Abstract
Ubiquitin-like proteins (UBLs) can change protein function, localization or turnover by covalent attachment to lysine residues1. Although UBLs achieve this conjugation through an intricate enzymatic cascade, their bacterial counterparts MoaD and ThiS function as sulphur carrier proteins2,3. Here we show that Urm1p, the most ancient UBL3, acts as a sulphur carrier in the process of eukaryotic transfer RNA (tRNA) modification, providing a possible evolutionary link between UBL and sulphur transfer. Moreover, we identify Uba4p, Ncs2p, Ncs6p and Yor251cp as components of this conserved pathway. Using in vitro assays, we show that Ncs6p binds to tRNA, whereas Uba4p first adenylates and then directly transfers sulphur onto Urm1p. Finally, functional analysis reveals that the thiolation function of Urm1p is critical to regulate cellular responses to nutrient starvation and oxidative stress conditions, most likely by increasing translation fidelity.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 51 print issues and online access
$199.00 per year
only $3.90 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Haglund, K. & Dikic, I. Ubiquitylation and cell signaling. EMBO J. 24, 3353–3359 (2005)
Jentsch, S. & Pyrowolakis, G. Ubiquitin and its kin: how close are the family ties? Trends Cell Biol. 10, 335–342 (2000)
Xu, J. et al. Solution structure of Urm1 and its implications for the origin of protein modifiers. Proc. Natl Acad. Sci. USA 103, 11625–11630 (2006)
Furukawa, K., Mizushima, N., Noda, T. & Ohsumi, Y. A protein conjugation system in yeast with homology to biosynthetic enzyme reaction of prokaryotes. J. Biol. Chem. 275, 7462–7465 (2000)
Goehring, A. S., Rivers, D. M. & Sprague, G. F. Attachment of the ubiquitin-related protein Urm1p to the antioxidant protein Ahp1p. Eukaryot. Cell 2, 930–936 (2003)
Goehring, A. S., Rivers, D. M. & Sprague, G. F. Urmylation: a ubiquitin-like pathway that functions during invasive growth and budding in yeast. Mol. Biol. Cell 14, 4329–4341 (2003)
Rubio-Texeira, M. Urmylation controls Nil1p and Gln3p-dependent expression of nitrogen-catabolite repressed genes in Saccharomyces cerevisiae. FEBS Lett. 581, 541–550 (2007)
Jeong, J. S., Kwon, S. J., Kang, S. W., Rhee, S. G. & Kim, K. Purification and characterization of a second type thioredoxin peroxidase (type II TPx) from Saccharomyces cerevisiae. Biochemistry 38, 776–783 (1999)
Huang, B., Lu, J. & Bystrom, A. S. A genome-wide screen identifies genes required for formation of the wobble nucleoside 5-methoxycarbonylmethyl-2-thiouridine in Saccharomyces cerevisiae. RNA 14, 2183–2194 (2008)
Frohloff, F., Fichtner, L., Jablonowski, D., Breunig, K. D. & Schaffrath, R. Saccharomyces cerevisiae Elongator mutations confer resistance to the Kluyveromyces lactis zymocin. EMBO J. 20, 1993–2003 (2001)
Fichtner, L. et al. Elongator’s toxin-target (TOT) function is nuclear localization sequence dependent and suppressed by post-translational modification. Mol. Microbiol. 49, 1297–1307 (2003)
Lu, J., Huang, B., Esberg, A., Johansson, M. J. & Bystrom, A. S. The Kluyveromyces lactis gamma-toxin targets tRNA anticodons. RNA 11, 1648–1654 (2005)
Ashraf, S. S. et al. Single atom modification (O→S) of tRNA confers ribosome binding. RNA 5, 188–194 (1999)
Esberg, A., Huang, B., Johansson, M. J. & Bystrom, A. S. Elevated levels of two tRNA species bypass the requirement for elongator complex in transcription and exocytosis. Mol. Cell 24, 139–148 (2006)
Igloi, G. L. Interaction of tRNAs and of phosphorothioate-substituted nucleic acids with an organomercurial. Probing the chemical environment of thiolated residues by affinity electrophoresis. Biochemistry 27, 3842–3849 (1988)
Umeda, N. et al. Mitochondria-specific RNA-modifying enzymes responsible for the biosynthesis of the wobble base in mitochondrial tRNAs. Implications for the molecular pathogenesis of human mitochondrial diseases. J. Biol. Chem. 280, 1613–1624 (2005)
