Characterization of the biochemical activity and tumor-promoting role of the dual protein methyltransferase METL-13/METTL13 in Caenorhabditis elegans.

The methyltransferase-like protein 13 (METTL13) methylates the eukaryotic elongation factor 1 alpha (eEF1A) on two locations: the N-terminal amino group and lysine 55. The absence of this methylation leads to reduced protein synthesis and cell proliferation in human cancer cells. Previous studies showed that METTL13 is dispensable in non-transformed cells, making it potentially interesting for cancer therapy. However, METTL13 has not been examined yet in whole animals. Here, we used the nematode Caenorhabditis elegans as a simple model to assess the functions of METTL13. Using methyltransferase assays and mass spectrometry, we show that the C. elegans METTL13 (METL-13) methylates eEF1A (EEF-1A) in the same way as the human protein. Crucially, the cancer-promoting role of METL-13 is also conserved and depends on the methylation of EEF-1A, like in human cells. At the same time, METL-13 appears dispensable for animal growth, development, and stress responses. This makes C. elegans a convenient whole-animal model for studying METL13-dependent carcinogenesis without the complications of interfering with essential wild-type functions.

Human seven-ß-strand (METTL) methyltransferases - conquering the universe of protein lysine methylation.

Lysine methylation is an abundant posttranslational modification, which has been most intensively studied in the context of histone proteins, where it represents an important epigenetic mark. Lysine methylation of histone proteins is primarily catalyzed by SET-domain methyltransferases (MTases). However, it has recently become evident that also another MTase family, the so-called seven-ß-strand (7BS) MTases, often denoted METTLs (methyltransferase-like), contains several lysine (K)-specific MTases (KMTs). These enzymes catalyze the attachment of up to three methyl groups to lysine residues in specific substrate proteins, using S-adenosylmethionine (AdoMet) as methyl donor. About a decade ago, only a single human 7BS KMT was known, namely the histone-specific DOT1L, but 15 additional 7BS KMTs have now been discovered and characterized. These KMTs typically target a single nonhistone substrate that, in most cases, belongs to one of the following three protein groups: components of the cellular protein synthesis machinery, mitochondrial proteins, and molecular chaperones. This article provides an extensive overview and discussion of the human 7BS KMTs and their biochemical and biological roles.

The human methyltransferase ZCCHC4 catalyses N6-methyladenosine modification of 28S ribosomal RNA.

RNA methylations are essential both for RNA structure and function, and are introduced by a number of distinct methyltransferases (MTases). In recent years, N6-methyladenosine (m6A) modification of eukaryotic mRNA has been subject to intense studies, and it has been demonstrated that m6A is a reversible modification that regulates several aspects of mRNA function. However, m6A is also found in other RNAs, such as mammalian 18S and 28S ribosomal RNAs (rRNAs), but the responsible MTases have remained elusive. 28S rRNA carries a single m6A modification, found at position A4220 (alternatively referred to as A4190) within a stem-loop structure, and here we show that the MTase ZCCHC4 is the enzyme responsible for introducing this modification. Accordingly, we found that ZCCHC4 localises to nucleoli, the site of ribosome assembly, and that proteins involved in RNA metabolism are overrepresented in the ZCCHC4 interactome. Interestingly, the absence of m6A4220 perturbs codon-specific translation dynamics and shifts gene expression at the translational level. In summary, we establish ZCCHC4 as the enzyme responsible for m6A modification of human 28S rRNA, and demonstrate its functional significance in mRNA translation.

Human FAM173A is a mitochondrial lysine-specific methyltransferase that targets adenine nucleotide translocase and affects mitochondrial respiration.

