Fig. 5

RP11 regulates Siah1 and Fbxo45 expression by forming the RP11-hnRNPA2B1-mRNA complex. a RNA pull-down analysis and MS identified hnRNPA2B1 as the specific protein interacting with RP11 in both HCT-15 and HCT-15 RP11 stable overexpression cells. The red arrow shows the position of hnRNPA2B1. b The secondary structure of RP11 was predicted (http://rna.tbi.univie.ac.at/). The red colour indicates strong confidence for the prediction of each base. c RNA pull-down detection of the interaction between hnRNPA2B1 and RP11, Siah1, or Fbxo45 in HCT-15 cells. d hnRNPA2B1 expression in the cytoplasmic and nuclear fractions of HCT-15 RP11 stable overexpression and control cells were analysed by western blot. e HCT-15 cells were transfected with pcDNA (vector) or pcDNA/hnRNPA2B1 for 24 h, and the expression of Siah1 and Fbxo45 was verified by western blot analysis. f &g The computational prediction of the interaction between RP11 and the Siah1 (f) or Fbxo45 (g) mRNA based on IntaRNA 2.0 (http://rna.informatik.uni-freiburg.de/IntaRNA/Input.jsp) [53]. h After in vitro transcription to generate biotin-labelled RP-11 and RP-11 AS, RIP-PCR was performed to analyse the relative enrichment of Siah1 or Fbxo45 mRNA on RP11 in HCT-15 cells. i & j After treatment with Act-D for the indicated times, the mRNA levels of Siah1 (i) or Fbxo45 (j) in HCT15 RP11 stable overexpression and control cells were measured by qRT-PCR. k Binding between hnRNPA2B1 and Siah1 mRNA or between hnRNPA2B1 and Fbxo45 mRNA in HCT-15 RP11 stable overexpression and control cells was analysed by RIP-PCR. Data are presented as the mean ± SD from three independent experiments. **p < 0.01 compared with control