rRNA loss induced by rifampicin addition in Escherichia coli reflects extraction artifacts rather than in vivo degradation.

When measuring RNA stability in Escherichia coli, RNA synthesis is usually stopped using rifampicin. Although rRNAs are considered stable in vivo, Hamouche et al. Ribosomal RNA degradation induced by the bacterial RNA polymerase inhibitor rifampicin. RNA 27, 946-958 (2021) recently presented experimental evidence that rifampicin treatment leads to a 50% decrease in total RNA levels due to degradation of 23S and 16S rRNA. Since most mRNA decay in E. coli is initiated by RNase E, which also contributes to the degradation of 23S and 16S rRNA, this suggests that mRNA and rRNA may compete for RNase E in vivo, potentially affecting cell physiology. Here, we show that in exponential growing cells, the decrease in 23S and 16S rRNA levels is an artefact of the RNA extraction protocol. We measured rRNA levels after rifampicin addition using four different protocols. Whereas the gentle lysis employed by Hamouche et al. did indeed lead to loss of rRNA, rRNA levels were stable under stronger lysis. We also measured mRNA levels after rifampicin addition with the different protocols. Our results indicate that 23S, 16S and 5S rRNAs are not degraded during the mRNA degradation period in rifampicin-treated cells. In exponential growing cells therefore, rRNAs are stable in vivo and do not compete with other RNAs for degradation by RNase E. Whether rRNA is degraded and competes with RNA for RNase E under other physiological conditions, particularly stress, remains an open question.