Upf1 stimulates degradation of the product derived from aberrant messenger RNA containing a specific nonsense mutation by the proteasome

Aberrant messenger RNAs containing a premature termination codon (PTC) are eliminated by the nonsense-mediated mRNA decay (NMD) pathway. Here, we show that a crucial NMD factor, up frameshift 1 protein (Upf1), is required for rapid proteasome-mediated degradation of an aberrant protein (PTC product) derived from a PTC-containing mRNA. Western blot and pulse–chase analyses revealed that Upf1 stimulates the degradation of specific PTC products by the proteasome. Moreover, the Upf1-dependent, proteasome-mediated degradation of the PTC product was also stimulated by mRNAs harbouring a faux 3′ untranslated region (3′-UTR). These results indicate that protein stability might be regulated by an aberrant mRNA 3′-UTR

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

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

Translation Inhibitors Induce Formation of Cholesterol Ester-Rich Lipid Droplets

<div><p>Lipid droplets (LDs) in non-adipocytes contain triglycerides (TG) and cholesterol esters (CE) in variable ratios. TG-rich LDs are generated when unsaturated fatty acids are administered, but the conditions that induce CE-rich LD formation are less well characterized. In the present study, we found that protein translation inhibitors such as cycloheximide (CHX) induced generation of CE-rich LDs and that TIP47 (perilipin 3) was recruited to the LDs, although the expression of this protein was reduced drastically. Electron microscopy revealed that LDs formed in CHX-treated cells possess a distinct electron-dense rim that is not found in TG-rich LDs, whose formation is induced by oleic acid. CHX treatment caused upregulation of mTORC1, but the CHX-induced increase in CE-rich LDs occurred even when rapamycin or Torin1 was given along with CHX. Moreover, the increase in CE was seen in both wild-type and autophagy-deficient Atg5-null mouse embryonic fibroblasts, indicating that mTORC1 activation and suppression of autophagy are not necessary to induce the observed phenomenon. The results showed that translation inhibitors cause a significant change in the lipid ester composition of LDs by a mechanism independent of mTORC1 signaling and autophagy.</p> </div

CHX treatment caused an increase in cellular cholesterol ester content.

<p>(A) Thin layer chromatography (TLC) of the total lipid extract from 3Y1 cells. Cholesterol esters (CE) increased significantly after treatment with 10 µg/ml CHX for 18 hr, whereas triglycerides (TG) did not show a significant change. In contrast, in cells treated with 0.4 mM OA, TG rather than CE increased. Lipids extracted from cells with an equal protein content were compared. (B) Quantification of TG, CE, and free cholesterol (FC) in 3Y1 cells. The experimental conditions were the same as in (A). CHX increased CE significantly, whereas OA induced a prominent increase in TG. The FC content was equivalent in the three samples. Mean ± standard deviation (SD) is shown. The difference from the control sample was examined by Student’s <i>t</i> test (*<i>p</i><0.01, **<i>p</i><0.05). (C) Time course of the CE increase after CHX treatment. The increase in CE in 3Y1 cells was detectable by means of TLC as early as 4 hr after treatment with 10 µg/ml CHX. (D) TLC of the total lipid extract from Huh7 and 293A cells treated without or with 10 µg/ml CHX for 18 hr. The increase in CE was observed in both cell types. (E) Quantification of CE and FC in Huh7 cells that were treated without or with 0.25 mM methyl-β-cyclodextrin-cholesterol complex (MβCD-FC) or with 10 µg/ml CHX for 18 hr. Both treatments increased CE without affecting the FC content. Mean ± SD is shown. The difference from the control sample was examined by Student’s <i>t</i> test (*<i>p</i><0.01).</p

CHX caused the CE increase even in autophagy-deficient cells.

<p>(A) 3Y1 cells were treated without or with either 10 µg/ml CHX or 0.4 mM OA for 18 hr. CHX caused a significant increase in phospho-S6K. (B) The autophagic flux was examined by incubating 3Y1 cells with 20 µM chloroquine for 1 hr immediately before sample preparation. Chloroquine caused a significant increase in LC3-II in the control, but not in cells pretreated with 10 µg/ml CHX for 18 hr. (C) Wild-type and Atg5-null MEF were treated without or with 10 µg/ml CHX for 18 hr. The increase in phospho-S6K was observed in a comparable degree in both cell lines. GAPDH was probed as a loading control. (D) Wild-type and Atg5-null MEF were treated without or with 10 µg/ml CHX for 18 hr. CE increased significantly in response to CHX treatment in both cell lines. Mean ± SD is shown. The difference from the respective control was examined by Student’s <i>t</i> test (*<i>p</i><0.01).</p

CHX induced increases in LDs and CE even in the presence of mTORC1 inhibitors.

<p>(A) 3Y1 cells were treated without or with 10 µg/ml CHX, 10 µg/ml CHX and 0.4 µM rapamycin, or 10 µg/ml CHX and 0.25 µM Torin1 for 8 hr. Addition of rapamycin or Torin1 decreased phospho-S6K significantly. GAPDH is shown as a loading control. Each lane was loaded with 20 µg protein. (B) 3Y1 cells were treated with 10 µg/ml CHX alone or together with 0.4 µM rapamycin or 0.25 µM Torin1 for 18 hr. LDs increased to a similar degree irrespective of the presence of rapamycin or Torin1. Bar, 10 µm. (C) 3Y1 cells were treated without or with 10 µg/ml CHX, 10 µg/ml CHX and 0.4 µM rapamycin, or 10 µg/ml CHX and 0.25 µM Torin1 for 18 hr. CE increased in response to CHX treatment even when rapamycin or Torin1 was given simultaneously. Mean ± SD is shown. The difference from the control sample was examined by Student’s <i>t</i> test (*<i>p</i><0.01). (D) 3Y1 cells were treated in the same manner as in Fig. 5A. The autophagic flux was examined by adding 20 µM chloroquine for 1 hr immediately before sample preparation. A low level of LC3-II increase was caused by chloroquine in samples treated with CHX alone or CHX and rapamycin, but not in samples treated with CHX and Torin1. GAPDH is shown as a loading control. Each lane was loaded with 50 µg protein.</p