An RNA-seq method for defining endoribonuclease cleavage specificity identifies dual rRNA substrates for toxin MazF-mt3

Jason M. Schifano; Irina O. Vvedenskaya; Jared G. Knoblauch; Ming Ouyang; Bryce E. Nickels; Nancy A. Woychik

doi:10.1038/ncomms4538

An RNA-seq method for defining endoribonuclease cleavage specificity identifies dual rRNA substrates for toxin MazF-mt3

Jason M. Schifano, Irina O. Vvedenskaya, Jared G. Knoblauch, Ming Ouyang, Bryce E. Nickels, Nancy A. Woychik

Research output: Contribution to journal › Article › peer-review

75 Scopus citations

Abstract

Toxin-antitoxin (TA) systems are widespread in prokaryotes. Among these, the mazEF TA system encodes an endoribonucleolytic toxin, MazF, that inhibits growth by sequence-specific cleavage of single-stranded RNA. Defining the physiological targets of a MazF toxin first requires the identification of its cleavage specificity, yet the current toolkit is antiquated and limited. We describe a rapid genome-scale approach, MORE (mapping by overexpression of an RNase in Escherichia coli) RNA-seq, for defining the cleavage specificity of endoribonucleolytic toxins. Application of MORE RNA-seq to MazF-mt3 from Mycobacterium tuberculosis reveals two critical ribosomal targets-the essential, evolutionarily conserved helix/loop 70 of 23S rRNA and the anti-Shine-Dalgarno (aSD) sequence of 16S rRNA. Our findings support an emerging model where both ribosomal and messenger RNAs are principal targets of MazF toxins and suggest that, as in E. coli, removal of the aSD sequence by a MazF toxin modifies ribosomes to selectively translate leaderless mRNAs in M. tuberculosis.

Original language	English (US)
Article number	3538
Pages (from-to)	3538
Number of pages	1
Journal	Nature communications
Volume	5
DOIs	https://doi.org/10.1038/ncomms4538
State	Published - 2014

All Science Journal Classification (ASJC) codes

Chemistry(all)
Biochemistry, Genetics and Molecular Biology(all)
Physics and Astronomy(all)

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10.1038/ncomms4538

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@article{90bc0f43edd64001ae7fc529a39738f1,

title = "An RNA-seq method for defining endoribonuclease cleavage specificity identifies dual rRNA substrates for toxin MazF-mt3",

abstract = "Toxin-antitoxin (TA) systems are widespread in prokaryotes. Among these, the mazEF TA system encodes an endoribonucleolytic toxin, MazF, that inhibits growth by sequence-specific cleavage of single-stranded RNA. Defining the physiological targets of a MazF toxin first requires the identification of its cleavage specificity, yet the current toolkit is antiquated and limited. We describe a rapid genome-scale approach, MORE (mapping by overexpression of an RNase in Escherichia coli) RNA-seq, for defining the cleavage specificity of endoribonucleolytic toxins. Application of MORE RNA-seq to MazF-mt3 from Mycobacterium tuberculosis reveals two critical ribosomal targets-the essential, evolutionarily conserved helix/loop 70 of 23S rRNA and the anti-Shine-Dalgarno (aSD) sequence of 16S rRNA. Our findings support an emerging model where both ribosomal and messenger RNAs are principal targets of MazF toxins and suggest that, as in E. coli, removal of the aSD sequence by a MazF toxin modifies ribosomes to selectively translate leaderless mRNAs in M. tuberculosis. ",

author = "Schifano, {Jason M.} and Vvedenskaya, {Irina O.} and Knoblauch, {Jared G.} and Ming Ouyang and Nickels, {Bryce E.} and Woychik, {Nancy A.}",

