title: Reconstitution and structural analysis of the yeast box H/ACA RNA-guided pseudouridine synthase.
abstract: Box H/ACA ribonucleoprotein particles (RNPs) mediate pseudouridine synthesis, ribosome formation, and telomere maintenance. The structure of eukaryotic H/ACA RNPs remains poorly understood. We reconstituted functional Saccharomyces cerevisiae H/ACA RNPs with recombinant proteins Cbf5, Nop10, Gar1, and Nhp2 and a two-hairpin H/ACA RNA; determined the crystal structure of a Cbf5, Nop10, and Gar1 ternary complex at 1.9 Å resolution; and analyzed the structure-function relationship of the yeast complex. Although eukaryotic H/ACA RNAs have a conserved two-hairpin structure, isolated single-hairpin RNAs are also active in guiding pseudouridylation. Nhp2, unlike its archaeal counterpart, is largely dispensable for the activity, reflecting a functional adaptation of eukaryotic H/ACA RNPs to the variable RNA structure that Nhp2 binds. The N-terminal extension of Cbf5, a hot spot for dyskeratosis congenita mutation, forms an extra structural layer on the PUA domain. Gar1 is distinguished from the assembly factor Naf1 by containing a C-terminal extension that controls substrate turnover and the Gar1-Naf1 exchange during H/ACA RNP maturation. Our results reveal significant novel features of eukaryotic H/ACA RNPs.
authors.affiliation: National Institute of Biological Sciences, Beijing, China.
authors.forename: Shuang
authors.initials: S
authors.lastname: Li
authors.forename: Jingqi
authors.initials: J
authors.lastname: Duan
authors.forename: Dandan
authors.initials: D
authors.lastname: Li
authors.forename: Bing
authors.initials: B
authors.lastname: Yang
authors.forename: Mengqiu
authors.initials: M
authors.lastname: Dong
authors.forename: Keqiong
authors.initials: K
authors.lastname: Ye
mesh_terms: D000595:Amino Acid Sequence; D006836:Hydro-Lyases / Q000737:chemistry / Q000235:genetics; D019751:Intramolecular Transferases; D008869:Microtubule-Associated Proteins / Q000737:chemistry / Q000235:genetics; D008958:Models, Molecular; D046912:Multiprotein Complexes / Q000737:chemistry; D009154:Mutation; D009687:Nuclear Proteins / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D011560:Pseudouridine / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017411:Ribonucleoproteins, Small Nuclear / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020718:Ribonucleoproteins, Small Nucleolar / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment
chemical_list: D008869:Microtubule-Associated Proteins; D046912:Multiprotein Complexes; C117016:NOP10 protein, S cerevisiae; D009687:Nuclear Proteins; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D017411:Ribonucleoproteins, Small Nuclear; D020718:Ribonucleoproteins, Small Nucleolar; D029701:Saccharomyces cerevisiae Proteins; C069628:NHP2 protein, S cerevisiae; D011560:Pseudouridine; C073180:GAR1 protein, S cerevisiae; D006836:Hydro-Lyases; C082278:CBF5 protein, S cerevisiae; D019751:Intramolecular Transferases; C096995:pseudouridine synthases
references.citation: Mol Cell Biol. 2002 Oct;22(20):7053-65
references.pmid: 12242285
references.citation: EMBO J. 1999 Jan 15;18(2):457-69
references.pmid: 9889201
references.citation: EMBO J. 2004 Apr 21;23(8):1857-67
references.pmid: 15044956
references.citation: Proteins. 2003 Feb 15;50(3):437-50
references.pmid: 12557186
references.citation: Methods Enzymol. 1997;276:307-26
references.pmid: 27754618
references.citation: Nucleic Acids Res. 2006 Feb 02;34(3):826-39
references.pmid: 16456033
references.citation: Curr Opin Genet Dev. 2005 Jun;15(3):249-57
references.pmid: 15917199
references.citation: Nature. 2006 Sep 21;443(7109):302-7
references.pmid: 16943774
references.citation: Genes Dev. 2005 May 15;19(10):1238-48
