Translation of the poly(A) tail plays crucial roles in nonstop mRNA surveillance via translation repression and protein destabilization by proteasome in yeast

Abstract

mRNA surveillance system represses the expression of nonstop mRNA by rapid mRNA degradation and translation repression. Here we show that the level of protein product of nonstop mRNA containing a poly(A) tail was reduced 100-fold, and this reduction was due to rapid mRNA degradation, translation repression, and protein destabilization, at least in part, by the proteasome. Insertion of a poly(A) tract upstream of a termination codon resulted in translation repression and protein destabilization, but not rapid mRNA decay. We propose that translation of the poly(A) tail plays crucial roles in nonstop mRNA surveillance via translation repression and protein destabilization.

Figure 1.

Multiple steps are involved in the repression of nonstop gene expression. (A) Schematic drawing of the construction of fusion genes. The boxes indicate the ORFs and the stretch of A indicates a poly(A) tail on reporter mRNAs. (B) Translation repression and post-translational regulation are involved in the reduced expression of nonstop mRNA. W303 cells were transformed with pGPDp-GFP-HIS3 (WT, −2A) or pGPDp-GFP-HIS3-NS (NS, −2A) or pGPDp-GFP-2A-HIS3 (WT, +2A) or pGPDp-GFP-2A-HIS3-NS (NS, +2A). Cells were grown in SC-Ura, and samples were prepared as described previously (Inada and Aiba 2005). The levels of proteins were analyzed by Western blotting with anti-GFP antibody (left panel) or anti-Flag antibody (right panel). The samples were applied after 10-fold dilution when indicated. (C) The quantification of GFP-2A and GFP-2A-His3 products derived from pGPDp-GFP-2A-HIS3 (WT) or pGPDp-GFP-2A-HIS3-NS (NS) genes. (Left) Protein samples prepared from cells harboring indicated plasmids were diluted fourfold and analyzed by Western blotting with anti-GFP antibody. (Right) The relative levels of each product normalized to the level of GFP-2A-His3 as 100% are shown as the mean values of three independent experiments with standard deviations. (D) The insertion of 2A sequence has little effect on the nonstop mRNA level. RNA samples prepared from cells shown in B were diluted twofold (5–1.25 μg) and analyzed by Northern blotting with GFP and SCR probes. (Lane 1) Numbers immediately below the figure represent the level of mRNA normalized to the level of GFP-HIS3 mRNA as 100%. The numbers are shown as the mean values of three independent experiments.

Figure 2.

Protein product of nonstop mRNA is destabilized by the proteasome. (A) The product of nonstop mRNA is unstable. (Top panels) Samples of W303 cells harboring an indicated plasmid were prepared at indicated times after the addition of cycloheximide (0.1 mg/mL). The levels of the remaining proteins were determined by Western blotting. The samples of cells harboring pGPDp-GFP-2A-HIS3 (WT) were applied after 10-fold dilution. (Bottom panels) The relative levels of proteins were quantified and shown as a function of time after the inhibition of translation by cycloheximide (CHX). Relative amounts are shown as the mean values of three independent experiments with standard deviations. (B) MG132, a proteasome-specific inhibitor, stabilizes the products of nonstop mRNA. (Top panels) W303erg6Δ cells harboring an indicated plasmid were cultured and samples were prepared 2 h after the addition of MG132 at the indicated concentration. The samples of cells harboring pGPDp-GFP-2A-HIS3 (WT) were applied after 10-fold dilution. (Bottom panels) The qualification of GFP-2A-His3 and GFP-2A-His3-NS levels. The relative numbers are shown as the mean values of three independent experiments with standard deviations.

Figure 3.

