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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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The CCA sequence at the 3′-termini of all tRNAs is first cleaved by angiogenin in vitro.(A) Angiogenin (1 µM) digestion of total HeLa tRNA radioactively labeled at either 5′- or 3′-end. (B) 3′-radioactively labeled HeLa tRNAs treated with 1 µM angiogenin for different times and their intact 3′-termini were visualized with tRNA macroarrays. Only tRNAs (or fragments of them) with intact 3′-ends are visible on the microarrays. Two exemplary tRNAs (Ala-IGC, green and Gln-yTG, red) are marked. Probes for each tRNA are arranged in clusters of six replicates. (C) Analysis of the integrity of the 3′-CCA end of full-length tRNAs with the specific oligonucleotide-ligation approach after angiogenin (0.2 µM) treatment for various times. The gel was visualized with SYBR Green. Note, to better resolve the kinetics of cleavage we decreased the concentration of angiogenin to 0.2 µM; thus the time points here are not directly comparable with the time points in panels A, B. The numbers on the left denote the DNA ladder in nt. Multiple bands for tiRNAs, full-length tRNAs, ligated and unligated tRNAs are detected (panels A and C) due to the natural variations in tRNAs length.
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pgen-1003767-g002: The CCA sequence at the 3′-termini of all tRNAs is first cleaved by angiogenin in vitro.(A) Angiogenin (1 µM) digestion of total HeLa tRNA radioactively labeled at either 5′- or 3′-end. (B) 3′-radioactively labeled HeLa tRNAs treated with 1 µM angiogenin for different times and their intact 3′-termini were visualized with tRNA macroarrays. Only tRNAs (or fragments of them) with intact 3′-ends are visible on the microarrays. Two exemplary tRNAs (Ala-IGC, green and Gln-yTG, red) are marked. Probes for each tRNA are arranged in clusters of six replicates. (C) Analysis of the integrity of the 3′-CCA end of full-length tRNAs with the specific oligonucleotide-ligation approach after angiogenin (0.2 µM) treatment for various times. The gel was visualized with SYBR Green. Note, to better resolve the kinetics of cleavage we decreased the concentration of angiogenin to 0.2 µM; thus the time points here are not directly comparable with the time points in panels A, B. The numbers on the left denote the DNA ladder in nt. Multiple bands for tiRNAs, full-length tRNAs, ligated and unligated tRNAs are detected (panels A and C) due to the natural variations in tRNAs length.

Mentions: Intrigued by the different time scales of the stress-induced alterations of cellular tRNAs, we next analyzed the kinetics of 3′-CCA end cleavage and tiRNA generation in vitro. Total tRNA was isolated from confluent, non-stressed HeLa cells, radioactively labeled at their 5′- or the 3′-end and subsequently subjected to angiogenin treatment. Strikingly, while 5′-labeled tRNAs are still visible at 120 min of incubation with angiogenin, the 3′-labeled tRNAs completely disappeared after 30 min (Figure 2A). The fast decay of the signal of the 3′-labeled full length tRNAs than the 5′-labeled tRNAs (Figure 2A) is consistent with a preferred and much faster cleavage in the 3′-CCA termini of the tRNAs. All tRNAs were equally sensitive to angiogenin cleavage at the 3′-CCA termini; the signal for all 3′-radioactively labeled tRNAs decayed almost simultaneously during the angiogenin treatment (Figure 2B and S3). In the fluorescent oligonucleotide-ligation approach (Figure 1B, schematic inset), we observed a clear progressive decrease of the yield of the oligonucleotide ligated to the 3′-CCA end of the tRNAs upon angiogenin treatment (Figure 2C). Notably, fragments migrating at the height of the tiRNAs appeared much later (Figure 2C), suggesting that angiogenin degraded the 3′-CCA ends more rapidly than it cleaved tRNAs in the anticodon loop, thus recapitulating the observations in HeLa cells (Figure 1). To define the exact cleavage site in the 3′-CCA end, we used internally radioactively labeled variants of tRNAPhe(GAA) with intact 3′-CCA and truncated 3′-CC end. tRNAPhe(GAA) lacks the CA motif in the anticodon loop and hence, only the 3′-CCA terminus is susceptible to angiogenin cleavage. Angiogenin cleaved endonucleolytically within the 3′-CCA motif between the C and A nucleotide and removed exclusively the adenosine residue (Figure S4), implying a high CA-dependent endonucleolytic activity of angiogenin.


