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.
Highlights
Multiple pathways in multicellular organisms have evolved to induce and execute cell death in defective, infected, or extraneous cells, and dysregulation of numerous steps in each pathway contribute to diseases and tissue damage[1,2]
RIPK1 ubiquitylation can be restricted by cIAP inhibition or by the deubiquitylase activity of CYLD, which is recruited to the complex-I through the LUBAC-binding protein SPATA217-22
We generated a transgenic cell line stably expressing Cas[9] and validated that co-expression of a sgRNA targeting Mlkl reduced MLKL protein to undetectable levels and conferred nearly complete resistance to necroptosis within 8 days (Figure S1). We used this cell line to screen a novel whole-genome sgRNA library evenly partitioned in nine pools targeting an average of 2203 genes, with 93% of genes targeted by eight sgRNAs (Figure S2). sgRNA sequences were selected using an algorithm that designs sgRNAs and prioritizes them at the gene level on the basis of the number of splice isoforms targeted, proximity to the 5′ end of the coding region, and the number of predicted off-target sites
Summary
Multiple pathways in multicellular organisms have evolved to induce and execute cell death in defective, infected, or extraneous cells, and dysregulation of numerous steps in each pathway contribute to diseases and tissue damage[1,2]. In a kinase-independent manner, ubiquitylated RIPK1 recruits and activates IKK and TAK1-TAB2/3 complexes, stimulating NF-κB, JNK, p38, and ERK pathways to transcriptionally activate antiapoptotic and pro-inflammatory genes, including CFLAR (cFLIP) encoding the catalytically inactive homolog of caspase-8. RIPK1 ubiquitylation can be restricted by cIAP inhibition or by the deubiquitylase activity of CYLD, which is recruited to the complex-I through the LUBAC-binding protein SPATA217-22. If caspase-8 expression is limited or its activity is blocked by pan-caspase inhibitors such as zVAD-fmk, complex-II can further develop to form the necrosome complex as RIPK1 recruits and activates RIPK3, leading to MLKL-dependent necroptosis[3,9,10,15]
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