Atlas of quantitative single-base-resolution N6-methyl-adenine methylomes

Abstract

Various methyltransferases and demethylases catalyse methylation and demethylation of N6-methyladenosine (m6A) and N6,2′-O-dimethyladenosine (m6Am) but precise methylomes uniquely mediated by each methyltransferase/demethylase are still lacking. Here, we develop m6A-Crosslinking-Exonuclease-sequencing (m6ACE-seq) to map transcriptome-wide m6A and m6Am at quantitative single-base-resolution. This allows for the generation of a comprehensive atlas of distinct methylomes uniquely mediated by every individual known methyltransferase or demethylase. Our atlas reveals METTL16 to indirectly impact manifold methylation targets beyond its consensus target motif and highlights the importance of precision in mapping PCIF1-dependent m6Am. Rather than reverse RNA methylation, we find that both ALKBH5 and FTO instead maintain their regulated sites in an unmethylated steady-state. In FTO’s absence, anomalous m6Am disrupts snRNA interaction with nuclear export machinery, potentially causing aberrant pre-mRNA splicing events.

METTL4 catalyzes m6Am methylation inU2 snRNAto regulate pre-mRNA splicing

“…We note that the HPLC-MS/MS m6Am signal was derived internally from U2 snRNA as we did not use any decapping enzyme in digesting U2 snRNA, which would prevent the quantification of any cap-adjacent RNA nucleotide. Furthermore, previous and current RNA methylomes have demonstrated that U2 snRNA lacks a TSS-associated adenosine N 6 -methylation ( Figure 1C) (27). Altogether, this demonstrates that METTL4 is necessary for m6Am formation within U2 snRNA.…”

“…By overexpressing recombinant METTL4, we demonstrated the necessity of METTL4's catalytic site in its methylation activity and that METTL4 is able to directly methylate U2 snRNA both in vitro and in vivo. N 6 -methylation writers such as METTL3 and PCIF1 have multiple in vivo targets and thus by comparing the difference in precise RNA methylomes before and after depleting these writers in cells, we can determine the in vivo target RNA preference for each writer (27). METTL4 however, only has 1 clear in vivo target, making such an analysis non-trivial.…”

“…Mettl4 is one of a diverse family of proteins that are homologous to the MT-A70 subunit of the m6A writer Mettl3 (29). In order to assess Mettl4 as a potential writer, we performed m6A-Crosslinking-Exonuclease-sequencing (m6ACE-seq), which is capable of quantitatively mapping precise N 6 -methyladenine methylomes in the form of m6A or m6Am ( Figure S1A) (27). Comparison of the RNA methylomes between wildtype (WT) and Mettl4-KO HEK293T cells revealed a single site that exhibited almost 70 fold reduced relative methylation level (RML) in the absence of METTL4 ( Figure 1A,1B).…”

“…m6ACE-seq analysis was performed as previously described (27): Fastq sequences were first filtered for a quality score of 20, then trimmed of 5' and 3' adapter sequences and poly(A) tails using Cutadapt (53). The 8-mer UMI located at the first 8 nucleotides of read 1 was registered and trimmed.…”

“…Given its predicted N 6 -methyladenine methyltransferase domain and its interaction with the phosphorylated C-terminal tail of RNA polymerase II during RNA transcription, PCIF1 was a strong candidate as a N 6 -methyladenine writer (21,22). Subsequently, PCIF1 was established as the writer responsible for the N 6 -methylation of Am at the transcriptional start site (TSS) to generate m6Am (23)(24)(25)(26)(27). This facilitated the characterization of TSS-associated m6Am as a regulator of mRNA resistance to DCP2-mediated decapping, and potentially mRNA translation and cell growth (19,23,25,26).…”