FIGURE 5.

Stabilization of non-translated mRNA by tethered Pab1–34Cp in a ski2Δ mutant requires a cap structure but not a poly(A) tail. The cells were grown in SC-Gal UraLeu medium, and RNA samples were prepared and analyzed by Northern blotting with a DIG labeled MPT4 probe. The stability of the mRNAs was measured as described in Fig. 3a. Relative quantities are shown as the mean values of three independent experiments. The half-lives of mRNAs are shown as the mean values with S.D. a, W303ski2Δ cells harboring pIT2071 (No-AUG-FLAG-MS) or pIT2070 (No-AUG-FLAG) were transformed with a control p415TEF1p-FLAG-MS2 plasmid or with a plasmid that encodes the FLAG-MS2-Pab1–34Cp fusion protein. b, W303ski2Δ cells harboring pIT2069 (AUG-FLAG-MS) or pIT2068 (AUG-FLAG) were transformed with a control p415TEF1p-FLAG-MS2 plasmid or with a plasmid that encodes the FLAG-MS2-Pab1–34Cp fusion protein. c, schematic drawing of the reporter mRNAs lacking a poly(A) tail or cap structure. The filled box indicates the open reading frame of MPT4ΔN, and the dark gray box indicates the FLAG tag. These boxes are shaded because these regions are not translated. The lines represent non-translated regions. The 3′-UTR region of the reporter genes contains the PGK1 3′-UTR in which two tandem MS2 binding sites were inserted. Rz indicates the sequences of a hammerhead ribozyme. d, W303ski2Δ cells harboring pIT2085 (No-AUG-FLAG-MS2-Rz) or pIT2084 (No-AUG-FLAG-Rz) were transformed with a control p415TEF1p-FLAG-MS2 plasmid or with a plasmid that encodes the FLAG-MS2-Pab1–34Cp fusion protein. e, W303ski2Δ cells harboring pIT2087 (Rz-No-AUG-FLAG-MS2-Rz) or the pIT2086 (Rz-No-AUG-FLAG-Rz) were transformed with a control p415TEF1p-FLAG-MS2 plasmid or with a plasmid that encodes the FLAG-MS2-Pab1–34Cp fusion protein.