FIGURE 2.

miRNA-targeted messages are bound to ER-attached polysomes. A, schematic representation of the isolation of polysomes attached to the rER. B, levels of Ago2 and miRNA in KCl-puromycin extract or rER. Ribosomal protein S3 (Rib. protein S3), eIF4E, eEF2, and ER integral membrane protein calnexin were used to monitor the extraction of rER-attached ribosome without an effect in ER integrity. C, level of miRNA-targeted message in KCl-puromycin extract and residual pellet fraction. D, 15–55% sucrose density gradient fractions of isolated microsomes of HEK293 cells expressing FLAG-HA-Ago2 (FA-Ago2). Endogenous let-7a level and FH-Ago2 were detected in different fractions by Northern and Western blot, respectively. The relative presence of 28s RNA was determined in individual fractions by semi-quantitative RT-PCR, and absorptions at 260 nm were plotted. Positions of the fractions with polysomes are marked. Frac. No., fraction number. E, levels of RL-HMGA2-3′-UTR or RL-3×bulge-let-7a mRNAs were quantified by RT-qPCR in isolated polysomal fractions in HEK293 cells. RL-HMGA2 3′-UTR-mut or RL-con were taken as a control. In all RT-qPCR experiments, 18s rRNA served as the endogenous control. RT-qPCR results from three independent experiments ± S.D. are shown, and the values of control are normalized to 1 (*, p < 0.05; **, p < 0.01; ***, p < 0.001).