title: Functional CRISPR screening identifies the ufmylation pathway as a regulator of SQSTM1/p62.
abstract: SQSTM1 is an adaptor protein that integrates multiple cellular signaling pathways and whose expression is tightly regulated at the transcriptional and post-translational level. Here, we describe a forward genetic screening paradigm exploiting CRISPR-mediated genome editing coupled to a cell selection step by FACS to identify regulators of SQSTM1. Through systematic comparison of pooled libraries, we show that CRISPR is superior to RNAi in identifying known SQSTM1 modulators. A genome-wide CRISPR screen exposed MTOR signalling and the entire macroautophagy machinery as key regulators of SQSTM1 and identified several novel modulators including HNRNPM, SLC39A14, SRRD, PGK1 and the ufmylation cascade. We show that ufmylation regulates SQSTM1 by eliciting a cell type-specific ER stress response which induces SQSTM1 expression and results in its accumulation in the cytosol. This study validates pooled CRISPR screening as a powerful method to map the repertoire of cellular pathways that regulate the fate of an individual target protein.
authors.affiliation: Developmental and Molecular Pathways, Novartis Institutes for BioMedical Research, Cambridge, United States.
authors.forename: Rowena
authors.initials: R
authors.lastname: DeJesus
authors.affiliation: Developmental and Molecular Pathways, Novartis Institutes for BioMedical Research, Basel, Switzerland.
authors.forename: Francesca
authors.initials: F
authors.lastname: Moretti
authors.affiliation: Developmental and Molecular Pathways, Novartis Institutes for BioMedical Research, Cambridge, United States.
authors.forename: Gregory
authors.initials: G
authors.lastname: McAllister
authors.affiliation: Developmental and Molecular Pathways, Novartis Institutes for BioMedical Research, Cambridge, United States.
authors.forename: Zuncai
authors.initials: Z
authors.lastname: Wang
authors.affiliation: Developmental and Molecular Pathways, Novartis Institutes for BioMedical Research, Cambridge, United States.
authors.forename: Phil
authors.initials: P
authors.lastname: Bergman
authors.affiliation: Developmental and Molecular Pathways, Novartis Institutes for BioMedical Research, Cambridge, United States.
authors.forename: Shanming
authors.initials: S
authors.lastname: Liu
authors.affiliation: Developmental and Molecular Pathways, Novartis Institutes for BioMedical Research, Cambridge, United States.
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Frias
authors.affiliation: Developmental and Molecular Pathways, Novartis Institutes for BioMedical Research, Cambridge, United States.
authors.forename: John
authors.initials: J
authors.lastname: Alford
authors.affiliation: Developmental and Molecular Pathways, Novartis Institutes for BioMedical Research, Cambridge, United States.
authors.forename: John S
authors.initials: JS
authors.lastname: Reece-Hoyes
authors.affiliation: Developmental and Molecular Pathways, Novartis Institutes for BioMedical Research, Cambridge, United States.
authors.forename: Alicia
authors.initials: A
authors.lastname: Lindeman
authors.affiliation: Developmental and Molecular Pathways, Novartis Institutes for BioMedical Research, Cambridge, United States.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Kelliher
authors.affiliation: Developmental and Molecular Pathways, Novartis Institutes for BioMedical Research, Cambridge, United States.
authors.forename: Carsten
authors.initials: C
authors.lastname: Russ
authors.affiliation: Developmental and Molecular Pathways, Novartis Institutes for BioMedical Research, Basel, Switzerland.
authors.forename: Judith
authors.initials: J
authors.lastname: Knehr
authors.affiliation: Developmental and Molecular Pathways, Novartis Institutes for BioMedical Research, Basel, Switzerland.
authors.forename: Walter
authors.identifier: 0000-0001-6150-8295
authors.initials: W
authors.lastname: Carbone
authors.affiliation: Developmental and Molecular Pathways, Novartis Institutes for BioMedical Research, Basel, Switzerland.
authors.forename: Martin
authors.initials: M
authors.lastname: Beibel
authors.affiliation: Developmental and Molecular Pathways, Novartis Institutes for BioMedical Research, Basel, Switzerland.
authors.forename: Guglielmo
authors.initials: G
authors.lastname: Roma
authors.affiliation: Gastrointestinal Unit and Center for the Study of Inflammatory Bowel Disease, Massachusetts General Hospital, Harvard Medical School, Boston, United States.
authors.forename: Aylwin
authors.initials: A
authors.lastname: Ng
authors.affiliation: Developmental and Molecular Pathways, Novartis Institutes for BioMedical Research, Cambridge, United States.
authors.forename: John A
authors.initials: JA
authors.lastname: Tallarico
authors.affiliation: Developmental and Molecular Pathways, Novartis Institutes for BioMedical Research, Cambridge, United States.
authors.forename: Jeffery A
authors.initials: JA
authors.lastname: Porter
authors.affiliation: Gastrointestinal Unit and Center for the Study of Inflammatory Bowel Disease, Massachusetts General Hospital, Harvard Medical School, Boston, United States.
authors.forename: Ramnik J
authors.initials: RJ
authors.lastname: Xavier
authors.affiliation: Developmental and Molecular Pathways, Novartis Institutes for BioMedical Research, Cambridge, United States.
authors.forename: Craig
authors.initials: C
authors.lastname: Mickanin
authors.affiliation: Developmental and Molecular Pathways, Novartis Institutes for BioMedical Research, Cambridge, United States.
authors.forename: Leon O
authors.initials: LO
authors.lastname: Murphy
authors.affiliation: Developmental and Molecular Pathways, Novartis Institutes for BioMedical Research, Cambridge, United States.
authors.forename: Gregory R
authors.initials: GR
authors.lastname: Hoffman
authors.affiliation: Developmental and Molecular Pathways, Novartis Institutes for BioMedical Research, Basel, Switzerland.
authors.forename: Beat
authors.identifier: 0000-0003-0624-9571
authors.initials: B
authors.lastname: Nyfeler
mesh_terms: D001343:Autophagy; D002460:Cell Line; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D005434:Flow Cytometry; D005786:Gene Expression Regulation; D018390:Gene Targeting; D005820:Genetic Testing; D006801:Humans; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000378:metabolism*; D000071456:Sequestosome-1 Protein / Q000378:metabolism*; D015398:Signal Transduction; D058570:TOR Serine-Threonine Kinases / Q000378:metabolism
chemical_list: D011506:Proteins; C099718:SQSTM1 protein, human; D000071456:Sequestosome-1 Protein; C502094:UFM1 protein, human; C546842:MTOR protein, human; D058570:TOR Serine-Threonine Kinases
keywords: CRISPR; ER stress; SQSTM1; autophagy; biochemistry; cell biology; human