Figure 4.

ZFAS1 cooperates with PABP2 to stabilize SREBP1 mRNA

(A) Nuclear and cytoplasmic RNA fractions were isolated from SW480 and RKO cells. The distribution of ZFAS1 was examined by qRT-PCR. GAPDH was used as a cytoplasmic control, and U1 was used as a nuclear control. (B) RNA pulldown assays were performed with biotin-labeled sense or antisense (negative control) full-length ZFAS1 RNA in SW480 cells. Precipitated proteins were subjected to SDS-PAGE and stained with silver. (C) Protein levels in immunoprecipitates were determined by western blot assay. The expression level of PABP2 protein is presented. RIP experiments were performed in SW480 and RKO cells, and the precipitated RNA was subjected to qRT-PCR analysis of ZFAS1. The fold enrichment of ZFAS1 in PABP2 is relative to the IgG control. (D) The PABP2 and SREBP1 mRNA expression levels in SW480 and RKO cells transfected with PABP2 siRNAs or negative control siRNA were examined by qRT-PCR. (E) SW480 and RKO cells were transfected with ZFAS1, PABP2, or negative control siRNA. Forty-eight hours later, the cells were incubated with actinomycin D (2.5 μg/mL) for the indicated periods of time. The SREBP1 mRNA levels were then analyzed by real-time RT-PCR. (F) RIP experiments were performed in SW480 and RKO cells, and the precipitated RNA was subjected to qRT-PCR analysis of SREBP1. The fold enrichment of SREBP1 in PABP2 is relative to the IgG control. (G) RIP experiments were performed in SW480 and RKO cells transfected with ZFAS1 or negative control siRNA, and the precipitated RNA was subjected to qRT-PCR analysis of SREBP1. Three independent experiments were performed. Data are shown as the mean ± SD. ∗p < 0.05, ∗∗p < 0.01.