Construction of a reporter gene that expresses mRNA, which contains no AUG codon in any reading frame. a, shown is a schematic drawing of the reporter mRNAs constructed. The filled box indicates the open reading frame of MPT4ΔN, the black box indicates the FLAG tag, the lines represent non-translated regions, and the tract of As denotes the poly(A) tail. AUG-MS2, No-AUG-MS2, and SL-AUG-MS2 reporter genes all contain the 3′-UTR region of PGK1 into which two tandem MS2 binding sites were inserted. In No-AUG-MS2, the codon UAC replaces AUG. In SL-AUG-MS2, a stable stem-loop structure for inhibition of translation (SL) was introduced into the 5′-UTR region. b, shown are expression levels of the reporter proteins. W303 cells harboring pIT2069 (AUG-FLAG-MS2, (AUG)) pIT2071 (No-AUG-FLAG-MS2, (No-AUG)) or pIT2073 (SL-AUG-FLAG-MS2, (SL-AUG)) plasmids were grown on SC-UraLeu medium containing 2% galactose. Samples were analyzed by Western blotting with anti-FLAG antibodies. c, shown is a schematic drawing of the reporter mRNAs containing FLAG sequences at the carboxyl terminal region of the open reading frame. d, shown are expression levels of the reporter proteins. W303 cells harboring pIT2074 (AUG-MPT4ΔN-FLAG-MS2, (AUG-C-FLAG-MS2)), pIT2075 (No-AUG-MPT4ΔN-FLAG-MS2, (No-AUG-C-FLAG-MS2)), or pIT2076 (SL-AUG-MPT4ΔN-FLAG-MS2, (SL-AUG-C-FLAG-MS2)) plasmids were grown on SC-UraLeu medium containing 2% galactose. Samples were analyzed by Western blotting with anti-FLAG antibodies. When indicated, cells were grown in the presence of 0.2 mm MG132. e, No-AUG-C-FLAG-MS2 and SL-AUG-C-FLAG-MS2 mRNAs are poorly translated. W303 cells harboring pIT2074 (AUG-C-FLAG-MS2), pIT2075 (No-AUG-C-FLAG-MS2), or pIT2076 (SL-AUG-C-FLAG-MS2) plasmids were grown on SC-UraLeu medium containing 2% galactose but lacking methionine and cysteine. The cells were pulse-labeled with ¹⁴C amino acids, and then immunoprecipitates were analyzed for associated radioactivity. Radioactivity was determined relative to the counts for AUG-C-FLAG-MS2, which were designated as 100%.

Stabilization of No-AUG-FLAG-MS2 mRNA by tethered Pab1p does not require an interaction between Pab1p and eIF4G. a, tethering of Pab1p without the RRM1–2 domains stabilizes non-translated mRNA containing no AUG codon. W303 cells harboring pIT2071 (No-AUG-FLAG-MS2, left) or pIT2070 (No-AUG-FLAG, right) were transformed with a control p415TEF1p-FLAG-MS2 plasmid or with plasmids expressing the indicated FLAG-MS2-Pab1p fusion proteins. The cells were grown in SC-UraLeu medium containing 2% galactose (SC-Gal UraLeu medium). The cells were re-suspended in medium containing 2% glucose to inhibit transcription from the GAL1 promoter and harvested at the indicated times. RNA samples were prepared, and the levels of the remaining mRNA were determined using Northern blot analysis with DIG-labeled MPT4 probe. The relative mRNA levels of the samples were determined by comparison with a series of 2-fold dilutions of samples time 0 (first through third lanes). These dilutions were used as standard curves as described previously (25). b, tethering of Pab1p stabilizes non-translated mRNA. W303 cells harboring pIT2071 (No-AUG-FLAG-MS2) or pIT2070 (No-AUG-FLAG) were transformed with the control p415TEF1p-FLAG-MS2 plasmid or with plasmids expressing the indicated FLAG-MS2-Pab1p fusion proteins. The cells were grown in SC-Gal UraLeu medium, and mRNA stability was measured as described in a. Relative quantities are shown as the mean values of three independent experiments. The half-lives of mRNAs are shown as the mean values of three independent experiments with S.D. c, tethering of Pab1p stabilizes translated mRNA. W303 cells harboring pIT2069 (AUG-FLAG-MS2) or pIT2068 (AUG-FLAG) were transformed with the control p415TEF1p-FLAG-MS2 plasmid or with plasmids expressing the indicated FLAG-MS2-Pab1p fusion proteins. The cells were grown in SC-Gal UraLeu medium, and mRNA stability was measured as described in a. d, tethering of Pab1–34Cp stabilizes non-translated mRNA in eIF4G1-ΔN300 mutant YAS2071 (W303tif4631⁻tif4632⁻ ptif4631-ΔN300TRPCEN) cells. YAS2071 cells, harboring pIT2071 (No-AUG-FLAG-MS2) or pIT2070 (No-AUG-FLAG), were transformed with a plasmid containing a FLAG-MS2-Pab1–34C fusion gene or with a control p415TEF1p-FLAG-MS2 plasmid. The cells were grown in SC-Gal UraLeu medium, and mRNA stability was measured as in a. e, tethering of Pab1–34Cp stabilizes translated mRNA in eIF4G1-ΔN300 mutant. YAS2071 (W303tif4631⁻tif4632⁻ ptif4631-ΔN300TRPCEN) cells harboring pIT2069 (AUG-FLAG-MS2) or pIT2068 (AUG-FLAG) plasmids were transformed with plasmids encoding the FLAG-MS2-Pab1–34C fusion gene or with the control p415TEF1p-FLAG-MS2 plasmid. The cells were grown in SC-Gal UraLeu medium, and mRNA stability was measured as described in a.

