Characterization of mazF-mediated sequence-specific RNA cleavage in pseudomonas putida using massive parallel sequencing

Tatsuki Miyamoto, Yuka Kato, Yuji Sekiguchi, Satoshi Tsuneda, Naohiro Noda

    Research output: Contribution to journalArticle

    5 Citations (Scopus)

    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.

    Original languageEnglish
    Article numbere0149494
    JournalPLoS One
    Volume11
    Issue number2
    DOIs
    Publication statusPublished - 2016 Feb 1

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    RNA Cleavage
    Pseudomonas putida
    RNA
    Endoribonucleases
    endoribonucleases
    Fluorescence Resonance Energy Transfer
    energy transfer
    Escherichia coli
    translation (genetics)
    Assays
    fluorescence
    microorganisms
    assays
    methodology

    ASJC Scopus subject areas

    • Agricultural and Biological Sciences(all)
    • Biochemistry, Genetics and Molecular Biology(all)
    • Medicine(all)

    Cite this

    Characterization of mazF-mediated sequence-specific RNA cleavage in pseudomonas putida using massive parallel sequencing. / Miyamoto, Tatsuki; Kato, Yuka; Sekiguchi, Yuji; Tsuneda, Satoshi; Noda, Naohiro.

    In: PLoS One, Vol. 11, No. 2, e0149494, 01.02.2016.

    Research output: Contribution to journalArticle

    Miyamoto, Tatsuki ; Kato, Yuka ; Sekiguchi, Yuji ; Tsuneda, Satoshi ; Noda, Naohiro. / Characterization of mazF-mediated sequence-specific RNA cleavage in pseudomonas putida using massive parallel sequencing. In: PLoS One. 2016 ; Vol. 11, No. 2.
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