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Identification of collateral RNA cleavage mediated by Type VI CRISPR-Cas system from Leptotrichia shahiii

ABSTRACT: We investigated the in vivo and in vitro specificity of collateral RNA degradation mediated by LshCas13a protein encoded by Type VI CRISPR-Cas system derived from L. shahii. In our experimental system, we used Escherichia coli strain harboring Type VI spacer matching inducible transcript ("targeting" cells) or strain that does not encode spacers matching any E. coli transcripts ("nontargeting" cells). After the induction of the target transcription, cells were collected, total RNA samples were extracted from the cells and subjected to high throughput RNA sequencing with a specific protocol allowing to capture RNA degradation products. To investigate the influence of RNase toxins encoded in E. coli genome on the observed RNA degradation pattern, we repeated the described experiment on E. coli strain lacking ten known ribonuclease-encoding toxin-antitoxin loci. To investigate in vitro specificity of LshCas13a protein, we analyzed products of the degradation on total E. coli RNA by purified LshCas13a supplemented with crRNA and targeted or nontargeted transcripts. The obtained samples were processed as it was described above. All experiments were independently performed in triplicate. To detect transcript degradation sites, the obtained read pairs were filtered using trimmomatic tool and then mapped onto reference sequences using bowtie2. Next, for each nucleotide position of each strand of reference sequences the number of 5’ ends of aligned fragments were counted producing corresponding tables. The differences between the numbers of mapped 5’ ends in targeting and nontargeting samples were analyzed using edgeR package. Using this data, we identified target preferences for collateral RNA degradation by LshCas13a effectors.

ORGANISM(S): Escherichia coli

PROVIDER: GSE183061 | GEO | 2022/08/31

REPOSITORIES: GEO

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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.

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Identification of collateral RNA cleavage mediated by Type VI CRISPR-Cas system from Leptotrichia shahiii

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