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PMID: 17537823 Published · ppublish English Journal Article Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, Non-P.H.S.

Proteomic analysis of in vivo-assembled pre-mRNA splicing complexes expands the catalog of participating factors.

Nucleic acids research ·Vol. 35 ·No. 12 ·2007-00-00 ·Pages 3928-44

Chen YI, Moore RE, Ge HY, Young MK, Lee TD, Stevens SW

Abstract

Previous compositional studies of pre-mRNA processing complexes have been performed in vitro on synthetic pre-mRNAs containing a single intron. To provide a more comprehensive list of polypeptides associated with the pre-mRNA splicing apparatus, we have determined the composition of the bulk pre-mRNA processing machinery in living cells. We purified endogenous nuclear pre-mRNA processing complexes from human and chicken cells comprising the massive (>200S) supraspliceosomes (a.k.a. polyspliceosomes). As expected, RNA components include a heterogeneous mixture of pre-mRNAs and the five spliceosomal snRNAs. In addition to known pre-mRNA splicing factors, 5' end binding factors, 3' end processing factors, mRNA export factors, hnRNPs and other RNA binding proteins, the protein components identified by mass spectrometry include RNA adenosine deaminases and several novel factors. Intriguingly, our purified supraspliceosomes also contain a number of structural proteins, nucleoporins, chromatin remodeling factors and several novel proteins that were absent from splicing complexes assembled in vitro. These in vivo analyses bring the total number of factors associated with pre-mRNA to well over 300, and represent the most comprehensive analysis of the pre-mRNA processing machinery to date.

MeSH Terms
Animals Cell Line Chickens/metabolism Cyclophilins/analysis HeLa Cells Heterogeneous-Nuclear Ribonucleoproteins/analysis Humans Mass Spectrometry Nuclear Proteins/analysis Peptides/analysis,isolation & purification Proteomics RNA Helicases/analysis RNA Precursors/isolation & purification,metabolism RNA Splicing RNA, Messenger/isolation & purification,metabolism RNA, Small Nuclear/analysis,isolation & purification RNA-Binding Proteins/analysis Ribonucleoproteins/analysis,isolation & purification Ribonucleoproteins, Small Nuclear/analysis,biosynthesis Serine-Arginine Splicing Factors Spliceosomes/chemistry
Chemicals
Heterogeneous-Nuclear Ribonucleoproteins Nuclear Proteins Peptides RNA Precursors RNA, Messenger RNA, Small Nuclear RNA-Binding Proteins Ribonucleoproteins Ribonucleoproteins, Small Nuclear Serine-Arginine Splicing Factors RNA Helicases Cyclophilins
Authors & Affiliations
6 authors, click to expand affiliations / ORCID
Chen Yen-I G
Graduate program in Microbiology, City of Hope Beckman Research Institute, Duarte, CA 91010, USA.
Moore Roger E
Ge Helen Y
Young Mary K
Lee Terry D
Stevens Scott W
References (103)
103 references, click to expand
  1. Rat1p and Xrn1p are functionally interchangeable exoribonucleases that are restricted to and required in the nucleus and cytoplasm, respectively.
    Mol Cell Biol. 1997 Oct;17(10):6122-30 PMID: 9315672
  2. Actin and myosin I in the nucleus: what next?
    Nat Struct Mol Biol. 2005 Sep;12(9):742-6 PMID: 16142228
  3. PUF60: a novel U2AF65-related splicing activity.
    RNA. 1999 Dec;5(12):1548-60 PMID: 10606266
  4. Requirement of the RNA helicase-like protein PRP22 for release of messenger RNA from spliceosomes.
    Nature. 1991 Feb 7;349(6309):487-93 PMID: 1992352
  5. Mammalian PRP4 kinase copurifies and interacts with components of both the U5 snRNP and the N-CoR deacetylase complexes.
