Figure 5

Tethering Stau2 to the 3'-UTR increases mRNA levels in 293F cells. (A) Schematic representation of the GFP-8xMS2bs mRNA. The 293F-GFP-8xMS2bs cell line stably expresses a reporter mRNA (driven by the TK promoter) encoding GFP with 8xMS2bs in its 3' UTR (B) Either MS2 or MS2-Stau2 was expressed in 293F-GFP-8xMS2bs cells. The cells were harvested at 48 h or 72 h after transfection and analyzed by Western blotting using anti-GFP and anti-Tubulin-α antibodies. The intensity of the GFP signal was normalized to that of Tubulin-α and is presented relative to MS2. (C) The expression of GFP in 293F-GFP-8xMS2bs cells transfected with an MS2 or MS2-Stau2 expression plasmid was observed by fluorescence microscopy at 72 h after transfection. The GFP signal intensities were analyzed and shown by surface plot. (D) MS2-fusion proteins were expressed in 293F-GFP-8xMS2bs cells. At 48 h after transfection, the cells were harvested and proteins and RNAs were extracted. The proteins were analyzed by Western blotting using anti-HA and anti-GAPDH antibodies. The GFP and GAPDH mRNAs were quantitated by Northern blotting using radiolabeled probes. (E) The GFP-8xMS2bs mRNA signal was normalized to that of GAPDH mRNA, and the normalized level of the MS2 transfection was defined as 1. Values are expressed as the means ± SEM of three independent experiments.