Cap-specific, terminal N⁶-methylation by a mammalian m⁶Am methyltransferase

Dear Editor,

Dynamic and reversible N⁶-methyladenosine (m⁶A) RNA methylation has been found to greatly impact gene expression, leading to the field of epitranscriptomics.¹ Unlike m⁶A that is an internal modification, a terminal modification at mRNA cap in higher eukaryotes exists, termed as N⁶,2′-O-dimethyladenosine (m⁶Am) (Fig. 1a). The first and sometimes the second nucleotide after the N⁷-methylguanosine (m⁷G) cap can be methylated at the 2′-hydroxyl group; and when the first nucleotide is 2′-O-methyladenosine (Am), it can be further methylated at the N⁶ position to become m⁶Am. m⁶Am was first identified in animal cells and virus mRNA in 1975²; several years later the methyltransferase was partially purified and was proposed to be a species whose molecular weight is ~65 KD.³ Only very recently, m⁶Am was found to be reversible as well: the first m⁶A demethylase FTO also catalyzed the demethylation of m⁶Am, depending on its sub-cellular localizations.^4,5 By changing FTO levels, m⁶Am at mRNA cap was also suggested to impair DCP2-mediated mRNA decapping.⁴ However, the methyltransferase of m⁶Am is not unambiguously identified, significantly hindering the functional and mechanistic study of m⁶Am.

Fig. 1.

Identification of N⁶,2′-O-dimethyladenosine methyltransferase. a Chemical structure of m⁶Am, which is adjacent to the m⁷G mRNA cap. b Cartoon view of the predicted domain structure of PCIF1, with the conserved “NPPF” motif in the zoom-in view. A sequence alignment is shown below to highlight the high conservation of the key residues for PCIF1 orthologues. Residue 43–77 (blue segment) represents the WW domain, and the red segment denotes the putative catalytic methyltransferase domain. c LC-MS/MS quantification of the m⁶Am/A ratios of HEK293T polyA + RNA treated with control or PCIF1 siRNA (n = 3). d LC-MS/MS quantification of the m⁶A/A ratios of HEK293T polyA+ RNA treated with control or PCIF1 siRNA (n = 3). e Quantification of the m⁶Am/(Am + m⁶Am) ratios in RNA probes starting with different cap structure (n = 3). f Quantification of the methylation activity of WT and mutant PCIF1 proteins (n = 3). g Distribution of enriched m⁶A/m⁶Am peak density across mRNA segments of control and PCIF1 knockdown samples using an m⁶A-seq protocol with random priming. Each segment was normalized according to its average length in Ref-seq annotation. h One representative transcript harboring m⁶Am and m⁶A peaks. The m⁶Am peak at the 5′-terminal is significantly decreased upon PCIF1 knockdown, while the m⁶A peak at the 3′-UTR stays the same. The grey line denotes “Input”, and the red line denotes “IP”. i Boxplot of log₂fold change of peak score in PCIF1 knockdown and control mRNA. Enriched peaks are classified into three groups: m⁶Am peak (near TSS and without GGACH motif), m⁶A peak (not in TSS and with GGACH motif), and the rest (potentially m⁶Am + m⁶A). j A proposed model for mammalian mRNA m⁶Am modification mediated by PCIF1. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant

To clearly identify the methyltransferase, we fractioned the cell lysates of HEK293 cells, which contain robust N⁶-methylation activity (Supplementary information, Fig.S1a). This activity was assayed by incubating the column fractions with a 25 nt, synthetic vaccinia virus RNA probe (Probe-1, see Supplementary information) that begins with m⁷GpppAm. We modified the purification route of cell lysates, based on the procedure originally reported³ (Supplementary information, Fig.S1b), and subjected the fractions of high N⁶-methylation activity to protein identification by sensitive mass spectrometry. We then searched for proteins with putative methyltransferase domain or sequence motif in the list of more than 100 proteins detected by MS, and found a protein named “phosphorylated CTD-interacting factor 1” (or PCIF1) (Fig. 1b; Supplementary information, Fig.S1c), which was bioinformatically proposed to be a DNA/RNA N⁶-adenosine methyltransferase.⁶ PCIF1 was originally identified and named due to its ability to directly bind to the phosphorylated C-terminal domain of RNA polymerase II via its WW domain⁷; hence it was speculated to play a role in mRNA biogenesis. However, no enzymatic activity has been reported for PCIF1.