    Mol Cell Biol. 2002 Jul;22(14):5141-56 PMID: 12077342
  6. Prp43 is an essential RNA-dependent ATPase required for release of lariat-intron from the spliceosome.
    J Biol Chem. 2002 May 17;277(20):17743-50 PMID: 11886864
  7. Composition and functional characterization of the yeast spliceosomal penta-snRNP.
    Mol Cell. 2002 Jan;9(1):31-44 PMID: 11804584
  8. Proteomics analysis reveals stable multiprotein complexes in both fission and budding yeasts containing Myb-related Cdc5p/Cef1p, novel pre-mRNA splicing factors, and snRNAs.
    Mol Cell Biol. 2002 Apr;22(7):2011-24 PMID: 11884590
  9. A cold-sensitive mRNA splicing mutant is a member of the RNA helicase gene family.
    Genes Dev. 1991 Apr;5(4):629-41 PMID: 2010088
  10. The human 18S U11/U12 snRNP contains a set of novel proteins not found in the U2-dependent spliceosome.
    RNA. 2004 Jun;10(6):929-41 PMID: 15146077
  11. Identification of Prp40, a novel essential yeast splicing factor associated with the U1 small nuclear ribonucleoprotein particle.
    Mol Cell Biol. 1996 Mar;16(3):960-7 PMID: 8622699
  12. Three-dimensional image reconstruction of large nuclear RNP (lnRNP) particles by automated electron tomography.
    J Mol Biol. 1997 Apr 4;267(3):570-83 PMID: 9126839
  13. PRP19: a novel spliceosomal component.
    Mol Cell Biol. 1993 Mar;13(3):1876-82 PMID: 8441419
  14. Characterization of cDNAs encoding the polypyrimidine tract-binding protein.
    Genes Dev. 1991 Jul;5(7):1224-36 PMID: 1906035
  15. The identification and characterization of a novel splicing protein, Isy1p, of Saccharomyces cerevisiae.
    RNA. 1999 Mar;5(3):360-8 PMID: 10094305
  16. The yeast Rat1 exonuclease promotes transcription termination by RNA polymerase II.
    Nature. 2004 Nov 25;432(7016):517-22 PMID: 15565157
  17. Three protein factors (SF1, SF3 and U2AF) function in pre-splicing complex formation in addition to snRNPs.
    EMBO J. 1991 Jun;10(6):1503-9 PMID: 1827409
  18. Purification of the yeast U4/U6.U5 small nuclear ribonucleoprotein particle and identification of its proteins.
    Proc Natl Acad Sci U S A. 1999 Jun 22;96(13):7226-31 PMID: 10377396
  19. PRP5: a helicase-like protein required for mRNA splicing in yeast.
    Proc Natl Acad Sci U S A. 1990 Jun;87(11):4236-40 PMID: 2349233
  20. TAP, the human homolog of Mex67p, mediates CTE-dependent RNA export from the nucleus.
    Mol Cell. 1998 Apr;1(5):649-59 PMID: 9660949
  21. Deletion of MUD2, the yeast homolog of U2AF65, can bypass the requirement for sub2, an essential spliceosomal ATPase.
    Genes Dev. 2001 Jan 1;15(1):42-9 PMID: 11156604
  22. Commitment of yeast pre-mRNA to the splicing pathway requires a novel U1 small nuclear ribonucleoprotein polypeptide, Prp39p.
    Mol Cell Biol. 1994 Jun;14(6):3623-33 PMID: 8196608
  23. Native spliceosomes assemble with pre-mRNA to form supraspliceosomes.
    J Mol Biol. 2006 Mar 3;356(4):955-66 PMID: 16386271
  24. The human SWI/SNF subunit Brm is a regulator of alternative splicing.
    Nat Struct Mol Biol. 2006 Jan;13(1):22-9 PMID: 16341228
  25. Assembly of the nuclear transcription and processing machinery: Cajal bodies (coiled bodies) and transcriptosomes.
    Mol Biol Cell. 1999 Dec;10(12):4385-402 PMID: 10588665
  26. A new cyclophilin and the human homologues of yeast Prp3 and Prp4 form a complex associated with U4/U6 snRNPs.
    RNA. 1997 Dec;3(12):1374-87 PMID: 9404889
  27. A supraspliceosome model for large nuclear ribonucleoprotein particles based on mass determinations by scanning transmission electron microscopy.
