A Helicase Unwinds Hexanucleotide Repeat RNA G‑Quadruplexes and Facilitates Repeat-Associated Non-AUG Translation

The
expansion of a hexanucleotide repeat GGGGCC (G4C2) in the C9orf72 gene is the most common cause of amyotrophic lateral
sclerosis (ALS) and frontotemporal dementia (FTD). The G4C2 expansion
leads to repeat-associated non-AUG (RAN) translation and the production
of toxic dipeptide repeat (DPR) proteins, but the mechanisms of RAN
translation remain enigmatic. Here, we report that the RNA helicase
DHX36 is a robust positive regulator of C9orf72 RAN
translation. DHX36 has a high affinity for the G4C2 repeat RNA, preferentially
binds to the repeat RNA’s G-quadruplex conformation, and efficiently
unwinds the G4C2 G-quadruplex structures. Native DHX36 interacts with
the G4C2 repeat RNA and is essential for effective RAN translation
in the cell. In induced pluripotent stem cells and differentiated
motor neurons derived from C9orf72-linked ALS patients, reducing DHX36
significantly decreased the levels of endogenous DPR proteins. DHX36
is also aberrantly upregulated in tissues of C9orf72-linked ALS patients. These results indicate that DHX36 facilitates C9orf72 RAN translation by resolving repeat RNA G-quadruplex
structures and may be a potential target for therapeutic intervention.

C9orf72基因内六核苷酸重复序列GGGGCC（G4C2）的扩增，是肌萎缩侧索硬化症（amyotrophic lateral sclerosis, ALS）与额颞叶痴呆（frontotemporal dementia, FTD）最常见的致病诱因。G4C2扩增可引发重复相关非AUG（repeat-associated non-AUG, RAN）翻译，并产生毒性二肽重复（dipeptide repeat, DPR）蛋白，但RAN翻译的具体机制仍未明晰。本研究发现，RNA解旋酶DHX36（RNA helicase DHX36）是C9orf72 RAN翻译的强效正向调控因子。DHX36对G4C2重复RNA具有高亲和力，可优先结合该重复RNA的G-四链体（G-quadruplex）构象，并有效解开G4C2的G-四链体结构。天然状态的DHX36可与G4C2重复RNA相互作用，且对细胞内高效的RAN翻译不可或缺。在携带C9orf72致病突变的ALS患者诱导多能干细胞（induced pluripotent stem cells）及其分化获得的运动神经元中，敲低DHX36可显著降低内源性DPR蛋白的表达水平。此外，C9orf72相关ALS患者的组织样本中，DHX36存在异常上调现象。上述结果表明，DHX36可通过解开重复RNA的G-四链体结构，促进C9orf72的RAN翻译，有望成为治疗干预的潜在靶点。