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Background Reversible enzymatic methylation of mammalian mRNA is widespread and serves crucial regulatory functions, but little is known to what degree chemical alkylators mediate overlapping modifications and whether cells distinguish aberrant from canonical methylations. Methods Here we use quantitative mass spectrometry to determine the fate of chemically induced methylbases in the mRNA of human cells. Concomitant alteration in the mRNA binding proteome was analyzed by SILAC mass spectrometry. Results MMS induced prominent direct mRNA methylations that were chemically identical to endogenous methylbases. Transient loss of 40S ribosomal proteins from isolated mRNA suggests that aberrant methylbases mediate arrested translational initiation and potentially also no-go decay of the affected mRNA. Four proteins (ASCC3, YTHDC2, TRIM25 and GEMIN5) displayed increased mRNA binding after MMS treatment. ASCC3 is a binding partner of the DNA/RNA demethylase ALKBH3 and was recently shown to promote disassembly of collided ribosomes as part of the ribosome quality control (RQC) trigger complex. We find that ASCC3-deficient cells display delayed removal of MMS-induced 1-methyladenosine (m1A) and 3-methylcytosine (m3C) from mRNA and impaired formation of MMS-induced P-bodies. Conclusions Our findings conform to a model in which ASCC3-mediated disassembly of collided ribosomes allows demethylation of aberrant m1A and m3C by ALKBH3. Our findings constitute first evidence of selective sanitation of aberrant mRNA methylbases over their endogenous counterparts and warrant further studies on RNA-mediated effects of chemical alkylators commonly used in the clinic.

A large number of RNA modifications are known to affect processing and function of rRNA, tRNA and mRNA 1. The N4-acetylcytidine (ac4C) is the only known RNA acetylation event and is known to occur on rRNA, tRNA and mRNA 2,3. RNA modification by acetylation affects a number of biological processes, including translation and RNA stability 2. For a few RNA methyl modifications, a reversible nature has been demonstrated where specific writer proteins deposit the modification and eraser proteins can remove them by oxidative demethylation 4–6. The functionality of RNA modifications is often mediated by interaction with reader proteins that bind dependent on the presence of specific modifications 1. The NAT10 acetyltransferase has been firmly identified as the main writer of acetylation of cytidine ribonucleotides, but so far neither readers nor erasers of ac4C have been identified 2,3. Here we show, that ac4C is bound by the nucleolar protein NOP58 and deacetylated by SIRT7, for the first time demonstrating reversal by another mechanism than oxidative demethylation. NOP58 and SIRT7 are involved in snoRNA function and pre-ribosomal RNA processing 7–10, and using a NAT10 deficient cell line we can show that the reduction in ac4C levels affects both snoRNA sub-nuclear localization and pre-rRNA processing. SIRT7 can deacetylate RNA in vitro and endogenous levels of ac4C on snoRNA increase in a SIRT7 deficient cell line, supporting its endogenous function as an RNA deacetylase. In summary, we identify the first eraser and reader proteins of the RNA modification ac4C, respectively, and suggest an involvement of RNA acetylation in snoRNA function and pre-rRNA processing.

Summary The N6-methyladenosine (m6A) RNA modification is used widely to alter the fate of mRNAs. Here we demonstrate that the C. elegans writer METT-10 (the ortholog of mouse METTL16) deposits an m6A mark on the 3′ splice site (AG) of the S-adenosylmethionine (SAM) synthetase pre-mRNA, which inhibits its proper splicing and protein production. The mechanism is triggered by a rich diet and acts as an m6A-mediated switch to stop SAM production and regulate its homeostasis. Although the mammalian SAM synthetase pre-mRNA is not regulated via this mechanism, we show that splicing inhibition by 3′ splice site m6A is conserved in mammals. The modification functions by physically preventing the essential splicing factor U2AF35 from recognizing the 3′ splice site. We propose that use of splice-site m6A is an ancient mechanism for splicing regulation., Graphical abstract, Highlights • m6A deposited at 3′ splice site by worm METT-10 inhibits splicing • Methylation blocks 3′ splice site recognition by splicing factor U2AF35 • Methylation and splicing inhibition is a response to change in worm diet • Splicing inhibition by 3′ splice site m6A is conserved in mammals, m6A methylation of a 3ʹ splice site blocks its recognition by splicing factors to inhibit pre-mRNA splicing in nematodes and mammals. In worms, this mechanism is used to modulate splicing in response to a change in diet.

Mechanistic and functional studies by gene disruption or editing approaches often suffer from confounding effects like compensatory cellular adaptations generated by clonal selection. These issues become particularly relevant when studying factors directly involved in genetic or epigenetic maintenance. To provide a genetic tool for functional and mechanistic investigation of DNA-repair mediated active DNA demethylation, we generated experimental models in mice and murine embryonic stem cells (ESCs) based on a minigene of the thymine-DNA glycosylase (TDG). The loxP-flanked miniTdg is rapidly and reliably excised in mice and ESCs by tamoxifen-induced Cre activation, depleting TDG to undetectable levels within 24 hours. We describe the functionality of the engineered miniTdg in mouse and ESCs (TDGiKO ESCs) and validate the pluripotency and differentiation potential of TDGiKO ESCs as well as the phenotype of induced TDG depletion. The controlled and rapid depletion of TDG allows for a precise manipulation at any point in time of multistep experimental procedures as presented here for neuronal differentiation in vitro. Thus, we provide a tested and well-controlled genetic tool for the functional and mechanistic investigation of TDG in active DNA (de)methylation and/or DNA repair with minimal interference from adaptive effects and clonal selection.

