Modification at the 5'-end of RNA was thought to be a distinguishing feature of eukaryotes. Eukaryotic mRNA is known to possess a 7-methylguanosine cap (m7G) at the 5'-end, which protects the transcript from degradation and plays an essential role in its maturation, localization, and transcription [1]. However, it is now established that prokaryotes have a diverse 5'-end modification repertoire that comprises 30 different molecules. One of the most abundant 5'-RNA conjugates in Escherichia coli is nicotinamide adenine dinucleotide (NAD) [2]. The main known function of this non-canonical cap in prokaryotes is the protection of RNA from degradation [3], but only a few proteins have been found capable of regulating 5'-NAD capping.
Members of the Nudix superfamily, previously shown to have decapping activity on the canonical eukaryotic m7G cap, are known to be among the proteins in prokaryotes capable of removing the 5'-NAD-RNA cap. In Escherichia coli bacteria Nudix hydrolase NudC can hydrolyze 5'-NAD-RNA to 5'-P-RNA, initiating RNA degradation by RNase E [4]. However, as it is unclear how this protein interacts with RNA, our laboratory identified one of the potential partners cold-shock DEAD-box RNA helicase – DeaD. Although the molecular function of these proteins has been extensively studied, further research is necessary to fully comprehend their significance on bacterial physiology and the importance of this noncanonical cap.
In this study, we aimed to extend current knowledge about 5'-NAD-RNA hydrolysis-associated proteins by analyzing the impact of E. coli Nudix hydrolase NudC and its partner DeaD on bacterial growth (i), motility (ii), and the quantity of 5'-NAD-modified and unmodified RNA (iii). The effect of these protein on bacterial growth was examined by growing E. coli strains at optimal and cold shock temperature and it was found that helicase DeaD is crucial for bacteria growth at low temperature. For the first time, we demonstrated that hydrolase NudC can decrease bacterial swimming intensity at both optimal and low temperatures by carefully analysing the E. coli strain movement phenotype in a semi-liquid medium. Using Northern hybridization method, we successfully showed that both proteins and their complex affect not only the quantity of 5'-NAD cap-modified sRNA, but also the amount of total sRNA. Knowing that selective RNA degradation is one of the key factors in gene expression which affects rapid cell response to environmental change, deeper understanding of the specific mechanisms by which 5'-NAD-RNA capping contributes to gene expression would provide further insight into the regulatory networks underlying bacterial physiology and the broader implications of RNA modifications in cellular processes.