Home LiteratureArticle Details
PMID: 8474454 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

A U5 small nuclear ribonucleoprotein particle protein involved only in the second step of pre-mRNA splicing in Saccharomyces cerevisiae.

Molecular and cellular biology ·Vol. 13 ·No. 5 ·1993-05-00 ·Pages 2959-70

Horowitz DS, Abelson J

Abstract

The PRP18 gene, which had been identified in a screen for pre-mRNA splicing mutants in Saccharomyces cerevisiae, has been cloned and sequenced. Yeast strains bearing only a disrupted copy of PRP18 are temperature sensitive for growth; even at a low temperature, they grow extremely slowly and do not splice pre-mRNA efficiently. This unusual temperature sensitivity can be reproduced in vitro; extracts immunodepleted of PRP18 are temperature sensitive for the second step of splicing. The PRP18 protein has been overexpressed in active form in Escherichia coli and has been purified to near homogeneity. Antibodies directed against PRP18 precipitate the U4/U5/U6 small nuclear ribonucleoprotein particle (snRNP) from yeast extracts. From extracts depleted of the U6 small nuclear RNA (snRNA), the U4 and U5 snRNAs can be immunoprecipitated, while no snRNAs can be precipitated from extracts depleted of the U5 snRNA. PRP18 therefore appears to be primarily associated with the U5 snRNP. The antibodies against PRP18 inhibit the second step of pre-mRNA splicing in vitro. Together, these results imply that the U5 snRNP plays a role in the second step of splicing and suggest a model for the action of PRP18.

