Figure 5

TINCR-STAU1 complex binds to CDKN2B mRNA and regulates their stability. (a) The abundance of CDKN2B mRNA was elevated upon TINCR and STAUI depletion in GC cells, detected by qRT-PCR. Error bars represent S.D., n=3. **P<0.01. (b) Interaction of CDKN2B mRNA with STAU1, detected by RIP assay (the relative ARF1 enrichment served as a positive control, and the GAPDH as a negative control that did not interact with STAU1). Error bars represent S.D., n=3. **P<0.01. (c) IP of STAU1-FLAG. MGC803 cells were transiently co-transfected with (1) STAU1-FLAG expression plasmid; (2) RLuc-CDKN2B 3′-UTR; (3) phCMV-MUP, which encodes MUP mRNA that lacks an SBS and serves as a negative control for STAU1-FLAG binding; and (4) Rluc-ARF1 SBS, which contains an ARF1 SBS downstream of the translation termination codon of C-terminally deleted renilla luciferase and serves as a positive control for STAU1-FLAG binding. After cell lysis, total RNA and protein were purified from the lysate before and after IP using FLAG antibody or nonspecific rabbit (r) IgG. The three leftmost lanes represent two-fold serial dilutions of RNA and demonstrate that the RT-PCR is semiquantitative. Schematic representations of the pLUC-CDKN2B 3′-UTR and pLUC-ARF1 SBS test plasmids (above). RT-PCR analysis demonstrates that CDKN2B 3′-UTRs, endogenous TINCR, and ARF1 SBS bind STAU1-FLAG, whereas MUP mRNA does not (below). Results are representative of three independently performed experiments. (d) Inhibiting CDKN2B mRNA interacting with STAU1 upon TINCR depletion, detected by RIP experiments. MGC803 cells were transfected with control (Scrambled) or si-TINCR, and cellular extract was prepared for RIP assay using SATU1 antibody 24 h after transfection. Error bars represent S.D, n=3. *P<0.05. (e) Biotinylated TINCR RNA pulls down the full-length CDKN2B mRNA detected by RT-PCR analysis. A nonspecific RNA (GAPDH) is shown as a control. (f) STAU1 depletion reduced the interaction between TINCR with CDKN2B mRNA. MGC803 cells were transfected with control (Scrambled) or si-STAU1, and cell lysates were incubated with biotin-labeled TINCR; after pull-down, mRNAs were extracted and assessed by qRT-PCR. Error bars represent S.D., n=3.*P<0.05; **P<0.01. (g) TINCR or STAU1 control CDKN2B mRNA stability. RNA stability assays were performed in MGC803 cells using Actinomycin D to disrupt RNA synthesis degradation rates of the mRNA CDKN2B over 12 h. *P<0.05; **P<0.01