Identification by mass spectrometry and functional analysis of novel proteins of the yeast [U4/U6·U5] tri‐snRNP

The 25S [U4/U6·U5] tri‐snRNP (small nuclear ribonucleoprotein) is a central unit of the nuclear pre‐mRNA splicing machinery. The U4, U5 and U6 snRNAs undergo numerous rearrangements in the spliceosome, and knowledge of all of the tri‐snRNP proteins is crucial to the detailed investigation of the RNA dynamics during the spliceosomal cycle. Here we characterize by mass spectrometric methods the proteins of the purified [U4/U6·U5] tri‐snRNP from the yeast Saccharomyces cerevisiae. In addition to the known tri–snRNP proteins (only one, Lsm3p, eluded detection), we identified eight previously uncharacterized proteins. These include four Sm‐like proteins (Lsm2p, Lsm5p, Lsm6p and Lsm7p) and four specific proteins named Snu13p, Dib1p, Snu23p and Snu66p. Snu13p comprises a putative RNA‐binding domain. Interestingly, the Schizosaccharomyces pombe orthologue of Dib1p, Dim1p, was previously assigned a role in cell cycle progression. The role of Snu23p, Snu66p and, additionally, Spp381p in pre‐mRNA splicing was investigated in vitro and/or in vivo. Finally, we show that both tri‐snRNPs and the U2 snRNP are co‐precipitated with protein A‐tagged versions of Snu23p, Snu66p and Spp381p from extracts fractionated by glycerol gradient centrifugation. This suggests that these proteins, at least in part, are also present in a [U2·U4/U6·U5] tetra‐snRNP complex.

[1]  J D Beggs,et al.  Characterization of Sm‐like proteins in yeast and their association with U6 snRNA , 1999, The EMBO journal.

[2]  S. Stevens,et al.  Purification of the yeast U4/U6.U5 small nuclear ribonucleoprotein particle and identification of its proteins. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[3]  B. Séraphin,et al.  Partial purification of the yeast U2 snRNP reveals a novel yeast pre‐mRNA splicing factor required for pre‐spliceosome assembly , 1999, The EMBO journal.

[4]  Bertrand Séraphin,et al.  Sm and Sm‐like proteins assemble in two related complexes of deep evolutionary origin , 1999, The EMBO journal.

[5]  Lynne D. Berry,et al.  The Schizosaccharomyces pombe dim1+ Gene Interacts with the Anaphase-Promoting Complex or Cyclosome (APC/C) Component lid1+ and Is Required for APC/C Function , 1999, Molecular and Cellular Biology.

[6]  K. Nagai,et al.  Crystal Structures of Two Sm Protein Complexes and Their Implications for the Assembly of the Spliceosomal snRNPs , 1999, Cell.

[7]  V. Brown,et al.  Elevated Levels of a U4/U6.U5 snRNP-Associated Protein, Spp381p, Rescue a Mutant Defective in Spliceosome Maturation , 1999, Molecular and Cellular Biology.

[8]  M. Mann,et al.  Mapping of phosphorylation sites of gel-isolated proteins by nanoelectrospray tandem mass spectrometry: potentials and limitations. , 1999, Analytical chemistry.

[9]  S. Wolin,et al.  A role for the yeast La protein in U6 snRNP assembly: evidence that the La protein is a molecular chaperone for RNA polymerase III transcripts , 1998, The EMBO journal.

[10]  C. Kaiser,et al.  A Link between Secretion and Pre-mRNA Processing Defects in Saccharomyces cerevisiae and the Identification of a Novel Splicing Gene, RSE1 , 1998, Molecular and Cellular Biology.

[11]  M. Mann,et al.  Cbf5p, a potential pseudouridine synthase, and Nhp2p, a putative RNA-binding protein, are present together with Gar1p in all H BOX/ACA-motif snoRNPs and constitute a common bipartite structure. , 1998, RNA.

[12]  M. Caizergues-Ferrer,et al.  Nhp2p and Nop10p are essential for the function of H/ACA snoRNPs , 1998, The EMBO journal.

[13]  R. Lührmann,et al.  The Human U5-220kD Protein (hPrp8) Forms a Stable RNA-Free Complex with Several U5-Specific Proteins, Including an RNA Unwindase, a Homologue of Ribosomal Elongation Factor EF-2, and a Novel WD-40 Protein , 1998, Molecular and Cellular Biology.

[14]  B. Séraphin,et al.  New constructs and strategies for efficient PCR‐based gene manipulations in yeast , 1998, Yeast.

[15]  A. Sachs,et al.  Capped mRNA Degradation Intermediates Accumulate in the Yeast spb8-2 Mutant , 1998, Molecular and Cellular Biology.

[16]  C. Guthrie,et al.  RNA unwinding in U4/U6 snRNPs requires ATP hydrolysis and the DEIH-box splicing factor Brr2 , 1998, Current Biology.

