The ubiquitous coenzyme nicotinamide adenine dinucleotide (NAD) decorates various RNAs in different organisms. In the proteobacterium Escherichia coli, the NAD-cap confers stability against RNA degradation. To date, NAD-RNAs have not been identified in any other bacterial microorganism. Here, we report the identification of NAD-RNA in the firmicute Bacillus subtilis. In the late exponential growth phase, predominantly mRNAs are NAD modified. NAD is incorporated de novo into RNA by the cellular RNA polymerase using non-canonical transcription initiation. The incorporation efficiency depends on the -1 position of the promoter but is independent of sigma factors or mutations in the rifampicin binding pocket. RNA pyrophosphohydrolase BsRppH is found to decap NAD-RNA. In vitro, the decapping activity is facilitated by manganese ions and single-stranded RNA 5' ends. Depletion of BsRppH influences the gene expression of ∼13% of transcripts in B. subtilis. The NAD-cap stabilizes RNA against 5'-to-3'-exonucleolytic decay by RNase J1.

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.

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 ∼7 d, excluding preparatory steps, sequencing and bioinformatic analysis.

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Background Human dermal mesenchymal stromal cells (MSCs) expressing the ATP-binding cassette (ABC) efflux transporter ABCB5 represent an easily accessible MSC population that, based on preclinical and first-in-human data, holds significant promise to treat a broad spectrum of conditions associated not only with skin-related but also systemic inflammatory and/or degenerative processes. Methods We have developed a validated Good Manufacturing Practice-compliant expansion and manufacturing process by which ABCB5+ MSCs derived from surgical discard skin tissues are processed to an advanced-therapy medicinal product (ATMP) for clinical use. Enrichment for ABCB5+ MSCs is achieved in a three-step process involving plastic adherence selection, expansion in a highly efficient MSC-selecting medium, and immunomagnetic isolation of the ABCB5+ cells from the mixed culture. Results Product Quality Review data covering 324 cell expansions, 728 ABCB5+ MSC isolations, 66 ABCB5+ MSC batches, and 85 final drug products reveal high process robustness and reproducible, reliable quality of the manufactured cell therapy product. Conclusion We have successfully established an expansion and manufacturing process that enables the generation of homogenous ABCB5+ MSC populations of proven biological activity manufactured as a standardized, donor-independent, highly pure, and highly functional off-the-shelf available ATMP, which is currently tested in multiple clinical trials.