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Identification and Analysis of Proteins Interacting with Nudix Hydrolase NudC Currently, there are more than 100 different RNA modifications identified, that modulate the structure of RNA molecules as well as its properties and functions. For a long time it was thought that only the eukaryotic RNAs can have a unique structure at their 5' end – a 7-methylguanosine cap that stabilizes the mRNA, promotes its transfer to the cytosol and translation (Nogalska ir Kiledjian, 2016). However, since 2009, a great number of articles have appeared showing that prokaryotic RNAs, besides triphospate modification, at 5' end can also be modified with a cap-like structure – nicotinamide adenine dinucleotide (NAD+). The major known function of this modification is the protection of RNA from degradation (Cahova et al., 2015). Escherichia coli Nudix hydrolase NudC is the only known protein in prokaryotes that hydrolyses 5‘-NAD-RNA to its monophosphate form (Zhang et al., 2016). In order to find out if this hydrolase works individually or together with other proteins, methods of protein purification under mild conditions, mass spectrometry as well as bacterial adenylate cyclase two-hybrid system were applied. Results confirmed that in vitro, in situ and in vivo NudC could form a complex with an additional protein just like other members of Nudix superfamily such as, Dcp2 and RppH, which hydrolyse the traditional 5' modifications (Hofer et al., 2016). It has also been noticed that interaction between two proteins does not depend on the catalytic activity of NudC, but rather on its C-terminal domain. To this day it is known that half of all molecules containing NAD+ modification are small non-coding RNAs, sRNAs (Cahova et al., 2015; Vvedenskaya et al., 2018). Having that in mind we were keen to see what kind of effect the complex has on known NAD+ capped sRNAs, DsrA, GcvB and McaS. Northern blot analysis showed that both proteins, NudC and is partner, could function in one complex and affect sRNA in two different ways, to stabilize or to destabilize it. During the bachelor's work, we have also created a method for 5'-NAD-RNA identification based on Nudix hydrolase NudC catalytic properties and RNA fractionation in an ABP reagent containing plyacrylamide gel. We hope that the created method will ensure successful further analysis of the selected known NAD+ containing sRNAs. |
