Fig. 6

HnRNPC binding to XB130 mRNA impedes recruitment of nucleases XRN1 and DIS3L2. A-C: Either control siRNA (NC siRNA) or gene-specific siRNA was transfected into hnRNPC-silenced cells. RT-qPCR and Western blotting assessed the silencing levels of each nuclease (A) and expression levels of XB130 mRNA (B) and protein (C) in the cells. GAPDH was used as an internal control. D: Control (psiCHECK-2) or dual-luciferase reporter recombinant vectors containing the 3’UTR segment 113–230 or 503–660 of XB130 mRNA, along with either NC siRNA or specific siRNA targeting XRN1 or DIS3L2, were co-transfected into control and hnRNPC-silenced cells. Dual-luciferase activity was measured 48 h post-transfection. hluc served as an internal control. E: A recombinant vector overexpressing Flag-tagged DIS3L2 was transfected into control or hnRNPC-silenced cells. RIP assays were then performed to evaluate interactions of DIS3L2 protein with XB130 and Hsp90 mRNA. IgG-bound RNA was used as a negative control. DCP2: decapping mRNA 2; DIS3L: DIS3 like exosome 3’-5’ exoribonuclease; EXOSC4: exosome component 4; CNOT1: CCR4-NOT transcription complex subunit 1; PARN: poly A-specific ribonuclease; PAN3: poly A specific ribonuclease subunit PAN3. * p < 0.05, ** p < 0.01, *** p < 0.001