RNA capping and decapping are thought to be distinctive features of eukaryotes. Recently, the redox cofactor NAD was 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-RNA over NAD and binds to a diverse set of cellular RNAs in an unspecific manner.
In prokaryotic organisms, certain regulatory RNAs have recently been found to be linked to the ubiquitous redox cofactor nicotinamide adenine dinucleotide (NAD) at their 5'-ends. Biochemical and structural investigations of this new caplike RNA modification require synthetic access to pure NAD-RNA. Here we report a chemoenzymatic approach to generate 5'-NAD-capped RNA in high yields and purity under mild conditions. This approach uses unprotected 5'-monophosphate RNA synthesized either chemically or enzymatically, 5',5'-pyrophosphate bond formation by phosphorimidazolide chemistry, and an enzymatic cleanup step. Thus, 5'-NAD-modified RNA can be synthesized independent of length, structure, and nucleotide sequence.
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’-RNA cap is essential for the initiation of RNA degradation. In addition to the canonical 5’-N7-methyl guanosine cap in eukaryotes, the ubiquitous redox cofactor nicotinamide adenine dinucleotide (NAD) was identified as a new 5’-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.
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