Jager, G., Leipuviene, R., Pollard, M. G., Qian, Q. & Bjork, G. R. The conserved Cys-X1–X2-Cys motif present in the TtcA protein is required for the thiolation of cytidine in position 32 of tRNA from Salmonella enterica serovar Typhimurium. J. Bacteriol. 186, 750–757 (2004)
Bjork, G. R., Huang, B., Persson, O. P. & Bystrom, A. S. A conserved modified wobble nucleoside (mcm5s2U) in lysyl-tRNA is required for viability in yeast. RNA 13, 1245–1255 (2007)
Dewez, M. et al. The conserved Wobble uridine tRNA thiolase Ctu1-Ctu2 is required to maintain genome integrity. Proc. Natl Acad. Sci. USA 105, 5459–5464 (2008)
Shigi, N., Sakaguchi, Y., Suzuki, T. & Watanabe, K. Identification of two tRNA thiolation genes required for cell growth at extremely high temperatures. J. Biol. Chem. 281, 14296–14306 (2006)
Kambampati, R. & Lauhon, C. T. MnmA and IscS are required for in vitro 2-thiouridine biosynthesis in Escherichia coli. Biochemistry 42, 1109–1117 (2003)
Mueller, E. G. & Palenchar, P. M. Using genomic information to investigate the function of ThiI, an enzyme shared between thiamin and 4-thiouridine biosynthesis. Protein Sci. 8, 2424–2427 (1999)
Leimkuhler, S., Wuebbens, M. M. & Rajagopalan, K. V. Characterization of Escherichia coli MoeB and its involvement in the activation of molybdopterin synthase for the biosynthesis of the molybdenum cofactor. J. Biol. Chem. 276, 34695–34701 (2001)
Palenchar, P. M., Buck, C. J., Cheng, H., Larson, T. J. & Mueller, E. G. Evidence that ThiI, an enzyme shared between thiamin and 4-thiouridine biosynthesis, may be a sulfurtransferase that proceeds through a persulfide intermediate. J. Biol. Chem. 275, 8283–8286 (2000)
Taylor, S. V. et al. Thiamin biosynthesis in Escherichia coli. Identification of this thiocarboxylate as the immediate sulfur donor in the thiazole formation. J. Biol. Chem. 273, 16555–16560 (1998)
Schmitz, J. et al. The sulfurtransferase activity of Uba4 presents a link between ubiquitin-like protein conjugation and activation of sulfur carrier proteins. Biochemistry 47, 6479–6489 (2008)
Pedrioli, P. G. et al. Automated identification of SUMOylation sites using mass spectrometry and SUMmOn pattern recognition software. Nature Methods 3, 533–539 (2006)
Marelja, Z., Stocklein, W., Nimtz, M. & Leimkuhler, S. A novel role for human Nfs1 in the cytoplasm: Nfs1 acts as a sulfur donor for MOCS3, a protein involved in molybdenum cofactor biosynthesis. J. Biol. Chem. 283, 25178–25185 (2008)
Williams, C., van den Berg, M., Sprenger, R. R. & Distel, B. A conserved cysteine is essential for Pex4p-dependent ubiquitination of the peroxisomal import receptor Pex5p. J. Biol. Chem. 282, 22534–22543 (2007)
Isel, C. et al. Specific initiation and switch to elongation of human immunodeficiency virus type 1 reverse transcription require the post-transcriptional modifications of primer tRNA3Lys. EMBO J. 15, 917–924 (1996)
Tong, A. H. et al. Systematic genetic analysis with ordered arrays of yeast deletion mutants. Science 294, 2364–2368 (2001)
Tong, A. H. et al. Global mapping of the yeast genetic interaction network. Science 303, 808–813 (2004)
Tong, A. H. Y., Boone, C., Stansfield, I. & Michael, J. R. S. 16 High-throughput strain construction and systematic synthetic lethal screening. Meths Microbiol. 36, 369–386, 706–707 (2007)
Garcia, O. et al. GOlorize: a Cytoscape plug-in for network visualization with Gene Ontology-based layout and coloring. Bioinformatics 23, 394–396 (2007)
Bjork, G. R. et al. A primordial tRNA modification required for the evolution of life? EMBO J. 20, 231–239 (2001)
Suzuki, T. & Suzuki, T. Chaplet column chromatography: isolation of a large set of individual RNAs in a single step. Meths Enzymol. 425, 231–239 (2007)