DNA modification is known to regulate experience-dependent gene expression. However, beyond cytosine methylation and its oxidated derivatives, very little is known about the functional importance of chemical modifications on other nucleobases in the brain. Here we report that in adult mice trained in fear extinction, the DNA modification N6-methyl-2'-deoxyadenosine (m6dA) accumulates along promoters and coding sequences in activated prefrontal cortical neurons. The deposition of m6dA is associated with increased genome-wide occupancy of the mammalian m6dA methyltransferase, N6amt1, and this correlates with extinction-induced gene expression. The accumulation of m6dA is associated with transcriptional activation at the brain-derived neurotrophic factor (Bdnf) P4 promoter, which is required for Bdnf exon IV messenger RNA expression and for the extinction of conditioned fear. These results expand the scope of DNA modifications in the adult brain and highlight changes in m6dA as an epigenetic mechanism associated with activity-induced gene expression and the formation of fear extinction memory.

Mre11 exonuclease activity removes gemcitabine from the nascent strand during DNA replication., The Mre11 nuclease is involved in early responses to DNA damage, often mediated by its role in DNA end processing. MRE11 mutations and aberrant expression are associated with carcinogenesis and cancer treatment outcomes. While, in recent years, progress has been made in understanding the role of Mre11 nuclease activities in DNA double-strand break repair, their role during replication has remained elusive. The nucleoside analog gemcitabine, widely used in cancer therapy, acts as a replication chain terminator; for a cell to survive treatment, gemcitabine needs to be removed from replicating DNA. Activities responsible for this removal have, so far, not been identified. We show that Mre11 3′ to 5′ exonuclease activity removes gemcitabine from nascent DNA during replication. This contributes to replication progression and gemcitabine resistance. We thus uncovered a replication-supporting role for Mre11 exonuclease activity, which is distinct from its previously reported detrimental role in uncontrolled resection in recombination-deficient cells.

DNA repair is essential to prevent the cytotoxic or mutagenic effects of various types of DNA lesions, which are sensed by distinct pathways to recruit repair factors specific to the damage type. Although biochemical mechanisms for repairing several forms of genomic insults are well understood, the upstream signalling pathways that trigger repair are established for only certain types of damage, such as double-stranded breaks and interstrand crosslinks. Understanding the upstream signalling events that mediate recognition and repair of DNA alkylation damage is particularly important, since alkylation chemotherapy is one of the most widely used systemic modalities for cancer treatment and because environmental chemicals may trigger DNA alkylation. Here we demonstrate that human cells have a previously unrecognized signalling mechanism for sensing damage induced by alkylation. We find that the alkylation repair complex ASCC (activating signal cointegrator complex) relocalizes to distinct nuclear foci specifically upon exposure of cells to alkylating agents. These foci associate with alkylated nucleotides, and coincide spatially with elongating RNA polymerase II and splicing components. Proper recruitment of the repair complex requires recognition of K63-linked polyubiquitin by the CUE (coupling of ubiquitin conjugation to ER degradation) domain of the subunit ASCC2. Loss of this subunit impedes alkylation adduct repair kinetics and increases sensitivity to alkylating agents, but not other forms of DNA damage. We identify RING finger protein 113A (RNF113A) as the E3 ligase responsible for upstream ubiquitin signalling in the ASCC pathway. Cells from patients with X-linked trichothiodystrophy, which harbour a mutation in RNF113A, are defective in ASCC foci formation and are hypersensitive to alkylating agents. Together, our work reveals a previously unrecognized ubiquitin-dependent pathway induced specifically to repair alkylation damage, shedding light on the molecular mechanism of X-linked trichothiodystrophy.

The 5' end of eukaryotic mRNAs is protected by the m

Our genome is constantly subject to damage from exogenous and endogenous sources, and cells respond to such damage by initiating a DNA damage response (DDR). Failure to induce an adequate DDR can result in increased mutation load, chromosomal aberrations and a variety of human diseases, including cancer. A rapidly growing body of evidence suggests that a large number of RNA binding proteins are involved in the DDR, and several canonical DNA repair factors have moonlighting functions in RNA metabolism. RNA polymerases and RNA itself have been implicated at various stages of the DDR, including damage sensing, recruitment of DNA repair factors and tethering of broken DNA ends. RNA may even serve as a template for DNA repair under certain conditions. Given the vast number of non-coding RNAs in cells, we have barely started to decipher their potential involvement in genomic maintenance and future research on the interrelationship between RNA and DNA repair may open entirely new treatment options for human disease.

