Moritz Weber (Marburg / DE), Dorothee Staiger (Bielefeld / DE), Katharina Höfer (Marburg / DE)
Introduction:
ADP-ribosyltransferases (ARTs) are found in all domains of life. A common function of ARTs is acting as a toxin in pathogen-host interactions. ARTs use NAD+ (NAD) as a substrate to attach ADP-ribose to their target proteins – a post-translational protein modification called ADP-ribosylation. Interestingly, NAD has also been discovered to be attached to specific transcripts at the 5´-terminus, forming "NAD-capped RNAs".
Recently, an ART of the T4 phage was shown to be used in addition to NAD and NAD-capped RNA as a substrate, resulting in a covalent connection between RNA and proteins in a reaction called RNAylation. This discovery connects the realms of protein- and RNA-modification. However, the biological role of RNAylation is still not fully understood. As RNAylation is a newly discovered reaction, it only describes the interaction of T4 phages and E. coli, where it was first discovered. However, the ubiquity of both ARTs and NAD-capped RNA raises the question of whether RNAylation occurs in other organisms or interactions.
Objectives:
This project addresses the question of whether RNAylation is a niche phenomenon or a widespread biological concept. We aim to screen for ARTs in various organisms or pathogen-host interactions beyond T4 phage-infected E. coli that can catalyse an RNAylation reaction. The focus is on bacterial ARTs, which modify host proteins. The ability of these enzymes to perform RNAylation in vitro and in vivo is investigated. Potentially occurring RNAylations are analysed for their target proteins, site selectivity, and their competition with ADP-ribosylations.
Materials and Methods:
To tackle these questions, we employ a combination of biochemical methods, enzyme assays using purified proteins and/or host lysates and proteomics.
Results:
We unveil the ability of a bacterial ART to not only ADP-ribosylate, but also RNAylate target-proteins in vitro. This discovery proves that more than one ART possesses RNAylation activity. Future studies will focus on the ability of this ART to perform RNAylations in host lysates to evaluate its biological significance.
References:
Wolfram-Schauerte et al., Nature 620.7976 (2023): 1054-1062.