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PMID: 26161500 Published · epublish English Journal Article Research Support, Non-U.S. Gov't

A composite double-/single-stranded RNA-binding region in protein Prp3 supports tri-snRNP stability and splicing.

eLife ·Vol. 4 ·2015-07-10 ·Pages e07320

Liu S, Mozaffari-Jovin S, Wollenhaupt J, Santos KF, Theuser M, Dunin-Horkawicz S, Fabrizio P, Bujnicki JM, Lührmann R, Wahl MC

Abstract

Prp3 is an essential U4/U6 di-snRNP-associated protein whose functions and molecular mechanisms in pre-mRNA splicing are presently poorly understood. We show by structural and biochemical analyses that Prp3 contains a bipartite U4/U6 di-snRNA-binding region comprising an expanded ferredoxin-like fold, which recognizes a 3'-overhang of U6 snRNA, and a preceding peptide, which binds U4/U6 stem II. Phylogenetic analyses revealed that the single-stranded RNA-binding domain is exclusively found in Prp3 orthologs, thus qualifying as a spliceosome-specific RNA interaction module. The composite double-stranded/single-stranded RNA-binding region assembles cooperatively with Snu13 and Prp31 on U4/U6 di-snRNAs and inhibits Brr2-mediated U4/U6 di-snRNA unwinding in vitro. RNP-disrupting mutations in Prp3 lead to U4/U6•U5 tri-snRNP assembly and splicing defects in vivo. Our results reveal how Prp3 acts as an important bridge between U4/U6 and U5 in the tri-snRNP and comparison with a Prp24-U6 snRNA recycling complex suggests how Prp3 may be involved in U4/U6 reassembly after splicing.

Keywords
S. cerevisiae X-ray crystallography biochemistry biophysics human pre-mRNA splicing protein–RNA interaction small nuclear ribonucleoprotein particle structural biology
MeSH Terms
Crystallography, X-Ray DNA Mutational Analysis Humans Models, Biological Models, Molecular Mutation Nuclear Proteins/chemistry,genetics,metabolism Protein Binding RNA/metabolism RNA Splicing Ribonucleoprotein, U4-U6 Small Nuclear/chemistry,genetics,metabolism Ribonucleoproteins, Small Nuclear/chemistry,metabolism
Chemicals
Nuclear Proteins PRPF3 protein, human Ribonucleoprotein, U4-U6 Small Nuclear Ribonucleoproteins, Small Nuclear RNA
Authors & Affiliations
10 authors, click to expand affiliations / ORCID
Liu Sunbin
Laboratory of Structural Biochemistry, Freie Universität Berlin, Berlin, Germany.
Mozaffari-Jovin Sina
Department of Cellular Biochemistry, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.
Wollenhaupt Jan
Laboratory of Structural Biochemistry, Freie Universität Berlin, Berlin, Germany.
Santos Karine F
Laboratory of Structural Biochemistry, Freie Universität Berlin, Berlin, Germany.
Theuser Matthias
Laboratory of Structural Biochemistry, Freie Universität Berlin, Berlin, Germany.
Dunin-Horkawicz Stanislaw
Laboratory of Bioinformatics and Protein Engineering, International Institute of Molecular and Cell Biology, Warsaw, Poland.
Fabrizio Patrizia
Department of Cellular Biochemistry, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.
Bujnicki Janusz M
Laboratory of Bioinformatics and Protein Engineering, International Institute of Molecular and Cell Biology, Warsaw, Poland.
Lührmann Reinhard
Department of Cellular Biochemistry, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.
Wahl Markus C
Laboratory of Structural Biochemistry, Freie Universität Berlin, Berlin, Germany.
References (75)
75 references, click to expand
  1. Insights into RNA biology from an atlas of mammalian mRNA-binding proteins.
    Cell. 2012 Jun 8;149(6):1393-406 PMID: 22658674
  2. The mRNA-bound proteome and its global occupancy profile on protein-coding transcripts.
    Mol Cell. 2012 Jun 8;46(5):674-90 PMID: 22681889
  3. EvolView, an online tool for visualizing, annotating and managing phylogenetic trees.
    Nucleic Acids Res. 2012 Jul;40(Web Server issue):W569-72 PMID: 22695796
  4. Structure determination by multiple-wavelength anomalous dispersion (MAD) at the Pr LIII edge.
    Acta Crystallogr Sect F Struct Biol Cryst Commun. 2012 Aug 1;68(Pt 8):981-4 PMID: 22869138
  5. Structural bioinformatics of the human spliceosomal proteome.
    Nucleic Acids Res. 2012 Aug;40(15):7046-65 PMID: 22573172
  6. Structural basis for functional cooperation between tandem helicase cassettes in Brr2-mediated remodeling of the spliceosome.
