title: From RNA and its NAD-cap: Exploring T4 phage infection from an epitranscriptomic perspective
Creators: name: Wolfram-Schauerte, Maik
Publisher: name: Philipps-Universität Marburg
Year: 2024
Subjects: bacteriophage
Chemistry &amp; allied sciences
Chemie
RNAylation
transcriptomics
proteomics
Epitranscriptomics
phage biology
T4 Phage
NAD-cap

description: Bacteriophages are viruses with the ability to specifically infect and kill bacteria. Bacteriophage research has shaped our understanding of fundamental biological principles and provided tools for molecular biology, but it has long been forgotten. Thus, phages are underestimated treasures of yet uncaptured biological potential and phenomena. Nowadays, while antimicrobial resistance is increasing, phage research is experiencing its renaissance, as phages present as a promising alternative to antibiotics to treat microbial infections with so-called phage therapy. Therefore, it is crucial to understand the molecular processes taking place in the intricate interplay of phages and their bacterial hosts. Bacteriophage T4 (T4 phage) represents a model phage that has been intensively studied in past decades to unravel the principles of phage infection. The T4 phage infects its prokaryotic host Escherichia coli and employs a myriad of strategies to hijack the host cell to efficiently produce phage progeny and kill the host cell. Yet, how the T4 phage accomplishes this highly efficient and fine-tuned gene expression program is not completely understood. Among others, many T4 phage genes are functionally uncharacterized, and most studies have so far focused on distinct gene expression events with targeted biochemical analyses. Consequently, comprehensive insights into the complex gene expression events and regulation during T4 phage infection are lacking.
One crucial layer of gene expression is the transcriptome, which encompasses RNA that is basically composed of four canonical nucleotides. However, RNA can be decorated by more than 160 chemical modifications referred to as the epitranscriptome. In E. coli, transcripts can possess a 5’-terminal modification with the ubiquitous redox cofactor nicotinamide adenine dinucleotide (NAD), serving as a cap-like structure (NAD-cap). The E. coli RNA polymerase caps RNA molecules with NAD during transcription, and the Nudix hydrolase NudC can decap NAD-capped RNA (NAD-RNA), destabilizing these transcripts. Despite the stabilizing effect of the NAD-cap on the RNA, it is yet unclear which function the NAD-cap serves. Further, the NAD-cap and RNA modifications in general have not been studied in the context of phage infection to this day.
Addressing these knowledge gaps, this thesis sets out to comprehensively study T4 phage infection of E. coli, focusing on the transcriptome and epitranscriptome to unveil novel regulatory mechanisms of phage infection. 
To realize this aim, Chapter 2 details a pioneering study that employs both time-resolved dual (phage and host) transcriptomics and dual-proteomics, offering an in-depth investigation into T4 phage infection processes. Here, these omics methods enable extensive and novel insights into phage and host gene expression on transcriptome and proteome levels during infection. Dual-transcriptomics reveals a yet unidentified set of four stable host transcripts in contrast to predominant host RNA degradation during infection and reproduces infection phase-specific T4 phage gene expression patterns on RNA level. The host proteome, on the other hand, is relatively stable, whilst T4 proteins are produced in a temporally controlled manner. Integration of the omics data reveals both temporal coupling and uncoupling of transcription and translation of specific T4 phage genes, pinpointing distinct post-transcriptional and translational regulation mechanisms. This time-resolved picture of T4 phage infection opens exciting perspectives for follow-up studies and demonstrates how state-of-the-art technologies can significantly boost our understanding of phage infections.
In Chapter 3, NAD-RNAs are identified and characterized during T4 phage infection, marking the first exploration of the dual epitranscriptome in phage biology. Both phage and host NAD-RNAs and their dynamic regulation during infection are discovered for the first time. While T4 phage NAD-RNAs dynamically appear according to their function in distinct infection phases, host NAD-RNAs mainly decrease in abundance. Moreover, their synthesis by the host RNA polymerase is demonstrated, and the first T4 phage-encoded NAD-RNA decapping enzyme, the Nudix hydrolase NudE.1, is identified and characterized. Catalytic inactivation of NudE.1 in the T4 phage decelerates infection, indicating its auxiliary role in boosting infection efficiency. Thereby, epitranscriptomics is showcased as an important research area in phage biology by comprehensively studying an RNA modification – the NAD-cap – during T4 phage infection.
Furthermore, a novel function for NAD-RNA is discovered in the context of T4 phage infection (Chapter 4). The T4 phage ADP-ribosyltransferase (ART) ModB usually accepts NAD as a substrate to ADP-ribosylate host proteins by post-translationally modifying them with ADP-ribose. Here, it is demonstrated that ModB also uses NAD-RNA as substrate, thereby covalently attaching entire RNA chains to host proteins, termed RNAylation. RNAylation by ModB is a post-translational modification of host proteins at specific arginine residues. ModB primarily RNAylates proteins of the host’s translational apparatus. Moreover, both phage and host transcripts serve as substrate RNAs for RNAylation during infection, as revealed by a specific capture and sequencing approach termed RNAylomeSeq. A T4 phage mutant of ModB displays reduced phage virulence, emphasizing the critical role of ModB for phage infection. Thus, this study presents a novel function for the NAD-cap – a post-translational protein modification creating RNA-protein conjugates, the RNAylation. It is a new way of protein-RNA interaction and presents a possible means to control critical processes during infection.
Moreover, this thesis focuses on concepts and prospects of RNA modifications in bacteria and bacteriophage-host interactions with a particular emphasis on NAD-RNAs. For one, established concepts of both internal and terminal mRNA modifications in bacteria are summarised, knowledge gaps regarding their existence, regulation and functions are highlighted, and perspectives towards their identification and characterization are provided (Chapter 5). Subsequently, the NAD-cap is highlighted as a particular RNA modification in all domains of life (Chapter 6). Therein, the identification of NAD-RNAs and the concepts of writing and erasing NAD-RNAs as well as their functions are illuminated. Challenges and future prospects – especially towards NAD-RNA functions – are discussed. Finally, the state-of-the-art in RNA modifications in the interaction of bacteriophages and their bacterial hosts is captured in Chapter 7, highlighting the rare focus of phage research on epitranscriptomics. On that basis, the putative roles of RNA modifications in phage infections are speculated.
In conclusion, this thesis provides extensive insights into bacteriophage infections from both a transcriptomic and epitranscriptomic perspective. Through this research, a fundamental and detailed temporal profile of gene expression events during T4 phage infection has been established. Based on this picture, this work defined the dynamic epitranscriptome of phage infection regarding the NAD-cap and unveiled a new function of NAD-RNAs – the RNAylation. These findings underscore the critical importance and vast potential of epitranscriptomic research in understanding phage infections, positioning this work at the forefront of an emerging interdisciplinary field.