Identification of pathological pathways centered on circRNA dysregulation associated with early pathogenic progression of Alzheimer’s disease

The introns flanking the two BSJ sites of a given circRNA need to base-pair and bring the BSJs into close proximity, which in turn allow circRNA biogenesis. In addition, specific RBPs often promote circRNA production by binding to the flanking intronic sequences and bring BSJs together via forming homodimers. siRNAs targeting the hnRNPL was transfected in Neuro 2A (N2a) cells. Depletion of the hnRNPL (Fig. 5h) caused a significant reduction of circRNA.

To experimentally validate circGigyf2-CPSF6 interactions, we performed circGigyf2 pulldown. A robust and specific CPSF6 enrichment was found in circGigyf2 pulldown (Fig. 7b). In contrast, QKI and TAR DNA-binding protein 43 (TDP-43), two RBPs that are not predicted to bind circGigyf2 strongly, were not detected in circGigyf2 pulldown (Fig. 7b). The non-RBP GAPDH was also absent (Fig. 7b).

western blots indicated marked elevation of PLAU protein levels in 7-month-old 5xFAD cortex as compared to age-matched littermate controls while no PLAU increase was observed in 5-month 5xFAD mouse (Fig. 7g).

特定环状RNA (circRNA) 的两侧内含子需进行碱基配对，进而将两个碱基跳跃位点 (BSJ) 带入邻近区域，此过程有利于环状RNA的生物合成。此外，特定的RNA结合蛋白 (RBP) 通过与相邻的内含子序列结合，并形成同源二聚体，通常能够促进环状RNA的产生。在神经2A (N2a) 细胞中，针对hnRNPL的siRNA进行了转染。hnRNPL的耗竭（图5h）导致环状RNA显著减少。为了实验验证circGigyf2与CPSF6之间的相互作用，我们进行了circGigyf2的pull-down实验。在circGigyf2 pull-down实验中，我们发现CPSF6的富集效果既稳健又具有特异性（图7b）。相反，QKI和TAR DNA结合蛋白43 (TDP-43)，这两种RNA结合蛋白并未被预测与circGigyf2强烈结合，在circGigyf2 pull-down实验中并未检测到（图7b）。非RNA结合蛋白GAPDH也不存在（图7b）。Western blot分析显示，与同龄的对照组相比，7个月大的5xFAD皮质中PLAU蛋白水平显著升高，而在5个月的5xFAD小鼠中并未观察到PLAU的增加（图7g）。