references.pmid: 15870259
references.citation: J Mol Biol. 2007 Aug 31;371(5):1338-53
references.pmid: 17612558
references.citation: Genes Dev. 1997 Apr 1;11(7):941-56
references.pmid: 9106664
references.citation: Nat Struct Mol Biol. 2005 Dec;12(12):1101-7
references.pmid: 16286935
references.citation: Curr Opin Struct Biol. 2007 Jun;17(3):287-92
references.pmid: 17574834
references.citation: Mol Cell Biol. 2000 May;20(9):3037-48
references.pmid: 10757788
references.citation: J Biol Chem. 2002 Nov 22;277(47):45235-42
references.pmid: 12228251
references.citation: Nat Rev Mol Cell Biol. 2007 Mar;8(3):209-20
references.pmid: 17318225
references.citation: Acta Crystallogr D Biol Crystallogr. 1999 Jan;55(Pt 1):247-55
references.pmid: 10089417
references.citation: RNA. 2009 Jan;15(1):176-86
references.pmid: 19033376
references.citation: Cell. 1996 Sep 6;86(5):823-34
references.pmid: 8797828
references.citation: Proc Natl Acad Sci U S A. 2002 May 28;99(11):7536-41
references.pmid: 12032318
references.citation: RNA. 2004 Jul;10(7):1034-46
references.pmid: 15208440
references.citation: RNA. 2002 Dec;8(12):1502-14
references.pmid: 12515383
references.citation: Mol Cell Biol. 1993 Aug;13(8):4884-93
references.pmid: 8336724
references.citation: Mol Cell. 2010 Mar 12;37(5):597-606
references.pmid: 20227365
references.citation: Mol Cell Biol. 1999 Jan;19(1):567-76
references.pmid: 9858580
references.citation: Chromosoma. 2005 May;114(1):1-14
references.pmid: 15770508
references.citation: J Biol Chem. 1994 Jul 15;269(28):18499-506
references.pmid: 8034598
references.citation: RNA. 1998 Dec;4(12):1549-68
references.pmid: 9848653
references.citation: Mol Cell Biol. 2005 Apr;25(8):3295-304
references.pmid: 15798213
references.citation: Nucleic Acids Res. 2001 Jul 1;29(13):2733-46
references.pmid: 11433018
references.citation: Mol Cell Biol. 2000 Dec;20(23):9028-40
references.pmid: 11074001
references.citation: RNA. 2004 Nov;10(11):1704-12
references.pmid: 15388873
references.citation: Nucleic Acids Res. 2005 Jun 02;33(10):3133-44
references.pmid: 15933208
references.citation: Nat Struct Mol Biol. 2007 Dec;14(12):1189-95
references.pmid: 18059286
references.citation: Biochemistry. 2008 Jun 10;47(23):6148-56
references.pmid: 18473479
references.citation: J Cell Biol. 2006 Apr 24;173(2):207-18
references.pmid: 16618814
references.citation: EMBO J. 2009 May 6;28(9):1260-70
references.pmid: 19322192
references.citation: RNA. 2006 Jan;12(1):40-52
references.pmid: 16373493
references.citation: Cell. 1997 May 30;89(5):799-809
references.pmid: 9182768
references.citation: Acta Crystallogr D Biol Crystallogr. 2004 Dec;60(Pt 12 Pt 1):2126-32
references.pmid: 15572765
references.citation: Mol Cell Biol. 2004 Feb;24(4):1769-78
references.pmid: 14749391
references.citation: Nat Struct Mol Biol. 2009 Jul;16(7):740-6
references.pmid: 19478803
references.citation: Nucleic Acids Res. 2003 Feb 1;31(3):869-77
references.pmid: 12560482
references.citation: J Mol Biol. 2011 Sep 2;411(5):927-42
references.pmid: 21708174
references.citation: Mol Cell Biol. 2005 Jul;25(13):5396-403
references.pmid: 15964797
references.citation: Br J Haematol. 2009 Apr;145(2):164-72
references.pmid: 19208095
references.citation: J Biol Chem. 2001 Oct 26;276(43):40313-8
references.pmid: 11483606
references.citation: Mol Cell Biol. 1993 Apr;13(4):2469-77
references.pmid: 8455623
references.citation: Mol Cell. 2006 Jan 20;21(2):249-60
references.pmid: 16427014
references.citation: Mol Cell. 2009 May 14;34(4):427-39
references.pmid: 19481523
references.citation: J Appl Crystallogr. 2007 Aug 1;40(Pt 4):658-674
references.pmid: 19461840
references.citation: Mol Cell Biol. 2002 Dec;22(24):8457-66
references.pmid: 12446766