DNA damage plays a central role in the cellular pathogenesis of polyglutamine (polyQ) diseases, including Huntington’s disease (HD). In this study, we showed that the expression of untranslatable expanded CAG RNA per se induced the cellular DNA damage response pathway. By means of RNA sequencing (RNA-seq), we found that expression of the Nudix hydrolase 16 (NUDT16) gene was down-regulated in mutant CAG RNA-expressing cells. The loss of NUDT16 function results in a misincorporation of damaging nucleotides into DNAs and leads to DNA damage. We showed that small CAG (sCAG) RNAs, species generated from expanded CAG transcripts, hybridize with CUG-containing NUDT16 mRNA and form a CAG-CUG RNA heteroduplex, resulting in gene silencing of NUDT16 and leading to the DNA damage and cellular apoptosis. These results were further validated using expanded CAG RNA-expressing mouse primary neurons and in vivo R6/2 HD transgenic mice. Moreover, we identified a bisamidinium compound, DB213, that interacts specifically with the major groove of the CAG RNA homoduplex and disfavors the CAG-CUG heteroduplex formation. This action subsequently mitigated RNA-induced silencing complex (RISC)-dependent NUDT16 silencing in both in vitro cell and in vivo mouse disease models. After DB213 treatment, DNA damage, apoptosis, and locomotor defects were rescued in HD mice. This work establishes NUDT16 deficiency by CAG repeat RNAs as a pathogenic mechanism of polyQ diseases and as a potential therapeutic direction for HD and other polyQ diseases.

DNA 损伤在多聚谷氨酰胺 (polyQ) 疾病，包括亨廷顿病 (HD) 的细胞发病机制中起着核心作用。在这项研究中，我们表明不可翻译的扩展 CAG RNA 的表达本身诱导了细胞 DNA 损伤反应途径。通过RNA测序（RNA-seq），我们发现Nudix水解酶16（NUDT16）基因在表达CAG RNA的突变细胞中表达下调。NUDT16 功能的丧失导致破坏性核苷酸错误掺入 DNA 并导致 DNA 损伤。我们发现小 CAG (sCAG) RNAs，从扩展的 CAG 转录物产生的物种，与含有 CUG 的NUDT16 mRNA杂交并形成 CAG-CUG RNA 异源双链，导致NUDT16 的基因沉默并导致 DNA 损伤和细胞凋亡。使用扩增的 CAG RNA 表达小鼠原代神经元和体内 R6/2 HD 转基因小鼠进一步验证了这些结果。此外，我们确定了一种双脒化合物 DB213，它与 CAG RNA 同源双链的大沟特异性相互作用，不利于 CAG-CUG 异源双链的形成。这一行动随后在体外细胞和体内小鼠疾病模型中减轻了 RNA 诱导的沉默复合物 (RISC) 依赖的NUDT16沉默。DB213 处理后，HD 小鼠的 DNA 损伤、细胞凋亡和运动缺陷得以挽救。这项工作将 CAG 重复 RNA 导致的 NUDT16 缺陷确立为 polyQ 疾病的致病机制以及 HD 和其他 polyQ 疾病的潜在治疗方向。