    J Mol Biol. 1998 Oct 23;283(2):383-94 PMID: 9769212
  28. A base-pairing interaction between U2 and U6 small nuclear RNAs occurs in > 150S complexes in HeLa cell extracts: implications for the spliceosome assembly pathway.
    Proc Natl Acad Sci U S A. 1993 Aug 1;90(15):7139-43 PMID: 8346227
  29. A new view of mRNA export: separating the wheat from the chaff.
    Nat Cell Biol. 2001 Sep;3(9):E201-4 PMID: 11533670
  30. The human La (SS-B) autoantigen interacts with DDX15/hPrp43, a putative DEAH-box RNA helicase.
    RNA. 2002 Nov;8(11):1428-43 PMID: 12458796
  31. The Glc7p nuclear phosphatase promotes mRNA export by facilitating association of Mex67p with mRNA.
    Mol Cell. 2004 Jan 30;13(2):201-12 PMID: 14759366
  32. The yeast U5 snRNP coisolated with the U1 snRNP has an unexpected protein composition and includes the splicing factor Aar2p.
    RNA. 2001 Nov;7(11):1554-65 PMID: 11720285
  33. The Prp19p-associated complex in spliceosome activation.
    Science. 2003 Oct 10;302(5643):279-82 PMID: 12970570
  34. Mass spectrometry and EST-database searching allows characterization of the multi-protein spliceosome complex.
    Nat Genet. 1998 Sep;20(1):46-50 PMID: 9731529
  35. Identification of novel genes required for yeast pre-mRNA splicing by means of cold-sensitive mutations.
    Genetics. 1996 May;143(1):67-80 PMID: 8722763
  36. RNA editing activity is associated with splicing factors in lnRNP particles: The nuclear pre-mRNA processing machinery.
    Proc Natl Acad Sci U S A. 2001 Jun 5;98(12):6571-6 PMID: 11381114
  37. ATPase/helicase activities of p68 RNA helicase are required for pre-mRNA splicing but not for assembly of the spliceosome.
    Mol Cell Biol. 2005 Sep;25(17):7484-93 PMID: 16107697
  38. 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
  39. Complex formation among the RNA export proteins Nup98, Rae1/Gle2, and TAP.
    J Biol Chem. 2003 Jun 6;278(23):20979-88 PMID: 12637516
  40. Yeast pre-mRNA splicing: methods, mechanisms, and machinery.
    Methods Enzymol. 2002;351:200-20 PMID: 12073346
  41. The tandem affinity purification (TAP) method: a general procedure of protein complex purification.
    Methods. 2001 Jul;24(3):218-29 PMID: 11403571
  42. Genetic and physical interactions between factors involved in both cell cycle progression and pre-mRNA splicing in Saccharomyces cerevisiae.
    Genetics. 2000 Dec;156(4):1503-17 PMID: 11102353
  43. Spliceosome assembly involves the binding and release of U4 small nuclear ribonucleoprotein.
    Proc Natl Acad Sci U S A. 1988 Jan;85(2):411-5 PMID: 2963332
  44. Characterization of novel SF3b and 17S U2 snRNP proteins, including a human Prp5p homologue and an SF3b DEAD-box protein.
    EMBO J. 2002 Sep 16;21(18):4978-88 PMID: 12234937
  45. Prp43: An RNA helicase-like factor involved in spliceosome disassembly.
    Proc Natl Acad Sci U S A. 1997 Oct 28;94(22):11798-802 PMID: 9342317
  46. PRP28, a 'DEAD-box' protein, is required for the first step of mRNA splicing in vitro.
    Nucleic Acids Res. 1994 Aug 11;22(15):3187-93 PMID: 7520570
  47. Characterization and molecular cloning of polypyrimidine tract-binding protein: a component of a complex necessary for pre-mRNA splicing.
