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.

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.

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.