Upf1 strongly downregulates the level of proteins produced from HIS3 genes containing a PTC at specific positions. (A) A schematic of the reporter FLAG–HIS3 gene that expresses mRNAs containing a PTC at the indicated codon number. The filled box indicates the open reading frame of HIS3 and the open box indicates the FLAG tag; lines represent untranslated regions, and AAAAA denotes the poly(A) tail. (B) The ability of Upf1 to downregulate aberrant mRNAs is increased, depending on the length of the 3′-UTR. The reporter mRNA levels were determined by northern blot analysis with a DIG-labelled HIS3 probe. The relative levels for each mRNA were normalized to the FLAG–HIS3 mRNA level in wild-type cells, which was assigned a value of 100, and SCR RNA levels were used as a loading control for RNA samples. The mean values of three independent experiments are shown. (C) Upf1 downregulates PTC product levels in a PTC position-specific manner. Protein levels were determined by quantitative western blot analysis using secondary antibodies conjugated to quantum dots (Invitrogen, Carlsbad, CA, USA). The relative levels of each product were normalized to FLAG–His3 protein levels in wild-type cells (assigned a value of 100). The mean values of three independent experiments are shown. (D) The strong downregulation of FLAG–His3-100 or FLAG–His3-157 protein levels by Upf1 is partly suppressed in the presence of MG132. W303 (+) or W303upf1Δ (Δ) cells harbouring the plasmids shown in (A) were grown in SC-Ura (BD Difco, Detroit, MI, USA) in the presence of 0.2 mM MG132. Protein levels were determined as described in (C). DIG, digoxigenin; mRNA, messenger RNA; PTC, premature termination codon; SCR, RNA subunit of signal recognition particle; SC-Ura, synthetic complete medium lacking uracil; Upf1, up frameshift protein 1; 3′-UTR, 3′ untranslated region; wt, wild type.

The stability of the FLAG–His3-100 protein is decreased in cells expressing Upf1. (A) MG132 treatment does not affect the stability of FLAG–his3-100 mRNA in wild-type and upf mutants. W303 or W303upf1Δ, W303upf2Δ or W303upf3Δ cells harbouring pGAL1p-his3-100 were grown in SC-Ura (BD Difco, Detroit, MI, USA) containing 2% galactose. Cells were collected at the indicated times, after the addition of glucose to inhibit transcription from the GAL1 promoter. RNA samples were analysed by northern blot, as shown in Fig 1B. Relative quantities are shown as the mean values of three independent experiments with s.d. values. (B) FLAG–His3-100 is more stable in upf mutants. Pulse–chase experiments were carried out using W303 or W303upf1Δ, W303upf2Δ or W303upf3Δ cells harbouring pGPD-FLAG–his3-100, as described in the Methods section. When indicated, cells were grown in the presence of 0.2 mM MG132. (C) The relative levels of protein were shown as a function of the chase time. Relative amounts are shown as the mean values of three independent experiments with s.d. values. GAL1, galactokinase; GPD, glycerol-3-phosphate dehydrogenase; mRNA, messenger RNA; SC-Ura, synthetic complete medium lacking uracil; SCR, RNA subunit of signal recognition particle; Upf1, up frameshift protein 1.

Upf1 downregulates His3-100 levels depending on faux 3′-UTR. (A) Schematic drawing of the mRNA derived from FLAG–HIS3 reporter genes. Filled boxes indicate the open reading frame, the open box indicates FLAG tag, lines represent UTRs and AAAAA denotes the poly(A) tail. (B) Upf1 downregulates aberrant mRNAs with long 3′-UTRs. W303 (+) or W303upf1Δ (Δ) cells harbouring the plasmids shown in (A) were grown and RNA samples were prepared. Relative mRNA levels were determined as described in Fig 1B. (C) Upf1 strongly reduces the levels of the FLAG–His3-100 protein in the presence of a long 3′-UTR. Relative protein levels were determined as described in Fig 1C. (D) The stability of the FLAG–His3-100 protein derived from pGPD-FLAG–his3-100-WUTR is unaffected by Upf1. Top: pulse–chase experiments were carried out using W303 or W303upf1Δ cells harbouring pGPD-FLAG–his3-100-WUTR. Bottom: relative levels of proteins were shown as a function of chase time. (E) Schematic for the mRNAs expressed from FLAG–PGK1 reporter genes constructed as described in the supplementary information online. (F) Upf1 downregulates aberrant PGK1 mRNAs with a long 3′-UTR. W303 or W303upf1Δ cells were transformed with the indicated plasmids. The levels of reporter mRNAs were analysed by northern blotting with a DIG-labelled anti-FLAG LNA oligonucleotide probe as described in the supplementary information online. The relative levels of each RNA product normalized to the FLAG–PGK1 mRNA level in wild-type cells (assigned a value of 100) are shown as mean values from three independent experiments. (G) Upf1 strongly reduces the level of the Pgk1-300 protein in a long 3′-UTR-dependent manner. W303 or W303upf1Δ cells containing indicated plasmids were grown in SC-Ura medium (BD Difco, Detroit, MI, USA). Samples were prepared, and protein levels were analysed by western blotting using a FLAG antibody. The relative levels of each protein product, normalized to the FLAG–Pgk1 protein level in wild-type cells (assigned a value of 100), are shown as mean values from three independent experiments. The asterisk indicates the endogenous products that are recognized by quantum dot streptavidin conjugates. DIG, digoxigenin; LNA, locked nucleic acid; mRNA, messenger RNA; PGK1, 3-phosphoglycerate kinase 1; SC-Ura, synthetic complete medium lacking uracil; SCR, RNA subunit of signal recognition particle; Upf1, up frameshift protein 1; 3′-UTR, 3′ untranslated region.

Upf1 destabilizes the Pgk1-300 protein depending on proteasome activity. (A) Upf1 destabilizes the FLAG–Pgk1-300 protein. Samples of W303 (top panel) or W303upf1Δ (bottom panel) cells containing a pGPD-FLAG-pgk1-300 or pGPD-FLAG-PGK1 plasmid were prepared at the indicated times after the addition of cycloheximide (0.5 mg/ml). The levels of the remaining proteins were determined by quantum dot western blotting using a FLAG antibody. The asterisk indicates the endogenous products that are recognized by quantum dot streptavidin conjugates. (B) The half-life of reporter proteins. The levels of remaining FLAG–Pgk1 or FLAG–Pgk1-300 protein after inhibition of translation elongation were determined. The mean±s.d. values obtained from at least three independent experiments are shown. (C) The Pgk1-300 protein is stabilized by the addition of MG132. W303 cells containing a pGPD-FLAG-pgk1-300 plasmid were grown in the presence of 0.2 mM MG132, and samples were prepared and analysed by western blot analysis. (D) Upf1 destabilizes the FLAG–Pgk1-300 protein depending on a faux 3′-UTR. The stability of the FLAG–Pgk1-300 protein derived from FLAG-pgk1-300-WUTR mRNA in W303 or W303upf1Δ cells was measured, as shown in (A). (E) The half-life of Pgk1-300 reporter proteins derived from pgk1-300-WUTR reporter gene. The levels of the remaining FLAG–Pgk1-300 protein after inhibition of translation elongation were determined, as shown in (A). GPD, glycerol-3-phosphate dehydrogenase; PGK1, 3-phosphoglycerate kinase 1; Upf1, up frameshift protein 1; 3′-UTR, 3′ untranslated region.