N6-methyladenosine (m6A) has recently been found abundantly on messenger RNA and shown to regulate most steps of mRNA metabolism. Several important m6A methyltransferases have been described functionally and structurally, but the enzymes responsible for installing one m6A residue on each subunit of human ribosomes at functionally important sites have eluded identification for over 30 years. Here, we identify METTL5 as the enzyme responsible for 18S rRNA m6A modification and confirm ZCCHC4 as the 28S rRNA modification enzyme. We show that METTL5 must form a heterodimeric complex with TRMT112, a known methyltransferase activator, to gain metabolic stability in cells. We provide the first atomic resolution structure of METTL5–TRMT112, supporting that its RNA-binding mode differs distinctly from that of other m6A RNA methyltransferases. On the basis of similarities with a DNA methyltransferase, we propose that METTL5–TRMT112 acts by extruding the adenosine to be modified from a double-stranded nucleic acid.

The error-free and efficient repair of DNA double-stranded breaks (DSBs) is extremely important for cell survival. RNA has been implicated in the resolution of DNA damage but the mechanism remains poorly understood. Here, we show that miRNA biogenesis enzymes, Drosha and Dicer, control the recruitment of repair factors from multiple pathways to sites of damage. Depletion of Drosha significantly reduces DNA repair by both homologous recombination (HR) and non-homologous end joining (NHEJ). Drosha is required within minutes of break induction, suggesting a central and early role for RNA processing in DNA repair. Sequencing of DNA:RNA hybrids reveals RNA invasion around DNA break sites in a Drosha-dependent manner. Removal of the RNA component of these structures results in impaired repair. These results show how RNA can be a direct and critical mediator of DNA damage repair in human cells.

Highlights d PCIF1 is the N6-adenosine methylase that produces m 6 Am in an m 7 G cap-dependent manner d PCIF1 depletion allows transcriptome-wide mapping of m 6 A and m 6 Am d m 6 Am mapping identifies alternative ''internal'' transcription start sites d m 6 Am increases stability of a subset of mRNAs and has no effect on translation

mRNAs are regulated by nucleotide modifications that influence their cellular fate. Two of the most abundant modified nucleotides are N 6 -methyladenosine (m 6 A), found within mRNAs, and N 6 ,2'-O-dimethyladenosine (m 6 Am), which is found at the first-transcribed nucleotide. A longstanding challenge has been distinguishing these similar modifications in transcriptome-wide mapping studies. Here we identify and biochemically characterize, PCIF1, the methyltransferase that generates m 6 Am. We find that PCIF1 binds and is dependent on the m 7 G cap. By depleting PCIF1, we definitively identified m 6 Am sites and generated transcriptomewide maps that are selective for m 6 Am and m 6 A. We find that m 6 A and m 6 Am misannotations largely arise from mRNA isoforms with alternate transcription-start sites. These isoforms contain m 6 Am that appear to map to "internal" sites, increasing the likelihood of misannotation. Using the new m 6 Am annotations, we find that depleting m 6 Am does not affect mRNA translation but reduces the stability of a subset of m 6 Am-annotated mRNAs. The discovery of PCIF1 and our accurate mapping technique will facilitate future studies to characterize m 6 Am's function.

Small nuclear RNAs (snRNAs) are core spliceosome components and mediate pre-mRNA splicing. Here we show that snRNAs contain a regulated and reversible nucleotide modification causing them to exist as two different methyl isoforms, m 1 and m 2 , reflecting the methylation state of the adenosine adjacent to the snRNA cap. We find that snRNA biogenesis involves the formation of an initial m 1-isoform with a single-methylated adenosine (2'-O-methyladenosine, Am), which is then converted to a dimethylated m 2-isoform (N 6 ,2'-O-dimethyladenosine, m 6 Am). The relative m 1-and m 2-isoform levels are determined by the RNA demethylase FTO, which selectively demethylates the m 2-isoform. We show FTO is inhibited by the oncometabolite D-2hydroxyglutarate, resulting in increased m 2-snRNA levels. Furthermore, cells that exhibit high m 2-snRNA levels show altered patterns of alternative splicing. Together, these data reveal that FTO controls a previously unknown central step of snRNA processing involving reversible methylation, and suggest that epitranscriptomic information in snRNA may influence mRNA splicing.