Lysine methylation is a common posttranslational modification of nuclear and cytoplasmic proteins but is also present in mitochondria. The human protein denoted "family with sequence similarity 173 member B" (FAM173B) was recently uncovered as a mitochondrial lysine (K)-specific methyltransferase (KMT) targeting the c-subunit of mitochondrial ATP synthase (ATPSc), and was therefore renamed ATPSc-KMT. We here set out to investigate the biochemical function of its yet uncharacterized paralogue FAM173A. We demonstrate that FAM173A localizes to mitochondria, mediated by a noncanonical targeting sequence that is partially retained in the mature protein. Immunoblotting analysis using methyllysine-specific antibodies revealed that FAM173A knock-out (KO) abrogates lysine methylation of a single mitochondrial protein in human cells. Mass spectrometry analysis identified this protein as adenine nucleotide translocase (ANT), represented by two highly similar isoforms ANT2 and ANT3. We found that methylation occurs at Lys-52 of ANT, which was previously reported to be trimethylated. Complementation of KO cells with WT or enzyme-dead FAM173A indicated that the enzymatic activity of FAM173A is required for ANT methylation at Lys-52 to occur. Both in human cells and in rat organs, Lys-52 was exclusively trimethylated, indicating that this modification is constitutive, rather than regulatory and dynamic. Moreover, FAM173A-deficient cells displayed increased mitochondrial respiration compared with FAM173A-proficient cells. In summary, we demonstrate that FAM173A is the long-sought KMT responsible for ANT methylation at Lys-52, and point out the functional significance of Lys-52 methylation in ANT. Based on the established naming nomenclature for KMTs, we propose to rename FAM173A to ANT-KMT (gene name ANTKMT).

Uncovering human METTL12 as a mitochondrial methyltransferase that modulates citrate synthase activity through metabolite-sensitive lysine methylation.

Lysine methylation is an important and much-studied posttranslational modification of nuclear and cytosolic proteins but is present also in mitochondria. However, the responsible mitochondrial lysine-specific methyltransferases (KMTs) remain largely elusive. Here, we investigated METTL12, a mitochondrial human S-adenosylmethionine (AdoMet)-dependent methyltransferase and found it to methylate a single protein in mitochondrial extracts, identified as citrate synthase (CS). Using several in vitro and in vivo approaches, we demonstrated that METTL12 methylates CS on Lys-395, which is localized in the CS active site. Interestingly, the METTL12-mediated methylation inhibited CS activity and was blocked by the CS substrate oxaloacetate. Moreover, METTL12 was strongly inhibited by the reaction product S-adenosylhomocysteine (AdoHcy). In summary, we have uncovered a novel human mitochondrial KMT that introduces a methyl modification into a metabolic enzyme and whose activity can be modulated by metabolic cues. Based on the established naming nomenclature for similar enzymes, we suggest that METTL12 be renamed CS-KMT (gene name CSKMT).

Protein lysine methylation by seven-ß-strand methyltransferases.

Methylation of biomolecules is a frequent biochemical reaction within the cell, and a plethora of highly specific methyltransferases (MTases) catalyse the transfer of a methyl group from S-adenosylmethionine (AdoMet) to various substrates. The posttranslational methylation of lysine residues, catalysed by numerous lysine (K)-specific protein MTases (KMTs), is a very common and important protein modification, which recently has been subject to intense studies, particularly in the case of histone proteins. The majority of KMTs belong to a class of MTases that share a defining 'SET domain', and these enzymes mostly target lysines in the flexible tails of histones. However, the so-called seven-ß-strand (7BS) MTases, characterized by a twisted beta-sheet structure and certain conserved sequence motifs, represent the largest MTase class, and these enzymes methylate a wide range of substrates, including small metabolites, lipids, nucleic acids and proteins. Until recently, the histone-specific Dot1/DOT1L was the only identified eukaryotic 7BS KMT. However, a number of novel 7BS KMTs have now been discovered, and, in particular, several recently characterized human and yeast members of MTase family 16 (MTF16) have been found to methylate lysines in non-histone proteins. Here, we review the status and recent progress on the 7BS KMTs, and discuss these enzymes at the levels of sequence/structure, catalytic mechanism, substrate recognition and biological significance.

Lysine Methylation of the Valosin-Containing Protein (VCP) Is Dispensable for Development and Survival of Mice.

