Logo of rnaThe RNA SocietyeTOC AlertsSubscriptionsJournal HomeCSHL PressRNA
RNA. Mar 2002; 8(3): 370–381.
PMCID: PMC1370258

A conserved family of Saccharomyces cerevisiae synthases effects dihydrouridine modification of tRNA.


Dihydrouridine modification of tRNA is widely observed in prokaryotes and eukaryotes, as well as in some archaea. In Saccharomyces cerevisiae every sequenced tRNA has at least one such modification, and all but one have two or more. We have used a biochemical genomics approach to identify the gene encoding dihydrouridine synthase 1 (Dus1, ORF YML080w), using yeast pre-tRNA(Phe) as a substrate. Dus1 is a member of a widespread family of conserved proteins, three other members of which are found in yeast: YNR015w, YLR405w, and YLR401c. We show that one of these proteins, Dus2, encoded by ORF YNR015w, has activity with two other substrates: yeast pre-tRNA(Tyr) and pre-tRNA(Leu). Both Dus1 and Dus2 are active as a single subunit protein expressed and purified from Escherichia coli, and the activity of both is stimulated in the presence of flavin adenine dinucleotide. Dus1 modifies yeast pre-tRNA(Phe) in vitro at U17, one of the two positions that are known to bear this modification in vivo. Yeast extract from a dus1-A strain is completely defective in modification of yeast pre-tRNAPhe, and RNA isolated from dus1-delta and dus2-delta strains is significantly depleted in dihydrouridine content.

