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. 2011 Sep;7(9):e1002258.
doi: 10.1371/journal.pgen.1002258. Epub 2011 Sep 1.

Elongator complex influences telomeric gene silencing and DNA damage response by its role in wobble uridine tRNA modification

Changchun Chen  1 , Bo Huang, Mattias Eliasson, Patrik Rydén, Anders S Byström
Affiliations

Elongator complex influences telomeric gene silencing and DNA damage response by its role in wobble uridine tRNA modification

Changchun Chen et al. PLoS Genet. 2011 Sep.

Abstract

Elongator complex is required for formation of the side chains at position 5 of modified nucleosides 5-carbamoylmethyluridine (ncm⁵U₃₄), 5-methoxycarbonylmethyluridine (mcm⁵U₃₄), and 5-methoxycarbonylmethyl-2-thiouridine (mcm⁵s²U₃₄) at wobble position in tRNA. These modified nucleosides are important for efficient decoding during translation. In a recent publication, Elongator complex was implicated to participate in telomeric gene silencing and DNA damage response by interacting with proliferating cell nuclear antigen (PCNA). Here we show that elevated levels of tRNA(Lys)(s²UUU), tRNA(Gln)(s²UUG), and tRNA(Glu)(s²UUC), which in a wild-type background contain the mcm⁵s²U nucleoside at position 34, suppress the defects in telomeric gene silencing and DNA damage response observed in the Elongator mutants. We also found that the reported differences in telomeric gene silencing and DNA damage response of various elp3 alleles correlated with the levels of modified nucleosides at U₃₄. Defects in telomeric gene silencing and DNA damage response are also observed in strains with the tuc2Δ mutation, which abolish the formation of the 2-thio group of the mcm⁵s²U nucleoside in tRNA(Lys)(mcm⁵s²UUU), tRNA(Gln)(mcm⁵s²UUG), and tRNA(Glu)(mcm⁵s²UUC). These observations show that Elongator complex does not directly participate in telomeric gene silencing and DNA damage response, but rather that modified nucleosides at U₃₄ are important for efficient expression of gene products involved in these processes. Consistent with this notion, we found that expression of Sir4, a silent information regulator required for assembly of silent chromatin at telomeres, was decreased in the elp3Δ mutants.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Increased levels of tRNALys s2 UUU, tRNAGln s2 UUG, and tRNAGlu s2 UUC suppress the telomeric silencing defect of Elongator mutants.
(A) The wild type (UMY2584) and elp3Δ (UMY3790) strains were 10-fold diluted, spotted on SC, SC-Ura and SC+5-FOA plates, and incubated at 30°C for 2 days. (B) The elp3Δ strain (UMY3790) with plasmids, pRS315-ELP3, pRS425-tK-tQ-tE or pRS425, were 10-fold diluted, spotted on SC-Leu, SC-Leu-Ura and SC-Leu+5-FOA plates, and incubated at 30°C for 2 days. (C) The wild type (UMY2584), elp1Δ (UMY3788), elp2Δ (UMY3789), elp4Δ (UMY3791), elp5Δ (UMY3792) and elp6Δ (UMY3793) with plasmids pRS425-tK-tQ-tE or pRS425 were treated as described in (B). Abbreviations for the tRNA genes encoding formula image, formula image and formula image are tK, tQ and tE, respectively.
Figure 2
Figure 2. Elevated levels of tRNALys s2 UUU, tRNAGln s2 UUG, and tRNAGlu s2 UUC suppress the HU sensitivity induced by Elongator mutants.
(A) The elp3Δ strain (UMY2843) carrying plasmids pRS315-ELP3, pRS425-tK-tQ-tE or pRS425 were 10-fold diluted, spotted on SC-Leu and SC-Leu+50 mM HU plates, and incubated at 30°C for 2 days. (B) The wild type (W303-1A), elp1Δ (UMY3783), elp2Δ (UMY3784), elp4Δ (UMY3785), elp5Δ (UMY3786) and elp6Δ (UMY3787) strains transformed with plasmids pRS425-tK-tQ-tE or pRS425 were assayed as described in (A). Abbreviations for the tRNA genes encoding tRNALys UUU, tRNAGln UUG, and tRNAGlu UUC are tK, tQ and tE, respectively.
Figure 3
Figure 3. Increased levels of tRNALys s2 UUU, tRNAGln s2 UUG, and tRNAGlu s2 UUC bypass the phenotypes of asf1Δ elp3Δ double mutants.
(A) The asf1Δ elp3Δ strain (UMY3805) was transformed with pRS315-ELP3, pRS425-tK-tQ-tE or pRS425. Transformants were streaked on SC-Leu plates and incubated at 30°C or 37°C for 2 days. (B) Ten fold dilutions of elp3Δ (UMY2843), asf1Δ (UMY3800) and asf1Δ elp3Δ (UMY3805) strains carrying either pRS425-tK-tQ-tE or pRS425 were spotted on SC-Leu and SC-Leu+50 mM HU plates, and incubated 4 days at 30°C. The elp3Δ (UMY2843) transformed with pRS315-ELP3 was used as control. Abbreviations for the tRNA genes encoding tRNALys UUU, tRNAGln UUG, and tRNAGlu UUC are tK, tQ and tE, respectively.
Figure 4
Figure 4. Strains carrying different ELP3 mutant alleles show decreased telomeric gene silencing and increased HU sensitivity.