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)

The CCA sequence at the 3′-termini of all tRNAs is first cleaved by angiogenin in vitro.(A) Angiogenin (1 µM) digestion of total HeLa tRNA radioactively labeled at either 5′- or 3′-end. (B) 3′-radioactively labeled HeLa tRNAs treated with 1 µM angiogenin for different times and their intact 3′-termini were visualized with tRNA macroarrays. Only tRNAs (or fragments of them) with intact 3′-ends are visible on the microarrays. Two exemplary tRNAs (Ala-IGC, green and Gln-yTG, red) are marked. Probes for each tRNA are arranged in clusters of six replicates. (C) Analysis of the integrity of the 3′-CCA end of full-length tRNAs with the specific oligonucleotide-ligation approach after angiogenin (0.2 µM) treatment for various times. The gel was visualized with SYBR Green. Note, to better resolve the kinetics of cleavage we decreased the concentration of angiogenin to 0.2 µM; thus the time points here are not directly comparable with the time points in panels A, B. The numbers on the left denote the DNA ladder in nt. Multiple bands for tiRNAs, full-length tRNAs, ligated and unligated tRNAs are detected (panels A and C) due to the natural variations in tRNAs length.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1003767-g002: The CCA sequence at the 3′-termini of all tRNAs is first cleaved by angiogenin in vitro.(A) Angiogenin (1 µM) digestion of total HeLa tRNA radioactively labeled at either 5′- or 3′-end. (B) 3′-radioactively labeled HeLa tRNAs treated with 1 µM angiogenin for different times and their intact 3′-termini were visualized with tRNA macroarrays. Only tRNAs (or fragments of them) with intact 3′-ends are visible on the microarrays. Two exemplary tRNAs (Ala-IGC, green and Gln-yTG, red) are marked. Probes for each tRNA are arranged in clusters of six replicates. (C) Analysis of the integrity of the 3′-CCA end of full-length tRNAs with the specific oligonucleotide-ligation approach after angiogenin (0.2 µM) treatment for various times. The gel was visualized with SYBR Green. Note, to better resolve the kinetics of cleavage we decreased the concentration of angiogenin to 0.2 µM; thus the time points here are not directly comparable with the time points in panels A, B. The numbers on the left denote the DNA ladder in nt. Multiple bands for tiRNAs, full-length tRNAs, ligated and unligated tRNAs are detected (panels A and C) due to the natural variations in tRNAs length.
Mentions: Intrigued by the different time scales of the stress-induced alterations of cellular tRNAs, we next analyzed the kinetics of 3′-CCA end cleavage and tiRNA generation in vitro. Total tRNA was isolated from confluent, non-stressed HeLa cells, radioactively labeled at their 5′- or the 3′-end and subsequently subjected to angiogenin treatment. Strikingly, while 5′-labeled tRNAs are still visible at 120 min of incubation with angiogenin, the 3′-labeled tRNAs completely disappeared after 30 min (Figure 2A). The fast decay of the signal of the 3′-labeled full length tRNAs than the 5′-labeled tRNAs (Figure 2A) is consistent with a preferred and much faster cleavage in the 3′-CCA termini of the tRNAs. All tRNAs were equally sensitive to angiogenin cleavage at the 3′-CCA termini; the signal for all 3′-radioactively labeled tRNAs decayed almost simultaneously during the angiogenin treatment (Figure 2B and S3). In the fluorescent oligonucleotide-ligation approach (Figure 1B, schematic inset), we observed a clear progressive decrease of the yield of the oligonucleotide ligated to the 3′-CCA end of the tRNAs upon angiogenin treatment (Figure 2C). Notably, fragments migrating at the height of the tiRNAs appeared much later (Figure 2C), suggesting that angiogenin degraded the 3′-CCA ends more rapidly than it cleaved tRNAs in the anticodon loop, thus recapitulating the observations in HeLa cells (Figure 1). To define the exact cleavage site in the 3′-CCA end, we used internally radioactively labeled variants of tRNAPhe(GAA) with intact 3′-CCA and truncated 3′-CC end. tRNAPhe(GAA) lacks the CA motif in the anticodon loop and hence, only the 3′-CCA terminus is susceptible to angiogenin cleavage. Angiogenin cleaved endonucleolytically within the 3′-CCA motif between the C and A nucleotide and removed exclusively the adenosine residue (Figure S4), implying a high CA-dependent endonucleolytic activity of angiogenin.

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