligand complexes suggests an in-line mechanism of methyl transfer, albeit without direct contacts between the enzyme and either the N7 atom of guanine (the attacking nucleophile), the methyl carbon of AdoMet, or the sulfur of AdoMet/AdoHcy (the leaving group). The structures indicate that catalysis of cap N7 methylation is accomplished by optimizing proximity and orientation of the substrates, assisted by a favorable electrostatic environment. The enzyme-ligand structures, together with new mutational data, fully account for the biochemical specificity of the cap guanine-N7 methylation reaction, an essential and defining step of eukaryotic mRNA synthesis.

Aqueous Ionization and Electron-Donating Properties of Dinucleotides: Sequence-Specific Electronic Effects on DNA Alkylation

He I, UV photoelectron data and results from self-consistent-field (SCF) and post-SCF molecular orbital calculations were used to evaluate multiple gas-phase ionization potentials (IPs) of the phosphorylated dinucleotide 5‘pGpAp both isolated and in a cluster with Na+ and H2O. SCF calculations with a split-valence basis set indicate that the ground-state valence orbital structure of 5‘pGpAp is generally localized on the base, sugar, or phosphate groups and that orbitals in the dinucleotide are similar to orbitals in the model compounds and anion, 1,9-dimethylguanine, 9-methyladenine, 3-hydroxytetrahydrofuran, and H2PO4-. This correlation parallels that in mononucleotides (Fernando, H.; Papadantonakis, G. A.; Kim, N. S.; LeBreton, P. R. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 5550−5555; Kim, N. S.; LeBreton, P. R. J. Am. Chem. Soc. 1996, 118, 3694−3707) and permits the correction of SCF ionization potentials using experimental IPs for the model compounds and theoretical IPs from post-SCF calculations on H2PO4-. A comparison of IPs of 5‘pGpAp, 5‘pGpA, 2‘-deoxyguanosine 5‘-phosphate (5‘-dGMP-), and 2‘-deoxyadenosine 5‘-phosphate (5‘-dAMP-), both isolated and in gas-phase clusters with Na+ and H2O, indicates that the electrostatic influence of Na+ and the anionic phosphate groups is great. IPs decrease significantly as the number of phosphate groups increases. For 5‘-dGMP-, 5‘pGpA, and 5‘pGpAp, the lowest adiabatic guanine ionization potentials are 5.2, 3.1, and 1.6 eV, respectively; the lowest phosphate vertical IPs are 5.1, 3.1, and 1.9 eV. Gas-phase IPs were combined with hydration energies obtained with a Langevin dipole relaxation model to evaluate the 22 lowest aqueous ionization energies in 5‘pGpAp as well as ionization energies in the other nucleotides with and without counterions. The electrostatic dependence of the gas-phase IPs on the number of phosphate groups is modulated in aqueous solution. For 5‘-dGMP-, 5‘pGpA, and 5‘pGpAp, the aqueous guanine and phosphate ionization energies lie in the ranges 4.2−4.9 eV and 5.5−6.1 eV, respectively. Solvent relaxation similarly modulates Na+ electrostatic effects. It also alters the relative energies of ionization events. In the gas phase, the first adiabatic base IP is equal to or larger than the lowest phosphate vertical IP. In water, the base ionization energies are 1.2−1.3 eV smaller than the phosphate ionization energies. Guanine gas-phase π ionization energies in five different B-DNA oligomer models, each containing six stacked base pairs, were compared with site-specific reactivity data for methylation at the guanine N7 and O6 atoms by the carcinogen, N-methyl-N-nitrosourea (MNU). O6 methylation is associated with MNU carcinogenesis; N7 methylation is the principal DNA reaction pathway. At both guanine atoms, reactivity increases as the lowest guanine π ionization potential decreases. This is consistent with a description of transition states in which activation barriers are lowered as nucleotide base π polarizability increases.

High resolution nuclear magnetic resonance studies of platinum(II) – guanosine-5′-monophosphate interactions in aqueous solutions

A detailed 400 MHz 1H nmr study was carried out on platinum complexation products of GMP by K2PtCl4. Coupling constant values show that platination on N7 induces a conformational change on the ribose moiety: the 3E form of the ring and the gt conformer about the C(4′)—C(5′) bond are favoured upon complexation. The results are compared to the conformational changes induced by N7 protonation and methylation.