[17]  J. Xie,et al.  Progression through the spliceosome cycle requires Prp38p function for U4/U6 snRNA dissociation , 1998, The EMBO journal.

[18]  M. Mann,et al.  A comprehensive biochemical and genetic analysis of the yeast U1 snRNP reveals five novel proteins. , 1998, RNA.

[19]  B. Séraphin,et al.  Interactions within the Yeast Sm Core Complex: from Proteins to Amino Acids , 1998, Molecular and Cellular Biology.

[20]  C. Guthrie,et al.  Mechanical Devices of the Spliceosome: Motors, Clocks, Springs, and Things , 1998, Cell.

[21]  M. Marahiel,et al.  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. , 1998, RNA.

[22]  T. Nilsen,et al.  RNA-RNA Interactions in Nuclear Pre-mRNA Splicing , 1998 .

[23]  M. Ares,et al.  Conservation of structure and subunit interactions in yeast homologues of splicing factor 3b (SF3b) subunits. , 1998, RNA.

[24]  A. Krainer,et al.  A new cyclophilin and the human homologues of yeast Prp3 and Prp4 form a complex associated with U4/U6 snRNPs. , 1997, RNA.

[25]  W. Zhang,et al.  Multiple protein: protein interactions between the snRNP common core proteins. , 1997, Experimental cell research.

[26]  C. Will,et al.  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. , 1997, RNA.

[27]  J. Woolford,et al.  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. , 1997, RNA.

[28]  P. Legrain,et al.  Toward a functional analysis of the yeast genome through exhaustive two-hybrid screens , 1997, Nature Genetics.

[29]  J. Lauber,et al.  An evolutionarily conserved U5 snRNP‐specific protein is a GTP‐binding factor closely related to the ribosomal translocase EF‐2 , 1997, EMBO Journal.

[30]  J. Woolford,et al.  Prp31p promotes the association of the U4/U6 x U5 tri-snRNP with prespliceosomes to form spliceosomes in Saccharomyces cerevisiae , 1997, Molecular and cellular biology.

[31]  K. Gould,et al.  Fission Yeast dim1 + Encodes a Functionally Conserved Polypeptide Essential for Mitosis , 1997, The Journal of cell biology.

[32]  C. Will,et al.  Protein functions in pre-mRNA splicing. , 1997, Current opinion in cell biology.

[33]  M. Mann,et al.  Cell biology and the genome projects a concerted strategy for characterizing multiprotein complexes by using mass spectrometry. , 1997, Trends in cell biology.

[34]  J. Lauber,et al.  The [U4/U6.U5] tri-snRNP-specific 27K protein is a novel SR protein that can be phosphorylated by the snRNP-associated protein kinase. , 1997, RNA.

[35]  J. Banroques,et al.  Mutations within the yeast U4/U6 snRNP protein Prp4 affect a late stage of spliceosome assembly. , 1997, RNA.

[36]  M. Mann,et al.  Identification of the proteins of the yeast U1 small nuclear ribonucleoprotein complex by mass spectrometry. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[37]  A. Krainer,et al.  A human protein required for the second step of pre-mRNA splicing is functionally related to a yeast splicing factor. , 1997, Genes & development.

[38]  A. Podtelejnikov,et al.  Linking genome and proteome by mass spectrometry: large-scale identification of yeast proteins from two dimensional gels. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[39]  R. Bordonné,et al.  The yeast SME1 gene encodes the homologue of the human E core protein. , 1996, Gene.

[40]  D. Tollervey,et al.  One-step PCR mediated strategy for the construction of conditionally expressed and epitope tagged yeast proteins. , 1996, Nucleic acids research.

[41]  J. Lauber,et al.  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. , 1996, The EMBO journal.

[42]  J. Rossi,et al.  Identification and characterization of yeast mutants that overcome an experimentally introduced block to splicing at the 3' splice site. , 1996, RNA.

[43]  J. Friesen,et al.  An RNA-dependent ATPase associated with U2/U6 snRNAs in pre-mRNA splicing , 1996, Nature.

[44]  C. Guthrie,et al.  Identification of novel genes required for yeast pre-mRNA splicing by means of cold-sensitive mutations. , 1996, Genetics.

[45]  R. Lührmann,et al.  The snRNP core assembly pathway: identification of stable core protein heteromeric complexes and an snRNP subcore particle in vitro. , 1996, The EMBO journal.

[46]  A. Shevchenko,et al.  Femtomole sequencing of proteins from polyacrylamide gels by nano-electrospray mass spectrometry , 1996, Nature.

[47]  R. Reed,et al.  Evidence that sequence-independent binding of highly conserved U2 snRNP proteins upstream of the branch site is required for assembly of spliceosomal complex A. , 1996, Genes & development.