Related Genes
MUQ1 PRP18 STF2
MeSH Terms
Amino Acid Sequence Base Sequence Chromatography, Ion Exchange Cloning, Molecular Escherichia coli/genetics Fungal Proteins/genetics,isolation & purification,metabolism Genes, Fungal Kinetics Molecular Sequence Data Nuclear Proteins Open Reading Frames RNA Precursors/genetics,metabolism RNA Splicing RNA, Fungal/genetics,metabolism Recombinant Proteins/isolation & purification,metabolism Restriction Mapping Ribonucleoprotein, U5 Small Nuclear/isolation & purification,metabolism Saccharomyces cerevisiae/genetics,metabolism Saccharomyces cerevisiae Proteins Temperature
Chemicals
Fungal Proteins Nuclear Proteins PRP18 protein, S cerevisiae RNA Precursors RNA, Fungal Recombinant Proteins Ribonucleoprotein, U5 Small Nuclear Saccharomyces cerevisiae Proteins
Authors & Affiliations
2 authors, click to expand affiliations / ORCID
Horowitz D S
Division of Biology, California Institute of Technology, Pasadena 91125.
Abelson J
References (72)
72 references, click to expand
  1. PRP4 (RNA4) from Saccharomyces cerevisiae: its gene product is associated with the U4/U6 small nuclear ribonucleoprotein particle.
    Mol Cell Biol. 1989 Sep;9(9):3698-709 PMID: 2528686
  2. PRP4: a protein of the yeast U4/U6 small nuclear ribonucleoprotein particle.
    Mol Cell Biol. 1989 Sep;9(9):3710-9 PMID: 2528687
  3. Isolation and characterization of pre-mRNA splicing mutants of Saccharomyces cerevisiae.
    Genes Dev. 1989 Aug;3(8):1206-16 PMID: 2676722
  4. U5 small nuclear ribonucleoprotein: RNA structure analysis and ATP-dependent interaction with U4/U6.
    Mol Cell Biol. 1989 Aug;9(8):3350-9 PMID: 2552294
  5. The mammalian analogue of the yeast PRP8 splicing protein is present in the U4/5/6 small nuclear ribonucleoprotein particle and the spliceosome.
    Proc Natl Acad Sci U S A. 1989 Nov;86(22):8742-6 PMID: 2479028
  6. Evidence from complementation assays in vitro that U5 snRNP is required for both steps of mRNA splicing.
    EMBO J. 1989 Oct;8(10):3105-12 PMID: 2531074
  7. PRP18, a protein required for the second reaction in pre-mRNA splicing.
    Mol Cell Biol. 1990 Jan;10(1):324-32 PMID: 2403639
  8. Conservation between yeast and man of a protein associated with U5 small nuclear ribonucleoprotein.
    Nature. 1989 Dec 14;342(6251):819-21 PMID: 2532307
  9. Self-splicing group II and nuclear pre-mRNA introns: how similar are they?
    Trends Biochem Sci. 1990 Sep;15(9):351-4 PMID: 2238045
  10. The yeast PRP6 gene encodes a U4/U6 small nuclear ribonucleoprotein particle (snRNP) protein, and the PRP9 gene encodes a protein required for U2 snRNP binding.
    Mol Cell Biol. 1990 Dec;10(12):6417-25 PMID: 2147224
  11. Characterization of VPS34, a gene required for vacuolar protein sorting and vacuole segregation in Saccharomyces cerevisiae.
    Mol Cell Biol. 1990 Dec;10(12):6742-54 PMID: 2247081
  12. Structure of spliceosomal snRNPs and their role in pre-mRNA splicing.
    Biochim Biophys Acta. 1990 Nov 30;1087(3):265-92 PMID: 2147394
  13. Mutations in yeast U5 snRNA alter the specificity of 5' splice-site cleavage.
    Cell. 1991 Apr 5;65(1):115-23 PMID: 2013092
  14. Suppressors of a U4 snRNA mutation define a novel U6 snRNP protein with RNA-binding motifs.
    Genes Dev. 1991 May;5(5):773-85 PMID: 1827420
  15. Specific labeling of 3' termini of RNA with T4 RNA ligase.
    Methods Enzymol. 1980;65(1):65-74 PMID: 6154874
  16. The intrinsic ATPase of DNA gyrase.
    J Biol Chem. 1980 Jul 10;255(13):6299-306 PMID: 6248518
  17. The effect of temperature-sensitive RNA mutants on the transcription products from cloned ribosomal protein genes of yeast.
    Cell. 1981 Jun;24(3):679-86 PMID: 7018694
  18. Transformation of intact yeast cells treated with alkali cations.
    J Bacteriol. 1983 Jan;153(1):163-8 PMID: 6336730
  19. Isolation of small nuclear ribonucleoproteins containing U1, U2, U4, U5, and U6 RNAs.
    J Biol Chem. 1983 Feb 25;258(4):2604-13 PMID: 6185498
  20. Fractionation and characterization of human small nuclear ribonucleoproteins containing U1 and U2 RNAs.
    J Biol Chem. 1983 Jun 10;258(11):7181-9 PMID: 6189835
  21. One-step gene disruption in yeast.
    Methods Enzymol. 1983;101:202-11 PMID: 6310324
