ABSTRACT

RNAs are highly regulated at the post-transcriptional level in neurodegenerative diseases and just a few mutations can significantly affect the fate of neuronal cells. To date, the impact of G-quadruplex (G4) regulation in neurodegenerative diseases like Parkinson’s disease (PD) has not been analysed. In this study, in silico potential G4s located in deregulated genes related to the nervous system were initially identified and were found to be significantly enriched. Several G4 sequences found in the 5ʹ untranslated regions (5ʹUTR) of mRNAs associated with Parkinson’s disease were demonstrated to in fact fold in vitro by biochemical assays. Subcloning of the full-length 5ʹUTRs of these candidates upstream of a luciferase reporter system led to the demonstration that the G4s of both Parkin RBR E3 Ubiquitin Protein Ligase (PRKN) and Vacuolar Protein Sorting-Associated Protein 35 (VPS35) significantly repressed the translation of both genes in SH-SY5Y cells. Subsequently, a strategy of using label-free RNA affinity purification assays with either of these two G4 sequences as bait isolated the Guanine Nucleotide-Binding Protein-Like 1 (GNL1). The latter was shown to have a higher affinity for the G4 sequences than for their mutated version. This study sheds light on new RNA G-quadruplexes located in genes dysregulated in Parkinson disease and a new G4-binding protein, GNL1.

摘要

RNA 在神经退行性疾病的转录后水平受到高度调节，只有少数突变可以显着影响神经元细胞的命运。迄今为止，尚未分析 G-四链体 (G4) 调节对帕金森病 (PD) 等神经退行性疾病的影响。在这项研究中，最初鉴定了位于与神经系统相关的失调基因中的计算机潜在 G4，并发现其显着富集。在与帕金森病相关的 mRNA 的 5'非翻译区 (5'UTR) 中发现的几个 G4 序列实际上在体外折叠通过生化分析。在荧光素酶报告系统上游对这些候选物的全长 5'UTR 进行亚克隆导致证明 Parkin RBR E3 泛素蛋白连接酶 (PRKN) 和液泡蛋白分选相关蛋白 35 (VPS35) 的 G4 显着抑制了SH-SY5Y 细胞中的两个基因。随后，使用以这两个 G4 序列中的任何一个作为诱饵的无标记 RNA 亲和纯化测定的策略分离了鸟嘌呤核苷酸结合蛋白样 1 (GNL1)。后者显示出对 G4 序列的亲和力高于对其突变版本的亲和力。这项研究揭示了位于帕金森病失调基因中的新 RNA G-四链体和新的 G4 结合蛋白 GNL1。