BAR YAACOV LAB

Adenosine-to-inosine (A-to-I) mRNA editing changes the genetic information post-transcriptionally and was only recently reported to occur in bacteria. Here, we examined whether endonuclease V (EndoV; encoded by nfi) cleaves inosine-containing RNAs in vivo and thereby influences the abundance and fate of A-to-I-edited mRNAs in Escherichia coli. We generated an nfi loss-of-function mutant carrying a premature stop codon and performed RNA sequencing alongside the isogenic wild-type strain. We observed that global and site-specific editing occurrence or levels in both mRNAs and tRNAs were indistinguishable between strains. Moreover, overexpression of EndoV did not affect the number of edited sites, motif enrichment, or editing levels compared with a control strain overexpressing mCherry. Our findings suggest that, in contrast to human EndoV, bacterial EndoV does not regulate the steady-state pool of edited mRNAs in E. coli under nutrient-rich, exponential-growth conditions in vivo.IMPORTANCEAdenosine-to-inosine (A-to-I) mRNA editing is an emerging regulatory layer in bacteria, but the factors that act on edited transcripts are largely unknown. Endonuclease V (EndoV) was a prime candidate because it cleaves inosine-containing nucleic acids and can act on inosine-containing RNA in vitro. By combining loss-of-function and overexpression of EndoV with genome-wide RNA editing measurements, we show that EndoV does not measurably influence TadA-dependent A-to-I mRNA editing in Escherichia coli under standard laboratory conditions. This negative result is important because it rules out a natural effector candidate and redirects attention to other bacterial pathways that may process edited RNAs. Our work, therefore, sharpens mechanistic models for bacterial RNA editing and helps focus future searches for its regulators and physiological roles.