C9orf72 Hexanucleotide Repeat RNA Drives Transcriptional Dysregulation Through Genome-wide Hybrid G-quadruplexes [RAP]

A hexanucleotide repeat expansion in C9orf72 is the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. While repeat RNAs are implicated in disease pathogenesis, their mechanisms of actions remain incompletely understood. Here we show that GGGGCC repeat RNA engages chromatin genome-wide preferentially at promoter regions in patient cells. This interaction obstructs RNA polymerase II and transcription factors with GC-rich motifs, leading to broad transcriptional repression. Biochemical assays, single-molecule imaging, and native bisulfite sequencing analyses demonstrate that GGGGCC repeat RNA intrinsically forms DNA:RNA hybrid G-quadruplexes with cognate DNA, providing a structural basis for transcriptional interference. Stabilization of these G-quadruplex structures exacerbates neuronal vulnerability to metabolic stress in patient-derived motor neurons and cortical organoids, whereas restoring key gene dysregulation improves neuronal resistance to stress. These findings uncover a previously unrecognized trans-acting mechanism whereby repetitive RNAs form hybrid structures with genomic DNA, disrupt gene regulation, and contribute to neurodegeneration. Overall design: RAP-DNA profiling of GGGGCC repeat RNA-associated DNA regions in C9-ALS iMNs and Isogenic control iMNs.

C9orf72基因的六核苷酸重复扩增（hexanucleotide repeat expansion）是肌萎缩侧索硬化（amyotrophic lateral sclerosis, ALS）与额颞叶痴呆（frontotemporal dementia, FTD）最常见的遗传致病因素。尽管重复RNA（repeat RNAs）已被证实与疾病发病机制相关，但其具体作用机制仍未完全阐明。本研究发现，GGGGCC重复RNA在患者细胞的全基因组范围内，优先结合启动子区域。该相互作用会阻断RNA聚合酶II以及携带GC富集基序的转录因子，进而引发广泛的转录抑制。生化实验、单分子成像及原生亚硫酸氢盐测序分析表明，GGGGCC重复RNA可与同源DNA内在形成DNA:RNA杂合G-四链体（DNA:RNA hybrid G-quadruplexes），为转录干扰提供了结构基础。稳定此类G-四链体结构会加剧患者来源的运动神经元与皮质类器官对代谢应激的易感性，而恢复关键基因的表达失调则可提升神经元对应激的抵抗能力。本研究揭示了一种此前未被发现的反式作用机制：重复RNA可与基因组DNA形成杂合结构，破坏基因调控并参与神经退行性病变的发生。总体实验设计：在C9-ALS诱导多能干细胞衍生运动神经元（induced motor neurons, iMNs）及同基因对照iMNs中，对与GGGGCC重复RNA结合的DNA区域开展RAP-DNA谱分析。