Characterization of a Deinococcus radiodurans MazF: A UACA‐specific RNA endoribonuclease

Abstract

Microbes are known to withstand environmental stresses by using chromosomal toxin–antitoxin systems. MazEF is one of the most extensively studied toxin–antitoxin systems. In stressful environments, MazF toxins modulate translation by cleaving single‐stranded RNAs in a sequence‐specific fashion. Previously, a chromosomal gene located at DR0417 in Deinococcus radiodurans was predicted to code for a MazF endoribonuclease (MazFDR 0417); however, its function remains unclear. In the present study, we characterized the molecular function of MazFDR 0417. Analysis of MazFDR 0417‐cleaved RNA sites using modified massively parallel sequencing revealed a unique 4‐nt motif, UACA, as a potential cleavage pattern. The activity of MazFDR 0417 was also assessed in a real‐time fluorometric assay, which revealed that MazFDR 0417 strictly recognizes the unique tetrad UACA. This sequence specificity may allow D. radiodurans to alter its translation profile and survive under stressful conditions.

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

“…Subsets of toxins sharing similar target sequences are boxed with different colors. Protein identifiers and cleavage specificity were taken from: E. coli MazF (ChpAK) (NP_417262.1) (Culviner and Laub, 2018;Mets et al, 2019), ChpBK (NP_418646.1) (Zhang et al, 2005), Kid (PemK) (YP_003937673.1) (Munoz-Gomez et al, 2004;Zhang et al, 2004); Bacillus subtilis MazF bs (AOR96854.1) (Park et al, 2011); Bacillus anthracis MoxT (NP_842807.1) (Verma and Bhatnagar, 2014); Staphylococcus aureus MazF sa (BBJ19047.1) (Zhu et al, 2009); Staphylococcus equorum MazF seq (AFV93478.1) (Schuster et al, 2013); Clostridium difficile MazF cd (YP_001089981.1) (Rothenbacher et al, 2012); Legionella pneumophila MazF lp (CCD10720.1) (Shaku et al, 2018); Deinococcus radiodurans MazF DR0417 (AAF09995.1) (Miyamoto et al, 2017); Haloquadratum walsbyi MazF hw (WP_048066888.1) (Yamaguchi et al, 2012); Pseudomonas putida MazF pp (NP_742932.1) (Miyamoto et al, 2016a); Myxococcus xanthus MazF mx (SDX28280.1) (Nariya and Inouye, 2008); Methanohalobium evestigatum MazF me (WP_013195679.1); Nitrosomonas europaea MazF ne1 (WP_011111532.1) (Miyamoto et al, 2018), MazF NE1181 (CAD85092.1) (Miyamoto et al, 2016b); Mycobacterium tuberculosis MazF-mt1 (NP_217317.1) (Zhu et al, 2006), MazF-mt3 (NP_216507.1) (Zhu et al, 2008;Schifano et al, 2014), MazF-mt6 (NP_215618.1) (Schifano et al, 2013), MazF-mt7 (NP_216011.1) (Zhu et al, 2008), MazF-mt9 (YP_004837055.2) (Barth et al, 2019).…”

Nitrosomonas europaea MazF Specifically Recognises the UGG Motif and Promotes Selective RNA Degradation

“…The cleavage sequence was identified using the protocols described in our previous study ( Miyamoto et al, 2016a , 2017 ). Briefly, 0.625 pmol of eight RNA mixtures (500-2, 1000-1, 1000-2, 1000-3, 1000-4, 1000-5, 1500-1, and 2000-1) were incubated with 50 ng of MazFne1 at 37°C for 30 min in MazF reaction buffer.…”

“…Briefly, 0.625 pmol of eight RNA mixtures (500-2, 1000-1, 1000-2, 1000-3, 1000-4, 1000-5, 1500-1, and 2000-1) were incubated with 50 ng of MazFne1 at 37°C for 30 min in MazF reaction buffer. Phosphorylation, barcode ligation, and sequencing library construction were performed as described previously ( Miyamoto et al, 2016a , 2017 ). Sequencing was performed using the MiSeq platform with the MiSeq 500 cycles reagent kit v2 (Illumina, San Diego, CA, United States) according to the manufacturer’s protocol.…”

“…Sequence data were analysed using CLC Genomics 7.5.1 (Qiagen, Venlo, The Netherlands). The parameters described in our previous study ( Miyamoto et al, 2016a , 2017 ) were used for the analysis. We added pseudo-counts to the nucleotide positions whose coverages were 0, and calculated the relative coverage increase, the value defined as the coverage at the ( n + 1)th position divided by the coverage at the n th position.…”

“…MazF homologues are widely distributed in the prokaryotic domain ( Chopra et al, 2013 ), with a hallmark of these enzymes being their sequence-specificities ( Masuda and Inouye, 2017 ; Schifano and Woychik, 2017 ). Typically, MazF toxins recognise three to seven specific-sequences (i.e., ACA, UACAU, and UUACUCA), suggesting that the biological roles of MazF homologues are diverse and differ across microbial species ( Zhu et al, 2006 , 2008 , 2009 ; Park et al, 2011 ; Rothenbacher et al, 2012 ; Schifano et al, 2013 , 2014 ; Schuster et al, 2013 ; Verma and Bhatnagar, 2014 ; Miyamoto et al, 2016b , 2017 ; Li et al, 2017 ).…”