In prokaryotes, the 5’ NAD+ modification is established by bacterial RNAP during transcription initiation and has been shown to display functions analogous to those of the eukaryotic 5’ cap. In-vitro transcribed NAD-modified RNA was shown to be more resistant to RNase E, the main enzyme in the decay pathway of E. coli. NAD-modification further was shown to decelerate RNA processing by RNA pyrophosphohydrolase (RppH), which is known to trigger RNase-E-mediated decay through the conversion of 5′-triphosphate-RNA to 5′-monophosphate-RNA. Nudc, a nudix phosphohydrolase, can decap NAD-RNA through hydrolyzing NAD(H) into NMN(H) and AMP, causing RNase-E-mediated decay, but is inactive against 5′-triphosphate-RNA. This 5’ modification allows for the selective initiation of degradation for a subset of RNAs as the NAD-capped RNAs are stabilized in the presence of RppH, but are decapped by Nudc, while the 5′-triphosphate-RNAs are susceptible to RppH but not Nudc.
Next generation sequencing (NGS) of the NAD-RNA conjugates in E. coli revealed an abundance of a specific group of small regulatory RNAs (sRNAs) which are known to be involved in stress response systems, as well as enzymes involved in cellular metabolism. The small number of RNA transcripts with a NAD cap might allow the cell to selectively degrade these RNAs separate from other pathways. Considering that the stress responses are known to affect NAD+ concentration, this finding further supports the possibility of undiscovered pathways linking the energetic state of a cell to mRNA turnover.
NAD capping has also been suggested to recruit specific proteins to the 5’ end of the RNA as NAD is one of the most common protein ligands. NAD-binding pockets are well characterized in many proteins and could help the localization of the RNA to an enzyme or receptor. Many NAD-utilizing metabolic enzymes can also bind to RNA, presenting the possibility of unknown ribonucleoprotein complexes.