    Proc Natl Acad Sci U S A. 2012 Oct 23;109(43):17418-23 PMID: 23045696
  7. Inhibition of RNA helicase Brr2 by the C-terminal tail of the spliceosomal protein Prp8.
    Science. 2013 Jul 5;341(6141):80-4 PMID: 23704370
  8. RNA Bricks--a database of RNA 3D motifs and their interactions.
    Nucleic Acids Res. 2014 Jan;42(Database issue):D123-31 PMID: 24220091
  9. Crystal structures of the Lsm complex bound to the 3' end sequence of U6 small nuclear RNA.
    Nature. 2014 Feb 6;506(7486):116-20 PMID: 24240276
  10. Core structure of the U6 small nuclear ribonucleoprotein at 1.7-Å resolution.
    Nat Struct Mol Biol. 2014 Jun;21(6):544-51 PMID: 24837192
  11. U4 small nuclear RNA dissociates from a yeast spliceosome and does not participate in the subsequent splicing reaction.
    Mol Cell Biol. 1991 Nov;11(11):5571-7 PMID: 1833635
  12. The U1 small nuclear RNA-protein complex selectively binds a 5' splice site in vitro.
    Cell. 1983 Jun;33(2):509-18 PMID: 6190573
  13. The 5' terminus of the RNA moiety of U1 small nuclear ribonucleoprotein particles is required for the splicing of messenger RNA precursors.
    Cell. 1984 Aug;38(1):299-307 PMID: 6235919
  14. Recognition of the TACTAAC box during mRNA splicing in yeast involves base pairing to the U2-like snRNA.
    Cell. 1987 Apr 24;49(2):229-39 PMID: 3552247
  15. Interactions between small nuclear ribonucleoprotein particles in formation of spliceosomes.
    Cell. 1987 Jun 19;49(6):763-74 PMID: 2953438
  16. An ordered pathway of snRNP binding during mammalian pre-mRNA splicing complex assembly.
    EMBO J. 1987 Aug;6(8):2415-24 PMID: 2959470
  17. Spliceosome assembly in yeast.
    Genes Dev. 1987 Nov;1(9):1014-27 PMID: 2962902
  18. A compensatory base change in human U2 snRNA can suppress a branch site mutation.
    Genes Dev. 1989 Oct;3(10):1545-52 PMID: 2612904
  19. Mammalian pre-mRNA branch site selection by U2 snRNP involves base pairing.
    Genes Dev. 1989 Oct;3(10):1553-61 PMID: 2558966
  20. In vitro assembly of yeast U6 snRNP: a functional assay.
    Genes Dev. 1989 Dec;3(12B):2137-50 PMID: 2560755
  21. Multiple roles for U6 snRNA in the splicing pathway.
    Genes Dev. 1990 Dec;4(12B):2264-77 PMID: 2149118
  22. Reconstituted mammalian U4/U6 snRNP complements splicing: a mutational analysis.
    EMBO J. 1992 Jan;11(1):345-59 PMID: 1740113
  23. Functional interaction of a novel 15.5kD [U4/U6.U5] tri-snRNP protein with the 5' stem-loop of U4 snRNA.
    EMBO J. 1999 Nov 1;18(21):6119-33 PMID: 10545122
  24. Characterization of U6 snRNA-protein interactions.
    RNA. 1999 Nov;5(11):1470-81 PMID: 10580475
  25. DaliLite workbench for protein structure comparison.
    Bioinformatics. 2000 Jun;16(6):566-7 PMID: 10980157
  26. Crystal structure of the cell division protein FtsA from Thermotoga maritima.
    EMBO J. 2000 Oct 16;19(20):5300-7 PMID: 11032797
  27. Crystal structure of the spliceosomal 15.5kD protein bound to a U4 snRNA fragment.
    Mol Cell. 2000 Dec;6(6):1331-42 PMID: 11163207
  28. p110, a novel human U6 snRNP protein and U4/U6 snRNP recycling factor.