Valosin-containing protein (VCP) is a homohexameric ATPase involved in a multitude cellular processes and it was recently shown that VCP is trimethylated at lysine 315 by the VCP lysine methyltransferase (VCPKMT). Here, we generated and validated a constitutive knockout mouse by targeting exon 1-4 of the Vcpkmt gene. We show that Vcpkmt is ubiquitously expressed in all tissues examined and confirm the sub-cellular localization to the cytoplasm. We show by (I) mass spectrometric analysis, (II) VCPKMT-mediated in vitro methylation of VCP in cell extracts and (III) immunostaining with a methylation specific antibody, that in Vcpkmt-/- mice the methylation of lysine 315 in VCP is completely abolished. In contrast, VCP is almost exclusively trimethylated in wild-type mice. Furthermore, we investigated the specificity of VCPKMT with in vitro methylation assays using as source of substrate protein extracts from Vcpkmt-/- mouse organs or three human Vcpkmt-/- cell lines. The results show that VCPKMT is a highly specific enzyme, and suggest that VCP is its sole substrate. The Vcpkmt-/- mice were viable, fertile and had no obvious pathological phenotype. Their body weight, life span and acute endurance capacity were comparable to wild-type controls. Overall the results show that VCPKMT is an enzyme required for methylation of K315 of VCP in vivo, but VCPKMT is not essential for development or survival under unstressed conditions.

Hsp70 (HSPA1) Lysine Methylation Status as a Potential Prognostic Factor in Metastatic High-Grade Serous Carcinoma.

Cellular proteins are subject to frequent methylation on lysine residues, introduced by specific methyltransferases, and each lysine residue can receive up to three methyl groups. Histone methylations, which are key determinants of chromatin state and transcriptional status, have been subject to particularly intense studies, but methylations on non-histone protein substrates are also abundant and biologically significant. Numerous studies have addressed lysine methylation in the realm of cancer biology. A recent study used an antibody-based approach to investigate the methylation of Lys-561 of the stress-inducible Hsp70 protein HSPA1, focusing exclusively on dimethylated HSPA1, concluding that it was elevated in cancer [Cho et al. (2012), Nat. Commun.,3, 1072]. In the present study, we have performed a more extensive analysis of HSPA1 methylation status in cancer samples, using protein mass spectrometry. We found that the four methylation states of Lys561 on HSPA1 (un-, mono-, di- and trimethylated) could be measured accurately and reproducibly in samples from carcinomas. We investigated HSPA1 methylation in 70 effusions, representing 53 high-grade serous ovarian carcinomas and 17 breast carcinomas. Notably, we found the trimethylated form of HSPA1 to be predominant in the cancer samples. HSPA1 methylation was studied for association with clinicopathologic parameters, including chemotherapy response and survival. The trimethylated form was more prevalent in breast carcinoma effusions (p = 0.014), whereas the dimethylated (p = 0.025), monomethylated (p = 0.004) and unmethylated (p = 0.021) forms were overrepresented in the ovarian carcinomas. For the ovarian carcinomas, the monomethylated (p = 0.028) and unmethylated (p = 0.007) forms were significantly related to the presence of higher residual disease volume, while the unmethylated form was significantly associated with poor overall (p = 0.015) and progression-free (p = 0.012) survival. In conclusion, lysine methylation of HSPA1 differs between metastatic breast and ovarian carcinoma, and unmethylated HSPA1 shows potential as a prognostic marker in high-grade serous carcinoma.

Differential repair of etheno-DNA adducts by bacterial and human AlkB proteins.

