Department of Neurology, University of Michigan, Ann Arbor, Michigan, USA; Cellular and Molecular Biology Graduate Program, University of Michigan, Ann Arbor, Michigan, USA.
Department of Biology, Ball State University, Muncie, Indiana, USA.
J Biol Chem. 2021 Aug;297(2):100914. doi: 10.1016/j.jbc.2021.100914. Epub 2021 Jun 24.
GGGGCC (GC) 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 GC 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 GC 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 GC repeat translation, providing potential implications for therapeutic development in nucleotide repeat expansion disorders.
GGGGCC（GC）六核苷酸重复扩展在内体运输基因 C9orf72 中是 ALS 和额颞叶痴呆的最常见遗传原因。通过近同源起始密码子使用和内部核糖体进入，这种扩展的重复相关非 AUG（RAN）翻译会产生毒性蛋白，这些蛋白在患者大脑中积累并导致疾病发病机制。解旋酶蛋白 DEAH 盒解旋酶 36（DHX36-G4R1）在细胞中 RNA 和 DNA G-四链体（G4）的解旋中发挥积极作用。由于已知 GC 重复在体外形成 G4 结构，我们试图确定操纵 DHX36 表达对重复转录和 RAN 翻译的影响。使用一系列在细胞内和体外的荧光素酶报告基因检测，我们发现 DHX36 耗竭以重复长度依赖性方式抑制 RAN 翻译，而 DHX36 的过表达增强 GC 报告 RNA 中的 RAN 翻译。此外，由整合应激反应激活引发的 RAN 翻译的上调被 DHX36 的缺失所阻止。这些结果表明 DHX36 积极参与调节 GC 重复翻译，为核苷酸重复扩展障碍的治疗开发提供了潜在的意义。