In most mammalian cells, DNA replication occurs once, and only once between cell divisions. Replication initiation is a highly regulated process with redundant mechanisms that prevent errant initiation events. In lower eukaryotes, replication is initiated from a defined consensus sequence, whereas a consensus sequence delineating mammalian origin of replication has not been identified. Here we show that 5-hydroxymethylcytosine (5hmC) is present at mammalian replication origins. Our data support the hypothesis that 5hmC has a role in cell cycle regulation. We show that 5hmC level is inversely proportional to proliferation; indeed, 5hmC negatively influences cell division by increasing the time a cell resides in G1. Our data suggest that 5hmC recruits replication-licensing factors, then is removed prior to or during origin firing. Later we propose that TET2, the enzyme catalyzing 5mC to 5hmC conversion, acts as barrier to rereplication. In a broader context, our results significantly advance the understating of 5hmC involvement in cell proliferation and disease states. © The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/)

The wobble hypothesis was proposed to explain the presence of fewer tRNAs than possible codons. The wobble nucleoside position in the anticodon stem-loop undergoes a number of modifications that help maintain the efficiency and fidelity of translation. AlkB homolog 8 (ALKBH8) is an atypical member of the highly conserved AlkB family of dioxygenases and is involved in the formation of mcm5s2U, (S)-mchm5U, (R)-mchm5U, mcm5U, and mcm5Um at the anticodon wobble uridines of specific tRNAs. In two multiplex consanguineous families, we identified two homozygous truncating ALKBH8 mutations causing intellectual disability. Analysis of tRNA derived from affected individuals showed the complete absence of these modifications, consistent with the presumptive loss of function of the variants. Our results highlight the sensitivity of the brain to impaired wobble modification and expand the list of intellectual-disability syndromes caused by mutations in genes related to tRNA modification.

// Alexey Zatula 1, * , Aida Dikic 1, * , Celine Mulder 1, 2, * , Animesh Sharma 1, 3 , Cathrine B. Vagbo 1, 3 , Mirta M. L. Sousa 1 , Anders Waage 1, 4 , Geir Slupphaug 1, 3 1 Department of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology, NTNU, Trondheim, Norway 2 Present address: University of Utrecht, Utrecht, Holland 3 PROMEC Core Facility for Proteomics and Metabolomics, Norwegian University of Science and Technology, NTNU, Trondheim, and the Central Norway Regional Health Authority, Stjordal, Norway 4 Department of Hematology, St. Olav’s Hospital, Trondheim, Norway * These authors have contributed equally to this work Correspondence to: Geir Slupphaug, email: geir.slupphaug@ntnu.no Keywords: multiple myeloma, secondary plasma cell leukemia, super-SILAC, quantitative proteomics Received: September 18, 2016 Accepted: November 30, 2016 Published: December 27, 2016 ABSTRACT Plasma cell leukemia is a rare and aggressive plasma cell neoplasm that may either originate de novo (primary PCL) or by leukemic transformation of multiple myeloma (MM) to secondary PCL (sPCL). The prognosis of sPCL is very poor, and currently no standard treatment is available due to lack of prospective clinical studies. In an attempt to elucidate factors contributing to transformation, we have performed super-SILAC quantitative proteome profiling of malignant plasma cells collected from the same patient at both the MM and sPCL stages of the disease. 795 proteins were found to be differentially expressed in the MM and sPCL samples. Gene ontology analysis indicated a metabolic shift towards aerobic glycolysis in sPCL as well as marked down-regulation of enzymes involved in glycan synthesis, potentially mediating altered glycosylation of surface receptors. There was no significant change in overall genomic 5-methylcytosine or 5-hydroxymethylcytosine at the two stages, indicating that epigenetic dysregulation was not a major driver of transformation to sPCL. The present study constitutes the first attempt to provide a comprehensive map of the altered protein expression profile accompanying transformation of MM to sPCL in a single patient, identifying several candidate proteins that can be targeted by currently available small molecule drugs. Our dataset furthermore constitutes a reference dataset for further proteomic analysis of sPCL transformation.