AlkB proteins are evolutionary conserved Fe(II)/2-oxoglutarate-dependent dioxygenases, which remove alkyl and highly promutagenic etheno(ɛ)-DNA adducts, but their substrate specificity has not been fully determined. We developed a novel assay for the repair of ɛ-adducts by AlkB enzymes using oligodeoxynucleotides with a single lesion and specific DNA glycosylases and AP-endonuclease for identification of the repair products. We compared the repair of three ɛ-adducts, 1,N(6)-ethenoadenine (ɛA), 3,N(4)-ethenocytosine (ɛC) and 1,N(2)-ethenoguanine (1,N(2)-ɛG) by nine bacterial and two human AlkBs, representing four different structural groups defined on the basis of conserved amino acids in the nucleotide recognition lid, engaged in the enzyme binding to the substrate. Two bacterial AlkB proteins, MT-2B (from Mycobacterium tuberculosis) and SC-2B (Streptomyces coelicolor) did not repair these lesions in either double-stranded (ds) or single-stranded (ss) DNA. Three proteins, RE-2A (Rhizobium etli), SA-2B (Streptomyces avermitilis), and XC-2B (Xanthomonas campestris) efficiently removed all three lesions from the DNA substrates. Interestingly, XC-2B and RE-2A are the first AlkB proteins shown to be specialized for ɛ-adducts, since they do not repair methylated bases. Three other proteins, EcAlkB (Escherichia coli), SA-1A, and XC-1B removed ɛA and ɛC from ds and ssDNA but were inactive toward 1,N(2)-ɛG. SC-1A repaired only ɛA with the preference for dsDNA. The human enzyme ALKBH2 repaired all three ɛ-adducts in dsDNA, while only ɛA and ɛC in ssDNA and repair was less efficient in ssDNA. ALKBH3 repaired only ɛC in ssDNA. Altogether, we have shown for the first time that some AlkB proteins, namely ALKBH2, RE-2A, SA-2B and XC-2B can repair 1,N(2)-ɛG and that ALKBH3 removes only ɛC from ssDNA. Our results also suggest that the nucleotide recognition lid is not the sole determinant of the substrate specificity of AlkB proteins.

Human METTL20 is a mitochondrial lysine methyltransferase that targets the ß subunit of electron transfer flavoprotein (ETFß) and modulates its activity.

Proteins are frequently modified by post-translational methylation of lysine residues, catalyzed by S-adenosylmethionine-dependent lysine methyltransferases (KMTs). Lysine methylation of histone proteins has been extensively studied, but it has recently become evident that methylation of non-histone proteins is also abundant and important. The human methyltransferase METTL20 belongs to a group of 10 established and putative human KMTs. We here found METTL20 to be associated with mitochondria and determined that recombinant METTL20 methylated a single protein in extracts from human cells. Using an methyltransferase activity-based purification scheme, we identified the ß-subunit of the mitochondrially localized electron transfer flavoprotein (ETFß) as the substrate of METTL20. Furthermore, METTL20 was found to specifically methylate two adjacent lysine residues, Lys(200) and Lys(203), in ETFß both in vitro and in cells. Interestingly, the residues methylated by METTL20 partially overlap with the so-called "recognition loop" in ETFß, which has been shown to mediate its interaction with various dehydrogenases. Accordingly, we found that METTL20-mediated methylation of ETFß in vitro reduced its ability to receive electrons from the medium chain acyl-CoA dehydrogenase and the glutaryl-CoA dehydrogenase. In conclusion, the present study establishes METTL20 as the first human KMT localized to mitochondria and suggests that it may regulate cellular metabolism through modulating the interaction between its substrate ETFß and dehydrogenases. Based on the previous naming of similar enzymes, we suggest the renaming of human METTL20 to ETFß-KMT.

Identification and characterization of a novel human methyltransferase modulating Hsp70 protein function through lysine methylation.

Hsp70 proteins constitute an evolutionarily conserved protein family of ATP-dependent molecular chaperones involved in a wide range of biological processes. Mammalian Hsp70 proteins are subject to various post-translational modifications, including methylation, but for most of these, a functional role has not been attributed. In this study, we identified the methyltransferase METTL21A as the enzyme responsible for trimethylation of a conserved lysine residue found in several human Hsp70 (HSPA) proteins. This enzyme, denoted by us as HSPA lysine (K) methyltransferase (HSPA-KMT), was found to catalyze trimethylation of various Hsp70 family members both in vitro and in vivo, and the reaction was stimulated by ATP. Furthermore, we show that HSPA-KMT exclusively methylates 70-kDa proteins in mammalian protein extracts, demonstrating that it is a highly specific enzyme. Finally, we show that trimethylation of HSPA8 (Hsc70) has functional consequences, as it alters the affinity of the chaperone for both the monomeric and fibrillar forms of the Parkinson disease-associated protein α-synuclein.

