Fig. 3.

TOP2A mRNA interacts with components of the microRNA decay machinery and with miR-548c-3p. (A) Forty-eight hours after transfection of either Ctrl siRNA or HuR siRNA, the interaction of TOP2A mRNA with Ago2-containing complexes was assessed by RNP IP (see Materials and Methods). (Right) Western blot analysis of Ago2 in the IP samples. (B) Schematic of plasmids expressing MS2-tagged RNA [pMS2, pMS2-TOP2A(3′)] and expressing a fluorescent reporter protein (pMS2-YFP). (C) HeLa cells were transfected with the siRNAs indicated; 48 h later, cells were transfected with the plasmids shown in panel B, and 24 h after that, cells were fixed for analysis. Fluorescent signals from MS2-YFP (green) as well as from DCP1a (red, detected by immunofluorescence) (see Materials and Methods) were analyzed by confocal microscopy. Merged images show the overlap between MS2-YFP and DCP1a; foci of colocalized signals (yellow) are indicated with arrowheads. (Graph) The numbers of cells showing overlapping MS2-YFP and DCP1a signals in each transfection group were counted and plotted. (D) In cells transfected as described for panel C, the interaction of the indicated microRNAs was tested by RNP IP analysis using anti-YFP antibody. The levels of microRNAs in IP samples were assessed by RT-qPCR analysis and plotted as enrichment in the pMS2-TOP2A(3′)-transfected cells relative to pMS2-transfected cells. (E) TOP2A 3′UTR depicting the miR-548c-3p site (red) and the HuR CLIP sites (blue); box, complementarity between TOP2A mRNA and miR-548c-3p. In panels A, C, and D, data are the means + SD from 3 independent experiments. *, P < 0.05; **, P < 0.01.