Ogg1 and Mutyh DNA glycosylases cooperate to prevent mutations caused by 8-oxoG, a major premutagenic DNA lesion associated with cognitive decline. We have examined behavior and cognitive function in mice deficient of these glycosylases. Ogg1−/−Mutyh−/− mice were more active and less anxious, with impaired learning ability. In contrast, Mutyh−/− mice showed moderately improved memory. We observed no apparent change in genomic 8-oxoG levels, suggesting that Ogg1 and Mutyh play minor roles in global repair in adult brain. Notably, transcriptome analysis of hippocampus revealed that differentially expressed genes in the mutants belong to pathways known to be involved in anxiety and cognition. Esr1 targets were upregulated, suggesting a role of Ogg1 and Mutyh in repression of Esr1 signaling. Thus, beyond their involvement in DNA repair, Ogg1 and Mutyh regulate hippocampal gene expression related to cognition and behavior, suggesting a role for the glycosylases in regulating adaptive behavior. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Understanding the biologic role of N6-methyladenosine (m6A) RNA modifications in mRNA requires an understanding of when and where in the life of a pre-mRNA transcript the modifications are made. We found that HeLa cell chromatin-associated nascent pre-mRNA (CA-RNA) contains many unspliced introns and m6A in exons but very rarely in introns. The m6A methylation is essentially completed upon the release of mRNA into the nucleoplasm. Furthermore, the content and location of each m6A modification in steady-state cytoplasmic mRNA are largely indistinguishable from those in the newly synthesized CA-RNA or nucleoplasmic mRNA. This result suggests that quantitatively little methylation or demethylation occurs in cytoplasmic mRNA. In addition, only ∼10% of m6As in CA-RNA are within 50 nucleotides of 5′ or 3′ splice sites, and the vast majority of exons harboring m6A in wild-type mouse stem cells is spliced the same in cells lacking the major m6A methyltransferase Mettl3. Both HeLa and mouse embryonic stem cell mRNAs harboring m6As have shorter half-lives, and thousands of these mRNAs have increased half-lives (twofold or more) in Mettl3 knockout cells compared with wild type. In summary, m6A is added to exons before or soon after exon definition in nascent pre-mRNA, and while m6A is not required for most splicing, its addition in the nascent transcript is a determinant of cytoplasmic mRNA stability.

Cytotoxic 1-methyladenine (1-meA) and 3-methylcytosine (3-meC) lesions induced in DNA and RNA in vitro and in pre-damaged DNA and RNA bacteriophages in vivo are repaired by the Escherichia coli (E. coli) protein AlkB and a human homolog, ALKBH3. However, it is not known whether endogenous RNA is repaired in vivo by repair proteins present at physiological concentrations. The concept of RNA repair as a biologically relevant process has therefore remained elusive. Here, we demonstrate AlkB-mediated repair of endogenous RNA in vivo by measuring differences in lesion-accumulation in two independent AlkB-proficient and deficient E. coli strains during exposure to methyl methanesulfonate (MMS). Repair was observed both in AlkB-overproducing strains and in the wild-type strains after AlkB induction. RNA repair appeared to be highest in RNA species below 200 nucleotides in size, mainly comprising tRNAs. Strikingly, at least 10-fold more lesions were repaired in RNA than in DNA. This may be a consequence of some 30-fold higher levels of aberrant methylation in RNA than in DNA after exposure to MMS. A high primary kinetic isotope effect (>10) was measured using a deuterated methylated RNA substrate, D3-1me(rA), demonstrating that it is the catalytic step, and not the search step that is rate-limiting. Our results demonstrate that RNA repair by AlkB takes place in endogenous RNA as part of an adaptive response in wild-type E. coli cells.

Sertoli cells have dual roles during the cells’ lifetime. In the juvenile mammal, Sertoli cells proliferate and create the structure of the testis, and during puberty they cease to proliferate and take on the adult role of supporting germ cells through spermatogenesis. Accordingly, many genes expressed in Sertoli cells during testis formation are repressed during spermatogenesis. 5-Hydroxymethylcytosine (5hmC) is a DNA modification enzymatically generated from 5mC and present in all investigated mammalian tissues at varying levels. Using mass spectrometry and immunofluorescence staining we identified a substantial Sertoli cell-specific global 5hmC increase during rat puberty. Chemical labeling, pull-down and sequencing of 5hmC-containing genomic DNA from juvenile and adult rat Sertoli cells revealed that genes that lose or gain 5hmC belong to different functional pathways and mirror the functions of the cells in the two different states. Loss of 5hmC is associated with genes involved in development and cell structure, whereas gain of 5hmC is associated with genes involved in cellular pathways pertaining to the function of the adult Sertoli cells. This redistribution during maturation shows that 5hmC is a dynamic nucleotide modification, correlated to gene expression. © The Author(s) 2017. This work is licensed under a Creative Commons Attribution 4.0 International License.

More than 100 structurally distinct RNA modifications have been identified in all kingdoms of life.1 These post-transcriptional modifications are widely present in various RNAs, including ribosomal RNA (rRNA), transfer RNA (tRNA), messenger RNA (mRNA), long non-coding RNA (lncRNA), etc. We have shown that the methylation of N6-methyladenine (m6A) can be reversed through the discovery of the first RNA demethylase, the human fat mass and obesity-associated protein, FTO, in 2011.2 Most recently, we have identified a new mammalian RNA demethylase, ALKBH5, which is also able to remove the methyl group of m6A from RNA both in vitro and in vivo (Fig. 1A). The ALKBH5 protein colocalizes with nuclear speckles where pre-mRNA processing occurs. This protein is actively involved in mRNA export regulation, in which its demethylation activity seems to play an important role, as well as in RNA synthesis. A knockout of the Alkbh5 gene in mice resulted in impaired male fertility due to compromised spermatogenesis. Importantly, increased m6A levels were observed in mRNA isolated from the Alkbh5-knockout mouse organs compared to those from wild-type littermates. RNA-Seq results indicate aberrant gene expression in spermatogenic cells of the seminoferous tubulus of testes from Alkbh5-deficient mice, thereby showing that the loss of the m6A demethylase influences gene expression, which, in turn, leads to defects in spermatogenesis and increased apoptosis of meiotic cells. Thus, the discovery of FTO and this new RNA demethylase strongly suggests that the methylation of RNA, like DNA and histone modifications, is dynamically regulated and likely to play broad roles in mammalian cells.

