Abstract
The mechanisms by which viruses hijack their host’s genetic machinery are of current interest. When bacteriophage T4 infects Escherichia coli, three different ARTs (ADP-ribosyltransferases) reprogram the host’s transcriptional and translational apparatus through ADP-ribosylation using nicotinamide adenine dinucleotide (NAD) as substrate 1,2. Recently, NAD was identified as a 5’-modification of cellular RNAs 3–5. Here, we report that T4 ART ModB accepts not only NAD but also NAD-capped RNA (NAD-RNA) as substrate and attaches entire RNA chains to acceptor proteins in an “RNAylation” reaction. ModB specifically RNAylates ribosomal proteins rS1 and rL2 at defined arginine residues, and a specific group of E. coli and T4 phage RNAs is linked to rS1 in vivo. T4 phages that express an inactive mutant of ModB show a decreased burst size and slowed lysis of E. coli. Our findings reveal a distinct biological role of NAD-RNA, namely activation of the RNA for enzymatic transfer to proteins. The attachment of specific RNAs to ribosomal proteins might provide a strategy for the phage to modulate the host’s translation machinery. This work exemplifies the first direct connection between RNA modification and post-translational protein modification. As ARTs play important roles far beyond viral infections 6, RNAylation may have far-reaching implications.
Competing Interest Statement
The authors declare the following competing interests: K.H. and A.J. filed a PCT application (PCT/EP2021/071295). The remaining authors declare no competing interests.
Footnotes
1. Using CRISPR-Cas-based technology, we generated a T4 phage mutant that expresses the inactive ModB R73A, G74A, allowing us to investigate the functional role of ModB during phage infection. We compared the phenotypes of T4 WT and T4 ModB R73A, G74A and observed a reduced burst size as well as a delayed lysis time. Thus, ModB inactivation significantly affects phage propagation properties. 2. We developed an RNAylomeSeq technology that enabled the identification of the RNAs that are covalently linked to rS1 in vivo. We identified specific E. coli and T4 phage RNAs. Importantly, several of the E. coli transcripts (mRNAs and sRNAs) have been previously reported to be 5'-NAD-capped in E. coli, consistent with their use as substrates by ModB. 3. We improved our LC-MS/MS analytics to detect a trinucleotide (ADP-ribose + cytidine) which clearly marks RNAylation to have occurred and which enables us to differentiate between RNAylation and ADP-ribosylation. 4. We discovered that ModB is a target-specific ART that RNAylates proteins of the translational apparatus. We identified ribosomal proteins rS1 and rL2 to be RNAylated at specific arginine residues in their RNA-binding regions.