Dataset Information

Toxins MazF and MqsR cleave Escherichia coli rRNA precursors at multiple sites.

ABSTRACT: The endoribonuclease toxins of the E. coli toxin-antitoxin systems arrest bacterial growth and protein synthesis by targeting cellular mRNAs. As an exception, E. coli MazF was reported to cleave also 16S rRNA at a single site and separate an anti-Shine-Dalgarno sequence-containing RNA fragment from the ribosome. We noticed extensive rRNA fragmentation in response to induction of the toxins MazF and MqsR, which suggested that these toxins can cleave rRNA at multiple sites. We adapted differential RNA-sequencing to map the toxin-cleaved 5'- and 3'-ends. Our results show that the MazF and MqsR cleavage sites are located within structured rRNA regions and, therefore, are not accessible in assembled ribosomes. Most of the rRNA fragments are located in the aberrant ribosomal subunits that accumulate in response to toxin induction and contain unprocessed rRNA precursors. We did not detect MazF- or MqsR-cleaved rRNA in stationary phase bacteria and in assembled ribosomes. Thus, we conclude that MazF and MqsR cleave rRNA precursors before the ribosomes are assembled and potentially facilitate the decay of surplus rRNA transcripts during stress.

SUBMITTER: Mets T

PROVIDER: S-EPMC5270532 | biostudies-literature | 2017 Jan

REPOSITORIES: biostudies-literature

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Toxins MazF and MqsR cleave Escherichia coli rRNA precursors at multiple sites.

Mets Toomas T Lippus Markus M Schryer David D Liiv Aivar A Kasari Villu V Paier Anton A Maiväli Ülo Ü Remme Jaanus J Tenson Tanel T Kaldalu Niilo N

RNA biology 20161118 1

The endoribonuclease toxins of the E. coli toxin-antitoxin systems arrest bacterial growth and protein synthesis by targeting cellular mRNAs. As an exception, E. coli MazF was reported to cleave also 16S rRNA at a single site and separate an anti-Shine-Dalgarno sequence-containing RNA fragment from the ribosome. We noticed extensive rRNA fragmentation in response to induction of the toxins MazF and MqsR, which suggested that these toxins can cleave rRNA at multiple sites. We adapted differential ...[more]

PMID: 27858580

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Project description:Bacterial toxin-antitoxin systems help bacteria to reduce their metabolism in various stressful conditions and also play a part in generating antibiotic tolerant bacterial subpopulation called persisters. Many toxins of the bacterial toxin-antitoxin systems are ribonucleases. Such toxins have been mostly viewed as degraders of mRNA, however recently it was demonstrated that they are also capable of cleaving non-coding RNA. Current libraries are a part of a project which aims to identify the major RNA cleavage sites (in mRNA, rRNA and regulatory non-coding RNA) of MazF and MqsR toxins in E. coli. We were also interested in the activity of promoters during toxin overexpression. We used a targeted RNA-sequencing approach that allowed us to map the distinct 5- and 3-ends of toxin-cleaved RNA and also the beginnings of transcripts. We extracted total RNA from cultures where the expression of MazF or MqsR was induced; RNA from log phase culture was used as the control. In addition, RNA extracted from stationary phase culture was used to test for the possible toxin cleavage in natural stress conditions. The cleaved RNA is rapidly recycled making it possible that we miss important cleavage sites due to RNA degradation during the prolonged stationary phase. Therefore, in addition to wt E. coli we also used stationary phase RNA from exoribonuclease deficient strain where RNA cleavage products accumulate. Identification of the 5 ends is based on ligation of RNA adapters to the cellular RNA molecules, which requires 5 -monophosphates. Mapping of the 3 -ends is based on poly(A) tailing of RNA, which requires 3 -OH groups. Transcription initiation, ordinary cellular RNases, and toxin endonucleases all produce different types of RNA ends. These can be enzymatically converted to 5 -phosphates and 3 -OHs, which allows one to separately quantify the RNA ends produced by each of these processes from a single biological sample. Briefly, (i) primary transcripts have 5 -triphosphate ends that can be converted to monophosphate by Tobacco Acid Pyrophosphatase (TAP), and 3 -hydroxyl ends that can be directly polyadenylated by poly(A) polymerase. (ii) Most cellular RNases (excluding RNase I and the toxins) produce 5 -monophosphates and 3 -hydroxyls, which are directly usable for ligation/polyadenylation. (iii) RNase I, MazF, and several other toxins produce 5 -hydroxyl ends that need to be phosphorylated by T4 Polynucleotide Kinase (PNK) prior to ligation, and 2,3 -cyclic phosphates that need to be dephosphorylated and converted to 3 -hydroxyls, also by T4 PNK. In short, for each of our RNA samples three 5 end and two 3 end libraries were made. The PNK treated libraries contain reads mapping specifically to 5- or 3-ends left by toxin (or RNaseI) cleavage and TAP treated libraries have extra reads mapping to beginnings of the transcripts.

Dataset Information

Toxins MazF and MqsR cleave Escherichia coli rRNA precursors at multiple sites.

Publications

Toxins MazF and MqsR cleave Escherichia coli rRNA precursors at multiple sites.

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