Single-strand-selective monofunctional uracil-DNA glycosylase 1 (SMUG1) is a base excision repair enzyme that removes uracil and oxidised pyrimidines from DNA. We show that SMUG1 interacts with the pseudouridine synthase Dyskerin (DKC1) and colocalizes with DKC1 in nucleoli and Cajal bodies. As DKC1 functions in RNA processing, we tested whether SMUG1 excised modified bases in RNA and demonstrated that SMUG1 has activity on single-stranded RNA containing 5-hydroxymethyldeoxyuridine, but not pseudouridine, the nucleoside resulting from isomerization of uridine by DKC1. Moreover, SMUG1 associates with the 47S rRNA precursor processed by DKC1, and depletion of SMUG1 leads to a reduction in the levels of mature rRNA accompanied by an increase in polyadenylated rRNA. Depletion of SMUG1, and, in particular, the combined loss of SMUG1 and DKC1, leads to accumulation of 5-hydroxymethyluridine in rRNA. In conclusion, SMUG1 is a DKC1 interaction partner that contributes to rRNA quality control, partly by regulating 5-hydroxymethyluridine levels.

Here we report that the recently discovered mammalian DNA modification N6-methyl-2’-deoxyadenosine (m6dA) is dynamically regulated in primary cortical neurons, and accumulates along promoters and coding sequences within the genome of activated prefrontal cortical neurons of adult C57/BI6 mice in response to fear extinction learning. The deposition of m6dA is generally associated with increased genome-wide occupancy of the mammalian m6dA methyltransferase, N6amt1, and this correlates with fear extinction learning-induced gene expression. Of particular relevance for fear extinction memory, the accumulation of m6dA is associated with an active chromatin state and the recruitment of transcriptional machinery to the brain-derived neurotrophic factor (Bdnf) P4 promoter, which is required for Bdnf exon IV mRNA expression and for the extinction of conditioned fear. These results expand the scope of DNA modifications in the adult brain and highlight changes in m6dA as a novel neuroepigenetic mechanism associated with activity-induced gene expression and the formation of fear extinction memory.

Background Hematopoietic stem cell renewal and differentiation are regulated through epigenetic processes. The conversion of 5-methylcytosine into 5-hydroxymethylcytosine (5hmC) by ten-eleven-translocation enzymes provides new insights into the epigenetic regulation of gene expression during development. Here, we studied the potential gene regulatory role of 5hmC during human hematopoiesis. Results We used reduced representation of 5-hydroxymethylcytosine profiling (RRHP) to characterize 5hmC distribution in CD34+ cells, CD4+ T cells, CD19+ B cells, CD14+ monocytes and granulocytes. In all analyzed blood cell types, the presence of 5hmC at gene bodies correlates positively with gene expression, and highest 5hmC levels are found around transcription start sites of highly expressed genes. In CD34+ cells, 5hmC primes for the expression of genes regulating myeloid and lymphoid lineage commitment. Throughout blood cell differentiation, intragenic 5hmC is maintained at genes that are highly expressed and required for acquisition of the mature blood cell phenotype. Moreover, in CD34+ cells, the presence of 5hmC at enhancers associates with increased binding of RUNX1 and FLI1, transcription factors essential for hematopoiesis. Conclusions Our study provides a comprehensive genome-wide overview of 5hmC distribution in human hematopoietic cells and new insights into the epigenetic regulation of gene expression during human hematopoiesis. © 2016 The Author(s). This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/)