The insertion of a poly(A) tract upstream of a termination codon reduced the expression. (A) Translation repression and protein destabilization by the insertion of a poly(A) tract upstream of a termination codon. (Left panel) The levels of proteins in W303 cells harboring indicated plasmid were analyzed as described in Figure 1B. (Lane 1) pGPDp-GFP-HIS. (Lane 2) pGPDp-GFP-HIS3-K12PGDGS. (Lane 3) pGPDp-GFP-HIS3-K12. (Lane 4) pGPDp-GFP-2A-HIS3. (Lane 5) pGPDp-GFP-2A-HIS3-K12PG DGS. (Lane 6) pGPDp-GFP-2A-HIS3-K12. (Middle panel) Protein samples prepared from cells harboring pGPDp-GFP-2A-HIS3 (WT) or pGPDp-GFP-2A-HIS3-K12 (K12) were diluted fourfold and analyzed by Western blotting with anti-GFP antibody. (Right panel) The relative levels of each product normalized to the level of GFP-2A-His3 as 100% are shown as the mean values of three independent experiments with standard deviations. (B) The insertion of the poly(A) tract does not affect the mRNA level. W303 cells described in A were cultured, and the levels of reporter mRNAs were determined by Northern blotting with GFP probe. (C) Effect of the number of inserted lysine residues at C terminus on the expression. W303 cells were transformed with a series of reporter genes (pGPDp-Flag-HIS3-Kn) encoding proteins bearing an indicated number of lysine residues at C terminus. The levels of protein products were determined with Western blotting, and numbers immediately below the figure represent the level of protein normalized to the level of His3p as 100%. (D) An amino acid sequence is a determinant to reduce the level of protein produced from mRNA containing the poly(A) tract in a coding region. (Left) The levels of proteins in W303 cells harboring pGPDp-GFP-2A-HIS3 (WT), pGPDp-GFP-2A-HIS3-K12 [K12(AAA)], pGPDp-GFP-2A-HIS3-K12(AAG) [K12(AAG)], or pGPDp-GFP-2A-HIS3-K12(AAG)FS [K12(AAG)FS] plasmid were analyzed as described in Figure 1C. (Right) The relative levels of each product normalized to the level of GFP-2A-His3 as 100% are shown as the mean values of three independent experiments with standard deviations.

Figure 4.

Translation of the poly(A) sequence results in the destabilization of products by the proteasome. (A) MG132 increased the level of the protein bearing a polylysine. Samples of W303erg6Δ cells harboring pGPDp-GFP-2A-HIS3-K12 were prepared 2 h after the addition of MG132 at the indicated concentration. The relative levels of GFP-2A-His3-K12 are shown as the mean values of three independent experiments with standard deviations. (B) The induction of tUb6 increased the levels of GFP-2A-His3-NS and GFP-2A-His3-K12 products. W303 cells harboring pKK43 (p424GAL1p-tUB6) were transformed with pGPDp-GFP-2A-HIS3 (WT) pGPDp-GFP-2A-HIS3-NS (NS), or pGPDp-GFP-2A-HIS3-K12 (K12). Cells were grown in SC medium containing 2% raffinose until OD₆₀₀ = 0.8, and samples were prepared for Western blotting. When indicated (+), galactose was added to induce the expression of tUb6 that inhibits the proteasome activity. Numbers immediately below the figure represent the ratio of the full-length protein levels under tUb6 induction condition to noninduction condition. The level of GFP-2A derived from GFP-2A-HIS3-NS was increased 1.5-fold by tUb6 overproduction. The numbers are shown as the mean values of three independent experiments. (C) Stability of GFP-2A-His3-K12 protein measured by Western blotting after the inhibition of translation. Samples of W303 cells harboring a pGPDp-GFP-2A-HIS3-K12 plasmid were prepared at indicated times after the addition of cycloheximide (0.1 mg/mL). The levels of the remaining proteins were determined by Western blotting. (D) Stability of translation products of GFP-2A-HIS3-K12 measured by pulse-chase analysis. W303 cells harboring pGPDp-GFP-2A-HIS3 (WT) or pGPDp-GFP-2A-HIS3-K12 (K12) were labeled with [³⁵S]methionine and cysteine (25 μCi/mL) for 1 min and chase for 20 min. Cells were harvested and cell extracts were prepared for immunoprecipitation with anti-GFP antibody. Samples were subjected to SDS-PAGE, and the radioactivity was measured using Typhoon9400 (GE).