    Genes Dev. 1991 Jul;5(7):1237-51 PMID: 1906036
  48. The human U5 snRNP-specific 100-kD protein is an RS domain-containing, putative RNA helicase with significant homology to the yeast splicing factor Prp28p.
    RNA. 1997 Nov;3(11):1313-26 PMID: 9409622
  49. Abundant nuclear ribonucleoprotein form of CAD RNA.
    Mol Cell Biol. 1985 Mar;5(3):569-75 PMID: 3990684
  50. The 20kD protein of human [U4/U6.U5] tri-snRNPs is a novel cyclophilin that forms a complex with the U4/U6-specific 60kD and 90kD proteins.
    RNA. 1998 Feb;4(2):127-41 PMID: 9570313
  51. Protein components specifically associated with prespliceosome and spliceosome complexes.
    Genes Dev. 1992 Oct;6(10):1986-2000 PMID: 1398075
  52. A nuclear function of Hu proteins as neuron-specific alternative RNA processing regulators.
    Mol Biol Cell. 2006 Dec;17(12):5105-14 PMID: 17035636
  53. Distributions of exons and introns in the human genome.
    In Silico Biol. 2004;4(4):387-93 PMID: 15217358
  54. Luc7p, a novel yeast U1 snRNP protein with a role in 5' splice site recognition.
    Genes Dev. 1999 Sep 15;13(18):2425-38 PMID: 10500099
  55. Yeast ortholog of the Drosophila crooked neck protein promotes spliceosome assembly through stable U4/U6.U5 snRNP addition.
    RNA. 1999 Aug;5(8):1042-54 PMID: 10445879
  56. RNA unwinding in U4/U6 snRNPs requires ATP hydrolysis and the DEIH-box splicing factor Brr2.
    Curr Biol. 1998 Jul 16;8(15):847-55 PMID: 9705931
  57. Increased ratio of targeted to random integration after transfection of chicken B cell lines.
    Cell. 1991 Oct 4;67(1):179-88 PMID: 1913816
  58. The Saccharomyces cerevisiae Prp5 protein has RNA-dependent ATPase activity with specificity for U2 small nuclear RNA.
    J Biol Chem. 1996 Dec 27;271(52):33261-7 PMID: 8969184
  59. The ends of the affair: capping and polyadenylation.
    Nat Struct Biol. 2000 Oct;7(10):838-42 PMID: 11017188
  60. An NMD pathway in yeast involving accelerated deadenylation and exosome-mediated 3'-->5' degradation.
    Mol Cell. 2003 May;11(5):1405-13 PMID: 12769863
  61. Isolation and characterization of RAT1: an essential gene of Saccharomyces cerevisiae required for the efficient nucleocytoplasmic trafficking of mRNA.
    Genes Dev. 1992 Jul;6(7):1173-89 PMID: 1628825
  62. Large-scale proteomic analysis of the human spliceosome.
    Genome Res. 2002 Aug;12(8):1231-45 PMID: 12176931
  63. TREX is a conserved complex coupling transcription with messenger RNA export.
    Nature. 2002 May 16;417(6886):304-8 PMID: 11979277
  64. Identification of HuR as a protein implicated in AUUUA-mediated mRNA decay.
    EMBO J. 1997 Apr 15;16(8):2130-9 PMID: 9155038
  65. Functional analyses of interacting factors involved in both pre-mRNA splicing and cell cycle progression in Saccharomyces cerevisiae.
