title: 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.
authors.affiliation: Department of Life Science and Medical Bioscience, Waseda University, 2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo, 162-8480, Japan.|Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, Ibaraki, 305-8566, Japan.
authors.forename: Tatsuki
authors.initials: T
authors.lastname: Miyamoto
authors.affiliation: Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, Ibaraki, 305-8566, Japan.
authors.forename: Yuka
authors.initials: Y
authors.lastname: Kato
authors.affiliation: Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, Ibaraki, 305-8566, Japan.
authors.forename: Yuji
authors.initials: Y
authors.lastname: Sekiguchi
authors.affiliation: Department of Life Science and Medical Bioscience, Waseda University, 2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo, 162-8480, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Tsuneda
authors.affiliation: Department of Life Science and Medical Bioscience, Waseda University, 2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo, 162-8480, Japan.|Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, Ibaraki, 305-8566, Japan.
authors.forename: Naohiro
authors.initials: N
authors.lastname: Noda
mesh_terms: D001483:Base Sequence; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004722:Endoribonucleases / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000378:metabolism; D005470:Fluorometry; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D009841:Oligonucleotides / Q000378:metabolism; D016958:Pseudomonas putida / Q000378:metabolism*; D059367:RNA Cleavage
chemical_list: D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; C422955:MazF protein, E coli; D009841:Oligonucleotides; D004722:Endoribonucleases