Cap-like structures in bacterial RNA and epitranscriptomic modification

The absence of capped RNA is considered as a hallmark of prokaryotic gene expression. Recent developments combine next-generation sequencing with a chemo-enzymatic capture step that allows the enrichment of rare 5'-modified RNA from bacteria. This approach identified covalent cap-like linkage of a specific set of small RNAs to the ubiquitous redox cofactor NAD, and a profound influence of this modification on RNA turnover. The modification revealed an unexpected connection between redox biology and RNA processing. We discuss possible roles of the NAD modification as well as broader implications for structurally related cofactors and metabolites which may also be linked to RNAs, leading to a new epitranscriptomic layer of information encoded in the chemical structure of the attached cofactors

Capture and sequencing of NAD-capped RNA sequences with NAD captureSeq

Here we describe a protocol for NAD captureSeq that allows for the identification of nicotinamide-adenine dinucleotide (NAD)-capped RNA sequences in total RNA samples from different organisms. NAD-capped RNA is first chemo-enzymatically biotinylated with high efficiency, permitting selective capture on streptavidin beads. Then, a highly efficient library preparation protocol tailored to immobilized, 5'-modified RNA is applied, with adaptor ligation to the RNA's 3' terminus and reverse transcription (RT) performed on-bead. Then, cDNA is released into solution, tailed, ligated to a second adaptor and PCR-amplified. After next-generation sequencing (NGS) of the DNA library, enriched sequences are identified by comparison with a control sample in which the first step of chemo-enzymatic biotinylation is omitted. Because the downstream protocol does not necessarily rely on NAD-modified but on 'clickable' or biotin-modified RNA, it can be applied to other RNA modifications or RNA-biomolecule interactions. The central part of this protocol can be completed in approximately 7 d, excluding preparatory steps, sequencing and bioinformatic analysis

Structure and function of the bacterial decapping enzyme NudC

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