Characterization of MazF-Mediated Sequence-Specific RNA Cleavage in Pseudomonas putida Using Massive Parallel Sequencing

Abstract

Under environmental stress, microbes are known to alter their translation patterns using sequence-specific endoribonucleases that we call RNA interferases. However, there has been limited insight regarding which RNAs are specifically cleaved by these RNA interferases, hence their physiological functions remain unknown. In the current study, we developed a novel method to effectively identify cleavage specificities with massive parallel sequencing. This approach uses artificially designed RNAs composed of diverse sequences, which do not form extensive secondary structures, and it correctly identified the cleavage sequence of a well-characterized Escherichia coli RNA interferase, MazF, as ACA. In addition, we also determined that an uncharacterized MazF homologue isolated from Pseudomonas putida specifically recognizes the unique triplet, UAC. Using a real-time fluorescence resonance energy transfer assay, the UAC triplet was further proved to be essential for cleavage in P. putida MazF. These results highlight an effective method to determine cleavage specificity of RNA interferases.

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

“…To the best of our knowledge, this is the first MazF that specifically recognizes the U^ACA sequence (Masuda & Inouye, ; Schifano & Woychik, ). Recently, Zorzini et al., () determined the crystal structure of E. coli MazF (MazFec) in complex with the substrate analogue d(A 1 U 2 A 3 C 4 A 5 U 6 A 7 ), and reinforced the notion that MazFec recognizes ACA triplet strictly and that MazFec cleaves the substrate at the position of ^ACA and A^CA (Miyamoto et al., ; Vesper et al., ; Zhang, Zhang, Hara, Kato, & Inouye, a; Zhang et al., ). They mentioned that the MazFec recognition site could be divided into two different regions: first, the one where dU 2 is located, which is called the upstream binding site; and second, the one that accommodates d(A 3 C 4 A 5 U 6 ), which is called the downstream binding groove.…”

“…Phosphorylation, barcode ligation, and sequencing were performed as described previously (Miyamoto et al 2016a). The sequence data were analyzed with CLC Genomics 7.5.1., using the same parameters outlined in our previous work (Miyamoto et al 2016a).…”

“…Phosphorylation, barcode ligation, and sequencing were performed as described previously (Miyamoto et al 2016a). The sequence data were analyzed with CLC Genomics 7.5.1., using the same parameters outlined in our previous work (Miyamoto et al 2016a). Relative coverage increase, which is defined as the coverage at the (n + 1) th position divided by the coverage at the n th position, was calculated for all reference samples.…”

“…Although the functions of prokaryotic MazF toxins remain unclear, they are thought to have various biological roles such as in virulence (Rothenbacher et al., ; Tiwari et al., ; Zhu et al., ), phage defense systems (Alawneh, Qi, Yonesaki, & Otsuka, ; Hazan & Engelberg‐Kulka, ), persister generation (Tripathi, Dewan, Siddique, & Varadarajan, ), and programmed cell death (Nariya & Inouye, ); an important characteristic of MazF endoribonucleases that may contribute to these physiological functions is their sequence‐specificities. In most cases, MazF toxins require strict sequences for RNA cleavages, which are typically 3–7‐nucleotide (nt) motifs (Miyamoto, Kato, Sekiguchi, Tsuneda & Noda, ; Miyamoto, Yokota, Tsuneda, Noda et al. ; Nariya & Inouye, ; Rothenbacher et al., ; Schifano et al., ; Schuster et al., ; Verma & Bhatnagar, ; Yamaguchi & Inouye, ; Yamaguchi, Nariya, Park, & Inouye, ; Zhang et al., ; Zhu et al., , , ).…”

Identification of MazF Homologue in <b><i>Legionella pneumophila</i></b> Which Cleaves RNA at the AACU Sequence

“…A large number of MazF homologues have subsequently been identified from bacteria to archaea, with some bacteria having more than one MazF homologue. The MazF homologues so far identified specifically cleave mRNAs at either 3-, 5-, or 7-base recognition sequences [Zhang et al, 2003a;Zhu et al, 2006Zhu et al, , 2008Zhu et al, , 2009Nariya and Inouye, 2008;Park et al, 2011;Miyamoto et al, 2016]. It was indicated that the physiological role of these MazF homologues differs in each species, making the deciphering of cleavage sequence important to reveal the detailed mechanism of MazF.…”