    EMBO J. 2002 Jun 3;21(11):2724-35 PMID: 12032085
  29. Central region of the human splicing factor Hprp3p interacts with Hprp4p.
    J Biol Chem. 2002 Jun 28;277(26):23764-72 PMID: 11971898
  30. The conserved central domain of yeast U6 snRNA: importance of U2-U6 helix Ia in spliceosome assembly.
    RNA. 2002 Aug;8(8):997-1010 PMID: 12212854
  31. The 5' and 3' domains of yeast U6 snRNA: Lsm proteins facilitate binding of Prp24 protein to the U6 telestem region.
    RNA. 2002 Aug;8(8):1011-33 PMID: 12212846
  32. Hierarchical, clustered protein interactions with U4/U6 snRNA: a biochemical role for U4/U6 proteins.
    EMBO J. 2002 Oct 15;21(20):5527-38 PMID: 12374753
  33. A conserved Lsm-interaction motif in Prp24 required for efficient U4/U6 di-snRNP formation.
    RNA. 2002 Nov;8(11):1378-92 PMID: 12458792
  34. MUSCLE: multiple sequence alignment with high accuracy and high throughput.
    Nucleic Acids Res. 2004;32(5):1792-7 PMID: 15034147
  35. Lsm proteins promote regeneration of pre-mRNA splicing activity.
    Curr Biol. 2004 Aug 24;14(16):1487-91 PMID: 15324666
  36. ALSCRIPT: a tool to format multiple sequence alignments.
    Protein Eng. 1993 Jan;6(1):37-40 PMID: 8433969
  37. Conformational changes of U6 RNA during the spliceosome cycle: an intramolecular helix is essential both for initiating the U4-U6 interaction and for the first step of slicing.
    Genes Dev. 1993 Jul;7(7B):1377-89 PMID: 8330741
  38. Disruption of base pairing between the 5' splice site and the 5' end of U1 snRNA is required for spliceosome assembly.
    Cell. 1993 Dec 3;75(5):863-73 PMID: 8252623
  39. A stem/loop in U6 RNA defines a conformational switch required for pre-mRNA splicing.
    Genes Dev. 1994 Jan;8(2):221-33 PMID: 8299941
  40. Crystal structure at 1.92 A resolution of the RNA-binding domain of the U1A spliceosomal protein complexed with an RNA hairpin.
    Nature. 1994 Dec 1;372(6505):432-8 PMID: 7984237
  41. SCOP: a structural classification of proteins database for the investigation of sequences and structures.
    J Mol Biol. 1995 Apr 7;247(4):536-40 PMID: 7723011
  42. Mutational analysis of human U6 RNA: stabilizing the intramolecular helix blocks the spliceosomal assembly pathway.
    Biochim Biophys Acta. 1995 Jul 25;1263(1):39-44 PMID: 7632731
  43. Crystal structure of common type acylphosphatase from bovine testis.
    Structure. 1997 Jan 15;5(1):69-79 PMID: 9016712
  44. The yeast Prp3 protein is a U4/U6 snRNP protein necessary for integrity of the U4/U6 snRNP and the U4/U6.U5 tri-snRNP.
    RNA. 1997 Oct;3(10):1143-52 PMID: 9326489
  45. PDBsum: a Web-based database of summaries and analyses of all PDB structures.
    Trends Biochem Sci. 1997 Dec;22(12):488-90 PMID: 9433130
  46. A spliceosomal recycling factor that reanneals U4 and U6 small nuclear ribonucleoprotein particles.
    Science. 1998 Feb 6;279(5352):857-60 PMID: 9452384
  47. Functional and structural characterization of the prp3 binding domain of the yeast prp4 splicing factor.
    J Mol Biol. 1998 Dec 4;284(3):673-87 PMID: 9826507
  48. The 2.2 A structure of a permanganate-sensitive DNA site bound by the Epstein-Barr virus origin binding protein, EBNA1.
    J Mol Biol. 1998 Dec 18;284(5):1273-8 PMID: 9878348
  49. A doughnut-shaped heteromer of human Sm-like proteins binds to the 3'-end of U6 snRNA, thereby facilitating U4/U6 duplex formation in vitro.
    EMBO J. 1999 Oct 15;18(20):5789-802 PMID: 10523320
  50. Protein production by auto-induction in high density shaking cultures.
    Protein Expr Purif. 2005 May;41(1):207-34 PMID: 15915565
  51. Lsm proteins and RNA processing.
    Biochem Soc Trans. 2005 Jun;33(Pt 3):433-8 PMID: 15916535
  52. Structure of a bacterial BLUF photoreceptor: insights into blue light-mediated signal transduction.
    Proc Natl Acad Sci U S A. 2005 Aug 30;102(35):12350-5 PMID: 16107542
  53. RNA structure and RNA-protein interactions in purified yeast U6 snRNPs.
    J Mol Biol. 2006 Mar 10;356(5):1248-62 PMID: 16410014
  54. Repositioning of the reaction intermediate within the catalytic center of the spliceosome.
    Mol Cell. 2006 Feb 17;21(4):543-53 PMID: 16483935
  55. Protein composition and electron microscopy structure of affinity-purified human spliceosomal B complexes isolated under physiological conditions.