[48]  A. Krämer,et al.  The structure and function of proteins involved in mammalian pre-mRNA splicing. , 1996, Annual review of biochemistry.

[49]  B. Séraphin.  Sm and Sm‐like proteins belong to a large family: identification of proteins of the U6 as well as the U1, U2, U4 and U5 snRNPs. , 1995, The EMBO journal.

[50]  L. Johnston,et al.  Identification and characterization of Uss1p (Sdb23p): a novel U6 snRNA‐associated protein with significant similarity to core proteins of small nuclear ribonucleoproteins. , 1995, The EMBO journal.

[51]  C. Guthrie,et al.  A novel role for a U5 snRNP protein in 3' splice site selection. , 1995, Genes & development.

[52]  J. Woolford,et al.  Structurally related but functionally distinct yeast Sm D core small nuclear ribonucleoprotein particle proteins , 1995, Molecular and cellular biology.

[53]  C. Will,et al.  Nuclear Pre-mRNA Splicing , 1995 .

[54]  M. Wilm,et al.  Error-tolerant identification of peptides in sequence databases by peptide sequence tags. , 1994, Analytical chemistry.

[55]  Peter Roepstorff,et al.  Improved resolution and very high sensitivity in MALDI TOF of matrix surfaces made by fast evaporation , 1994 .

[56]  C. Guthrie,et al.  PRP28, a 'DEAD-box' protein, is required for the first step of mRNA splicing in vitro. , 1994, Nucleic acids research.

[57]  C. Sander,et al.  A novel RNA-binding motif in omnipotent suppressors of translation termination, ribosomal proteins and a ribosome modification enzyme? , 1994, Nucleic acids research.

[58]  B. Kastner,et al.  Isolation of S. cerevisiae snRNPs: comparison of U1 and U4/U6.U5 to their human counterparts. , 1994, Science.

[59]  D. Horowitz,et al.  A U5 small nuclear ribonucleoprotein particle protein involved only in the second step of pre-mRNA splicing in Saccharomyces cerevisiae , 1993, Molecular and cellular biology.

[60]  Brian C. Rymond,et al.  Convergent transcripts of the yeast PRP38-SMD1 locus encode two essential splicing factors, including the D1 core polypeptide of small nuclear ribonucleoprotein particles. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[61]  P. Sharp,et al.  Splicing of precursors to mRNAs by the spliceosomes , 1993 .

[62]  K. Hall,et al.  The 5' splice site consensus RNA oligonucleotide induces assembly of U2/U4/U5/U6 small nuclear ribonucleoprotein complexes. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[63]  J. Steitz,et al.  Interactions of small nuclear RNA's with precursor messenger RNA during in vitro splicing. , 1992, Science.

[64]  Brian C. Rymond,et al.  PRP38 encodes a yeast protein required for pre-mRNA splicing and maintenance of stable U6 small nuclear RNA levels , 1992, Molecular and cellular biology.

[65]  D. Lilley,et al.  Nucleic Acids and Molecular Biology , 1992, Nucleic Acids and Molecular Biology.

[66]  P. Legrain,et al.  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 , 1990, Molecular and cellular biology.

[67]  J. Banroques,et al.  PRP4: a protein of the yeast U4/U6 small nuclear ribonucleoprotein particle , 1989, Molecular and cellular biology.

[68]  J. Beggs,et al.  PRP4 (RNA4) from Saccharomyces cerevisiae: its gene product is associated with the U4/U6 small nuclear ribonucleoprotein particle , 1989, Molecular and cellular biology.

[69]  R. Sikorski,et al.  A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. , 1989, Genetics.

[70]  M. Yamamoto.  Fission yeast. , 1989, Biotechnology.

[71]  P. Sharp,et al.  Association of U2, U4, U5, and U6 small nuclear ribonucleoproteins in a spliceosome-type complex in absence of precursor RNA. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[72]  R. Wagner,et al.  5S RNA structure and function. , 1988, Methods in enzymology.

[73]  J. Beggs,et al.  Identification of a yeast snRNP protein and detection of snRNP-snRNP interactions , 1987, Cell.

[74]  P. Sharp,et al.  Affinity chromatography of splicing complexes: U2, U5, and U4 + U6 small nuclear ribonucleoprotein particles in the spliceosome. , 1986, Science.

[75]  R. Lin,et al.  Yeast mRNA splicing in vitro. , 1985, The Journal of biological chemistry.

[76]  H. Weaver,et al.  Conservation of what? , 1966, Science.

[77]  Aagcttgacc Tgcatgggat Tcagcattag,et al.  PRP 4 ( RNA 4 ) from Saccharomyces cerevisiae : Its Gene Product Is Associated with the U 4 / U 6 Small Nuclear Ribonucleoprotein Particle , 2022 .