Siân Owen (@sianowen.bsky.social)

Nanopore direct RNA sequencing (DRS) of MS2 bacteriophages in E.coli throughout its life cycle reveals a complex transcriptional activity to control and maintain its growth The RNA bacteriophage MS2 is a positive single stranded RNA phage that infects the bacterium E. coli and is one of the most crucial and representative model organisms in molecular biology and virology mimicking eukaryotic viruses. Previous research revealed translational control and feedback inhibitions in the MS2 life cycle. However, the dynamics of its transcriptional activity and replication during the life cycle within the bacteria is still relatively unknown. Here, we employed Nanopore Direct RNA sequencing (DRS) to investigate the transcriptome and epitranscriptome landscape of the MS2 in infected E. coli throughout its life cycle and detailed the dynamics of MS2 replication in real-time. We discovered that MS2 phages sustain a high level of transcriptional activity within the bacterial cell. We found large amounts of subgenomic small RNA transcripts containing the coat−encoding region. We found hybrid reads and mutations due to the error−prone activity of the MS2 replicase complex. We evidenced that RNA modification is conserved throughout the entire life cycle in full−length transcripts without the acquisition of new modifications whereas small transcripts did acquire and remove modified sites. The conserved sequence (5″−ΨU−3″) and secondary structure (stem-loops) of Ψ installation sites were the most amenable to RNA modification. Our investigation reveals a more complex transcriptional dynamics of MS2 phages within E. coli to maintain its growth and replicate under host pressure. ### Competing Interest Statement The authors have declared no competing interest. https://www.biorxiv.org/content/10.1101/2025.03.30.646209v1