Global analysis of the specificities and targets of endoribonucleases from E. coli toxin-antitoxin systems

Abstract

Toxin-antitoxin systems are widely distributed genetic modules typically featuring toxins that can inhibit bacterial growth and antitoxins that can reverse inhibition. Although Escherichia coli encodes 11 toxins with known or putative endoribonuclease activity, the target of most of these toxins remain poorly characterized. Using a new RNA-seq pipeline that enables the mapping and quantification of RNA cleavage with single-nucleotide resolution, we characterize the targets and specificities of 9 endoribonuclease toxins from E. coli. We find that these toxins use low-information cleavage motifs to cut a significant proportion of mRNAs in E. coli, but not tRNAs or the rRNAs from mature ribosomes. However, all the toxins, including those that are ribosome-dependent and cleave only translated RNA, inhibit ribosome biogenesis. This inhibition likely results from the cleavage of ribosomal protein transcripts, which disrupts the stoichiometry and biogenesis of new ribosomes and causes the accumulation of aberrant ribosome precursors. Collectively, our results provide a comprehensive, global analysis of endoribonuclease-based toxin-antitoxin systems in E. coli and support the conclusion that, despite their diversity, each disrupts translation and ribosome biogenesis. ImportanceToxin-antitoxin systems are widespread genetic modules found in almost all bacteria that can regulate their growth and may play prominent roles in phage defense. Escherichia coli encodes 11 TA systems in which the toxin is a known or predicted endoribonuclease. The targets and cleavage specificities of these endoribonucleases have remained largely uncharacerized, precluding an understanding of how each impacts cell growth and an assessment of whether they have distinct or overlapping targets. Using a new and broadly applicable RNA-seq pipeline, we present a global analysis of 9 endoribonulease toxins from E. coli. We find that each uses a relatively low-information cleavage motif to cut a large proportion of mRNAs in E. coli, but not tRNAs or mature rRNAs. Notably, although the precise set of targets varies, each toxin efficiently disrupts ribosome biogenesis, primarily by cleaving the mRNAs of ribosomal proteins. In sum, the analyses presented provide new, comprehensive insights into the cleavage specificities and targets of almost all endoribonuclease toxins in E. coli. Despite different specificities, our work reveals a striking commonality in function as each toxin disrupts ribosome biogenesis and translation.