The Escherichia coli AlkB protein (EcAlkB) is a DNA repair enzyme which reverses methylation damage such as 1-methyladenine (1-meA) and 3-methylcytosine (3-meC). The mammalian AlkB homologues ALKBH2 and ALKBH3 display EcAlkB-like repair activity in vitro , but their substrate specificities are different, and ALKBH2 is the main DNA repair enzyme for 1-meA in vivo . The genome of the model plant Arabidopsis thaliana encodes several AlkB homologues, including the yet uncharacterized protein AT2G22260, which displays sequence similarity to both ALKBH2 and ALKBH3. We have here characterized protein AT2G22260, by us denoted ALKBH2, as both our functional studies and bioinformatics analysis suggest it to be an orthologue of mammalian ALKBH2. The Arabidopsis ALKBH2 protein displayed in vitro repair activities towards methyl and etheno adducts in DNA, and was able to complement corresponding repair deficiencies of the E. coli alkB mutant. Interestingly, alkbh2 knock-out plants were sensitive to the methylating agent methylmethanesulphonate (MMS), and seedlings from these plants developed abnormally when grown in the presence of MMS. The present study establishes ALKBH2 as an important enzyme for protecting Arabidopsis against methylation damage in DNA, and suggests its homologues in other plants to have a similar function. The Author(s) 2012. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Cytotoxicity of 5-fluorouracil (FU) and 5-fluoro-2′-deoxyuridine (FdUrd) due to DNA fragmentation during DNA repair has been proposed as an alternative to effects from thymidylate synthase (TS) inhibition or RNA incorporation. The goal of the present study was to investigate the relative contribution of the proposed mechanisms for cytotoxicity of 5-fluoropyrimidines. We demonstrate that in human cancer cells, base excision repair (BER) initiated by the uracil–DNA glycosylase UNG is the major route for FU–DNA repair in vitro and in vivo. SMUG1, TDG and MBD4 contributed modestly in vitro and not detectably in vivo. Contribution from mismatch repair was limited to FU:G contexts at best. Surprisingly, knockdown of individual uracil–DNA glycosylases or MSH2 did not affect sensitivity to FU or FdUrd. Inhibitors of common steps of BER or DNA damage signalling affected sensitivity to FdUrd and HmdUrd, but not to FU. In support of predominantly RNA-mediated cytotoxicity, FU-treated cells accumulated ~3000- to 15 000-fold more FU in RNA than in DNA. Moreover, FU-cytotoxicity was partially reversed by ribonucleosides, but not deoxyribonucleosides and FU displayed modest TS-inhibition compared to FdUrd. In conclusion, UNG-initiated BER is the major route for FU–DNA repair, but cytotoxicity of FU is predominantly RNA-mediated, while DNA-mediated effects are limited to FdUrd. The Author(s) 2011. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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.

Well-known epigenetic DNA modifications in mammals include the addition of a methyl group and a hydroxyl group to cytosine, resulting in 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) respectively. In contrast, the abundance and the functional implications of these modifications in invertebrate model organisms such as the honey bee (Apis mellifera) and the fruit fly (Drosophila melanogaster) are not well understood. Here we show that both adult honey bees and fruit flies contain 5mC and also 5hmC. Using a highly sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) technique, we quantified 5mC and 5hmC in different tissues of adult honey bee worker castes and in adult fruit flies. A comparison of our data with reports from human and mouse shed light on notable differences in 5mC and 5hmC levels between tissues and species., Highlights • Reporting cytosine modifications in uncharacterized tissues, phenotypes and species. • Quantification of 5mC and 5hmC suggests species-specific roles and turnover.​ • Low levels of 5hmC relative to 5mC and cytosine in honey bees compared to mammals. • Honey bee abdominal tissues are richer in5hmC than the brain. • We found a higher 5hmC to 5mC ratio in fruit flies as compared to the honey bee.

We adapted UV CLIP (cross-linking immunoprecipitation) to accurately locate tens of thousands of m6A residues in mammalian mRNA with single-nucleotide resolution. More than 70% of these residues are present in the 3′-most (last) exons, with a very sharp rise (sixfold) within 150–400 nucleotides of the start of the last exon. Two-thirds of last exon m6A and >40% of all m6A in mRNA are present in 3′ untranslated regions (UTRs); contrary to earlier suggestions, there is no preference for location of m6A sites around stop codons. Moreover, m6A is significantly higher in noncoding last exons than in next-to-last exons harboring stop codons. We found that m6A density peaks early in the 3′ UTR and that, among transcripts with alternative polyA (APA) usage in both the brain and the liver, brain transcripts preferentially use distal polyA sites, as reported, and also show higher proximal m6A density in the last exons. Furthermore, when we reduced m6A methylation by knocking down components of the methylase complex and then examined 661 transcripts with proximal m6A peaks in last exons, we identified a set of 111 transcripts with altered (approximately two-thirds increased proximal) APA use. Taken together, these observations suggest a role of m6A modification in regulating proximal alternative polyA choice.

Repair of DNA damage is essential for maintaining genome integrity, and repair deficiencies in mammals are associated with cancer, neurological disease and developmental defects1. Alkylation damage in DNA is repaired by at least three different mechanisms, including damage reversal by oxidative demethylation of 1-methyladenine and 3-methylcytosine by Escherichia coli AlkB2,3. By contrast, little is known about consequences and cellular handling of alkylation damage to RNA4. Here we show that two human AlkB homologues, hABH2 and hABH3, also are oxidative DNA demethylases and that AlkB and hABH3, but not hABH2, also repair RNA. Whereas AlkB and hABH3 prefer single-stranded nucleic acids, hABH2 acts more efficiently on double-stranded DNA. In addition, AlkB and hABH3 expressed in E. coli reactivate methylated RNA bacteriophage MS2 in vivo, illustrating the biological relevance of this repair activity and establishing RNA repair as a potentially important defence mechanism in living cells. The different catalytic properties and the different subnuclear localization patterns shown by the human homologues indicate that hABH2 and hABH3 have distinct roles in the cellular response to alkylation damage.

