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).