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.

Stabilization of non-translated mRNA by tethered Pab1–34Cp results in the accumulation of deadenylated mRNA. a, shown is a schematic drawing of the non-translated mRNAs and the antisense oligonucleotide used for the RNase H assay. The filled box indicates the open reading frame of MPT4ΔN, the black box indicates the FLAG tag, the lines represent non-translated regions, and the tract of As denotes the poly(A) tail. MS2 binding sites are indicated as stem-loop structures in the 3′-UTR. A stable stem-loop structure for inhibition of translation (SL) was introduced into the 5′-UTR of the AUG-FLAG-MS2. The probes for Northern hybridization are shown as dashed lines. The antisense oligonucleotide (AS1, 5′-CCAAAGATGGCAAGTTAGAAACGTCAATGT-3′) that was used for RNase H digestion is shown as a bold line. b, W303 cells harboring pIT2071 (No-AUG-FLAG-MS) or pIT2073 (SL-AUG-FLAG-MS2) were transformed with a control p415TEF1p-FLAG-MS2 plasmid or with a plasmid that encodes the FLAG-MS2-Pab1–34Cp fusion protein. Cells were grown in SC-Gal UraLeu medium, and RNA samples were prepared as in Fig. 3a. RNA samples of cells harboring a control p415TEF1p-FLAG-MS2 plasmid were prepared before (0 min) and 5 min (5′) after a shift to glucose medium. RNA samples of cells harboring a control a plasmid that encodes the FLAG-MS2-Pab1–34Cp fusion protein were prepared before (0 min) and 10 min (10′) after a shift to glucose medium. RNA samples were digested with RNase H after hybridization with the AS1 oligonucleotide (AS1) or with oligo dT (dT), and Northern blot analysis was subsequently performed with a DIG-labeled 3′ probe. The RNA size marker III (11373099910, Roche Applied Science) is loaded in lane 21.

Construction of MS2-Pab1 fusion proteins. a, shown is a schematic drawing of FLAG-MS2-Pab1p wild type and deletion mutant fusion proteins. The black box represents the FLAG tag. The white box appears to show MS2. The numbered boxes indicate the RRM domains of Pab1p, and the shaded box labeled with a C represents the carboxyl terminal domain of the poly(A)-binding protein. b, shown are expression levels of the FLAG-MS2-Pab1p fusion proteins. W303 cells harboring the indicated FLAG-MS2-Pab1p fusion genes, and pIT2069 (AUG-FLAG-MS2, (AUG)), pIT2071 (No-AUG-FLAG-MS2, (No-AUG)), or pIT2073 (SL-AUG-FLAG-MS2, (SL-AUG)) plasmids were grown on SC-UraLeu medium containing 2% galactose. Samples were analyzed by Western blotting using anti-FLAG antibodies.

Tethering of Pab1p stabilizes non-translated mRNAs without affecting association of the mRNA with the ribosome. W303 cells were co-transformed with the indicated reporter plasmids and a control p415TEF1p-MS2 plasmid or a plasmid that encodes the MS2-Pab1–34Cp fusion protein. Polysome analysis was then performed on cell extracts as described previously. RNA samples prepared from each fraction were analyzed using Northern blotting with a DIG labeled FLAG probe (5′-CTTGTCATCGTCGTCCTTGTAGTCCATACTAGT-3′).

Stabilization of non-translated mRNA by tethered Pab1p-34Cp in an xrn1Δ 5′ to 3′ mRNA degradation mutant requires a poly(A) tail. The cells were grown in SC-Gal UraLeu medium, and RNA samples were prepared and analyzed by Northern blotting 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, W303xrn1Δ 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, W303xrn1Δ cells harboring pIT2069 (AUG-FLAG-MS) or pIT2068 (AUG-FLAG) were transformed with control p415TEF1p-FLAG-MS2 plasmid or with a plasmid that encodes the FLAG-MS2-Pab1–34Cp fusion protein. c, W303xrn1Δ 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.

Tethering of Pab1p-34C stabilizes mRNAs that contain a stable stem-loop structure at the 5′-UTR. The cells were grown in SC-Gal UraLeu medium. RNA samples were prepared, and mRNA stability 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, W303 cells harboring the pIT2073 (SL-AUG-FLAG-MS2) plasmid 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 pIT2073 (SL-AUG-FLAG-MS2) were transformed with a control p415TEF1p-FLAG-MS2 plasmid or with a plasmid that encodes the FLAG-MS2-Pab1–34Cp fusion protein.