Identification of the m6Am Methyltransferase PCIF1 Reveals the Location and Functions of m6Am in the Transcriptome.

mRNAs are regulated by nucleotide modifications that influence their cellular fate. Two of the most abundant modified nucleotides are N6-methyladenosine (m6A), found within mRNAs, and N6,2'-O-dimethyladenosine (m6Am), which is found at the first transcribed nucleotide. Distinguishing these modifications in mapping studies has been difficult. Here, we identify and biochemically characterize PCIF1, the methyltransferase that generates m6Am. We find that PCIF1 binds and is dependent on the m7G cap. By depleting PCIF1, we generated transcriptome-wide maps that distinguish m6Am and m6A. We find that m6A and m6Am misannotations arise from mRNA isoforms with alternative transcription start sites (TSSs). These isoforms contain m6Am that maps to "internal" sites, increasing the likelihood of misannotation. We find that depleting PCIF1 does not substantially affect mRNA translation but is associated with reduced stability of a subset of m6Am-annotated mRNAs. The discovery of PCIF1 and our accurate mapping technique will facilitate future studies to characterize m6Am's function.

A second type of N7-guanine RNA cap methyltransferase in an unusual locus of a large RNA virus genome.

The order Nidovirales is a diverse group of (+)RNA viruses, with a common genome organization and conserved set of replicative and editing enzymes. In particular, RNA methyltransferases play a central role in mRNA stability and immune escape. However, their presence and distribution in different Nidovirales families is not homogeneous. In Coronaviridae, the best characterized family, two distinct methytransferases perform methylation of the N7-guanine and 2'-OH of the RNA-cap to generate a cap-1 structure (m7GpppNm). The genes of both of these enzymes are located in the ORF1b genomic region. While 2'-O-MTases can be identified for most other families based on conservation of both sequence motifs and genetic loci, identification of the N7-guanine methyltransferase has proved more challenging. Recently, we identified a putative N7-MTase domain in the ORF1a region (N7-MT-1a) of certain members of the large genome Tobaniviridae family. Here, we demonstrate that this domain indeed harbors N7-specific methyltransferase activity. We present its structure as the first N7-specific Rossmann-fold (RF) MTase identified for (+)RNA viruses, making it remarkably different from that of the known Coronaviridae ORF1b N7-MTase gene. We discuss the evolutionary implications of such an appearance in this unexpected location in the genome, which introduces a split-off in the classification of Tobaniviridae.

Access to High-Purity 7m G-cap RNA in Substantial Quantities by a Convenient All-Chemical Solid-Phase Method.

Given the importance of mRNA with 5'-cap, easy access to RNA substrates with different 7m G caps, of high quality and in large quantities is essential to elucidate the roles of RNA and the regulation of underlying processes. In addition to existing synthetic routes to 5'-cap RNA based on enzymatic, chemical or chemo-enzymatic methods, we present here an all-chemical method for synthetic RNA capping. The novelty of this study lies in the fact that the capping reaction is performed on solid-support after automated RNA assembly using commercial 2'-O-propionyloxymethyl ribonucleoside phosphoramidites, which enable final RNA deprotection under mild conditions while preserving both 7m G-cap and RNA integrity. The capping reaction is efficiently carried out between a 5'-phosphoroimidazolide RNA anchored on the support and 7m GDP in DMF in the presence of zinc chloride. Substantial amounts of 7m G-cap RNA (from 1 to 28 nucleotides in length and of any sequence with or without internal methylations) containing various cap structures (7m GpppA, 7m GpppAm , 7m Gpppm6 A, 7m Gpppm6 Am , 7m GpppG, 7m GpppGm ) were obtained with high purity after IEX-HPLC purification. This capping method using solid-phase chemistry is convenient to perform and provides access to valuable RNA substrates as useful research tools to unravel specific issues regarding cap-related processes.

FTO controls reversible m6Am RNA methylation during snRNA biogenesis.

Small nuclear RNAs (snRNAs) are core spliceosome components and mediate pre-mRNA splicing. Here we show that snRNAs contain a regulated and reversible nucleotide modification causing them to exist as two different methyl isoforms, m1 and m2, reflecting the methylation state of the adenosine adjacent to the snRNA cap. We find that snRNA biogenesis involves the formation of an initial m1 isoform with a single-methylated adenosine (2'-O-methyladenosine, Am), which is then converted to a dimethylated m2 isoform (N6,2'-O-dimethyladenosine, m6Am). The relative m1 and m2 isoform levels are determined by the RNA demethylase FTO, which selectively demethylates the m2 isoform. We show FTO is inhibited by the oncometabolite D-2-hydroxyglutarate, resulting in increased m2-snRNA levels. Furthermore, cells that exhibit high m2-snRNA levels show altered patterns of alternative splicing. Together, these data reveal that FTO controls a previously unknown central step of snRNA processing involving reversible methylation, and suggest that epitranscriptomic information in snRNA may influence mRNA splicing.

The methyltransferase domain of the Sudan ebolavirus L protein specifically targets internal adenosines of RNA substrates, in addition to the cap structure.

Mononegaviruses, such as Ebola virus, encode an L (large) protein that bears all the catalytic activities for replication/transcription and RNA capping. The C-terminal conserved region VI (CRVI) of L protein contains a K-D-K-E catalytic tetrad typical for 2'O methyltransferases (MTase). In mononegaviruses, cap-MTase activities have been involved in the 2'O methylation and N7 methylation of the RNA cap structure. These activities play a critical role in the viral life cycle as N7 methylation ensures efficient viral mRNA translation and 2'O methylation hampers the detection of viral RNA by the host innate immunity. The functional characterization of the MTase+CTD domain of Sudan ebolavirus (SUDV) revealed cap-independent methyltransferase activities targeting internal adenosine residues. Besides this, the MTase+CTD also methylates, the N7 position of the cap guanosine and the 2'O position of the n1 guanosine provided that the RNA is sufficiently long. Altogether, these results suggest that the filovirus MTases evolved towards a dual activity with distinct substrate specificities. Whereas it has been well established that cap-dependent methylations promote protein translation and help to mimic host RNA, the characterization of an original cap-independent methylation opens new research opportunities to elucidate the role of RNA internal methylations in the viral replication.

Combining Chemical Synthesis and Enzymatic Methylation to Access Short RNAs with Various 5' Caps.

Eukaryotic RNAs are heavily processed, including co- and post-transcriptional formation of various 5' caps. In small nuclear RNAs (snRNAs) or small nucleolar RNAs (snoRNAs), the canonical 7m G cap is hypermethylated at the N2 -position, whereas in higher eukaryotes and viruses 2'-O-methylation of the first transcribed nucleotide yields the cap1 structure. The function and potential dynamics of several RNA cap modifications have not been fully elucidated, which necessitates preparative access to these caps. However, the introduction of these modifications during chemical solid-phase synthesis is challenging and enzymatic production of defined short and uniform RNAs also faces difficulties. In this work, the chemical synthesis of RNA is combined with site-specific enzymatic methylation by using the methyltransferases human trimethylguanosine synthase 1 (hTgs1), trimethylguanosine synthase from Giardia lamblia (GlaTgs2), and cap methyltransferase 1 (CMTR1). It is shown that RNAs with di-and trimethylated caps, as well as RNAs with caps methylated at the 2'-O-position of the first transcribed nucleotide, can be conveniently prepared. These highly modified RNAs, with a defined and uniform sequence, are hard to access by in vitro transcription or chemical synthesis alone.