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Reversible and rapid transfer-RNA deactivation as a mechanism of translational repression in stress.

Czech A, Wende S, Mörl M, Pan T, Ignatova Z - PLoS Genet. (2013)

Bottom Line: Stress-induced changes of gene expression are crucial for survival of eukaryotic cells.This CCA deactivation is reversible and quickly repairable by the CCA-adding enzyme [ATP(CTP):tRNA nucleotidyltransferase].Through this mechanism the eukaryotic cell dynamically represses and reactivates translation at low metabolic costs.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biochemistry and Biology, University of Potsdam, Potsdam, Germany.

ABSTRACT
Stress-induced changes of gene expression are crucial for survival of eukaryotic cells. Regulation at the level of translation provides the necessary plasticity for immediate changes of cellular activities and protein levels. In this study, we demonstrate that exposure to oxidative stress results in a quick repression of translation by deactivation of the aminoacyl-ends of all transfer-RNA (tRNA). An oxidative-stress activated nuclease, angiogenin, cleaves first within the conserved single-stranded 3'-CCA termini of all tRNAs, thereby blocking their use in translation. This CCA deactivation is reversible and quickly repairable by the CCA-adding enzyme [ATP(CTP):tRNA nucleotidyltransferase]. Through this mechanism the eukaryotic cell dynamically represses and reactivates translation at low metabolic costs.

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Scanning-independent translation initiation is less influenced by low dose oxidative stress.(A) Polysomal profiles of untreated or arsenite treated HeLa cells. (B) Schematic of the plasmid used to monitor cap-dependent and IRES-dependent translation initiation. Inhibition of cap-dependent, Rluc (C) or IRES-mediated, Fluc (D) translation upon exposure to oxidative stress. HeLa cells expressing the bicistronic construct encoding Rluc under the CMV-promoter (scanning-dependent translation) and Fluc under the CrPV-IRES (non-scanning controlled translation) were exposed to different arsenite concentrations for various times. Addition of DMSO to the cells served as a control. Data in (C) and (D) ± SEM are normalized to the first data point for which the activity was set as 100.
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pgen-1003767-g004: Scanning-independent translation initiation is less influenced by low dose oxidative stress.(A) Polysomal profiles of untreated or arsenite treated HeLa cells. (B) Schematic of the plasmid used to monitor cap-dependent and IRES-dependent translation initiation. Inhibition of cap-dependent, Rluc (C) or IRES-mediated, Fluc (D) translation upon exposure to oxidative stress. HeLa cells expressing the bicistronic construct encoding Rluc under the CMV-promoter (scanning-dependent translation) and Fluc under the CrPV-IRES (non-scanning controlled translation) were exposed to different arsenite concentrations for various times. Addition of DMSO to the cells served as a control. Data in (C) and (D) ± SEM are normalized to the first data point for which the activity was set as 100.

Mentions: The 3′-CCA ends are indispensable for tRNA aminoacylation and subsequently for translation. What is the effect of angiogenin-induced deactivation of the 3′-CCA termini of cellular tRNAs on protein translation? Exposure of HeLa cells to acute oxidative stress (500 µM arsenite) altered the polysomal profile and shut down translation (Figure 4A). Importantly, at low arsenite concentration (100 µM) the cells retained some translation activity, detectable as a considerable polysomal fraction (Figure 4A). The most potent inhibition of translation is mediated by eIF2α phosphorylation upon oxidative stress via haem-regulated inhibitor kinase (HRI), which represses translation of mRNAs with scanning- or cap-dependent translation initiation [3]. By contrast, a sizeable subset of genes are translated through a cap-independent mechanism: internal ribosome-entry sites (IRES) direct translation initiation without the aid of canonical initiation factors and initiator Met-tRNA [28]. We hypothesized that cap-dependent translation will be influenced at much lower arsenite concentrations compared to mRNAs with scanning-independent initiation; the combined effect of oxidative stress on eIF2α phosphorylation and the deactivation of the 3′-CCA ends of all tRNAs will have much higher impact on mRNAs initiated in a scanning-dependent manner. In contrast, in the IRES-initiated translation, as only the 3′-CCA-end inactivation should play a role the effect should be less pronounced. We therefore tested the effect of two arsenite concentrations, representing severe (500 µM) and moderate (100 µM) oxidative stress using bicistronic mRNA encoding renilla luciferase (Rluc), initiated in a cap-controlled manner, and firefly luciferase (Fluc), initiated via cricket paralysis virus IRES (CrPV-IRES) (Figure 4B), an IRES sequence described to confer translation independent of any initiation factor [29]. At a low arsenite concentration (100 µM), the Fluc activity remained at >80%, while Rluc activity progressively decreased, indicating much potent inhibition of cap-dependent initiation compared to IRES-dependent initiation (Figure 4C,D). At a high arsenite concentration (500 µM), however, a similar decrease for both Rluc and Fluc activity was observed, implying that both IRES-dependent and scanning-controlled initiation were equally inhibited (Figure 4C,D). This cannot be attributed to the decrease of mRNA levels, since the mRNA expression levels of the bicistronic construct remained similar upon stress exposure (Figure S6). Variations in the transfection efficiency are not likely; transfection efficiency was equal in all experiments as assessed with fluorescent reporter. This suggests that under acute oxidative stress translation of all mRNAs is globally repressed, while moderate oxidative stress affects more strongly the cap-dependent than the IRES-controlled initiation due to the combined effect on eIF2α phosphorylation and the tRNAs deactivation.


