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.

The Variety in the Common Theme of Translation Inhibition by Type II Toxin–Antitoxin Systems

“…However, these particular 16S rRNA bases are paired in the 30S subunits, making the MazF-dependent cleavage very unlikely. It was later shown that indeed, MazF cleaves the rRNAs in their precursor states, before the 30S subunit biogenesis, and at multiple sites (Mets et al, 2017;Culviner and Laub, 2018). This is in agreement with structural data showing that MazF binds to single-stranded RNA (ssRNA) and interacts with the bases (Simanshu et al, 2013;Zorzini et al, 2016).…”

“…Most of the MazF toxins prefer U upstream of cleavage (at position −1) and AC downstream of cleavage (at positions +1 and +2, respectively) with less stringency outside of the two to four main recognized bases (Figure 2). The E. coli MazF cleaves in the coding as well as untranslated regions of mRNAs, independent from the reading frame, and in rRNA precursors (Mets et al, 2017;Culviner and Laub, 2018;Mets et al, 2019). Slight changes in sequence specificity of different MazF enzymes from different bacteria, correspond well to their amino acid sequence similarity (Figure 2).…”

“…Structural studies have shown that a MazF dimer binds to one mRNA molecule and covers at least seven bases (Simanshu et al, 2013;Zorzini et al, 2016), explaining the extent of possible recognition and necessity of an unstructured RNA substrate, i.e., solvent exposed bases. MazF cleavage leaves 2 , 3 -cyclic phosphate at the 3 -end and 5 -OH group at the 5 -end of the cleavage site, which are used as a signature to study the cleavage products generated by the in vivo overexpression of MazF toxins (Schifano et al, 2014;Mets et al, 2017).…”

Global Analysis of the E. coli Toxin MazF Reveals Widespread Cleavage of mRNA and the Inhibition of rRNA Maturation and Ribosome Biogenesis

“…Our results indicate that, although MazF does not significantly impact mature rRNA, it does have a major effect on rRNA precursors. One recent study of E. coli MazF had suggested that it may cleave precursor rRNAs (Mets et al, 2017) based on 5 0 -OH mapping that produced a peak in the 16S precursor and several peaks within structured regions of 16S and 23S rRNA. However, these experiments were conducted after MazF was expressed for 2 hr using the 5 0 -OH method, so whether these peaks indicate common products or rare but stable products is unclear.…”

“…Promoting translation through MazF cleavage of leader regions was proposed to be widespread in E. coli, with the MazF regulon suggested to include >300 transcripts (Sauert et al, 2016). Work by a different group also found that MazF triggered production of a 43-nt fragment corresponding to the 3 0 end of 16S rRNA but then provided evidence MazF could be cleaving rRNA precursors as well (Mets et al, 2017). However, that study did not formally separate or quantify MazF's effects on rRNA precursors and mature rRNA.…”