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.

Repair of DNA damage is essential for maintaining genome integrity, and repair deficiencies in mammals are associated with cancer, neurological disease and developmental defects. 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 AlkB. By contrast, little is known about consequences and cellular handling of alkylation damage to RNA. 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.

Understanding the biologic role of N 6 -methyladenosine (m 6 A) 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 m 6 A in exons but very rarely in introns. The m 6 A methylation is essentially completed upon the release of mRNA into the nucleoplasm. Furthermore, the content and location of each m 6 A 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 m 6 As in CA-RNA are within 50 nucleotides of 5 ′ or 3 ′ splice sites, and the vast majority of exons harboring m 6 A in wild-type mouse stem cells is spliced the same in cells lacking the major m 6 A methyltransferase Mettl3. Both HeLa and mouse embryonic stem cell mRNAs harboring m 6 As 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, m 6 A is added to exons before or soon after exon definition in nascent pre-mRNA, and while m 6 A is not required for most splicing, its addition in the nascent transcript is a determinant of cytoplasmic mRNA stability. Studying nascent RNA synthesis in cultured cells using very brief pulse labeling with radioactive nucleosides allowed a number of advances in understanding premRNA synthesis and processing in the era before rapid nucleic acid sequencing. Examples include polyA addition on pre-mRNA before completion of mRNA processing and cytoplasmic entry (Darnell et al. 1971;Edmonds et al. 1971) and locating the first known boundaries of eukaryotic polymerase II transcription units through studying labeled nascent adenovirus transcripts (Bachenheimer and Darnell 1975;Evans et al. 1977;Weber et al. 1977).These early experiments were joined by a cell fractionation technique originated by Wuarin and Schibler (1994) that uses a 1 M urea solution to liberate a "chromatin" fraction from nuclei. This fraction provides a stringent purification of growing nascent pre-mRNA chains, isolated as a chromatin-associated RNA fraction (referred to as CA-RNA), along with RNA polymerase II plus all nuclear DNA and associated histones. Using specific labeled DNA probes, Wuarin and Schibler (1994) demonstrated removal in liver cell nuclei of some, but not all, introns from two specific nascent pre-mRNAs: a transcription factor pre-mRNA involved in circadian rhythm and the HMG coA reductase pre-mRNA. Recently, Pandya-Jones and Black (2009) adapted this procedure to study the extent and order of intron removal in cultured human carcinoma cell nuclei, again showing that many, but not all, introns are removed in CA-RNA.

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.