Non-canonical open reading frames encode functional proteins essential for cancer cell survival

Although genomic analyses predict many non-canonical open reading frames (ORFs) in the human genome, it is unclear whether they encode biologically active proteins. Here, we experimentally interrogated 553 candidates selected from non-canonical ORF datasets. Of these, 57 induced viability defects wh...

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Published inNature biotechnology Vol. 39; no. 6; pp. 697 - 704
Main Authors Prensner, John R., Enache, Oana M., Luria, Victor, Krug, Karsten, Clauser, Karl R., Dempster, Joshua M., Karger, Amir, Wang, Li, Stumbraite, Karolina, Wang, Vickie M., Botta, Ginevra, Lyons, Nicholas J., Goodale, Amy, Kalani, Zohra, Fritchman, Briana, Brown, Adam, Alan, Douglas, Green, Thomas, Yang, Xiaoping, Jaffe, Jacob D., Roth, Jennifer A., Piccioni, Federica, Kirschner, Marc W., Ji, Zhe, Root, David E., Golub, Todd R.
Format Journal Article
LanguageEnglish
Published 28.01.2021
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Summary:Although genomic analyses predict many non-canonical open reading frames (ORFs) in the human genome, it is unclear whether they encode biologically active proteins. Here, we experimentally interrogated 553 candidates selected from non-canonical ORF datasets. Of these, 57 induced viability defects when knocked out in human cancer cell lines. Upon ectopic expression, 257 showed evidence of protein expression and 401 induced gene expression changes. CRISPR tiling and start codon mutagenesis indicated that their biological effects required translation as opposed to RNA-mediated effects. We found that one of these ORFs, G029442 — renamed GREP1 (Glycine-Rich Extracellular Protein-1) — encodes a secreted protein highly expressed in breast cancer, and its knock-out in 263 cancer cell lines showed preferential essentiality in breast cancer–derived lines. The secretome of GREP1-expressing cells has an increased abundance of the oncogenic cytokine GDF15, and GDF15 supplementation mitigated the growth inhibitory effect of GREP1 knock-out. Our experiments suggest that non-canonical ORFs can express biologically active proteins that are potential therapeutic targets.
Bibliography:Author Contributions
J.R.P. and T.R.G. conceived the project, designed experimental approaches, supervised the study, and analyzed data. J.R.P. selected ORFs for screening and developed ORF prioritization methods. J.R.P. and X.Y. designed and generated the ORF cDNA library. J.R.P performed ORF library screening, in vitro CRISPR experiments, siRNA experiments, western blots, cell culture assays, and all GREP1 functional experiments. B.F. executed arrayed ORF screen for L1000. O.M.E. and N.J.L. performed gene expression profiling and analyzed L1000 gene expression data. Z.J. contributed ORF predictions and assisted in analysis of ORF candidates. V.L., A.K., M.K. and J.R.P. performed protein evolutionary analyses and analyzed phylostratigraphy data. K.K., K.R.C., and J.D.J. performed proteomic identification of ORFs from datasets. J.R.P., F.P., and D.E.R. designed and analyzed CRISPR screens. T.G., D.A., and A.B. assisted with sgRNA design. A.G. and Z.K. performed cell line CRISPR screens. L.W., K.S., G.B. and J.A.R. performed pooled CRISPR screening. V.M.W. and J.M.D. analyzed pooled CRISPR screen data. J.M.D. performed comparative analyses of ORF CRISPR data with publicly available CRISPR screens. J.R.P. and T.R.G. wrote the manuscript draft and all authors contributed to editing the manuscript draft.
ISSN:1087-0156
1546-1696
DOI:10.1038/s41587-020-00806-2