Identification of the m6Am methyltransferase PCIF1 reveals the location and functions of m6Am in the transcriptome

Abstract

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 emerging role of RNA modifications in the regulation of mRNA stability

“…First, m 6 Am is generated by the methylation of Am, which is primarily located in the first nucleotide position adjacent to the m 7 G cap structure of mRNA [3][4][5][6][7] . Second, a unique writer protein, phosphorylated CTD-interacting factor 1, is responsible for generating the m 6 Am modification [61][62][63][64] , whereas a methyltransferase complex comprising METTL3, METTL14, WTAP, and KIAA1429 is involved in the generation of the m 6 A modification [22][23][24][25] . Finally, whereas m 6 A is largely demethylated by ALKBH5 but also by FTO, with a weak preference [29][30][31][32] , m 6 Am is preferentially and specifically demethylated by FTO 29,31,62 .…”

“…Although controversial 63,64 , findings from recent studies have revealed that m 6 Am-initiated mRNAs are in greater abundance and have longer half-lives than mRNAs with Am, Gm, Cm, or Um 31,62 . In vitro decapping experiments have shown that m 6 Am-initiated mRNAs are more resistant to decapping by decapping mRNA 2 (DCP2) 65 , resulting in the increased mRNA stability (Fig.…”

METTL4 is an snRNA m6Am methyltransferase that regulates RNA splicing

“…Protein coding mRNAs and non-coding RNAs (e.g., rRNAs, snRNAs and miRNAs) are extensively modified by chemical modifications , including methylation of the N 6 position of adenosine (m 6 A) 2 . While mRNA internal m 6 A methylation is mediated by METTL3 and METTL14, which are members of the MT-A70 family 2 , N 6 -methylation of cap-adjacent N 6 ,2´-O-dimethyladenosine (m 6 Am) is catalyzed by PCIF1 [3][4][5][6] .…”

So close, no matter how far: multiple paths connecting transcription to mRNA translation in eukaryotes

“…When this nucleotide is adenosine, it can be further methylated on its N6 position (Keith et al, 1978) to yield, together with the 2 0 -O methyl group, a modification termed m 6 Am. Although this modification is known for many years, the methylating enzyme, PCIF1, has been discovered only very recently by several groups (Akichika et al, 2019;Boulias et al, 2019;Sendinc et al, 2019;Sun et al, 2019). Despite such a remarkably simultaneous identification of the effector protein and the confirmation of its specificity for m 6 Am and not the m 6 A modification, the impact of this modification on translation remains unclear, as the experimental results significantly diverge.…”

“…Accordingly, knock-down of PCIF1 resulted in a significant upregulation of many (% 500) proteins. The third study has employed the CAGE approach to show the actual location of the m 6 Am sites to alternative TSSs (Boulias et al, 2019). Interestingly, the authors identified a substantial amount of m 6 Am deposited downstream of the annotated TSS, sometimes even within CDRs or 3 0 UTRs, possibly indicating pervasive transcription initiation.…”