Introduction: 5´-NAD-RNA capping was described as the first transcriptional modification in bacteria, in which the NAD-cap provides transcript protection against endonuclease degradation, like RNAse E.
In E. coli, NudC was originally described as a Nudix hydrolase, hydrolyzing NAD into AMP and NMN. However, NudC is the main NAD-decapping enzyme, since it has a stronger affinity towards 5´-NAD-RNA, hydrolyzing it into 5´- P-RNA and NMN.
Although the NudC structure has been resolved, no RNAbinding motif has been identified, and the mechanisms for the NudC-RNA interaction remain elusive.
Intrigued by these observations we hypothesized that the NudC-RNA interaction promotes NudC catalytic activity, such interaction might further result in the association with the RNA degradosome complex to facilitate RNA turnover.
Objectives:
• Identify the residues of NudC required for RNA interaction.
• Elucidate NudC localization and analyze changes upon RNA depletion.
• Demonstrate the association of NudC with the RNA degradosome.
Material and Methods:
• To identify residues required for NudC-RNA interaction, Hydrogen-Deuterium exchange (HDX) assays were performed. Site-directed mutagenesis was performed on the identified residues, recombinant proteins were purified and used for in vitro NAD-decapping assays.
• To analyze subcellular localization, NudC was fused to the fluorescent protein mNeongreen, and single molecule tracking (SMT) assays were performed.
Results: HDX assays led to identifying two main residues of NudC, which showed a significatively reduced activity towards NAD-RNA when these residue were substituted. Furthermore, it was observed that NudC displays precise confinement towards the membrane and a static behavior, which was severely modified upon RNA depletion. Both phenotypes were lost when NudC no longer had the ability to interact with RNA, displaying cytosol localization and fast diffusive behavior.
Conclusion: Altogether, the presented data demonstrate the mechanisms by which NudC interacts with RNA. Such interaction promotes its decapping activity and results in NudC confinement to the membrane and association with the RNA degradosome.