    RNA. 2000 Nov;6(11):1565-72 PMID: 11105756
  66. Isolation and visualization of large compact ribonucleoprotein particles of specific nuclear RNAs.
    Proc Natl Acad Sci U S A. 1989 Jan;86(2):466-70 PMID: 2521390
  67. Three-dimensional structure of the native spliceosome by cryo-electron microscopy.
    Mol Cell. 2004 Sep 10;15(5):833-9 PMID: 15350226
  68. The DEAH-box protein PRP22 is an ATPase that mediates ATP-dependent mRNA release from the spliceosome and unwinds RNA duplexes.
    EMBO J. 1998 May 15;17(10):2926-37 PMID: 9582286
  69. A nuclear cap-binding complex facilitates association of U1 snRNP with the cap-proximal 5' splice site.
    Genes Dev. 1996 Jul 1;10(13):1683-98 PMID: 8682298
  70. PRP38 encodes a yeast protein required for pre-mRNA splicing and maintenance of stable U6 small nuclear RNA levels.
    Mol Cell Biol. 1992 Sep;12(9):3939-47 PMID: 1508195
  71. Cotranscriptional coupling of splicing factor recruitment and precursor messenger RNA splicing in mammalian cells.
    Nat Struct Mol Biol. 2006 Sep;13(9):815-22 PMID: 16921380
  72. Comprehensive proteomic analysis of the human spliceosome.
    Nature. 2002 Sep 12;419(6903):182-5 PMID: 12226669
  73. Rapid protein identification using a microscale electrospray LC/MS system on an ion trap mass spectrometer.
    J Am Soc Mass Spectrom. 1998 Mar;9(3):194-201 PMID: 9879356
  74. How introns influence and enhance eukaryotic gene expression.
    Trends Biochem Sci. 2003 Apr;28(4):215-20 PMID: 12713906
  75. Identification by mass spectrometry and functional analysis of novel proteins of the yeast [U4/U6.U5] tri-snRNP.
    EMBO J. 1999 Aug 16;18(16):4535-48 PMID: 10449419
  76. Interactions of PRP2 protein with pre-mRNA splicing complexes in Saccharomyces cerevisiae.
    Nucleic Acids Res. 1990 Nov 25;18(22):6559-64 PMID: 2251118
  77. An extensive network of coupling among gene expression machines.
    Nature. 2002 Apr 4;416(6880):499-506 PMID: 11932736
  78. Human RNPS1 and its associated factors: a versatile alternative pre-mRNA splicing regulator in vivo.
    Mol Cell Biol. 2004 Feb;24(3):1174-87 PMID: 14729963
  79. A cooperative interaction between U2AF65 and mBBP/SF1 facilitates branchpoint region recognition.
    Genes Dev. 1998 Mar 15;12(6):858-67 PMID: 9512519
  80. The ever-increasing complexities of the exon junction complex.
    Curr Opin Cell Biol. 2004 Jun;16(3):279-84 PMID: 15145352
  81. The splicing factor PRP2, a putative RNA helicase, interacts directly with pre-mRNA.
    EMBO J. 1994 Feb 15;13(4):888-97 PMID: 8112302
  82. p54(nrb) associates with the 5' splice site within large transcription/splicing complexes.
    EMBO J. 2004 Apr 21;23(8):1782-91 PMID: 15057275
  83. An RNA-export mediator with an essential nuclear export signal.
    Nature. 1996 Sep 26;383(6598):357-60 PMID: 8848052
  84. The human Ski-interacting protein functionally substitutes for the yeast PRP45 gene.
    Biochem Biophys Res Commun. 2004 Jul 9;319(4):1105-9 PMID: 15194481
  85. hNMP 200: a novel human common nuclear matrix protein combining structural and regulatory functions.
    Exp Cell Res. 2000 Nov 25;261(1):166-79 PMID: 11082287
  86. Cef1p is a component of the Prp19p-associated complex and essential for pre-mRNA splicing.
    J Biol Chem. 1999 Apr 2;274(14):9455-62 PMID: 10092627
  87. The HeLa 200 kDa U5 snRNP-specific protein and its homologue in Saccharomyces cerevisiae are members of the DEXH-box protein family of putative RNA helicases.
