1. The RNA helicase DHX36–G4R1 modulates C9orf72 GGGGCC hexanucleotide repeat–associated translation
- Author
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Philip J. Smaldino, Amy Krans, Yi-Ju Tseng, Yuh-Hwa Wang, Adam E. Richardson, Katelyn M. Green, James P. Vaughn, Meredith E. Sharlow, Eric D. Routh, Peter K. Todd, Melissa A. Smaldino, Siara N. Sandwith, Antonio E. Chambers, Michael A. Reisinger, and Heather M Raimer
- Subjects
0301 basic medicine ,fragile X ,NLuc, nanoluciferase ,FFLuc, firefly luciferase ,Biochemistry ,DEAD-box RNA Helicases ,C9orf72 ,rDHX36, recombinant DHX36 ,dipeptide repeat proteins ,DNA Repeat Expansion ,G-quadruplex ,doxy, doxycycline ,Translation (biology) ,RNA Helicase A ,Cell biology ,FTD, frontal temporal dementia ,Frontotemporal Dementia ,G4C2, GGGGCC ,RNA Helicases ,Research Article ,RAN, repeat-associated non-AUG ,RNA helicase ,repeat-associated non-AUG translation ,DNA helicase ,Biology ,03 medical and health sciences ,Stress granule ,DHX36 ,Cell Line, Tumor ,HRE, hexanucleotide repeat expansion ,Humans ,Molecular Biology ,ALS (Lou Gehrig's disease) ,ISR, integrated stress response ,KD, knockdown ,030102 biochemistry & molecular biology ,C9orf72 Protein ,C9-NLuc, C9RAN translation–specific NLuc reporter ,Amyotrophic Lateral Sclerosis ,RNA ,Helicase ,Cell Biology ,SG, stress granule ,Ctrl, control ,G-Quadruplexes ,030104 developmental biology ,Protein Biosynthesis ,Ran ,Tg, thapsigargin ,biology.protein ,DHX36, DEAH-box helicase 36 ,Trinucleotide repeat expansion ,G4, G-quadruplex ,DHX36–G4R1–RHAU ,DPR, dipeptide repeat protein ,KCl, potassium chloride - Abstract
GGGGCC (G4C2) hexanucleotide repeat expansions in the endosomal trafficking gene C9orf72 are the most common genetic cause of ALS and frontotemporal dementia. Repeat-associated non-AUG (RAN) translation of this expansion through near-cognate initiation codon usage and internal ribosomal entry generates toxic proteins that accumulate in patients' brains and contribute to disease pathogenesis. The helicase protein DEAH-box helicase 36 (DHX36–G4R1) plays active roles in RNA and DNA G-quadruplex (G4) resolution in cells. As G4C2 repeats are known to form G4 structures in vitro, we sought to determine the impact of manipulating DHX36 expression on repeat transcription and RAN translation. Using a series of luciferase reporter assays both in cells and in vitro, we found that DHX36 depletion suppresses RAN translation in a repeat length–dependent manner, whereas overexpression of DHX36 enhances RAN translation from G4C2 reporter RNAs. Moreover, upregulation of RAN translation that is typically triggered by integrated stress response activation is prevented by loss of DHX36. These results suggest that DHX36 is active in regulating G4C2 repeat translation, providing potential implications for therapeutic development in nucleotide repeat expansion disorders.
- Published
- 2021