Abstract:
Nucleotide repeat expansion of GGGGCC (G4C2) in the non-coding region of C9orf72 is the most common genetic cause underlying amyotrophic lateral sclerosis and frontotemporal dementia. Transcripts harboring this repeat expansion undergo the translation of dipeptide repeats via a non-canonical process known as repeat-associated non-AUG (RAN) translation. In order to ascertain the essential components required for RAN translation, we successfully recapitulated G4C2-RAN translation using an in vitro reconstituted translation system comprising human factors, namely the human PURE system. Our findings conclusively demonstrate that the presence of fundamental translation factors is sufficient to mediate the elongation from the G4C2 repeat. Furthermore, the initiation mechanism proceeded in a 5\' cap-dependent manner, independent of eIF2A or eIF2D. In contrast to cell lysate-mediated RAN translation, where longer G4C2 repeats enhanced translation, we discovered that the expansion of the G4C2 repeats inhibited translation elongation using the human PURE system. These results suggest that the repeat RNA itself functions as a repressor of RAN translation. Taken together, our utilization of a reconstituted RAN translation system employing minimal factors represents a distinctive and potent approach for elucidating the intricacies underlying RAN translation mechanism.
摘要:
GGGGCC(G4C2)在C9orf72非编码区的核苷酸重复扩增是肌萎缩性侧索硬化症和额颞叶痴呆的最常见遗传原因。具有这种重复扩增的转录物通过称为重复相关非AUG(RAN)翻译的非规范过程进行二肽重复的翻译。为了确定RAN翻译所需的基本组件,我们成功地概述了G4C2-RAN翻译使用体外重组的翻译系统,包括人为因素,即人类纯净系统。我们的发现最终证明,基本翻译因子的存在足以介导G4C2重复序列的延伸。此外,启动机制以依赖于上限的方式进行,独立于eIF2A或eIF2D。与细胞裂解物介导的RAN翻译相反,更长的G4C2重复增强翻译,我们发现,使用人PURE系统,G4C2重复序列的扩增抑制了翻译延伸。这些结果表明重复RNA本身作为RAN翻译的阻遏物起作用。一起来看,我们利用采用最少因素的重组RAN翻译系统代表了一种独特而有效的方法来阐明RAN翻译机制的复杂性。