    EMBO J. 1996 Aug 1;15(15):4001-15 PMID: 8670905
  88. Pre-mRNA splicing: awash in a sea of proteins.
    Mol Cell. 2003 Jul;12(1):5-14 PMID: 12887888
  89. An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database.
    J Am Soc Mass Spectrom. 1994 Nov;5(11):976-89 PMID: 24226387
  90. Prp22, a DExH-box RNA helicase, plays two distinct roles in yeast pre-mRNA splicing.
    EMBO J. 1998 Apr 1;17(7):2086-94 PMID: 9524130
  91. Identification and characterization of Prp45p and Prp46p, essential pre-mRNA splicing factors.
    RNA. 2003 Jan;9(1):138-50 PMID: 12554883
  92. Phosphorylated Ser/Arg-rich proteins: limiting factors in the assembly of 200S large nuclear ribonucleoprotein particles.
    Proc Natl Acad Sci U S A. 1996 Aug 20;93(17):8830-5 PMID: 8799112
  93. Modulation of alternative pre-mRNA splicing in vivo by pinin.
    Biochem Biophys Res Commun. 2002 Jun 7;294(2):448-55 PMID: 12051732
  94. Electrophoretic separation of complexes involved in the splicing of precursors to mRNAs.
    Cell. 1986 Sep 12;46(6):845-55 PMID: 2944598
  95. The human Tap protein is a nuclear mRNA export factor that contains novel RNA-binding and nucleocytoplasmic transport sequences.
    Genes Dev. 1999 May 1;13(9):1126-39 PMID: 10323864
  96. Epitope tagging of proteins at the native chromosomal loci of genes in mice and in cultured vertebrate cells.
    J Mol Biol. 2006 Aug 18;361(3):412-9 PMID: 16859702
  97. Improvements in two-dimensional gel electrophoresis by utilizing a low cost "in-house" neutral pH sodium dodecyl sulfate-polyacrylamide gel electrophoresis system.
    Proteomics. 2005 Jun;5(9):2309-14 PMID: 15887185
  98. Identification and characterization of yUAP/Sub2p, a yeast homolog of the essential human pre-mRNA splicing factor hUAP56.
    Genes Dev. 2001 Jan 1;15(1):30-5 PMID: 11156602
  99. Improved staining of proteins in polyacrylamide gels including isoelectric focusing gels with clear background at nanogram sensitivity using Coomassie Brilliant Blue G-250 and R-250.
    Electrophoresis. 1988 Jun;9(6):255-62 PMID: 2466658
  100. Method for screening peptide fragment ion mass spectra prior to database searching.
    J Am Soc Mass Spectrom. 2000 May;11(5):422-6 PMID: 10790846
  101. Initial sequencing and analysis of the human genome.
    Nature. 2001 Feb 15;409(6822):860-921 PMID: 11237011
  102. Broad specificity of SR (serine/arginine) proteins in the regulation of alternative splicing of pre-messenger RNA.
    Prog Nucleic Acid Res Mol Biol. 2004;78:37-88 PMID: 15210328
  103. Multiple roles for the yeast SUB2/yUAP56 gene in splicing.
    Genes Dev. 2001 Jan 1;15(1):36-41 PMID: 11156603
Article Info
Journal
Nucleic acids research
Abbr.
Nucleic Acids Res
ISSN
1362-4962
DOI
10.1093/nar/gkm347
Published
2007-00-00
Epub
2007-00-30
Pages
3928-44
Language
English
Region
England
NLM ID
0411011
PMCID
PMC1919476
Subset
IM
Grants
NCI NIH HHS · P30 CA033572 · United States
NCI NIH HHS · P30 CA33572 · United States
External Links
PubMed source DOI: 10.1093/nar/gkm347 PMC Full Text View journal details
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