Considerable progress has been made in understanding the origins of genomic uracil and its role in genome stability and host defense; however, the main question concerning the basal level of uracil in DNA remains disputed. Results from assays designed to quantify genomic uracil vary by almost three orders of magnitude. To address the issues leading to this inconsistency, we explored possible shortcomings with existing methods and developed a sensitive LC/MS/MS-based method for the absolute quantification of genomic 2'-deoxyuridine (dUrd). To this end, DNA was enzymatically hydrolyzed to 2'-deoxyribonucleosides and dUrd was purified in a preparative HPLC step and analyzed by LC/MS/MS. The standard curve was linear over four orders of magnitude with a quantification limit of 5 fmol dUrd. Control samples demonstrated high inter-experimental accuracy (94.3%) and precision (CV 9.7%). An alternative method that employed UNG2 to excise uracil from DNA for LC/MS/MS analysis gave similar results, but the intra-assay variability was significantly greater. We quantified genomic dUrd in Ung(+/+) and Ung(-/-) mouse embryonic fibroblasts and human lymphoblastoid cell lines carrying UNG mutations. DNA-dUrd is 5-fold higher in Ung(-/-) than in Ung(+/+) fibroblasts and 11-fold higher in UNG2 dysfunctional than in UNG2 functional lymphoblastoid cells. We report approximately 400-600 dUrd per human or murine genome in repair-proficient cells, which is lower than results using other methods and suggests that genomic uracil levels may have previously been overestimated.

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 (S)-5-methoxycarbonylhydroxymethyluridine (mchm(5)U), hydroxylated forms of mcm(5)U, are present in mammalian tRNA(UCG)(Arg), and tRNA(UCC)(Gly), 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 tRNAUCCGly. 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.

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 GlyUCC , 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. © The Author(s) 2011. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited

AlkB PCNA-interacting motif (APIM) is present in >200 proteins and may mediate PCNA binding during genotoxic stress., Numerous proteins, many essential for the DNA replication machinery, interact with proliferating cell nuclear antigen (PCNA) through the PCNA-interacting peptide (PIP) sequence called the PIP box. We have previously shown that the oxidative demethylase human AlkB homologue 2 (hABH2) colocalizes with PCNA in replication foci. In this study, we show that hABH2 interacts with a posttranslationally modified PCNA via a novel PCNA-interacting motif, which we term AlkB homologue 2 PCNA-interacting motif (APIM). We identify APIM in >200 other proteins involved in DNA maintenance, transcription, and cell cycle regulation, and verify a functional APIM in five of these. Expression of an APIM peptide increases the cellular sensitivity to several cytostatic agents not accounted for by perturbing only the hABH2–PCNA interaction. Thus, APIM is likely to mediate PCNA binding in many proteins involved in DNA repair and cell cycle control during genotoxic stress.

The Escherichia coli AlkB protein and human homologs hABH2 and hABH3 are 2-oxoglutarate (2OG)/Fe(II)-dependent DNA/RNA demethylases that repair 1-methyladenine and 3-methylcytosine residues. Surprisingly, hABH1, which displays the strongest homology to AlkB, failed to show repair activity in two independent studies. Here, we show that hABH1 is a mitochondrial protein, as demonstrated using fluorescent fusion protein expression, immunocytochemistry, and Western blot analysis. A fraction is apparently nuclear and this fraction increases strongly if the fluorescent tag is placed at the N-terminal end of the protein, thus interfering with mitochondrial targeting. Molecular modeling of hABH1 based upon the sequence and known structures of AlkB and hABH3 suggested an active site almost identical to these enzymes. hABH1 decarboxylates 2OG in the absence of a prime substrate, and the activity is stimulated by methylated nucleotides. Employing three different methods we demonstrate that hABH1 demethylates 3-methylcytosine in single-stranded DNA and RNA in vitro. Site-specific mutagenesis confirmed that the putative Fe(II) and 2OG binding residues are essential for activity. In conclusion, hABH1 is a functional mitochondrial AlkB homolog that repairs 3-methylcytosine in single-stranded DNA and RNA.

Aberrant methylations in DNA are repaired by base excision repair (BER) and direct repair by a methyltransferase or by an oxidative demethylase of the AlkB type. Yang et al. [Nature 452 (2008) 961-966] have now solved the crystal structure of AlkB and human AlkB homolog 2 (hABH2) in complex with DNA using an ingenious crosslinking strategy to stabilize the DNA-protein complex. AlkB proteins have similar catalytic domains, but different DNA recognition motifs. Whereas AlkB mainly makes contact with the damaged strand, hABH2 makes numerous contacts with both strands. hABH2 flips out the damaged base and fills the vacant space by a hydrophobic amino acid residue similar to DNA glycosylases, essentially without distorting the double helix structure. In contrast, AlkB squeezes together the bases flanking the flipped-out base to maintain the base stack. This unprecedented flipping mechanism and the differences between AlkB and hABH2 in contacting the DNA strands explain their preferences for single stranded- and double stranded DNA, respectively.

