A quantitative shRNA screen identifies ATP1A1 as a gene that regulates cytotoxicity by aurilide B

Shohei Takase, Rumi Kurokawa, Daisuke Arai, Kind Kanemoto Kanto, Tatsufumi Okino, Yoichi Nakao, Tetsuo Kushiro, Minoru Yoshida, Ken Matsumoto

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Abstract

Genome-wide RNA interference (RNAi) with pooled and barcoded short-hairpin RNA (shRNA) libraries provides a powerful tool for identifying cellular components that are relevant to the modes/mechanisms of action (MoA) of bioactive compounds. shRNAs that affect cellular sensitivity to a given compound can be identified by deep sequencing of shRNA-specific barcodes. We used multiplex barcode sequencing technology by adding sample-specific index tags to PCR primers during sequence library preparation, enabling parallel analysis of multiple samples. An shRNA library screen with this system revealed that downregulation of ATP1A1, an α-subunit of Na+/K+ ATPase, conferred significant sensitivity to aurilide B, a natural marine product that induces mitochondria-mediated apoptosis. Combined treatment with ouabain which inhibits Na+/K+ ATPase by targeting α-subunits potentiated sensitivity to aurilide B, suggesting that ATP1A1 regulates mitochondria-mediated apoptosis. Our results indicate that multiplex sequencing facilitates the use of pooled shRNA library screening for the identification of combination drug therapy targets.

Original languageEnglish
Article number2002
JournalScientific reports
Volume7
Issue number1
DOIs
Publication statusPublished - 2017 Dec 1

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Cite this

Takase, S., Kurokawa, R., Arai, D., Kanto, K. K., Okino, T., Nakao, Y., Kushiro, T., Yoshida, M., & Matsumoto, K. (2017). A quantitative shRNA screen identifies ATP1A1 as a gene that regulates cytotoxicity by aurilide B. Scientific reports, 7(1), [2002]. https://doi.org/10.1038/s41598-017-02016-4