Full Text

The Full Text of this article is available as a PDF (11M).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 1997 Sep 1;25(17):3389–3402. [PMC free article] [PubMed]
  • Ansmant I, Massenet S, Grosjean H, Motorin Y, Branlant C. Identification of the Saccharomyces cerevisiae RNA:pseudouridine synthase responsible for formation of psi(2819) in 21S mitochondrial ribosomal RNA. Nucleic Acids Res. 2000 May 1;28(9):1941–1946. [PMC free article] [PubMed]
  • Ansmant I, Motorin Y, Massenet S, Grosjean H, Branlant C. Identification and characterization of the tRNA:Psi 31-synthase (Pus6p) of Saccharomyces cerevisiae. J Biol Chem. 2001 Sep 14;276(37):34934–34940. [PubMed]
  • Becker HF, Motorin Y, Planta RJ, Grosjean H. The yeast gene YNL292w encodes a pseudouridine synthase (Pus4) catalyzing the formation of psi55 in both mitochondrial and cytoplasmic tRNAs. Nucleic Acids Res. 1997 Nov 15;25(22):4493–4499. [PMC free article] [PubMed]
  • Bochner BR, Ames BN. Complete analysis of cellular nucleotides by two-dimensional thin layer chromatography. J Biol Chem. 1982 Aug 25;257(16):9759–9769. [PubMed]
  • Claros MG, Vincens P. Computational method to predict mitochondrially imported proteins and their targeting sequences. Eur J Biochem. 1996 Nov 1;241(3):779–786. [PubMed]
  • Corpet F. Multiple sequence alignment with hierarchical clustering. Nucleic Acids Res. 1988 Nov 25;16(22):10881–10890. [PMC free article] [PubMed]
  • Culver GM, McCraith SM, Consaul SA, Stanford DR, Phizicky EM. A 2'-phosphotransferase implicated in tRNA splicing is essential in Saccharomyces cerevisiae. J Biol Chem. 1997 May 16;272(20):13203–13210. [PubMed]
  • Dalluge JJ, Hashizume T, Sopchik AE, McCloskey JA, Davis DR. Conformational flexibility in RNA: the role of dihydrouridine. Nucleic Acids Res. 1996 Mar 15;24(6):1073–1079. [PMC free article] [PubMed]
  • Del Campo M, Kaya Y, Ofengand J. Identification and site of action of the remaining four putative pseudouridine synthases in Escherichia coli. RNA. 2001 Nov;7(11):1603–1615. [PMC free article] [PubMed]
  • Ellis SR, Morales MJ, Li JM, Hopper AK, Martin NC. Isolation and characterization of the TRM1 locus, a gene essential for the N2,N2-dimethylguanosine modification of both mitochondrial and cytoplasmic tRNA in Saccharomyces cerevisiae. J Biol Chem. 1986 Jul 25;261(21):9703–9709. [PubMed]
  • Etcheverry T, Colby D, Guthrie C. A precursor to a minor species of yeast tRNASer contains an intervening sequence. Cell. 1979 Sep;18(1):11–26. [PubMed]
  • Gehrke CW, Kuo KC. Ribonucleoside analysis by reversed-phase high-performance liquid chromatography. J Chromatogr. 1989 Jun 2;471:3–36. [PubMed]
  • Gillman EC, Slusher LB, Martin NC, Hopper AK. MOD5 translation initiation sites determine N6-isopentenyladenosine modification of mitochondrial and cytoplasmic tRNA. Mol Cell Biol. 1991 May;11(5):2382–2390. [PMC free article] [PubMed]
  • Gutgsell NS, Del Campo M, Raychaudhuri S, Ofengand J. A second function for pseudouridine synthases: A point mutant of RluD unable to form pseudouridines 1911, 1915, and 1917 in Escherichia coli 23S ribosomal RNA restores normal growth to an RluD-minus strain. RNA. 2001 Jul;7(7):990–998. [PMC free article] [PubMed]
  • Jiang HQ, Motorin Y, Jin YX, Grosjean H. Pleiotropic effects of intron removal on base modification pattern of yeast tRNAPhe: an in vitro study. Nucleic Acids Res. 1997 Jul 15;25(14):2694–2701. [PMC free article] [PubMed]
  • Knapp G, Beckmann JS, Johnson PF, Fuhrman SA, Abelson J. Transcription and processing of intervening sequences in yeast tRNA genes. Cell. 1978 Jun;14(2):221–236. [PubMed]
  • Kowalak JA, Bruenger E, McCloskey JA. Posttranscriptional modification of the central loop of domain V in Escherichia coli 23 S ribosomal RNA. J Biol Chem. 1995 Jul 28;270(30):17758–17764. [PubMed]
  • Kuchino Y, Borek E. Tumour-specific phenylalanine tRNA contains two supernumerary methylated bases. Nature. 1978 Jan 12;271(5641):126–129. [PubMed]
  • Lafontaine DL, Bousquet-Antonelli C, Henry Y, Caizergues-Ferrer M, Tollervey D. The box H + ACA snoRNAs carry Cbf5p, the putative rRNA pseudouridine synthase. Genes Dev. 1998 Feb 15;12(4):527–537. [PMC free article] [PubMed]
  • Lecointe F, Simos G, Sauer A, Hurt EC, Motorin Y, Grosjean H. Characterization of yeast protein Deg1 as pseudouridine synthase (Pus3) catalyzing the formation of psi 38 and psi 39 in tRNA anticodon loop. J Biol Chem. 1998 Jan 16;273(3):1316–1323. [PubMed]
  • Lee Y, Kindelberger DW, Lee JY, McClennen S, Chamberlain J, Engelke DR. Nuclear pre-tRNA terminal structure and RNase P recognition. RNA. 1997 Feb;3(2):175–185. [PMC free article] [PubMed]
  • Lo RY, Bell JB, Roy KL. Dihydrouridine-deficient tRNAs in Saccharomyces cerevisiae. Nucleic Acids Res. 1982 Feb 11;10(3):889–902. [PMC free article] [PubMed]
  • Martzen MR, McCraith SM, Spinelli SL, Torres FM, Fields S, Grayhack EJ, Phizicky EM. A biochemical genomics approach for identifying genes by the activity of their products. Science. 1999 Nov 5;286(5442):1153–1155. [PubMed]
  • McCraith SM, Phizicky EM. A highly specific phosphatase from Saccharomyces cerevisiae implicated in tRNA splicing. Mol Cell Biol. 1990 Mar;10(3):1049–1055. [PMC free article] [PubMed]
  • Motorin Y, Keith G, Simon C, Foiret D, Simos G, Hurt E, Grosjean H. The yeast tRNA:pseudouridine synthase Pus1p displays a multisite substrate specificity. RNA. 1998 Jul;4(7):856–869. [PMC free article] [PubMed]
  • O'Connor M, Lee WM, Mankad A, Squires CL, Dahlberg AE. Mutagenesis of the peptidyltransferase center of 23S rRNA: the invariant U2449 is dispensable. Nucleic Acids Res. 2001 Feb 1;29(3):710–715. [PMC free article] [PubMed]
  • Ogden RC, Beckman JS, Abelson J, Kang HS, Söll D, Schmidt O. In vitro transcription and processing of a yeast tRNA gene containing an intervening sequence. Cell. 1979 Jun;17(2):399–406. [PubMed]
  • Peebles CL, Gegenheimer P, Abelson J. Precise excision of intervening sequences from precursor tRNAs by a membrane-associated yeast endonuclease. Cell. 1983 Feb;32(2):525–536. [PubMed]
  • Phizicky Eric M, Martzen Mark R, McCraith Stephen M, Spinelli Sherry L, Xing Feng, Shull Neil P, Van Slyke Ceri, Montagne Rebecca K, Torres Francy M, Fields Stanley, et al. Biochemical genomics approach to map activities to genes. Methods Enzymol. 2002;350:546–559. [PubMed]
  • RajBhandary UL, Stuart A, Faulkner RD, Chang SH, Khorana HG. Nucleotide sequence studies on yeast phenylalanine sRNA. Cold Spring Harb Symp Quant Biol. 1966;31:425–434. [PubMed]
  • Reyes VM, Abelson J. A synthetic substrate for tRNA splicing. Anal Biochem. 1987 Oct;166(1):90–106. [PubMed]
  • Rinaldi T, Lande R, Bolotin-Fukuhara M, Frontali L. Additional copies of the mitochondrial Ef-Tu and aspartyl-tRNA synthetase genes can compensate for a mutation affecting the maturation of the mitochondrial tRNAAsp. Curr Genet. 1997 Jun;31(6):494–496. [PubMed]
  • Sprinzl M, Horn C, Brown M, Ioudovitch A, Steinberg S. Compilation of tRNA sequences and sequences of tRNA genes. Nucleic Acids Res. 1998 Jan 1;26(1):148–153. [PMC free article] [PubMed]
  • Steiger MA, Kierzek R, Turner DH, Phizicky EM. Substrate recognition by a yeast 2'-phosphotransferase involved in tRNA splicing and by its Escherichia coli homolog. Biochemistry. 2001 Nov 20;40(46):14098–14105. [PubMed]
  • Topp H, Duden R, Schöch G. 5,6-Dihydrouridine: a marker ribonucleoside for determining whole body degradation rates of transfer RNA in man and rats. Clin Chim Acta. 1993 Sep 17;218(1):73–82. [PubMed]
  • Zennaro E, Francisci S, Ragnini A, Frontali L, Bolotin-Fukuhara M. A point mutation in a mitochondrial tRNA gene abolishes its 3' end processing. Nucleic Acids Res. 1989 Jul 25;17(14):5751–5764. [PMC free article] [PubMed]

Articles from RNA are provided here courtesy of The RNA Society


Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...


Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...