NAD captureSeq indicates NAD as a bacterial cap for a subset of regulatory RNAs

Abstract

A distinctive feature of prokaryotic gene expression is the absence of 5'-capped RNA. In eukaryotes, 5',5'-triphosphate-linked 7-methylguanosine protects messenger RNA from degradation and modulates maturation, localization and translation. Recently, the cofactor nicotinamide adenine dinucleotide (NAD) was reported as a covalent modification of bacterial RNA. Given the central role of NAD in redox biochemistry, posttranslational protein modification and signalling, its attachment to RNA indicates that there are unknown functions of RNA in these processes and undiscovered pathways in RNA metabolism and regulation. The unknown identity of NAD-modified RNAs has so far precluded functional analyses. Here we identify NAD-linked RNAs from bacteria by chemo-enzymatic capture and next-generation sequencing (NAD captureSeq). Among those identified, specific regulatory small RNAs (sRNAs) and sRNA-like 5'-terminal fragments of certain mRNAs are particularly abundant. Analogous to a eukaryotic cap, 5'-NAD modification is shown in vitro to stabilize RNA against 5'-processing by the RNA-pyrophosphohydrolase RppH and against endonucleolytic cleavage by ribonuclease (RNase) E. The nudix phosphohydrolase NudC decaps NAD-RNA and thereby triggers RNase-E-mediated RNA decay, while being inactive against triphosphate-RNA. In vivo, ∼13% of the abundant sRNA RNAI is NAD-capped in the presence, and ∼26% in the absence, of functional NudC. To our knowledge, this is the first description of a cap-like structure and a decapping machinery in bacteria.

Identification of NAD + capped mRNAs in Saccharomyces cerevisiae

“…Consistent with this idea, NAD + addition to RNAs appears to occur during transcription initiation (9), as opposed to the more complex eukaryote 5′ capping, which occurs after transcription has commenced (10). Because the NAD + modification defines the 5′ end of NAD-RNAs, this modification can affect RNA stability in Escherichia coli (7,9).…”

“…To examine if NAD-RNA exists in yeast, we used the approach wherein NADRNAs (structurally depicted in Fig. 1A) are identified by the reaction of the NAD + moiety with ADP ribosylcyclase (ADPRC), thereby allowing biotin incorporation and purification of modified RNAs on streptavidin (7). Comparison of in vitro synthesized uncapped and 5′ NAD-RNA demonstrated that this method was specific for the purification of NAD-RNA (Fig.…”

“…From in vitro-transcribed RNAs. Following gel purification, in vitro-transcribed RNAs were manipulated basically as described (7). Equal radioactive counts of NAD-RNA or A-RNA were incubated with 0.4 μM ADPRC, 3 μL 4-pentyn-1-ol, 50 mM Hepes, and 5 mM MgCl 2 (pH 7) in a total of 60 μL at 37°C for 60 min.…”

“…One modification recently identified in bacteria is 5′ nicotinamide-adenine dinucleotide (NAD + )-linked RNA (7,8). Because canonical bacterial RNAs contain a 5′ triphosphate terminus, addition of NAD + to the 5′ end of RNAs represents a rudimentary "capping" mechanism, perhaps designed to impart specific properties for these RNAs by granting them a more structurally complex 5′ end.…”

Properties and reactivity of nucleic acids relevant to epigenomics, transcriptomics, and therapeutics

“…8, Please do not adjust margins Please do not adjust margins 169 Furthermore, it is likely that there are important modifications waiting to be discovered. 234 New chemistry will be required to sequence modified DNAs and RNAs and to determine their function. While the present review makes it clear how important selective chemistry is in the study of nucleic acids, it should also serve to highlight that continued research in nucleic acid chemistry is vital.…”