Figure 5. TTP is involved in Otud-6b mRNA rapid degradation after induction.
A. The sequence of Otud-6b 3′UTR. AUUUA pentamer or AUUUUA hexamer are underlined. Each rectangle indicates an AU-rich sequence site. The AU-rich sequence with U surrounding it is indicated as a black rectangle. B. pSUPER Mock, pSUPER siTTP-1 or 2 and HA-TTP vectors co-expressing Ba/F3 cells were seeded at 2×105 cells per ml. Cell extracts were immunoblotted with anti-HA antibody. GFP was used as a loading control. C. Otud-6b RNA level in Ba/F3 cells transfected with pSUPER siTTP-1,2 vectors after starvation and stimulation with 10 pM mouse IL-3 for the indicated times (0, 0.5, 1, 2, 4, 8, 12, 16, and 24 hours). D. Ba/F3 cells were transfected with pSUPER Mock, pSUPER siTTP-1 or 2 vectors. 24 later, cells were stimulated by 10 pM IL-3 and incubated with 15 ug/ml Act.D for the time indicated. The qRT-PCR was performed to detect Otud-6b mRNA remaining at each time point. E. Semi-quantitative RT-PCR analysis of Otud-6b mRNA remaining at each time point after Act.D adding. F. RT-PCR for Otud-6b mRNA in protein-mRNA immunoprecipitation complex. The mRNAs of Ba/F3 cells transfected with HA-TTP when response to IL-3 was immunoprecipitated with anti-HA and IgG serum. As a control, Input (10%) denoted that the RNA sample contained Otud-6b mRNA and didn't contain genomic DNA. G. TTP transfection had little effect on pGL3 control luciferase expression. Wild-type TTP transfection resulted in a significant decrease in full-length Otud-6b 3′-UTR luciferase reporter expression in 293T cells, while the TTP Zn-FM did not alter Otud-6b 3′-UTR-mediated expression. Different region of Otud-6b 3′UTR was inserted into downstream of luciferase coding region. The truncate 3′UTR vectors were constructed depending on the AU-rich sequence distribution. 293T cells were provided by the Cell Bank, Shanghai Institute of Biochemistry and Cell Biology, SIBS, CAS.