Native RNA nanopore sequencing reveals antibiotic-induced loss of rRNA modifications in the A- and P-sites

Abstract

The biological relevance and dynamics of mRNA modifications have been extensively studied in the past few years, revealing their key roles in major cellular processes, such as cellular differentiation or sex determination. However, whether rRNA modifications are dynamically regulated, and under which conditions, remains largely unclear. Here, we performed a systematic characterization of bacterial rRNA modification dynamics upon exposure to diverse antibiotics using native RNA nanopore sequencing. To identify significant rRNA modification changes, we developed NanoConsensus, a novel pipeline that integrates the estimates from multiple RNA modification detection algorithms, predicting differentially modified rRNA sites with very low false positive rates and high replicability. We showed that NanoConsensus is robust across RNA modification types, stoichiometries and coverage, and outperforms all individual algorithms tested. Using this approach, we identified multiple rRNA modifications that are lost upon the presence of antibiotics, showing that rRNA modification profiles are altered in an antibiotic-specific manner. We found that significantly altered rRNA modified sites upon antibiotic exposure are located in the vicinity of the A and P-sites of the ribosome, possibly contributing to antibiotic resistance. We then systematically examined whether loss of antibiotic-sensitive rRNA modifications may be sufficient to confer antibiotic resistance, finding that depletion of some rRNA modification enzymes guiding dysregulated rRNA modifications confers increased antibiotic resistance. Altogether, our work reveals that rRNA modification profiles can be rapidly altered in response to environmental exposures, and that nanopore sequencing can accurately identify dysregulated rRNA modifications, contributing to the mechanistic dissection of antibiotic resistance. Moreover, we provide a novel, robust workflow to study rRNA modification dynamics in any species using nanopore sequencing in a scalable and reproducible manner.