Figure 5.

OCC-1 RNA associates with HuR protein. (A) RNA pull-down assay followed by western blot confirmed HuR as a protein partner binding specifically to OCC-1 3′UTR. EGFP RNA was used as a RNA control. ACTB and GAPDH are protein controls. (B) RIP confirmed the association between OCC-1 and HuR in Caco-2 cells. GAPDH mRNA was used as a non-HuR target control. (C) Schematic of the 3′UTRs of OCC-1 orthologues showing the HuR-binding motifs. The HuR-binding sites (HuR CLIP site) identified previously by a CLIP experiment in human OCC-1 3′UTR were also indicated. (D) Venn diagram demonstrating the significant enrichment of OCC-1-repressed genes in the set of HuR targets determined by the previous CLIP experiment. About 74% of OCC-1-repressed genes identified by microarray analysis were also HuR targets. (E) RIP assay revealed that the mRNAs of the six selected OCC-1-repressed genes also interact with HuR in Caco-2 cells. (F) The mRNA and protein level of HuR were markedly reduced by a HuR-targeting shRNA as determined by RT-qPCR (left) and western blot (right). The density of protein bands was measured by Image J software and the relative level of HuR protein was calculated after normalizing to ACTB protein. (G) RT-qPCR analysis revealed that OCC-1 and all six selected OCC-1-repressed genes were downregulated by HuR knockdown. Images are the result of one representative experiment. Data are presented as mean± standard deviation of three independent experiments. **P < 0.01.