note = "Funding Information: We thank Ling Zhu for providing the pET-28a-mazF-mt3, pBAD33-mazF-mt3 and pET-28a-mazE-mt3 plasmids; Jared D. Sharp and Robert N. Husson for providing M. tuberculosis total RNA; Seth R. Goldman for suggesting the term MORE RNA-seq; and Ann Hochschild for comments on the manuscript. This work was supported in part by National Institutes of Health (NIH) grant R21 AI072399 and R01 GM095693 (to N.A.W.), NIH grant R01 GM088343 (to B.E.N.) and NIH training grant T32 AI007403, Virus–Host Interactions in Eukaryotic Cells (to J.M.S., awarded to G. Brewer).",

year = "2014",

doi = "10.1038/ncomms4538",

language = "English (US)",

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pages = "3538",

journal = "Nature communications",

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T1 - An RNA-seq method for defining endoribonuclease cleavage specificity identifies dual rRNA substrates for toxin MazF-mt3

AU - Schifano, Jason M.

AU - Vvedenskaya, Irina O.

AU - Knoblauch, Jared G.

AU - Ouyang, Ming

AU - Nickels, Bryce E.

AU - Woychik, Nancy A.

N1 - Funding Information: We thank Ling Zhu for providing the pET-28a-mazF-mt3, pBAD33-mazF-mt3 and pET-28a-mazE-mt3 plasmids; Jared D. Sharp and Robert N. Husson for providing M. tuberculosis total RNA; Seth R. Goldman for suggesting the term MORE RNA-seq; and Ann Hochschild for comments on the manuscript. This work was supported in part by National Institutes of Health (NIH) grant R21 AI072399 and R01 GM095693 (to N.A.W.), NIH grant R01 GM088343 (to B.E.N.) and NIH training grant T32 AI007403, Virus–Host Interactions in Eukaryotic Cells (to J.M.S., awarded to G. Brewer).

PY - 2014

Y1 - 2014

N2 - Toxin-antitoxin (TA) systems are widespread in prokaryotes. Among these, the mazEF TA system encodes an endoribonucleolytic toxin, MazF, that inhibits growth by sequence-specific cleavage of single-stranded RNA. Defining the physiological targets of a MazF toxin first requires the identification of its cleavage specificity, yet the current toolkit is antiquated and limited. We describe a rapid genome-scale approach, MORE (mapping by overexpression of an RNase in Escherichia coli) RNA-seq, for defining the cleavage specificity of endoribonucleolytic toxins. Application of MORE RNA-seq to MazF-mt3 from Mycobacterium tuberculosis reveals two critical ribosomal targets-the essential, evolutionarily conserved helix/loop 70 of 23S rRNA and the anti-Shine-Dalgarno (aSD) sequence of 16S rRNA. Our findings support an emerging model where both ribosomal and messenger RNAs are principal targets of MazF toxins and suggest that, as in E. coli, removal of the aSD sequence by a MazF toxin modifies ribosomes to selectively translate leaderless mRNAs in M. tuberculosis.

AB - Toxin-antitoxin (TA) systems are widespread in prokaryotes. Among these, the mazEF TA system encodes an endoribonucleolytic toxin, MazF, that inhibits growth by sequence-specific cleavage of single-stranded RNA. Defining the physiological targets of a MazF toxin first requires the identification of its cleavage specificity, yet the current toolkit is antiquated and limited. We describe a rapid genome-scale approach, MORE (mapping by overexpression of an RNase in Escherichia coli) RNA-seq, for defining the cleavage specificity of endoribonucleolytic toxins. Application of MORE RNA-seq to MazF-mt3 from Mycobacterium tuberculosis reveals two critical ribosomal targets-the essential, evolutionarily conserved helix/loop 70 of 23S rRNA and the anti-Shine-Dalgarno (aSD) sequence of 16S rRNA. Our findings support an emerging model where both ribosomal and messenger RNAs are principal targets of MazF toxins and suggest that, as in E. coli, removal of the aSD sequence by a MazF toxin modifies ribosomes to selectively translate leaderless mRNAs in M. tuberculosis.

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ER -

An RNA-seq method for defining endoribonuclease cleavage specificity identifies dual rRNA substrates for toxin MazF-mt3

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