An RNA-seq method for defining endoribonuclease cleavage specificity identifies dual rRNA substrates for toxin MazF-mt3.
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.
Department of Biochemistry and Molecular Biology, Rutgers University, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854, USA.
Jason M
JM
Schifano
1] Waksman Institute, Rutgers University, Piscataway, New Jersey 08854, USA [2] Department of Genetics, Rutgers University, Piscataway, New Jersey 08854, USA.
Irina O
IO
Vvedenskaya
1] Waksman Institute, Rutgers University, Piscataway, New Jersey 08854, USA [2] Department of Genetics, Rutgers University, Piscataway, New Jersey 08854, USA.
Jared G
JG
Knoblauch
Department of Computer Science, University of Massachusetts Boston, Boston, Massachusetts 02125, USA.
Ming
M
Ouyang
1] Waksman Institute, Rutgers University, Piscataway, New Jersey 08854, USA [2] Department of Genetics, Rutgers University, Piscataway, New Jersey 08854, USA.
Bryce E
BE
Nickels
Department of Biochemistry and Molecular Biology, Rutgers University, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854, USA.
Nancy A
NA
Woychik
D001426:Bacterial Proteins / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D004722:Endoribonucleases / Q000378:metabolism*; D004926:Escherichia coli; D029968:Escherichia coli Proteins / Q000378:metabolism; D009169:Mycobacterium tuberculosis; D017433:Protein Structure, Secondary; D059367:RNA Cleavage; D012333:RNA, Messenger / Q000378:metabolism*; D012336:RNA, Ribosomal, 16S / Q000378:metabolism*; D012338:RNA, Ribosomal, 23S / Q000378:metabolism*; D012260:Ribonucleases / Q000378:metabolism; D017423:Sequence Analysis, RNA; D014118:Toxins, Biological
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C422955:MazF protein, E coli; D012333:RNA, Messenger; D012336:RNA, Ribosomal, 16S; D012338:RNA, Ribosomal, 23S; D014118:Toxins, Biological; D004722:Endoribonucleases; C000601629:MazF protein, Mycobacterium tuberculosis; D012260:Ribonucleases