PCIF1 Catalyzes m6Am mRNA Methylation to Regulate Gene Expression

Abstract

Highlights d PCIF1 is an evolutionarily conserved mRNA m6Am methyltransferase d Loss of PCIF1 leads to loss of m6Am, but m6A level or distribution is not affected d m6Am decreases cap-dependent translation; no effect on transcription nor mRNA stability d m6Am-Exo-Seq is a robust methodology that enables global m6Am mapping

CAP-MAP: Cap Analysis Protocol with Minimal Analyte Processing, a rapid and sensitive approach to analysing mRNA cap structures

“…The most common cap structures in liver mRNA were m7 GpppGm (19 fmol/µg), m7 GpppAm (4 fmol/µg), and m7 Gppp m6 Am (20 fmol/µg) ( Fig.5A); these are the major cap variants previously elucidated by mass spectrometry and thin layer chromatography of radiolabelled nucleotides [1,29]. In line with recent observations that m7 Gppp m6 Am is an abundant cap structure, m7 Gppp m6 Am constituted 75% of the caps when A is the first transcribed nucleotide [10][11][12]43]. A number of incompletely methylated cap structures were detected including GpppA, GpppAm, Gppp m6 Am, and GpppGm, which lack cap guanosine N-7 methylation; and m7 GpppA, and m7 GpppG which lack ribose O-2 methylation of the first transcribed nucleotide.…”

“…Notably CMTR1 binds directly to the RNAPII C-terminal domain whereas RNMT interacts indirectly with RNAPII predominantly via interactions with RNA and RNAPIIassociated proteins, which does not imply an obligate order of action [49,50]. CAPAM/PCIF1, the enzyme catalysing first transcribed nucleotide m6 A methylation has recently been identified and characterised [9][10][11][12]. m6 A methylation is the only cap methylation that has been demonstrated to be reversible leading to an interest in whether it may coordinate signalling events with translation or transcript stability [26].…”

“…Mammalian mRNA processing initiates co-transcriptionally with the addition of the inverted guanosine cap, catalysed by Capping Enzyme/RNA guanylyltransferase and 5' phosphatase (CE/RNGTT) [2,8]. The terminal cap guanosine is methylated by RNA cap methyltransferase (RNMT) on the N-7 position and the first two transcribed nucleotides are modified by cap-specific methyltransferases as follows: the O-2 position of the ribose of the first and second transcribed nucleotides are methylated by Cap-Methyltransferase 1 (CMTR1) and CMTR2 respectively, and if the first transcribed nucleotide is adenosine it is methylated on the N-6 position by Cap-specific Adenosine Methyltransferase (CAPAM, also known as PCIF1) [9][10][11][12] (Fig.1).…”

METTL4 catalyzes m6Am methylation inU2 snRNAto regulate pre-mRNA splicing

“…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).…”

Identification of m6A residues at single-nucleotide resolution using eCLIP and an accessible custom analysis pipeline

“…While m 6 A modifications have been identified throughout the transcriptome, they are most-often enriched around 3' UTRs and stop codons (Meyer et al 2012;Dominissini et al 2012;Ke et al 2015). In contrast, a similar RNA modification, N 6 , 2′-Odimethyladenosine (m 6 Am), is located on the 5' ends of mRNAs and is catalyzed by the methyltransferase PCIF1 (Boulias et al 2019;Sendinc et al 2019). Following methylation, m 6 A containing-transcripts are specifically recognized by 'reader' proteins, the most well characterized being members of the YTH domain family (Wu et al 2017).…”