Human ALKBH4 interacts with proteins associated with transcription.

The Fe(II)- and 2-oxoglutarate (2OG)-dependent dioxygenase AlkB from E. coli is a demethylase which repairs alkyl lesions in DNA, as well as RNA, through a direct reversal mechanism. Humans possess nine AlkB homologs (ALKBH1-8 and FTO). ALKBH2 and ALKBH3 display demethylase activities corresponding to that of AlkB, and both ALKBH8 and FTO are RNA modification enzymes. The biochemical functions of the rest of the homologs are still unknown. To increase our knowledge on the functions of ALKBH4 and ALKBH7 we have here performed yeast two-hybrid screens to identify interaction partners of the two proteins. While no high-confidence hits were detected in the case of ALKBH7, several proteins associated with chromatin and/or involved in transcription were found to interact with ALKBH4. For all interaction partners, the regions mediating binding to ALKBH4 comprised domains previously reported to be involved in interaction with DNA or chromatin. Furthermore, some of these partners showed nuclear co-localization with ALKBH4. However, the global gene expression pattern was only marginally altered upon ALKBH4 over-expression, and larger effects were observed in the case of ALKBH7. Although the molecular function of both proteins remains to be revealed, our findings suggest a role for ALKBH4 in regulation of gene expression or chromatin state.

Lysine methylation of VCP by a member of a novel human protein methyltransferase family.

Valosin-containing protein (VCP, also called p97) is an essential and highly conserved adenosine triphosphate-dependent chaperone implicated in a wide range of cellular processes in eukaryotes, and mild VCP mutations can cause severe neurodegenerative disease. Here we show that mammalian VCP is trimethylated on Lys315 in a variety of cell lines and tissues, and that the previously uncharacterized protein METTL21D (denoted here as VCP lysine methyltransferase, VCP-KMT) is the responsible enzyme. VCP methylation was abolished in three human VCP-KMT knockout cell lines generated with zinc-finger nucleases. Interestingly, VCP-KMT was recently reported to promote tumour metastasis, and indeed, VCP-KMT-deficient cells displayed reduced growth rate, migration and invasive potential. Finally, we present data indicating that VCP-KMT, calmodulin-lysine methyltransferase and eight uncharacterized proteins together constitute a novel human protein methyltransferase family. The present work provides new insights on protein methylation and its links to human disease.

Roles of Trm9- and ALKBH8-like proteins in the formation of modified wobble uridines in Arabidopsis tRNA.

Uridine at the wobble position of tRNA is usually modified, and modification is required for accurate and efficient protein translation. In eukaryotes, wobble uridines are modified into 5-methoxycarbonylmethyluridine (mcm(5)U), 5-carbamoylmethyluridine (ncm(5)U) or derivatives thereof. Here, we demonstrate, both by in vitro and in vivo studies, that the Arabidopsis thaliana methyltransferase AT1G31600, denoted by us AtTRM9, is responsible for the final step in mcm(5)U formation, thus representing a functional homologue of the Saccharomyces cerevisiae Trm9 protein. We also show that the enzymatic activity of AtTRM9 depends on either one of two closely related proteins, AtTRM112a and AtTRM112b. Moreover, we demonstrate that AT1G36310, denoted AtALKBH8, is required for hydroxylation of mcm(5)U to (S)-mchm(5)U in tRNA(Gly)(UCC), and has a function similar to the mammalian dioxygenase ALKBH8. Interestingly, atalkbh8 mutant plants displayed strongly increased levels of mcm(5)U, and also of mcm(5)Um, its 2'-O-ribose methylated derivative. This suggests that accumulated mcm(5)U is prone to further ribose methylation by a non-specialized mechanism, and may challenge the notion that the existence of mcm(5)U- and mcm(5)Um-containing forms of the selenocysteine-specific tRNA(Sec) in mammals reflects an important regulatory process. The present study reveals a role in for several hitherto uncharacterized Arabidopsis proteins in the formation of modified wobble uridines.

