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.

The 5′-cap structure, characteristic for RNA polymerase II-transcribed RNAs, plays important roles in RNA metabolism. In humans, RNA cap formation includes post-transcriptional modification of the first transcribed nucleotide by RNA cap1 methyltransferase (CMTr1). Here, we report that CMTr1 activity is hindered towards RNA substrates with highly structured 5′ termini. We found that CMTr1 binds ATP-dependent RNA DHX15 helicase and that this interaction, mediated by the G-patch domain of CMTr1, has an advantageous effect on CMTr1 activity towards highly structured RNA substrates. The effect of DHX15 helicase activity is consistent with the strength of the secondary structure that has to be removed for CMTr1 to access the 5′-terminal residues in a single-stranded conformation. This is, to our knowledge, the first demonstration of the involvement of DHX15 in post-transcriptional RNA modification, and the first example of a molecular process in which DHX15 directly affects the activity of another enzyme. Our findings suggest a new mechanism underlying the regulatory role of DHX15 in the RNA capping process. RNAs with highly structured 5′ termini constitute a significant fraction of the human transcriptome. Hence, CMTr1–DHX15 cooperation is likely to be important for the metabolism of RNA polymerase II-transcribed RNAs.This article is part of the theme issue ‘5′ and 3′ modifications controlling RNA degradation’.

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