SUMMARY N6-methyladenosine (m6A) is the most prevalent internal modification of messenger RNA (mRNA) in higher eukaryotes. Here we report ALKBH5 as another mammalian demethylase that oxidatively reverses m6A in mRNA in vitro and in vivo. This demethylation activity of ALKBH5 significantly affects mRNA export and RNA metabolism as well as the assembly of mRNA processing factors in nuclear speckles. Alkbh5-deficient male mice have increased m6A in mRNA and are characterized by impaired fertility resulting from apoptosis that affects meiotic metaphase-stage spermatocytes. In accordance with this defect, we have identified in mouse testes 1,551 differentially expressed genes that cover broad functional categories and include spermatogenesis-related mRNAs involved in the p53 functional interaction network. The discovery of this RNA demethylase strongly suggests that the reversible m6A modification has fundamental and broad functions in mammalian cells.

We adapted UV CLIP (cross-linking immunoprecipitation) to accurately locate tens of thousands of m 6 A 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 m 6 A and >40% of all m 6 A in mRNA are present in 3 ′ untranslated regions (UTRs); contrary to earlier suggestions, there is no preference for location of m 6 A sites around stop codons. Moreover, m 6 A is significantly higher in noncoding last exons than in next-to-last exons harboring stop codons. We found that m 6 A 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 m 6 A 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 m 6 A modification in regulating proximal alternative polyA choice.

FTO, the first RNA demethylase discovered, mediates the demethylation of internal N-methyladenosine (mA) and N, 2-O-dimethyladenosine (mA) at the +1 position from the 5' cap in mRNA. Here we demonstrate that the cellular distribution of FTO is distinct among different cell lines, affecting the access of FTO to different RNA substrates. We find that FTO binds multiple RNA species, including mRNA, snRNA, and tRNA, and can demethylate internal mA and cap mA in mRNA, internal mA in U6 RNA, internal and cap mA in snRNAs, and N-methyladenosine (mA) in tRNA. FTO-mediated demethylation has a greater effect on the transcript levels of mRNAs possessing internal mA than the ones with cap mA in the tested cells. We also show that FTO can directly repress translation by catalyzing mA tRNA demethylation. Collectively, FTO-mediated RNA demethylation occurs to mA and mA in mRNA and snRNA as well as mA in tRNA.

Two forms of DNA base excision‐repair (BER) have been observed: a ‘short‐patch’ BER pathway involving replacement of one nucleotide and a ‘long‐patch’ BER pathway with gap‐filling of several nucleotides. The latter mode of repair has been investigated using human cell‐free extracts or purified proteins. Correction of a regular abasic site in DNA mainly involves incorporation of a single nucleotide, whereas repair patches of two to six nucleotides in length were found after repair of a reduced or oxidized abasic site. Human AP endonuclease, DNA polymerase β and a DNA ligase (either III or I) were sufficient for the repair of a regular AP site. In contrast, the structure‐specific nuclease DNase IV (FEN1) was essential for repair of a reduced AP site, which occurred through the long‐patch BER pathway. DNase IV was required for cleavage of a reaction intermediate generated by template strand displacement during gap‐filling. XPG, a related nuclease, could not substitute for DNase IV. The long‐patch BER pathway was largely dependent on DNA polymerase β in cell extracts, but the reaction could be reconstituted with either DNA polymerase β or δ. Efficient repair of γ‐ray‐induced oxidized AP sites in plasmid DNA also required DNase IV. PCNA could promote the Pol β‐dependent long‐patch pathway by stimulation of DNase IV.

Although oxidative damage has long been associated with ageing and neurological disease, mechanistic connections of oxidation to these phenotypes have remained elusive. Here we show that the age-dependent somatic mutation associated with Huntington's disease occurs in the process of removing oxidized base lesions, and is remarkably dependent on a single base excision repair enzyme, 7,8-dihydro-8-oxoguanine-DNA glycosylase (OGG1). Both in vivo and in vitro results support a 'toxic oxidation' model in which OGG1 initiates an escalating oxidation-excision cycle that leads to progressive age-dependent expansion. Age-dependent CAG expansion provides a direct molecular link between oxidative damage and toxicity in post-mitotic neurons through a DNA damage response, and error-prone repair of single-strand breaks.