Reversible and rapid transfer-RNA deactivation as a mechanism of translational repression in stress.

Czech A, Wende S, Mörl M, Pan T, Ignatova Z - PLoS Genet. (2013)

Scanning-independent translation initiation is less influenced by low dose oxidative stress.(A) Polysomal profiles of untreated or arsenite treated HeLa cells. (B) Schematic of the plasmid used to monitor cap-dependent and IRES-dependent translation initiation. Inhibition of cap-dependent, Rluc (C) or IRES-mediated, Fluc (D) translation upon exposure to oxidative stress. HeLa cells expressing the bicistronic construct encoding Rluc under the CMV-promoter (scanning-dependent translation) and Fluc under the CrPV-IRES (non-scanning controlled translation) were exposed to different arsenite concentrations for various times. Addition of DMSO to the cells served as a control. Data in (C) and (D) ± SEM are normalized to the first data point for which the activity was set as 100.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3757041&req=5

pgen-1003767-g004: Scanning-independent translation initiation is less influenced by low dose oxidative stress.(A) Polysomal profiles of untreated or arsenite treated HeLa cells. (B) Schematic of the plasmid used to monitor cap-dependent and IRES-dependent translation initiation. Inhibition of cap-dependent, Rluc (C) or IRES-mediated, Fluc (D) translation upon exposure to oxidative stress. HeLa cells expressing the bicistronic construct encoding Rluc under the CMV-promoter (scanning-dependent translation) and Fluc under the CrPV-IRES (non-scanning controlled translation) were exposed to different arsenite concentrations for various times. Addition of DMSO to the cells served as a control. Data in (C) and (D) ± SEM are normalized to the first data point for which the activity was set as 100.
Mentions: The 3′-CCA ends are indispensable for tRNA aminoacylation and subsequently for translation. What is the effect of angiogenin-induced deactivation of the 3′-CCA termini of cellular tRNAs on protein translation? Exposure of HeLa cells to acute oxidative stress (500 µM arsenite) altered the polysomal profile and shut down translation (Figure 4A). Importantly, at low arsenite concentration (100 µM) the cells retained some translation activity, detectable as a considerable polysomal fraction (Figure 4A). The most potent inhibition of translation is mediated by eIF2α phosphorylation upon oxidative stress via haem-regulated inhibitor kinase (HRI), which represses translation of mRNAs with scanning- or cap-dependent translation initiation [3]. By contrast, a sizeable subset of genes are translated through a cap-independent mechanism: internal ribosome-entry sites (IRES) direct translation initiation without the aid of canonical initiation factors and initiator Met-tRNA [28]. We hypothesized that cap-dependent translation will be influenced at much lower arsenite concentrations compared to mRNAs with scanning-independent initiation; the combined effect of oxidative stress on eIF2α phosphorylation and the deactivation of the 3′-CCA ends of all tRNAs will have much higher impact on mRNAs initiated in a scanning-dependent manner. In contrast, in the IRES-initiated translation, as only the 3′-CCA-end inactivation should play a role the effect should be less pronounced. We therefore tested the effect of two arsenite concentrations, representing severe (500 µM) and moderate (100 µM) oxidative stress using bicistronic mRNA encoding renilla luciferase (Rluc), initiated in a cap-controlled manner, and firefly luciferase (Fluc), initiated via cricket paralysis virus IRES (CrPV-IRES) (Figure 4B), an IRES sequence described to confer translation independent of any initiation factor [29]. At a low arsenite concentration (100 µM), the Fluc activity remained at >80%, while Rluc activity progressively decreased, indicating much potent inhibition of cap-dependent initiation compared to IRES-dependent initiation (Figure 4C,D). At a high arsenite concentration (500 µM), however, a similar decrease for both Rluc and Fluc activity was observed, implying that both IRES-dependent and scanning-controlled initiation were equally inhibited (Figure 4C,D). This cannot be attributed to the decrease of mRNA levels, since the mRNA expression levels of the bicistronic construct remained similar upon stress exposure (Figure S6). Variations in the transfection efficiency are not likely; transfection efficiency was equal in all experiments as assessed with fluorescent reporter. This suggests that under acute oxidative stress translation of all mRNAs is globally repressed, while moderate oxidative stress affects more strongly the cap-dependent than the IRES-controlled initiation due to the combined effect on eIF2α phosphorylation and the tRNAs deactivation.

Bottom Line: Stress-induced changes of gene expression are crucial for survival of eukaryotic cells.This CCA deactivation is reversible and quickly repairable by the CCA-adding enzyme [ATP(CTP):tRNA nucleotidyltransferase].Through this mechanism the eukaryotic cell dynamically represses and reactivates translation at low metabolic costs.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biochemistry and Biology, University of Potsdam, Potsdam, Germany.

ABSTRACT
Stress-induced changes of gene expression are crucial for survival of eukaryotic cells. Regulation at the level of translation provides the necessary plasticity for immediate changes of cellular activities and protein levels. In this study, we demonstrate that exposure to oxidative stress results in a quick repression of translation by deactivation of the aminoacyl-ends of all transfer-RNA (tRNA). An oxidative-stress activated nuclease, angiogenin, cleaves first within the conserved single-stranded 3'-CCA termini of all tRNAs, thereby blocking their use in translation. This CCA deactivation is reversible and quickly repairable by the CCA-adding enzyme [ATP(CTP):tRNA nucleotidyltransferase]. Through this mechanism the eukaryotic cell dynamically represses and reactivates translation at low metabolic costs.

Show MeSH
Related in: MedlinePlus