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. 2019 Dec 21;7(4):1900949. doi: 10.1002/advs.201900949

Figure 4.

Figure 4

Circ‐MALAT1 obstructs PAX5 translation by binding to PAX5 coding sequence and ribosomes. A) RNA fluorescence in situ hybridization for circ‐MALAT1. Nuclei were stained with DAPI. Green staining signal against circ‐MALAT1 was localized in cytoplasm. Scale bar, 10 µm. B) The level of PAX5 mRNA was analyzed by qRT‐PCR. Circ‐MALAT1 overexpression (circ‐MALAT1 O/E) led to no significant difference in PAX5 mRNA level compared to the control (Vector). Control primers, GAPDH. C) An interaction model showing that circ‐MALAT1 recognized PAX5 mRNA via 11 bp bases paring (red site) and competitively inhibited PAX5 translation through IRESs (yellow sites). D) Left panel, RIP lysates prepared from circ‐MALAT1 overexpressed Huh7 cells were subjected to immunoprecipitation using either a normal rabbit IgG or anti‐RPS6 antibody. Purified RNA was analyzed by qRT‐PCR using primers specific for MALAT1, circ‐MALAT1 and PAX5, respectively. Middle panel, RIP was performed using lysates prepared from Huh7 cells overexpressing wild type (WT), IRES1‐mutated (IRES1 MUT) and IRES2‐mutated (IRES2 MUT) circ‐MALAT1, respectively. Purified RNA was analyzed by qRT‐PCR using primers specific for circ‐MALAT1. Right panel, the model above the dashed line (indicating the result in the left panel of (D) showed that circ‐MALAT1 with wild type of IRES (WT, “1” and “2” overlapped yellow sites) could bind to ribosome while the model below the dashed line (indicating the result in the middle panel of (D) showed that circ‐MALAT1 with mutated IRES (MUT) could not bind to ribosome. E) In vivo RNA pull‐down using PAX5 specific probes was performed in circ‐MALAT1 WT or 11 bp MUT overexpressed Huh7 cells, followed by qRT‐PCR to detect PAX5 (left panel) and circ‐MALAT1 (middle panel). Right panel, the interaction model between circ‐MALAT1 and the CDS region of PAX5 mRNA via 11 bp bases paring (red site). F) PAX5 and its downstream protein p53 were detected by western blot. Vector, empty vector overexpression; circ‐MALAT1 O/E, circ‐MALAT1 overexpression. G) PAX5 and p53 were analyzed in CSCs and adherent cells of Hep3B cell line by western blot. CSCs were enriched by the tumorsphere assay. H) PAX5 was detected in Huh7 cells overexpressing circ‐MALAT1 wild (WT O/E) or mutant type (11 bp MUT O/E, IRES1 MUT O/E, and IRES2 MUT O/E) or none (Vector) by western blot. Columns, means from three independent experiments; bars, SD. ** p < 0.01, * p < 0.05.