Lau, N. C., Lim, L. P., Weinstein, E. G. & Bartel, D. P. An abundant class of tiny RNAs with probable regulatory roles in Caenorhabditis elegans. Science 294, 858–862 (2001)
Kosower, N. S., Kosower, E. M., Wertheim, B. & Correa, W. S. Diamide, a new reagent for the intracellular oxidation of glutathione to the disulfide. Biochem. Biophys. Res. Commun. 37, 593–596 (1969)
Wanke, V. et al. Caffeine extends yeast lifespan by targeting TORC1. Mol. Microbiol. 69, 277–285 (2008)
Kunz, J. et al. Target of rapamycin in yeast, TOR2, is an essential phosphatidylinositol kinase homolog required for G1 progression. Cell 73, 585–596 (1993)
Braun, P. et al. Proteome-scale purification of human proteins from bacteria. Proc. Natl Acad. Sci. USA 99, 2654–2659 (2002)
Brenner, S. The genetics of Caenorhabditis elegans. Genetics 77, 71–94 (1974)
Gehrke, C. W., Kuo, K. C., McCune, R. A., Gerhardt, K. O. & Agris, P. F. Quantitative enzymatic hydrolysis of tRNAs: reversed-phase high-performance liquid chromatography of tRNA nucleosides. J. Chromatogr. 230, 297–308 (1982)
Pedrioli, P. G. et al. A common open representation of mass spectrometry data and its application to proteomics research. Nature Biotechnol. 22, 1459–1466 (2004)
Craig, R. & Beavis, R. C. TANDEM: matching proteins with tandem mass spectra. Bioinformatics 20, 1466–1467 (2004)
MacLean, B., Eng, J. K., Beavis, R. C. & McIntosh, M. General framework for developing and evaluating database scoring algorithms using the TANDEM search engine. Bioinformatics 22, 2830–2832 (2006)
Nesvizhskii, A. I., Keller, A., Kolker, E. & Aebersold, R. A statistical model for identifying proteins by tandem mass spectrometry. Anal. Chem. 75, 4646–4658 (2003)
Keller, A., Nesvizhskii, A. I., Kolker, E. & Aebersold, R. Empirical statistical model to estimate the accuracy of peptide identifications made by MS/MS and database search. Anal. Chem. 74, 5383–5392 (2002)
Acknowledgements
We thank C. Rupp for technical support, M. Sohrmann for yeast strains, G. Rabut for the gift of the pGR51 plasmid, G. L. Igloi and K.-H. Altmann for providing APM, S. Mitani for tut-1(tm1297), B. Casado-Fernandez and D. Hilvert for discussion and technical advice, and T. Kurz and J. D. Alfonzo for reading the manuscript. This work was supported by grants from EuroDyna (to S.L.), the Roche Research Foundation (to P.G.A.P.), ETHZ (to S.L. and M.P.), the SNF (to M.P.), the Canadian Institutes of Health Research (to C.B.) and Genome Canada through the Ontario Genomics Institute (to C.B.).
Author information
Authors and Affiliations
Corresponding authors
Supplementary information
Supplementary Information
This file contains Supplementary Figures S1-S8 and Supplementary Tables 1-5 (PDF 5933 kb)
Rights and permissions
About this article
Cite this article
Leidel, S., Pedrioli, P., Bucher, T. et al. Ubiquitin-related modifier Urm1 acts as a sulphur carrier in thiolation of eukaryotic transfer RNA. Nature 458, 228–232 (2009). https://doi.org/10.1038/nature07643
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/nature07643
This article is cited by
-
Modulation of translational decoding by m6A modification of mRNA
Nature Communications (2023)
-
Unconventional secretion of Magnaporthe oryzae effectors in rice cells is regulated by tRNA modification and codon usage control
Nature Microbiology (2023)
-
CHIKV infection reprograms codon optimality to favor viral RNA translation by altering the tRNA epitranscriptome
Nature Communications (2022)
-
A novel splice variant of Elp3/Kat9 regulates mitochondrial tRNA modification and function
Scientific Reports (2022)
-
Epigenetic activation of the elongator complex sensitizes gallbladder cancer to gemcitabine therapy
Journal of Experimental & Clinical Cancer Research (2021)
Comments
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.