Functional CRISPR screening identifies the ufmylation pathway as a regulator of SQSTM1/p62

Author:

DeJesus Rowena1,Moretti Francesca2,McAllister Gregory1,Wang Zuncai1,Bergman Phil1,Liu Shanming1,Frias Elizabeth1,Alford John1,Reece-Hoyes John S1,Lindeman Alicia1,Kelliher Jennifer1,Russ Carsten1,Knehr Judith2,Carbone Walter2ORCID,Beibel Martin2,Roma Guglielmo2,Ng Aylwin3,Tallarico John A1,Porter Jeffery A1,Xavier Ramnik J3,Mickanin Craig1,Murphy Leon O1,Hoffman Gregory R1,Nyfeler Beat2ORCID

Affiliation:

1. Developmental and Molecular Pathways, Novartis Institutes for BioMedical Research, Cambridge, United States

2. Developmental and Molecular Pathways, Novartis Institutes for BioMedical Research, Basel, Switzerland

3. Gastrointestinal Unit and Center for the Study of Inflammatory Bowel Disease, Massachusetts General Hospital, Harvard Medical School, Boston, United States

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.

Funder

Novartis

National Institutes of Health

Publisher

eLife Sciences Publications, Ltd

Subject

General Immunology and Microbiology,General Biochemistry, Genetics and Molecular Biology,General Medicine,General Neuroscience

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