Fig. 3.

Insulin-like growth factor 2 messenger RNA binding protein (IMP1) binds to urethral carcinoma-associated 1 (UCA1) and decreases UCA1 stability. a Upper: shows MS2-UCA1 fusion RNA. UCA1 with mutated IMP1 binding sites are shown by red arrow heads. Lower; UCA1 RNAs were pulled down by MBP-MCP; immunoblots show that interaction of IMP1 with UCA1 was greatly reduced when the IMP1 binding motifs were mutated. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a control. b Stability of UCA1 in T47D and T47D-IMP1/short hairpin RNA (shRNA) cells were measured after treatment with actinomycin D for 12 h. Relative UCA1 levels were determined by normalizing to GAPDH messenger RNA (mRNA) levels. Data are presented as means ± SD from three independent experiments: **P < 0.01 as determined by Student’s t test. c and d Vectors expressing a green fluorescent protein (GFP) and UCA1 or mutated (mut)-UCA1 were transiently transfected into 293 T and T47D cells for 24 h. Relative levels of UCA1 were analyzed by RT-qPCR and were nomalized to control GFP mRNA levels. The data are presented as mean ± SD from three independent experiments: *P < 0.05. d and e IMP1 interacts with the components of the RNA decay machinery. Immunoprecipitation was performed on MDA231/IMP1-GFP and MDA231/GFP cells with GFP antibodies. The representative immunoblots (IB) show that IMP1 and UCA1 co-precipitate with CNOT1