Abstract
The necroptotic cell death pathway is a key component of human pathogen defense that can become aberrantly derepressed during tissue homeostasis to contribute to multiple types of tissue damage and disease. While formation of the necrosome kinase signaling complex containing RIPK1, RIPK3, and MLKL has been extensively characterized, additional mechanisms of its regulation and effector functions likely remain to be discovered. We screened 19,883 mouse protein-coding genes by CRISPR/Cas9-mediated gene knockout for resistance to cytokine-induced necroptosis and identified 112 regulators and mediators of necroptosis, including 59 new candidate pathway components with minimal or no effect on cell growth in the absence of necroptosis induction. Among these, we further characterized the function of PTBP1, an RNA binding protein whose activity is required to maintain RIPK1 protein abundance by regulating alternative splice-site selection.
MeSH terms
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Alternative Splicing*
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Animals
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CRISPR-Associated Protein 9 / genetics
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CRISPR-Associated Protein 9 / metabolism
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CRISPR-Cas Systems*
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Clustered Regularly Interspaced Short Palindromic Repeats*
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Fibroblasts / enzymology*
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Fibroblasts / pathology
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Gene Expression Regulation, Enzymologic
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Gene Targeting / methods*
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HEK293 Cells
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HT29 Cells
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Heterogeneous-Nuclear Ribonucleoproteins / genetics
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Heterogeneous-Nuclear Ribonucleoproteins / metabolism*
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Humans
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Mice
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Necroptosis*
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Polypyrimidine Tract-Binding Protein / genetics
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Polypyrimidine Tract-Binding Protein / metabolism*
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Receptor-Interacting Protein Serine-Threonine Kinases / genetics
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Receptor-Interacting Protein Serine-Threonine Kinases / metabolism*
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Signal Transduction
Substances
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Heterogeneous-Nuclear Ribonucleoproteins
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Ptbp1 protein, mouse
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Polypyrimidine Tract-Binding Protein
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Receptor-Interacting Protein Serine-Threonine Kinases
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Ripk1 protein, mouse
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CRISPR-Associated Protein 9