CRISPR-Cas9 Screens Identify the RNA Helicase DDX3X as a Repressor of C9ORF72 (GGGGCC)n Repeat-Associated Non-AUG Translation

Weiwei Cheng; Shaopeng Wang; Zhe Zhang; David W Morgens; Lindsey R Hayes; Soojin Lee; Bede Portz; Yongzhi Xie; Baotram V Nguyen; Michael S Haney; Shirui Yan; Daoyuan Dong; Alyssa N Coyne; Junhua Yang; Fengfan Xian; Don W Cleveland; Zhaozhu Qiu; Jeffrey D Rothstein; James Shorter; Fen-Biao Gao; Michael C Bassik; Shuying Sun

doi:10.1016/j.neuron.2019.09.003

CRISPR-Cas9 Screens Identify the RNA Helicase DDX3X as a Repressor of C9ORF72 (GGGGCC)n Repeat-Associated Non-AUG Translation

Neuron. 2019 Dec 4;104(5):885-898.e8. doi: 10.1016/j.neuron.2019.09.003. Epub 2019 Oct 3.

Authors

Weiwei Cheng¹, Shaopeng Wang¹, Zhe Zhang¹, David W Morgens², Lindsey R Hayes³, Soojin Lee⁴, Bede Portz⁵, Yongzhi Xie¹, Baotram V Nguyen⁵, Michael S Haney², Shirui Yan¹, Daoyuan Dong¹, Alyssa N Coyne³, Junhua Yang⁶, Fengfan Xian¹, Don W Cleveland⁷, Zhaozhu Qiu⁶, Jeffrey D Rothstein³, James Shorter⁵, Fen-Biao Gao⁴, Michael C Bassik², Shuying Sun⁸

Affiliations

¹ Department of Pathology and Brain Science Institute, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
² Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305, USA.
³ Brain Science Institute and Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
⁴ Department of Neurology, University of Massachusetts Medical School, Worcester, MA 01605, USA.
⁵ Department of Biochemistry and Biophysics, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA 19104, USA.
⁶ Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
⁷ Ludwig Institute for Cancer Research and Department of Cellular and Molecular Medicine, University of California at San Diego, La Jolla, CA 92093, USA.
⁸ Department of Pathology and Brain Science Institute, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA. Electronic address: shuying.sun@jhmi.edu.

Abstract

Hexanucleotide GGGGCC repeat expansion in C9ORF72 is the most prevalent genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). One pathogenic mechanism is the aberrant accumulation of dipeptide repeat (DPR) proteins produced by the unconventional translation of expanded RNA repeats. Here, we performed genome-wide CRISPR-Cas9 screens for modifiers of DPR protein production in human cells. We found that DDX3X, an RNA helicase, suppresses the repeat-associated non-AUG translation of GGGGCC repeats. DDX3X directly binds to (GGGGCC)n RNAs but not antisense (CCCCGG)n RNAs. Its helicase activity is essential for the translation repression. Reduction of DDX3X increases DPR levels in C9ORF72-ALS/FTD patient cells and enhances (GGGGCC)n-mediated toxicity in Drosophila. Elevating DDX3X expression is sufficient to decrease DPR levels, rescue nucleocytoplasmic transport abnormalities, and improve survival of patient iPSC-differentiated neurons. This work identifies genetic modifiers of DPR protein production and provides potential therapeutic targets for C9ORF72-ALS/FTD.

Keywords: ALS; CRISPR-Cas9 screen; DDX3X; FTD; RAN translation; RNA; helicase; neurodegeneration; repeat expansion.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

MeSH terms

Amyotrophic Lateral Sclerosis / metabolism*
Animals
C9orf72 Protein / biosynthesis*
CRISPR-Cas Systems
DEAD-box RNA Helicases / metabolism*
Drosophila
Frontotemporal Dementia / metabolism*
Humans
Protein Biosynthesis / physiology
Repetitive Sequences, Nucleic Acid

Substances

C9orf72 Protein
C9orf72 protein, human
DDX3X protein, human
DEAD-box RNA Helicases

Abstract

Publication types

MeSH terms

Substances

Grants and funding