脊髓小脑性共济失调3型(SCA3)是一种主要的遗传性神经退行性疾病,由Ataxin-3(ATXN3)基因中的CAG(编码谷氨酰胺)重复扩增引起。我们先前已经证明ATXN3耗尽或致病性ATXN3表达细胞消除了多核苷酸激酶3'-磷酸酶(PNKP)活性。这里,我们报道ATXN3与RNA聚合酶II(RNAPII)和经典的非同源末端连接(C-NHEJ)蛋白相关,包括PNKP,以及生理条件下新生的RNA。值得注意的是,ATXN3耗竭显著降低全局转录,转录基因的修复,和无错误的双链断裂修复含3'-磷酸盐的末端间隙,线性化的报告质粒。通过使用内源性同源转录本作为模板,在对照细胞中的再循环质粒中恢复了末端断裂位点的缺失序列,表明ATXN3在PNKP介导的无错误C-NHEJ中的作用。此外,SCA3患者和小鼠的脑提取物显示出显著较低的PNKP活性,p53BP1水平升高,转录基因中更丰富的链断裂,和RNAPII相对于对照的降解。在表达突变ATXN3的果蝇幼虫的大脑和眼睛中,类似的RNAPII降解也很明显。重要的是,果蝇的SCA3表型完全适合PNKP互补。因此,挽救PNKP的活性可能是SCA3的一种有前途的治疗策略。
Spinocerebellar ataxia type 3 (SCA3) is a dominantly inherited neurodegenerative disease caused by CAG (encoding glutamine) repeat expansion in the Ataxin-3 (ATXN3) gene. We have shown previously that ATXN3-depleted or pathogenic ATXN3-expressing cells abrogate polynucleotide kinase 3\'-phosphatase (PNKP) activity. Here, we report that ATXN3 associates with RNA polymerase II (RNAP II) and the classical nonhomologous end-joining (C-NHEJ) proteins, including PNKP, along with nascent RNAs under physiological conditions. Notably, ATXN3 depletion significantly decreased global transcription, repair of transcribed genes, and error-free double-strand break repair of a 3\'-phosphate-containing terminally gapped, linearized reporter plasmid. The missing sequence at the terminal break site was restored in the recircularized plasmid in control cells by using the endogenous homologous transcript as a template, indicating ATXN3\'s role in PNKP-mediated error-free C-NHEJ. Furthermore, brain extracts from SCA3 patients and mice show significantly lower PNKP activity, elevated p53BP1 level, more abundant strand-breaks in the transcribed genes, and degradation of RNAP II relative to controls. A similar RNAP II degradation is also evident in mutant ATXN3-expressing Drosophila larval brains and eyes. Importantly, SCA3 phenotype in Drosophila was completely amenable to PNKP complementation. Hence, salvaging PNKP\'s activity can be a promising therapeutic strategy for SCA3.