ALKBH8-mediated formation of a novel diastereomeric pair of wobble nucleosides in mammalian tRNA.

Mammals have nine different homologues (ALKBH1-9) of the Escherichia coli DNA repair demethylase AlkB. ALKBH2 is a genuine DNA repair enzyme, but the in vivo function of the other ALKBH proteins has remained elusive. It was recently shown that ALKBH8 contains an additional transfer RNA (tRNA) methyltransferase domain, which generates the wobble nucleoside 5-methoxycarbonylmethyluridine (mcm(5)U) from its precursor 5-carboxymethyluridine (cm(5)U). In this study, we report that (R)- and 5-methoxycarbonylhydroxymethyluridine (mchm(5)U), hydroxylated forms of mcm(5)U, are present in mammalian tRNA-Arg(UCG), and tRNA-Gly(UCC), respectively, representing the first example of a diastereomeric pair of modified RNA nucleosides. Through in vitro and in vivo studies, we show that both diastereomers of mchm(5)U are generated from mcm(5)U, and that the AlkB domain of ALKBH8 specifically hydroxylates mcm(5)U into (S)-mchm(5)U in tRNA-Gly(UCC). These findings expand the function of the ALKBH oxygenases beyond nucleic acid repair and increase the current knowledge on mammalian wobble uridine modifications and their biogenesis.

Spectroscopic and magnetic studies of wild-type and mutant forms of the Fe(II)- and 2-oxoglutarate-dependent decarboxylase ALKBH4.

The Fe(II)/2OG (2-oxoglutarate)-dependent dioxygenase superfamily comprises proteins that couple substrate oxidation to decarboxylation of 2OG to succinate. A member of this class of mononuclear non-haem Fe proteins is the Escherichia coli DNA/RNA repair enzyme AlkB. In the present work, we describe the magnetic and optical properties of the yet uncharacterized human ALKBH4 (AlkB homologue). Through EPR and UV-visible spectroscopy studies, we address the Fe-binding environment of the proposed catalytic centre of wild-type ALKBH4 and an Fe(II)-binding mutant. We could observe a novel unusual Fe(III) high-spin EPR-active species in the presence of sulfide with a g(max) of 8.2. The Fe(II) site was probed with NO. An intact histidine-carboxylate site is necessary for productive Fe binding. We also report the presence of a unique cysteine-rich motif conserved in the N-terminus of ALKBH4 orthologues, and investigate its possible Fe-binding ability. Furthermore, we show that recombinant ALKBH4 mediates decarboxylation of 2OG in absence of primary substrate. This activity is dependent on Fe as well as on residues predicted to be involved in Fe(II) co-ordination. The present results demonstrate that ALKBH4 represents an active Fe(II)/2OG-dependent decarboxylase and suggest that the cysteine cluster is involved in processes other than Fe co-ordination.

Mammalian ALKBH8 possesses tRNA methyltransferase activity required for the biogenesis of multiple wobble uridine modifications implicated in translational decoding.