Elaborate repair pathways counteract the deleterious effects of DNA damage by mechanisms that are understood in reasonable detail. In contrast, repair of damaged RNA has not been widely explored. This may be because aberrant RNAs are generally assumed to be degraded rather than repaired. The reason for this view is well founded, since conserved surveillance mechanisms that degrade abnormal RNAs are thoroughly documented. Numerous proteins and protein-RNA complexes are involved in the metabolism of different RNA species, assuring correct transcription, splicing, posttranscriptional modifications, transport, translation and timely degradation of the molecule. However, like DNA, RNA is under constant attack of various environmental and endogenous agents that damage the molecule, such as alkylating agents, radiation and free radicals. Importantly, many DNA damaging drugs used in cancer therapy also modify RNA, presumably causing delayed or faulty translation. This may result in generation of inactive proteins, dominant negative proteins or toxic protein aggregates. Several lines of evidence indicate RNA repair as a possible cellular defence mechanism to cope with RNA damage. Thus, there are convincing examples of tRNA repair by elongation of truncated forms, and repair of cleaved tRNA by T4 phage proteins. In addition, in vitro repair of aberrant tRNA methylation by a methyl transferase has been reported. Finally, recent reports on repair of chemically methylated RNA by AlkB and a human homologue (hABH3) in vitro and in vivo strengthen the idea of RNA base repair as a cellular defence mechanism.

Methylating agents are ubiquitous in the environment, and central in cancer therapy. The 1-methyladenine and 3-methylcytosine lesions in DNA/RNA contribute to the cytotoxicity of such agents. These lesions are directly reversed by ABH3 (hABH3) in humans and AlkB in Escherichia coli. Here, we report the structure of the hABH3 catalytic core in complex with iron and 2-oxoglutarate (2OG) at 1.5 A resolution and analyse key site-directed mutants. The hABH3 structure reveals the beta-strand jelly-roll fold that coordinates a catalytically active iron centre by a conserved His1-X-Asp/Glu-X(n)-His2 motif. This experimentally establishes hABH3 as a structural member of the Fe(II)/2OG-dependent dioxygenase superfamily, which couples substrate oxidation to conversion of 2OG into succinate and CO2. A positively charged DNA/RNA binding groove indicates a distinct nucleic acid binding conformation different from that predicted in the AlkB structure with three nucleotides. These results uncover previously unassigned key catalytic residues, identify a flexible hairpin involved in nucleotide flipping and ss/ds-DNA discrimination, and reveal self-hydroxylation of an active site leucine that may protect against uncoupled generation of dangerous oxygen radicals.

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.

To determine the variability of coumarin 7- and 3-hydroxylation in a human population and to evaluate the evidence for the existence of genetic polymorphism in these pathways. 7-Hydroxylation of coumarin is considered to be a detoxication pathway, whilst 3-hydroxylation, which predominates in rats, leads to hepatotoxicity in the rat. Coumarin metabolic phenotypes could aid in refining the risk evaluation for humans of dietary and environmental exposure to coumarin and for the chronic use of coumarin in high doses as a drug to treat lymphoedema and certain cancers.Healthy male and female Jordanian volunteers (n = 103) were administered 2 mg coumarin by mouth and collected their 0-8-h urines. These, together with pre-dose blank urines, were analysed by selected-ion monitoring gas chromatography mass spectrometry for their content of the coumarin metabolites 7-hydroxycoumarin (70HC) and 2-hydroxyphenylacetic acid (2OHPAA), the latter arising from the 3-hydroxylation pathway.After coumarin administration, excretion of both 70HC and 2OHPAA was highly variable. A coumarin metabolic ratio (2OHPAA/7OHC) was suggestive of polymorphism. At least one subject had a metabolic response similar to an individual known to be both phenotypically and genotypically (CYP2A6 gene) 7-hydroxylation-deficient.In the light of the finding of high variability and possible polymorphism in both the 7- and 3-hydroxylation of coumarin in a human population. we recommend a reappraisal of the risk evaluation of human exposure to coumarin, particularly in pharmaceutical doses.

Recently, 5-hydroxymethylcytosine (5hmC) was identified in mammalian genomic DNA. The biological role of this modification remains unclear; however, identifying the genomic location of this modified base will assist in elucidating its function. We describe a method for the rapid and inexpensive identification of genomic regions containing 5hmC. This method involves the selective glucosylation of 5hmC residues by the b-glucosyltransferase from T4 bacteriophage creating b-glucosyl-5-hydroxymethylcytosine (b-glu-5hmC). The b-glu-5hmC modification provides a target that can be efficiently and selectively pulled down by J-binding protein 1 coupled to magnetic beads. DNA that is precipitated is suitable for analysis by quantitative PCR, microarray or sequencing. Furthermore, we demonstrate that the J-binding protein 1 pull down assay identifies 5hmC at the promoters of developmentally regulated genes in human embryonic stem cells. The method described here will allow for a greater understanding of the temporal and spatial effects that 5hmC may have on epigenetic regulation at the single gene level. The Author(s) 2011. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.