    Mol Cell Biol. 2006 Jul;26(14):5528-43 PMID: 16809785
  56. The network of protein-protein interactions within the human U4/U6.U5 tri-snRNP.
    RNA. 2006 Jul;12(7):1418-30 PMID: 16723661
  57. HKL-3000: the integration of data reduction and structure solution--from diffraction images to an initial model in minutes.
    Acta Crystallogr D Biol Crystallogr. 2006 Aug;62(Pt 8):859-66 PMID: 16855301
  58. The Cajal body: a meeting place for spliceosomal snRNPs in the nuclear maze.
    Chromosoma. 2006 Oct;115(5):343-54 PMID: 16575476
  59. RNA structural requirements for the association of the spliceosomal hPrp31 protein with the U4 and U4atac small nuclear ribonucleoproteins.
    J Biol Chem. 2006 Sep 22;281(38):28278-86 PMID: 16857676
  60. Solving structures of protein complexes by molecular replacement with Phaser.
    Acta Crystallogr D Biol Crystallogr. 2007 Jan;63(Pt 1):32-41 PMID: 17164524
  61. Binding of the human Prp31 Nop domain to a composite RNA-protein platform in U4 snRNP.
    Science. 2007 Apr 6;316(5821):115-20 PMID: 17412961
  62. Isolation of an active step I spliceosome and composition of its RNP core.
    Nature. 2008 Apr 17;452(7189):846-50 PMID: 18322460
  63. Quantitative analysis of protein-RNA interactions by gel mobility shift.
    Methods Mol Biol. 2008;488:99-115 PMID: 18982286
  64. The spliceosome: design principles of a dynamic RNP machine.
    Cell. 2009 Feb 20;136(4):701-18 PMID: 19239890
  65. The evolutionarily conserved core design of the catalytic activation step of the yeast spliceosome.
    Mol Cell. 2009 Nov 25;36(4):593-608 PMID: 19941820
  66. Reconstitution of both steps of Saccharomyces cerevisiae splicing with purified spliceosomal components.
    Nat Struct Mol Biol. 2009 Dec;16(12):1237-43 PMID: 19935684
  67. Molecular replacement with MOLREP.
    Acta Crystallogr D Biol Crystallogr. 2010 Jan;66(Pt 1):22-5 PMID: 20057045
  68. XDS.
    Acta Crystallogr D Biol Crystallogr. 2010 Feb;66(Pt 2):125-32 PMID: 20124692
  69. Features and development of Coot.
    Acta Crystallogr D Biol Crystallogr. 2010 Apr;66(Pt 4):486-501 PMID: 20383002
  70. Characterization of purified human Bact spliceosomal complexes reveals compositional and morphological changes during spliceosome activation and first step catalysis.
    RNA. 2010 Dec;16(12):2384-403 PMID: 20980672
  71. Web servers and services for electrostatics calculations with APBS and PDB2PQR.
    J Comput Chem. 2011 May;32(7):1488-91 PMID: 21425296
  72. REFMAC5 for the refinement of macromolecular crystal structures.
    Acta Crystallogr D Biol Crystallogr. 2011 Apr;67(Pt 4):355-67 PMID: 21460454
  73. Spliceosome structure and function.
    Cold Spring Harb Perspect Biol. 2011 Jul;3(7). pii: a003707. doi: 10.1101/cshperspect.a003707 PMID: 21441581
  74. Structural basis for the dual U4 and U4atac snRNA-binding specificity of spliceosomal protein hPrp31.
    RNA. 2011 Sep;17(9):1655-63 PMID: 21784869
  75. Towards automated crystallographic structure refinement with phenix.refine.
    Acta Crystallogr D Biol Crystallogr. 2012 Apr;68(Pt 4):352-67 PMID: 22505256
Article Info
Journal
eLife
Abbr.
Elife
ISSN
2050-084X
DOI
10.7554/eLife.07320
Published
2015-07-10
Epub
2015-00-10
Pages
e07320
Language
English
Region
England
NLM ID
101579614
PMCID
PMC4520091
Subset
IM
Databases
PDB
4YHU, 4YHV, 4YHW
External Links
PubMed source DOI: 10.7554/eLife.07320 PMC Full Text View journal details
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