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The mechanisms by which viruses hijack their host’s genetic machinery are of enormous current interest. One mechanism is adenosine diphosphate (ADP) ribosylation, where ADP-ribosyltransferases (ARTs) transfer an ADP-ribose fragment from the ubiquitous coenzyme nicotinamide adenine dinucleotide (NAD) to acceptor proteins. When bacteriophage T4 infects Escherichia coli, three different ARTs reprogram the host’s transcriptional and translational apparatus. Recently, NAD was identified as a 5’-modification of cellular RNA molecules in bacteria and higher organisms. Here, we report that bacteriophage T4 ARTs accept not only NAD, but also NAD-RNA as substrate, thereby covalently linking entire RNA chains to acceptor proteins in an “RNAylation” reaction. One of these ARTs, ModB, efficiently RNAylates its host protein target, ribosomal protein S1, at arginine residues and strongly prefers NAD-RNA over NAD. Mutation of a single arginine at position 139 abolishes ADP-ribosylation and RNAylation. Overexpression of mammalian ADP-ribosylarginine hydrolase 1 (ARH1), which cleaves arginine-phosphoribose bonds, shows a decelerated lysis of E. coli when infected with T4. Our findings not only challenge the established views of the phage replication cycle, but also reveal a distinct biological role of NAD-RNA, namely activation of the RNA for enzymatic transfer. Our work exemplifies the first direct connection between RNA modification and post-translational protein modification. As ARTs play important roles in different viral infections, as well as in antiviral defence by the host, RNAylation may have far-reaching implications.

The complexity of the transcriptome is governed by the intricate interplay of transcription, RNA processing, translocation, and decay. In eukaryotes, the removal of the 5&rsquo, RNA cap is essential for the initiation of RNA degradation. In addition to the canonical 5&rsquo, N7-methyl guanosine cap in eukaryotes, the ubiquitous redox cofactor nicotinamide adenine dinucleotide (NAD) was identified as a new 5&rsquo, RNA cap structure in prokaryotic and eukaryotic organisms. So far, two classes of NAD-RNA decapping enzymes have been identified, namely Nudix enzymes that liberate nicotinamide mononucleotide (NMN) and DXO-enzymes that remove the entire NAD cap. Herein, we introduce 8-(furan-2-yl)-substituted NAD-capped-RNA (FurNAD-RNA) as a new research tool for the identification and characterization of novel NAD-RNA decapping enzymes. These compounds are found to be suitable for various enzymatic reactions that result in the release of a fluorescence quencher, either nicotinamide (NAM) or nicotinamide mononucleotide (NMN), from the RNA which causes a fluorescence turn-on. FurNAD-RNAs allow for real-time quantification of decapping activity, parallelization, high-throughput screening and identification of novel decapping enzymes in vitro. Using FurNAD-RNAs, we discovered that the eukaryotic glycohydrolase CD38 processes NAD-capped RNA in vitro into ADP-ribose-modified-RNA and nicotinamide and therefore might act as a decapping enzyme in vivo. The existence of multiple pathways suggests that the decapping of NAD-RNA is an important and regulated process in eukaryotes.

RNA capping and decapping are thought to be distinctive features of eukaryotes. The redox cofactor NAD was recently discovered to be attached to small regulatory RNAs in bacteria in a cap-like manner, and Nudix hydrolase NudC was found to act as a NAD-decapping enzyme in vitro and in vivo. Here, crystal structures of Escherichia coli NudC in complex with substrate NAD and with cleavage product NMN reveal the catalytic residues lining the binding pocket and principles underlying molecular recognition of substrate and product. Biochemical mutation analysis identifies the conserved Nudix motif as the catalytic center of the enzyme, which needs to be homodimeric, as the catalytic pocket is composed of amino acids from both monomers. NudC is single-strand specific and has a purine preference for the 5'-terminal nucleotide. The enzyme strongly prefers NAD-linked RNA (NAD-RNA) over NAD and binds to a diverse set of cellular RNAs in an unspecific manner.