  22. Yeast contains small nuclear RNAs encoded by single copy genes.
    Cell. 1983 Dec;35(3 Pt 2):743-51 PMID: 6197183
  23. A comprehensive set of sequence analysis programs for the VAX.
    Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387-95 PMID: 6546423
  24. U4 and U6 RNAs coexist in a single small nuclear ribonucleoprotein particle.
    Nucleic Acids Res. 1984 Apr 11;12(7):3283-93 PMID: 6201826
  25. Pre-mRNA splicing in yeast.
    Trends Genet. 1991 Mar;7(3):79-85 PMID: 2031287
  26. mRNA-type introns in U6 small nuclear RNA genes: implications for the catalysis in pre-mRNA splicing.
    Genes Dev. 1991 Jun;5(6):1022-31 PMID: 2044950
  27. Antisense probes containing 2-aminoadenosine allow efficient depletion of U5 snRNP from HeLa splicing extracts.
    Nucleic Acids Res. 1991 Jun 25;19(12):3193-8 PMID: 1648201
  28. Genetic depletion indicates a late role for U5 snRNP during in vitro spliceosome assembly.
    Nucleic Acids Res. 1991 Jul 25;19(14):3857-60 PMID: 1830649
  29. Immunoaffinity purification of a [U4/U6.U5] tri-snRNP from human cells.
    Genes Dev. 1991 Aug;5(8):1439-52 PMID: 1831175
  30. Who's on first? The U1 snRNP-5' splice site interaction and splicing.
    Trends Biochem Sci. 1991 May;16(5):187-90 PMID: 1882420
  31. 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
  32. A putative zinc finger protein, Saccharomyces cerevisiae Vps18p, affects late Golgi functions required for vacuolar protein sorting and efficient alpha-factor prohormone maturation.
    Mol Cell Biol. 1991 Dec;11(12):5813-24 PMID: 1840635
  33. The yeast PRP8 protein interacts directly with pre-mRNA.
    Nucleic Acids Res. 1991 Oct 25;19(20):5483-9 PMID: 1945827
  34. DMSO-enhanced whole cell yeast transformation.
    Nucleic Acids Res. 1991 Oct 25;19(20):5791 PMID: 1945859
  35. U5 snRNA interacts with exon sequences at 5' and 3' splice sites.
    Cell. 1992 Feb 21;68(4):743-54 PMID: 1739979
  36. Cyclic 2',3'-phosphates and nontemplated nucleotides at the 3' end of spliceosomal U6 small nuclear RNA's.
    Science. 1992 Jan 17;255(5042):327-30 PMID: 1549778
  37. D-E-A-D protein family of putative RNA helicases.
    Mol Microbiol. 1992 Feb;6(3):283-91 PMID: 1552844
  38. A splicing factor that is inactivated during in vivo heat shock is functionally equivalent to the [U4/U6.U5] triple snRNP-specific proteins.
    Genes Dev. 1992 Apr;6(4):631-41 PMID: 1532785
  39. Biochemical mechanisms of constitutive and regulated pre-mRNA splicing.
    Annu Rev Cell Biol. 1991;7:559-99 PMID: 1839712
  40. PROSITE: a dictionary of sites and patterns in proteins.
    Nucleic Acids Res. 1992 May 11;20 Suppl:2013-8 PMID: 1598232
  41. Synthetic lethal mutations suggest interactions between U5 small nuclear RNA and four proteins required for the second step of splicing.
    Mol Cell Biol. 1992 Nov;12(11):5197-205 PMID: 1406691
  42. Stages in the second reaction of pre-mRNA splicing: the final step is ATP independent.
    Genes Dev. 1993 Feb;7(2):320-9 PMID: 8436300
  43. Messenger RNA splicing in yeast: clues to why the spliceosome is a ribonucleoprotein.
    Science. 1991 Jul 12;253(5016):157-63 PMID: 1853200
  44. Evidence for the existence of snRNAs U4 and U6 in a single ribonucleoprotein complex and for their association by intermolecular base pairing.
    EMBO J. 1984 Jun;3(6):1357-63 PMID: 6204860
  45. The "spliceosome": yeast pre-messenger RNA associates with a 40S complex in a splicing-dependent reaction.
    Science. 1985 May 24;228(4702):963-7 PMID: 3890181
  46. Stepwise assembly of a pre-mRNA splicing complex requires U-snRNPs and specific intron sequences.
    Cell. 1985 Aug;42(1):355-67 PMID: 3160483
  47. Yeast mRNA splicing in vitro.
    J Biol Chem. 1985 Nov 25;260(27):14780-92 PMID: 2997224
  48. The 3' splice site of pre-messenger RNA is recognized by a small nuclear ribonucleoprotein.
    Science. 1985 Dec 20;230(4732):1344-9 PMID: 2933810
  49. Nucleotide sequence and transcriptional mapping of the yeast pet56-his3-ded1 gene region.
    Nucleic Acids Res. 1985 Dec 9;13(23):8587-601 PMID: 3001645
  50. Affinity chromatography of splicing complexes: U2, U5, and U4 + U6 small nuclear ribonucleoprotein particles in the spliceosome.
    Science. 1986 Sep 19;233(4770):1294-9 PMID: 3638792