Uridines in the wobble position of tRNA are almost invariably modified. Modifications can increase the efficiency of codon reading, but they also prevent mistranslation by limiting wobbling. In mammals, several tRNAs have 5-methoxycarbonylmethyluridine (mcm5U) or derivatives thereof in the wobble position. Through analysis of tRNA from Alkbh8-/- mice, we show here that ALKBH8 is a tRNA methyltransferase required for the final step in the biogenesis of mcm5U. We also demonstrate that the interaction of ALKBH8 with a small accessory protein, TRM112, is required to form a functional tRNA methyltransferase. Furthermore, prior ALKBH8-mediated methylation is a prerequisite for the thiolation and 2'-O-ribose methylation that form 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U) and 5-methoxycarbonylmethyl-2'-O-methyluridine (mcm5Um), respectively. Despite the complete loss of all of these uridine modifications, Alkbh8-/- mice appear normal. However, the selenocysteine-specific tRNA (tRNASec) is aberrantly modified in the Alkbh8-/- mice, and for the selenoprotein Gpx1, we indeed observed reduced recoding of the UGA stop codon to selenocysteine.

AlkB homologue 2-mediated repair of ethenoadenine lesions in mammalian DNA.

Endogenous formation of the mutagenic DNA adduct 1,N(6)-ethenoadenine (epsilon A) originates from lipid peroxidation. Elevated levels of epsilon A in cancer-prone tissues suggest a role for this adduct in the development of some cancers. The base excision repair pathway has been considered the principal repair system for epsilon A lesions until recently, when it was shown that the Escherichia coli AlkB dioxygenase could directly reverse the damage. We report here kinetic analysis of the recombinant human AlkB homologue 2 (hABH2), which is able to repair epsilon A lesions in DNA. Furthermore, cation exchange chromatography of nuclear extracts from wild-type and mABH2(-/-) mice indicates that mABH2 is the principal dioxygenase for epsilon A repair in vivo. This is further substantiated by experiments showing that hABH2, but not hABH3, is able to complement the E. coli alkB mutant with respect to its defective repair of etheno adducts. We conclude that ABH2 is active in the direct reversal of epsilon A lesions, and that ABH2, together with the alkyl-N-adenine-DNA glycosylase, which is the most effective enzyme for the repair of epsilon A, comprise the cellular defense against epsilon A lesions.

Repair deficient mice reveal mABH2 as the primary oxidative demethylase for repairing 1meA and 3meC lesions in DNA.

Two human homologs of the Escherichia coli AlkB protein, denoted hABH2 and hABH3, were recently shown to directly reverse 1-methyladenine (1meA) and 3-methylcytosine (3meC) damages in DNA. We demonstrate that mice lacking functional mABH2 or mABH3 genes, or both, are viable and without overt phenotypes. Neither were histopathological changes observed in the gene-targeted mice. However, in the absence of any exogenous exposure to methylating agents, mice lacking mABH2, but not mABH3 defective mice, accumulate significant levels of 1meA in the genome, suggesting the presence of a biologically relevant endogenous source of methylating agent. Furthermore, embryonal fibroblasts from mABH2-deficient mice are unable to remove methyl methane sulfate (MMS)-induced 1meA from genomic DNA and display increased cytotoxicity after MMS exposure. In agreement with these results, we found that in vitro repair of 1meA and 3meC in double-stranded DNA by nuclear extracts depended primarily, if not solely, on mABH2. Our data suggest that mABH2 and mABH3 have different roles in the defense against alkylating agents.

AlkB restores the biological function of mRNA and tRNA inactivated by chemical methylation.

Deleterious 1-methyladenine (1-meA) and 3-methylcytosine (3-meC) lesions are introduced into nucleic acids by methylating agents. It was recently demonstrated that the E. coli AlkB protein and a human homolog, hABH3, can demethylate these lesions both in DNA and RNA. To elucidate the biological significance of the RNA repair, we have tested whether such repair can rescue the function of chemically methylated RNA. We demonstrate that a methylation-induced block in translation of an mRNA can be readily relieved by treatment with AlkB and hABH3 prior to translation. Furthermore, we show that chemical methylation of tRNAPhe inhibits aminoacylation and translation, but that the inhibition can be reversed by AlkB and hABH3. AlkB-mediated repair of 1-meA in tRNA was also observed in E. coli in vivo. Our data demonstrate that AlkB proteins can mediate functional recovery of RNA exposed to methylation damage, supporting the notion that RNA repair is important.