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.
Highlights
Target identification of bioactive compounds is a long-standing challenge for drug development, as well as for chemical biology studies aimed at elucidating cellular functions using compounds
The results revealed ATP1A1, which encodes a catalytic α-subunit of Na+/K+ ATPase, as a gene involved in survival of cells treated with aurilide B
We used the Decipher lentiviral and pooled short-hairpin RNA (shRNA) libraries to identify genes involved in mechanisms of action (MoA) or synthetic-lethal pathways of a compound of interest
Summary
Multiplex barcode sequencing following shRNA library screens. We used the Decipher lentiviral and pooled shRNA libraries to identify genes involved in MoA or synthetic-lethal pathways of a compound of interest. The barcode read counts of DNA spike-ins were increased in a dose-dependent manner, indicating that our method works quantitatively (Fig. 1C) Taken together, these results illustrate the suitability of our multiplex barcode sequencing for analysis of shRNA library screens. Combined treatment with ouabain did not sensitize HeLa S3 cells to etoposide or actinomycin D (Fig. 4H and I) These results clearly demonstrate that knockdown or inhibition of ATP1A1 sensitized cells to aurilide B. It is not likely that ATP1A1 plays a functional role apart from Na+/K+ ATPase complex, but given that more ATP1A1 molecules than β-subunits (ATP1B1 and ATP1B3) are present at the plasma membrane in HeLa cells[20], it is not outside the realm of possibility that ATP1A1 has specific functions that are inhibited by ouabain. The use of indexed PCR primers when amplifying barcodes, or shRNA or guide RNA inserts, will facilitate unbiased functional genomics
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