Fig. 5

TIAR specifically binds to the bulge and loop structures of 5’ epsilon element in pgRNA. a The schematic diagram of 5 RNA fragments of the HBV genome used in the pulldown assay. b HepG2 cell lysates were co-incubated with the indicated biotin-labeled RNA fragment (F1, F2, F3, F4 and F5) transcribed in vitro. TIAR in the RNA-protein complex was pulled down by streptavidin beads and detected by Western blot. Biotin-labeled Saccharomyces cerevisiae tRNA served as negative controls. c The schematic workflow of CLIP assay. d Western blot analysis was performed to verify the accuracy of the CLIP results by anti-flag. e pgRNA sequence was covered by primers 1 to 18. f HepAD38 cells were transfected with pCDH-TIAR-flag or pCDH-flag plasmid. CLIP followed by RT-qPCR was performed to clarify the sequence of pgRNA that bound to TIAR. Data are represented as mean ± SD (n = 3 per group), *P < 0.05, ns, no significance. g The schematic diagram of 5’ ɛ region of pgRNA and derived F1 RNA fragments with the indicated deletion of RNA structure element in the 5’ ɛ region. h HepG2 cell lysates were co-incubated with biotin-labeled F1 or the indicated F1- derived fragments (F1, ∆ε, ∆B, ∆L, ∆B&L) transcribed in vitro. TIAR in the RNA-protein complex pulled down by streptavidin beads was detected by Western blot. i Huh-7 cells were co-transfected with pcDNA3.1-pgRNA-PolΔC-flag or the mutant plasmid, and siTIAR or pCDH-TIAR-flag. Intracellular PolΔC-flag and Cp were detected by Western blot