(A) Schematic drawing of the protein structure of Elp3. Orange box represents the radical-S-adenosyl methionine (Radical-SAM) domain and blue box indicates the location of the histone acetyltransferase (HAT) domain. Cysteine residues at position 103, 108, 118 or 121 were substituted with alanines. Glycine residues at position 168 or 180 together with 181 were replaced by arginines. Two tyrosine residues, positions 540 or 541, in the HAT domain were substituted with alanines. (B) The wild type and the different mutant alleles of the ELP3 gene, located in LEU2 containing vector pRS315, were transformed into the elp3Δ strain (UMY3790). The elp3Δ strain (UMY3790) carrying a pRS315 without insertion serves as control. The transformed yeast cells were spotted on SC-Leu, SC-Leu-Ura and SC-Leu+5-FOA plates, and incubated at 30°C for 2 days. (C) The elp3Δ strain (UMY2843) transformed with the same set of plasmids as in (B) were spotted on SC-Leu, SC-Leu+50 mM hydroxyurea plates, and incubated at 30°C for 2 days.
Figure 5
Figure 5. HPLC analysis of total tRNAs isolated from mutants with different alleles of ELP3.
HPLC chromatograms of total tRNA isolated from SUP4 (UMY2894), elp3Δ SUP4 (UMY2915), elp3-C103A SUP4 (UMY3314), elp3-C108A SUP4 (UMY3315), elp3-C118A SUP4 (UMY3316), elp3-C121A SUP4 (UMY3317), elp3-G168R SUP4 (UMY3794), elp3-G180R G181R SUP4 (UMY3795), elp3-Y540A SUP4 (UMY3060) and elp3-Y541A SUP4 (UMY3061). Chromatograms were monitored at 314 nm. The parts of chromatograms between retention times 43 and 52 min are displayed. The arrows in B, D, E, F and H indicate the expected retention time of mcm5s2U.
Figure 6
Figure 6. U34 modification levels influence ochre stop codon read through by a suppressor tRNA.
(A) Schematic drawing of the dual luciferase reporter system constructed by Keeling et al . The sequence of the read through cassette between Renilla and firefly luciferase genes is shown. The XXX in red stands for either UAA in the assay plasmid or CAA in the control plasmid. (B) Read through levels of the UAA stop codon in SUP4 (UMY2894), elp3-C103A SUP4 (UMY3314), elp3-G168R SUP4 (UMY3794), elp3-Y541A SUP4 (UMY3060), elp3-Y541A SUP4 (UMY3061) and elp3Δ SUP4 (UMY2915). Values are ratios of Firefly to Renilla luciferase activities and based on three independent experiments. The error bars represent the standard deviation. Values were normalized to the wild type SUP4 (UMY2894), which was arbitrarily set to 1.
Figure 7
Figure 7. The tuc2Δ strain is deficient in telomeric gene silencing and show increased HU sensitivity.
(A) The wild type strain (UMY2584) harboring plasmid pRS425 and the tuc2Δ mutant (UMY3804) harboring plasmids pRS425-tK-tQ-tE or pRS425 were assayed as described in Figure 1. (B) The wild type strain (UMY2067) harboring plasmid pRS425 and the tuc2Δ mutant (UMY3442) harboring plasmids pRS425-tK-tQ-tE or pRS425 were assayed as described in Figure 2. Abbreviations for the tRNA genes encoding formula image, formula image and formula image are tK, tQ and tE, respectively.
Figure 8
Figure 8. Sir4 protein levels are decreased in the elp3Δ mutant.
(A). Western blot analysis of Sir4-Myc protein levels in the elp3Δ strain transformed with plasmids pRS315-ELP3, pRS425-tK-tQ-tE or pRS425. The ratios of Sir4-Myc to Actin signals were calculated. The values are shown relative to elp3Δ pRS315-ELP3 strain, which was arbitrarily set to 1, and are the average of two independent experiments. (B). Northern blot analysis of SIR4 mRNA. The elp3Δ strain was transformed with plasmids pRS315-ELP3, pRS425-tK-tQ-tE or pRS425. Signals of SIR4 mRNAs were normalized to the non-coding SCR1 transcript. The values are shown relative to elp3Δ pRS315-ELP3 strain, which was arbitrarily set to 1, and are the average of two independent experiments. (C). The wild type strain (UMY2584) transformed with plasmids pRS424-SIR4 or pRS424, and the elp3Δ mutant (UMY3790) with plasmids pRS424-SIR4 or pRS424 were assayed as described in Figure 1. For A, B and C, representative figures are shown. Abbreviations for the tRNA genes encoding formula image, formula image and formula image are tK, tQ and tE, respectively.
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References

    1. Otero G, Fellows J, Li Y, de Bizemont T, Dirac AM, et al. Elongator, a multisubunit component of a novel RNA polymerase II holoenzyme for transcriptional elongation. Mol Cell. 1999;3:109–118. - PubMed
    1. Winkler GS, Petrakis TG, Ethelberg S, Tokunaga M, Erdjument-Bromage H, et al. RNA polymerase II elongator holoenzyme is composed of two discrete subcomplexes. J Biol Chem. 2001;276:32743–32749. - PubMed
    1. Krogan NJ, Greenblatt JF. Characterization of a six-subunit holo-elongator complex required for the regulated expression of a group of genes in Saccharomyces cerevisiae. Mol Cell Biol. 2001;21:8203–8212. - PMC - PubMed
    1. Hawkes NA, Otero G, Winkler GS, Marshall N, Dahmus ME, et al. Purification and characterization of the human elongator complex. J Biol Chem. 2002;277:3047–3052. - PubMed
    1. Kim JH, Lane WS, Reinberg D. Human Elongator facilitates RNA polymerase II transcription through chromatin. Proc Natl Acad Sci U S A. 2002;99:1241–1246. - PMC - PubMed

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