  51. The yeast RNA gene products are essential for mRNA splicing in vitro.
    Cell. 1986 Dec 26;47(6):953-63 PMID: 3536128
  52. Electrophoresis of ribonucleoproteins reveals an ordered assembly pathway of yeast splicing complexes.
    Nature. 1986 Nov 27-Dec 3;324(6095):341-5 PMID: 3537805
  53. An essential yeast snRNA with a U5-like domain is required for splicing in vivo.
    Cell. 1987 Jun 5;49(5):613-24 PMID: 3555841
  54. Interactions between small nuclear ribonucleoprotein particles in formation of spliceosomes.
    Cell. 1987 Jun 19;49(6):763-74 PMID: 2953438
  55. An ordered pathway of snRNP binding during mammalian pre-mRNA splicing complex assembly.
    EMBO J. 1987 Aug;6(8):2415-24 PMID: 2959470
  56. The PRP4 (RNA4) protein of Saccharomyces cerevisiae is associated with the 5' portion of the U4 small nuclear RNA.
    Mol Cell Biol. 1990 Mar;10(3):1217-25 PMID: 2154681
  57. In vitro assembly of yeast U6 snRNP: a functional assay.
    Genes Dev. 1989 Dec;3(12B):2137-50 PMID: 2560755
  58. Use of T7 RNA polymerase to direct expression of cloned genes.
    Methods Enzymol. 1990;185:60-89 PMID: 2199796
  59. Cloning and expression of rat liver CTP: phosphocholine cytidylyltransferase: an amphipathic protein that controls phosphatidylcholine synthesis.
    Proc Natl Acad Sci U S A. 1990 Aug;87(16):6029-33 PMID: 2166941
  60. Isolation of a gene for a regulatory 15-kDa subunit of mitochondrial F1F0-ATPase and construction of mutant yeast lacking the protein.
    Eur J Biochem. 1990 Aug 28;192(1):49-53 PMID: 2169416
  61. Domains of yeast U4 spliceosomal RNA required for PRP4 protein binding, snRNP-snRNP interactions, and pre-mRNA splicing in vivo.
    Genes Dev. 1990 Jul;4(7):1185-96 PMID: 2145195
  62. Two domains of yeast U6 small nuclear RNA required for both steps of nuclear precursor messenger RNA splicing.
    Science. 1990 Oct 19;250(4979):404-9 PMID: 2145630
  63. Basic local alignment search tool.
    J Mol Biol. 1990 Oct 5;215(3):403-10 PMID: 2231712
  64. Identification of a yeast snRNP protein and detection of snRNP-snRNP interactions.
    Cell. 1987 Dec 24;51(6):1019-26 PMID: 2961458
  65. Molecular cloning and characterization of the gene encoding cholinephosphate cytidylyltransferase in Saccharomyces cerevisiae.
    Eur J Biochem. 1987 Dec 15;169(3):477-86 PMID: 2826147
  66. Spliceosome assembly in yeast.
    Genes Dev. 1987 Nov;1(9):1014-27 PMID: 2962902
  67. Synthesis and sequence-specific proteolysis of hybrid proteins produced in Escherichia coli.
    Methods Enzymol. 1987;153:461-81 PMID: 3323806
  68. Improved tools for biological sequence comparison.
    Proc Natl Acad Sci U S A. 1988 Apr;85(8):2444-8 PMID: 3162770
  69. Cloning of the RNA8 gene of Saccharomyces cerevisiae, detection of the RNA8 protein, and demonstration that it is essential for nuclear pre-mRNA splicing.
    Mol Cell Biol. 1988 Mar;8(3):1067-75 PMID: 2835658
  70. RNA11 protein is associated with the yeast spliceosome and is localized in the periphery of the cell nucleus.
    Mol Cell Biol. 1988 Jun;8(6):2379-93 PMID: 3043176
  71. Splicing a spliceosomal RNA.
    Nature. 1989 Jan 5;337(6202):14-5 PMID: 2909888
  72. 20S small nuclear ribonucleoprotein U5 shows a surprisingly complex protein composition.
    Proc Natl Acad Sci U S A. 1989 Aug;86(16):6038-42 PMID: 2527369
Article Info
Journal
Molecular and cellular biology
Abbr.
Mol Cell Biol
ISSN
0270-7306
DOI
10.1128/mcb.13.5.2959-2970.1993
Published
1993-05-00
Pages
2959-70
Language
English
Region
United States
NLM ID
8109087
PMCID
PMC359689
Subset
IM
Grants
NIGMS NIH HHS · GM32367 · United States
Databases
GENBANK
L03536
External Links
PubMed source DOI: 10.1128/mcb.13.5.2959-2970.1993 PMC Full Text View journal details
Analysis Services
Analysis Services

Contact

No. 2 Wenbo Road, Zhangqiu District, Jinan, Shandong

Qilu Normal University · Genelibs Bioinformatics Lab

750 Shunhua Rd, Jinan

2F, Bldg F, University Science Park

Tel: 0531-88819269

WeChat Official Account

Follow our WeChat subscription account for real-time updates and the latest in medical and biological research.


Business Email

E-mail: product@genelibs.com

Copyright © 2026.Genelibs Jinan Honghui Biotechnology Co., Ltd. 鲁ICP备20007159号-2