Internal bases in mRNA can be subjected to modifications that influence the fate of mRNA in cells. One of the most prevalent modified bases is found at the 5′ end of mRNA, at the first encoded nucleotide adjacent to the 7-methylguanosine cap. Here we show that this nucleotide, N6,2′-O-dimethyladenosine (m6Am), is a reversible modification that influences cellular mRNA fate. Using a transcriptome-wide map of m6Am we find that m6Am-initiated transcripts are markedly more stable than mRNAs that begin with other nucleotides. We show that the enhanced stability of m6Am-initiated transcripts is due to resistance to the mRNA-decapping enzyme DCP2. Moreover, we find that m6Am is selectively demethylated by fat mass and obesity-associated protein (FTO). FTO preferentially demethylates m6Am rather than N6-methyladenosine (m6A), and reduces the stability of m6Am mRNAs. Together, these findings show that the methylation status of m6Am in the 5′ cap is a dynamic and reversible epitranscriptomic modification that determines mRNA stability.

The Middle East respiratory syndrome coronavirus (MERS-CoV) nonstructural protein 16 (nsp16) is an S-adenosyl-L-methionine (SAM)-dependent 2=-Omethyltransferase (2=-O-MTase) that is thought to methylate the ribose 2=-OH of the first transcribed nucleotide (N 1 ) of viral RNA cap structures. This 2=-O-MTase activity is regulated by nsp10. The 2=-O methylation prevents virus detection by cell innate immunity mechanisms and viral translation inhibition by the interferon-stimulated IFIT-1 protein. To unravel the regulation of nsp10/nsp16 2=-O-MTase activity, we used purified MERS-CoV nsp16 and nsp10. First, we showed that nsp16 recruited N7-methylated capped RNA and SAM. The SAM binding promotes the assembly of the enzymatically active nsp10/nsp16 complex that converted 7m GpppG (cap-0) into 7m GpppG 2=Om (cap-1) RNA by 2=-OH methylation of N 1 in a SAM-dependent manner. The subsequent release of SAH speeds up nsp10/nsp16 dissociation that stimulates the reaction turnover. Alanine mutagenesis and RNA binding assays allowed the identification of the nsp16 residues involved in RNA recognition forming the RNA binding groove (K46, K170, E203, D133, R38, Y47, and Y181) and the cap-0 binding site (Y30, Y132, and H174). Finally, we found that nsp10/nsp16 2=-O-MTase activity is sensitive to known MTase inhibitors, such as sinefungin and cap analogues. This characterization of the MERS-CoV 2=-O-MTase is a preliminary step toward the development of molecules to inhibit cap 2=-O methylation and to restore the host antiviral response.IMPORTANCE MERS-CoV codes for a cap 2=-O-methyltransferase that converts cap-0 into cap-1 structure in order to prevent virus detection by cell innate immunity mechanisms. We report the biochemical properties of MERS-CoV 2=O-methyltransferase, which is stimulated by nsp10 acting as an allosteric activator of the nsp16 2=-Omethyltransferase possibly through enhanced RNA binding affinity. In addition, we show that SAM promotes the formation of the active nsp10/nsp16 complex. Conversely, after cap methylation, the reaction turnover is speeded up by cap-1 RNA release and nsp10/ nsp16 complex dissociation, at the low intracellular SAH concentration. These results suggest that SAM/SAH balance is a regulator of the 2=-O-methyltransferase activity and raises the possibility that SAH hydrolase inhibitors might interfere with CoV replication cycle. The enzymatic and RNA binding assays developed in this work were also used to identify nsp16 residues involved in cap-0 RNA recognition and to understand the action mode of known methyltransferase inhibitors.KEYWORDS RNA processing, RNA virus, biochemistry

Alphaviruses are known to possess a unique viral mRNA capping mechanism involving the viral nonstructural protein nsP1. This enzyme harbors methyltransferase (MTase) and nsP1 guanylylation (GT) activities catalyzing the transfer of the methyl group from S-adenosylmethionine (AdoMet) to the N7 position of a GTP molecule followed by the formation of an m 7 GMPnsP1 adduct. Subsequent transfer of m 7 GMP onto the 5= end of the viral mRNA has not been demonstrated in vitro yet. Here we report the biochemical characterization of Venezuelan equine encephalitis virus (VEEV) nsP1. We have developed enzymatic assays uncoupling the different reactions steps catalyzed by nsP1. The MTase and GT reaction activities were followed using a nonhydrolyzable GTP (GIDP) substrate and an original Western blot assay using anti-m 3 G/m 7 G-cap monoclonal antibody, respectively. The GT reaction is stimulated by S-adenosyl-L-homocysteine (Ado-Hcy), the product of the preceding MTase reaction, and metallic ions. The covalent linking between nsP1 and m 7 GMP involves a phosphamide bond between the nucleotide and a histidine residue. Final guanylyltransfer onto RNA was observed for the first time with an alphavirus nsP1 using a 5=-diphosphate RNA oligonucleotide whose sequence corresponds to the 5= end of the viral genome. Alanine scanning mutagenesis of residues H37, H45, D63, E118, Y285, D354, R365, N369, and N375 revealed their respective roles in MT and GT reactions. Finally, the inhibitory effects of sinefungin, aurintricarboxylic acid (ATA), and ribavirin triphosphate on MTase and capping reactions were investigated, providing possible avenues for antiviral research. IMPORTANCEEmergence or reemergence of alphaviruses represents a serious health concern, and the elucidation of their replication mechanisms is a prerequisite for the development of specific inhibitors targeting viral enzymes. In particular, alphaviruses are able, through an original reaction sequence, to add to their mRNA a cap required for their protection against cellular nucleases and initiation of viral proteins translation. In this study, the capping of a 5= diphosphate synthetic RNA mimicking the 5= end of an alphavirus mRNA was observed in vitro for the first time. The different steps for this capping are performed by the nonstructural protein 1 (nsP1). Reference compounds known to target the viral capping inhibited nsP1 enzymatic functions, highlighting the value of this enzyme in antiviral development. E mergence or reemergence of alphaviruses represents a serious health concern, as exemplified by the worldwide epidemics of Chikungunya virus during the last 10 years (1). The Alphavirus genus comprises 31 species that together with the genus Rubivirus forms the Togaviridae family. Alphaviruses have been classified on the basis of their geographical distribution. Alphaviruses circulating in the Old World (OW) most commonly cause febrile illness and painful arthralgias or polyarthralgias, particularly in the small joints (2). In contrast, New World (NW) alph...