To test whether PCIF1 possesses methyltransferase activity in vivo, we first knocked down PCIF1 in HEK293 cells by two independent siRNAs and confirmed the knockdown efficiency by qRT-PCR (Supplementary information, Fig.S2a). We then measured the level of m⁶Am in polyA + RNA fraction after decapping using LC-MS/MS. We were able to observe a reduction of m⁶Am level upon PCIF1 knockdown (Fig. 1c; Supplementary information, Fig.S2b); importantly, the level of the internal m⁶A modification remained unchanged (Fig. 1d), suggesting that PCIF1 is a specific methyltransferase for the terminal m⁶Am. Encouraged by the in vivo results, we then expressed and purified recombinant PCIF1 protein, and tested whether the single protein is capable of methylating RNA substrates under in vitro conditions (Supplementary information, Fig.S2c). The highest activity of PCIF1 was obtained with RNA Probe-1 beginning with a complete cap structure m⁷GpppAm; much lower activity was found with RNA beginning with GpppAm; and barely detectable activity was found with RNA beginning with pppAm or RNA Probe-2 with an internal Am (Fig. 1e). The above enzymatic preference was also supported by biochemical experiments using two different RNA probes (Probe-3 and Probe-4), which in addition showed that the ribose 2′-O-methylation is required for optimal methylation activity as well (Supplementary information, Fig.S2d). Moreover, we introduced point mutations in the highly conserved “NPPF” motif that is characteristic of adenosine methyltransferases, and found that the disruption of this motif reduced the methyltransferase activity of the mutant proteins (Fig. 1f; Supplementary information, Fig.S1d). Because PCIF1 is highly conserved among different species (Fig. 1b), we further tested whether the mouse PCIF1 protein is also functional. We knocked down mPcif1 by siRNA in mouse NIH-3T3 cells and also observed reduced m⁶Am level (Supplementary information, Fig.S3a, b). Additionally, mouse PCIF1 protein also exhibited a robust methylation activity in vitro (Supplementary information, Fig.S3c). Altogether, the evidence presented above demonstrated that PCIF1 is a novel mammalian m⁶Am writer, which is specific for the 5′-end capped RNA.

To identify the RNA targets of PCIF1, we performed m⁶A-seq experiments for PCIF1 knockdown and control cells using an anti-m⁶A antibody.^8,9 Because the antibody recognizes m⁶Am and m⁶A, both types of modifications were enriched and hence detected simultaneously.¹⁰ m⁶A modifications are known to be enriched around 3′-UTR, with a small portion also present internally in the 5′-UTR; while m⁶Am modifications localized at the 5′-end of RNA. We envisioned that the cap-specific PCIF1 should selectively alter the m⁶Am modification at the 5′-terminal region of transcripts. Indeed, we observed a reduction of modification peaks at the 5′-end but not the 3′-UTR regions of mRNAs upon PCIF1 knockdown (Fig. 1g; Supplementary information, Fig.S3d). One example is the TBRG4 transcript, for which we found a 5′-end peak and a 3′-UTR peak by m⁶A-seq (Fig. 1h); only the former peak underwent a clear reduction while the latter remained the same. We then grouped the enriched peaks into three categories and again observed significantly decreased signals for the m⁶Am peaks after PCIF1 knockdown when comparing to the m⁶A and m⁶A + m⁶Am categories (Fig. 1i; Supplementary information, Table S1). We further adopted a different m⁶A-seq procedure that can preserve the 5′-end information of polyA+ RNA, and again found a decrease of m⁶Am peak intensity after PCIF1 knockdown (Supplementary information, Fig.S3e). In addition, a motif analysis revealed that m⁶Am modification occurs at the transcription start sites, in accordance with the known m⁶Am pattern (Supplementary information, Fig.S3f).⁴ Thus, results from our m⁶A-seq experiments revealed the direct mRNA targets of PCIF1 inside of human cells (Fig. 1j).

Taken together, in this study we revealed the exact identity of the m⁶Am writer protein, characterized its biochemical property and substrate preference, and profiled its cellular targets using an epitranscriptomic sequencing approach. PCIF1 recognizes the positively charged cap structure m⁷GpppAm for optimal activity and is a “stand-alone” RNA methyltransferase. In comparison, the internal m⁶A is installed by a methyltransferase complex, the core components of which are composed of METTL3, METTL14 and WTAP. The m⁶A methyltransferase complex also recognizes internal adenosines, with a preference for those located within a RRACH consensus motif. Hence, while m⁶Am and m⁶A share a common eraser protein FTO, the writer proteins for the two modifications are orthogonal. Manipulating the protein levels of the writers could potentially separate the differential roles of FTO in demethylating m⁶Am and m⁶A. The functional study of m⁶A is greatly facilitated by the discovery and characterization of its regulation system involving the writer, reader and eraser proteins; we envision that the identification of PCIF1 as the m⁶Am writer will pave the path toward functional and mechanistic dissection of this dynamic and reversible epitranscriptomic mark in the future.

Author contributions

H.S. and M.Z. conceived all experiments under the guidance of C.Y., H.S. synthesized the RNA probes, purified the recombinant protein and performed in vitro methylation experiments. M.Z. developed the methylation assay, performed sequencing and cell biology experiments. K.L. performed the bioinformatics analysis with the help of C.Y., D.B. assisted in probe synthesis.

Competing interests

The authors declare no competing interests.

Electronic supplementary material

Supplementary information accompanies this paper at 10.1038/s41422-018-0117-4.