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Nucleic Acids Res. 2017 Mar 17;45(5):2329-2340. doi: 10.1093/nar/gkw1316.

Identification of endoribonuclease specific cleavage positions reveals novel targets of RNase III in Streptococcus pyogenes.

Author information

1
The Laboratory for Molecular Infection Sweden (MIMS), Umeå Centre for Microbial Research (UCMR), Department of Molecular Biology, Umeå University, S-90187 Umeå, Sweden.
2
Max Planck Institute for Infection Biology, Department of Regulation in Infection Biology, D-10117 Berlin, Germany.
3
Helmholtz Centre for Infection Research, Department of Regulation in Infection Biology, D-38124 Braunschweig, Germany.
4
Science for Life Laboratory, Department of Cell and Molecular Biology, Uppsala University, S-75123 Uppsala, Sweden.
5
Science for Life Laboratory, Department of Biochemistry and Biophysics, Stockholm University, Box 1031, SE-17121 Solna, Sweden.
6
Humboldt University, D-10115 Berlin, Germany.

Abstract

A better understanding of transcriptional and post-transcriptional regulation of gene expression in bacteria relies on studying their transcriptome. RNA sequencing methods are used not only to assess RNA abundance but also the exact boundaries of primary and processed transcripts. Here, we developed a method, called identification of specific cleavage position (ISCP), which enables the identification of direct endoribonuclease targets in vivo by comparing the 5΄ and 3΄ ends of processed transcripts between wild type and RNase deficient strains. To demonstrate the ISCP method, we used as a model the double-stranded specific RNase III in the human pathogen Streptococcus pyogenes. We mapped 92 specific cleavage positions (SCPs) among which, 48 were previously described and 44 are new, with the characteristic 2 nucleotides 3΄ overhang of RNase III. Most SCPs were located in untranslated regions of RNAs. We screened for RNase III targets using transcriptomic differential expression analysis (DEA) and compared those with the RNase III targets identified using the ISCP method. Our study shows that in S. pyogenes, under standard growth conditions, RNase III has a limited impact both on antisense transcripts and on global gene expression with the expression of most of the affected genes being downregulated in an RNase III deletion mutant.

PMID:
28082390
PMCID:
PMC5389636
DOI:
10.1093/nar/gkw1316
[Indexed for MEDLINE]
Free PMC Article

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