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. 2013 May 10;288(24):17832–17843. doi: 10.1074/jbc.M112.448977

FIGURE 4.

FIGURE 4.

Non-stop mRNA decay requires the exosome-Ski complex. A, down-regulation of Ski2 expression. HeLa cells were transfected with either control luciferase siRNA (lane 1) or Ski2 siRNA-1 (Ski2(#1); lane 2). Total cell lysate was analyzed by Western blotting (WB) using the indicated antibodies. B, down-regulation of Mtr4 expression. HeLa cells were transfected with control luciferase siRNA or Mtr4 siRNA-1 (Mtr4(#1)). Total RNA isolated from the cells was analyzed by real-time PCR. The levels of Mtr4 and GAPDH mRNAs were quantitated. Results are representative of three independently performed experiments. C, down-regulation of either Ski2 or Mtr4 expression partially inhibits fast degradation of non-stop mRNA. HeLa cells were transfected with p5×FLAG-EGFP; pT7-TR; either pFBG control (lanes 1–5, 11–15, and 21–25) or pFBG non-stop (lanes 6–10, 16–20, and 26–30); and control luciferase siRNA (lanes 1–10), Ski2 siRNA-1 (lanes 11–20), or Mtr4 siRNA-1 (lanes 21–30). The transcriptional pulse-chase analysis was performed as described in the legend to Fig. 3. D, the levels of FBG mRNAs were quantitated as described in the legend to Fig. 2. E, down-regulation of Ski2/Mtr4 expression inhibits fast degradation of non-stop mRNA. HeLa cells were transfected with p5×FLAG-EGFP, pT7-TR, either pFBG control (lanes 1–5 and 11–15) or pFBG non-stop (lanes 6–10 and 16–20), and either control luciferase siRNA (lanes 1–10) or Ski2 siRNA-1/Mtr4 siRNA-1 (lanes 11–20). The transcriptional pulse-chase analysis was performed as described in the legend to Fig. 2, except that the siRNAs were cotransfected. F, the levels of FBG mRNAs were quantitated as described in the legend to Fig. 2. G, down-regulation of Ski2/Mtr4 expression. HeLa cells were transfected with control luciferase siRNA (lane 1) or Ski2 siRNA-1/Mtr4 siRNA-1 (lane 2). Total cell lysate was analyzed by Western blotting using the indicated antibodies. H, the half-lives of FBG mRNAs were calculated. The asterisks denote the levels of statistical significance (Student's t test) between luciferase siRNA and either Ski2 or Mtr4 siRNA: *, p < 0.05; **, p < 0.01.