DNA damage generated by oxidant byproducts of cellular metabolism has been proposed as a key factor in cancer and aging. Oxygen free radicals cause predominantly base damage in DNA, and the most frequent mutagenic base lesion is 7,8-dihydro-8-oxoguanine (8-oxoG). This altered base can pair with A as well as C residues, leading to a greatly increased frequency of spontaneous G.C-->T.A transversion mutations in repair-deficient bacterial and yeast cells. Eukaryotic cells use a specific DNA glycosylase, the product of the OGG1 gene, to excise 8-oxoG from DNA. To assess the role of the mammalian enzyme in repair of DNA damage and prevention of carcinogenesis, we have generated homozygous ogg1(-/-) null mice. These animals are viable but accumulate abnormal levels of 8-oxoG in their genomes. Despite this increase in potentially miscoding DNA lesions, OGG1-deficient mice exhibit only a moderately, but significantly, elevated spontaneous mutation rate in nonproliferative tissues, do not develop malignancies, and show no marked pathological changes. Extracts of ogg1 null mouse tissues cannot excise the damaged base, but there is significant slow removal in vivo from proliferating cells. These findings suggest that in the absence of the DNA glycosylase, and in apparent contrast to bacterial and yeast cells, an alternative repair pathway functions to minimize the effects of an increased load of 8-oxoG in the genome and maintain a low endogenous mutation frequency.

Maternal-to-zygotic transition (MZT) is essential for the formation of a new individual, but is still poorly understood despite recent progress in analysis of gene expression and DNA methylation in early embryogenesis1–9. Dynamic histone modifications may have important roles in MZT10–13, but direct measurements of chromatin states have been hindered by technical difficulties in profiling histone modifications from small quantities of cells. Recent improvements allow for 500 cell-equivalents of chromatin per reaction, but require 10,000 cells for initial steps14 or require a highly specialized microfluidics device that is not readily available15. We developed a micro-scale chromatin immunoprecipitation and sequencing (μChlP-seq) method, which we used to profile genome-wide histone H3 lysine methylation (H3K4me3) and acetylation (H3K27ac) in mouse immature and metaphase II oocytes and in 2-cell and 8-cell embryos. Notably, we show that ~22% of the oocyte genome is associated with broad H3K4me3 domains that are anti-correlated with DNA methylation. The H3K4me3 signal becomes confined to transcriptional-start-site regions in 2-cell embryos, concomitant with the onset of major zygotic genome activation. Active removal of broad H3K4me3 domains by the lysine demethylases KDM5A and KDM5B is required for normal zygotic genome activation and is essential for early embryo development. Our results provide insight into the onset of the developmental program in mouse embryos and demonstrate a role for broad H3K4me3 domains in MZT.

Repair of a uracil‐guanine base pair in DNA has been reconstituted with the recombinant human proteins uracil‐DNA glycosylase, apurinic/apyrimidinic endonuclease, DNA polymerase beta and DNA ligase III. The XRCC1 protein, which is known to bind DNA ligase III, is not absolutely required for the reaction but suppresses strand displacement by DNA polymerase beta, allowing for more efficient ligation after filling of a single nucleotide patch. We show that XRCC1 interacts directly with DNA polymerase beta using far Western blotting, affinity precipitation and yeast two‐hybrid analyses. In addition, a complex formed between DNA polymerase beta and a double‐stranded oligonucleotide containing an incised abasic site was supershifted by XRCC1 in a gel retardation assay. The region of interaction with DNA polymerase beta is located within residues 84–183 in the N‐terminal half of the XRCC1 protein, whereas the C‐terminal region of XRCC1 is involved in binding DNA ligase III. These data indicate that XRCC1, which has no known catalytic activity, might serve as a scaffold protein during base excision‐repair. DNA strand displacement and excessive gap filling during DNA repair were observed in cell‐free extracts of an XRCC1‐deficient mutant cell line, in agreement with the results from the reconstituted system.