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. 2012 Jul;9(7):990-1001.
doi: 10.4161/rna.20531. Epub 2012 Jul 1.

Translational infidelity-induced protein stress results from a deficiency in Trm9-catalyzed tRNA modifications

Ashish Patil  1 , Clement T Y Chan, Madhu Dyavaiah, John P Rooney, Peter C Dedon, Thomas J Begley
Affiliations

Translational infidelity-induced protein stress results from a deficiency in Trm9-catalyzed tRNA modifications

Ashish Patil et al. RNA Biol. 2012 Jul.

Abstract

Correct codon-anticodon pairing promotes translational fidelity, with these interactions greatly facilitated by modified nucleosides found in tRNA. We hypothesized that wobble uridine modifications catalyzed by tRNA methyltransferase 9 (Trm9) are essential for translational fidelity. In support, we have used phenotypic, reporter and protein-based assays to demonstrate increased translational infidelity in trm9Δ Saccharomyces cerevisiae cells. Codon reengineering studies suggest that Trm9-catalyzed tRNA modifications promote fidelity during the translation of specific genes, those rich in arginine and glutamic acid codons from mixed boxes. Using quantitative tRNA modification analysis, we determined that trm9Δ cells are only deficient in 2 of 23 tRNA modifications, with those 2, 5-methoxycarbonylmethyluridine (mcm ( 5) U) and 5-methoxycarbonylmethyl-2-thiouridine (mcm ( 5) s ( 2) U), classified as key determinants of translational fidelity. We also show that in the absence of mcm ( 5) U and mcm ( 5) s ( 2) U, the resulting translational infidelity promotes protein errors and activation of unfolded protein and heat shock responses. These data support a model in which Trm9-catalyzed tRNA modifications promote fidelity during the translation of specific transcripts, with decreased wobble base modification leading to translational infidelity, protein errors and activation of protein stress response pathways.

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Figures

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Figure 1. Molecular and viability phenotypes associated with translational infidelity identified in trm9Δ cells. (A) Wild-type (By4741), trm9Δ and trm9Δ + TRM9 cells were grown overnight in YPD at 30°C. Cultures were serially (10-fold) diluted and plated on YPD, YPD + paromomycin (350 ug/ml) or YPD + G418 (20 ug/ml). Figure S1 contains similar data for cells from the CEN.PK2–1C background. (B) Wild-type and trm9Δ cells from the BY4741 background were grown to ~5x106 cells/ml and were either mock treated or treated with paromomycin (1000 ug/ml) for 1 h. The cells were spun down and RNA was extracted using an RNeasy mini kit and used for northern blot analysis. (C) Wild-type and trm9Δ cells, both containing an integrated Pnc1-Tap tagged construct, were grown to ~5 x 106 cells/ml and left untreated or treated with 20 μg/ml of G418 for 60 min. Proteins were extracted and western blot analysis of Pnc1-TAP and tubulin was performed as described in Materials and Methods.
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Figure 2. Increased translational infidelity observed in trm9Δ cells. (A) On the left, the misincorporation construct containing Renilla luciferase attached by a linker region (black arrow) in frame to firefly luciferase. Misincorporation of an arginine in place of serine will result in firefly luciferase activity. On the right are four mixed codon boxes, with each box specific to two different amino acids. Codons that have the potential to interact with Trm9-modified tRNA’s are shaded gray. (B-E) Wild-type (white bars) and trm9Δ cells (black bars) from the CEN.PK2 background were transformed with misincorporation reporter plasmids and grown to ~5 x 106 cells / ml. The population was divided into equal halves and either mock or paromomycin (200 μg / ml) treated for 60 min. Cells were then spun down and firefly and Renilla luciferase activity was determined. (F-G) The frameshifting reporter system was modified to contain four GAG codons in the linker region connecting Renilla and firefly constructs. The Firefly reporter is either in the + 1 or -1 orientation, with frameshifting putting it in frame with Renilla luciferase. Assays were preformed similar to described for B-E.
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Figure 3. Some targets of Trm9-dependent translation are prone to errors. (A) Wild-type and trm9Δ cells in which either YEF3 or RNR1 genes were Tap tagged were grown to ~5 x 106 cells/ml and then treated with 1000 μg/ml of paromomycin for 1 h. Western blots performed using anti-Tap and anti-tubulin antibodies. (B) Wild-type and trm9Δ cells containing RNR1 or a codon-optimized RNR1 gene (Opt) were grown to ~5 x 106 cells/ml and then left untreated or treated with 1000 μg/ml of paromomycin for 1 h. Western blots performed using anti-Rnr1 and anti-tubulin antibodies. For both panels, we show representative blots (n = 3).
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Figure 4. Protein stress transcripts are upregulated in trm9Δ cells. (A) A Venn diagram demonstrating that transcripts which are basally upregulated in trm9Δ cells are also regulated by agents that promote protein and ER stress. (B) northern blot analysis of UBI1 encoding ubiquitin and (C) the heat-shock marker SSA4, before and after paromomycin treatment. For both blots, ACT1 serves as a loading control. For both panels, we show representative blots (n = 3).
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Figure 5. Ire1-dependent activation of the UPR identified in trm9Δ cells. (A) northern blots analyses of KAR2, HAC1 and ACT1 were performed as described in materials and methods, using cells left untreated or treated with paromomycin (200 μg/ml) for 60 min. We show a representative blot (n = 3). (B) β-galactosidase activity is a measure of the unfolded protein response. (C) Cells were grown to ~107 cells/ml, centrifuged to remove the media, proteins were extracted and β-galactosidase activity was determined. (D) Cells were grown to ~5 x 106 cells/ml, treated with 1000 μg/ml of paromomycin for 1 h and β-galactosidase activity was determined. For panels C-D, average values are shown with standard deviations (n = 3).
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Figure 6. Model describing the link between Trm9-catalyzed tRNA modifications, translational infidelity and protein stress. In trm9Δ cells, the absence of the mcm5U modification causes the tRNAUCUArg to base pair with its near cognate AGC Serine or AGU Serine codons. This mispairing causes serine to arginine amino acid misincorporation. The higher level of amino acid misincorporation gives rise to increased unfolded proteins, thus activating the UPR and heat shock pathways in trm9Δ cells. Frameshifting (not shown) could also lead to protein errors.
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References

    1. Agris PF. Decoding the genome: a modified view. Nucleic Acids Res. 2004;32:223–38. doi: 10.1093/nar/gkh185. - DOI - PMC - PubMed
    1. Agris PF. Bringing order to translation: the contributions of transfer RNA anticodon-domain modifications. EMBO Rep. 2008;9:629–35. doi: 10.1038/embor.2008.104. - DOI - PMC - PubMed
    1. Huang B, Lu J, Byström AS. A genome-wide screen identifies genes required for formation of the wobble nucleoside 5-methoxycarbonylmethyl-2-thiouridine in Saccharomyces cerevisiae. RNA. 2008;14:2183–94. doi: 10.1261/rna.1184108. - DOI - PMC - PubMed
    1. Huang BO, Johansson MJO, Byström AS. An early step in wobble uridine tRNA modification requires the Elongator complex. RNA. 2005;11:424–36. doi: 10.1261/rna.7247705. - DOI - PMC - PubMed
    1. Leihne V, Kirpekar F, Vågbø CB, van den Born E, Krokan HE, Grini PE, et al. Roles of Trm9- and ALKBH8-like proteins in the formation of modified wobble uridines in Arabidopsis tRNA. Nucleic Acids Res. 2011;39:7688–701. doi: 10.1093/nar/gkr406. - DOI - PMC - PubMed

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