Pre-mRNA splicing: awash in a sea of proteins.

What's in a spliceosome? More than we ever imagined, according to recent reports employing proteomics techniques to analyze this multi-megadalton machine. As of 1999, around 100 splicing factors were identified (Burge et al., 1999); however, that number has now nearly doubled due primarily to improved purification of spliceosomes coupled with advances in mass spectrometry analyses of complex mixtures. Gratifyingly, most of the previously identified splicing factors were found in the recent mass spec studies. Nonetheless, the number of new proteins emerging with no prior connection to splicing was surprising. Without functional validation, it would be premature to label these proteins as bona fide splicing factors. Yet many were identified multiple times in complexes purified under diverse conditions or from different organisms. Another recurring theme regards the dynamic nature of spliceosomal complexes, which may be even more intricate than previously thought.

[1]  B. Séraphin,et al.  The tandem affinity purification (TAP) method: a general procedure of protein complex purification. , 2001, Methods.

[2]  S. Gygi,et al.  Absolute quantification of proteins and phosphoproteins from cell lysates by tandem MS , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[3]  K. Gould,et al.  Proteomics Analysis Reveals Stable Multiprotein Complexes in Both Fission and Budding Yeasts Containing Myb-Related Cdc5p/Cef1p, Novel Pre-mRNA Splicing Factors, and snRNAs , 2002, Molecular and Cellular Biology.

[4]  J. Steitz,et al.  Human Upf Proteins Target an mRNA for Nonsense-Mediated Decay When Bound Downstream of a Termination Codon , 2000, Cell.

[5]  Henning Urlaub,et al.  Characterization of novel SF3b and 17S U2 snRNP proteins, including a human Prp5p homologue and an SF3b DEAD‐box protein , 2002, The EMBO journal.

[6]  M. Wilm,et al.  Protein composition of human prespliceosomes isolated by a tobramycin affinity-selection method , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[7]  Kevin Struhl,et al.  TREX is a conserved complex coupling transcription with messenger RNA export , 2002, Nature.

[8]  A. Shyu,et al.  RNA surveillance by nuclear scanning? , 2002, Nature Cell Biology.

[9]  M. Hayman,et al.  The Ski oncoprotein interacts with Skip, the human homolog of Drosophila Bx42 , 1998, Oncogene.

[10]  T. Maniatis,et al.  An extensive network of coupling among gene expression machines , 2002, Nature.

[11]  R. Reed,et al.  Functional association of U2 snRNP with the ATP-independent spliceosomal complex E. , 2000, Molecular cell.

[12]  Juri Rappsilber,et al.  Mass spectrometry and EST-database searching allows characterization of the multi-protein spliceosome complex , 1998, Nature Genetics.

[13]  J. Yates,et al.  Proteasomal proteomics: identification of nucleotide-sensitive proteasome-interacting proteins by mass spectrometric analysis of affinity-purified proteasomes. , 2000, Molecular biology of the cell.

[14]  J. Vilardell,et al.  Pre‐spliceosome formation in S.pombe requires a stable complex of SF1–U2AF59–U2AF23 , 2002, The EMBO journal.

[15]  J. Friesen,et al.  Splicing Factor Slt11p and Its Involvement in Formation of U2/U6 Helix II in Activation of the Yeast Spliceosome , 2001, Molecular and Cellular Biology.

[16]  Gary D Bader,et al.  Systematic identification of protein complexes in Saccharomyces cerevisiae by mass spectrometry , 2002, Nature.

[17]  P. Bork,et al.  Functional organization of the yeast proteome by systematic analysis of protein complexes , 2002, Nature.

[18]  T. Nilsen.  The spliceosome: no assembly required? , 2002, Molecular cell.

[19]  W. Tsai,et al.  Functional and physical interactions between components of the Prp19p-associated complex. , 2002, Nucleic acids research.

[20]  J. Steitz,et al.  Communication of the Position of Exon-Exon Junctions to the mRNA Surveillance Machinery by the Protein RNPS1 , 2001, Science.

[21]  H. Le Hir,et al.  Pre-mRNA splicing alters mRNP composition: evidence for stable association of proteins at exon-exon junctions. , 2000, Genes & development.

[22]  P. Bork,et al.  REF, an evolutionary conserved family of hnRNP-like proteins, interacts with TAP/Mex67p and participates in mRNA nuclear export. , 2000, RNA.

[23]  Henning Urlaub,et al.  Small Nuclear Ribonucleoprotein Remodeling During Catalytic Activation of the Spliceosome , 2002, Science.

[24]  Ruedi Aebersold,et al.  The study of macromolecular complexes by quantitative proteomics , 2003, Nature Genetics.

[25]  M. Snead,et al.  A Tuftelin-interacting Protein (TIP39) Localizes to the Apical Secretory Pole of Mouse Ameloblasts* , 2000, The Journal of Biological Chemistry.

[26]  J. Beggs,et al.  Identification and characterization of Prp45p and Prp46p, essential pre-mRNA splicing factors. , 2003, RNA.

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

[28]  Steven P. Gygi,et al.  Comprehensive proteomic analysis of the human spliceosome , 2002, Nature.

[29]  B. Ganem.  RNA world , 1987, Nature.

[30]  R. Rando,et al.  Specific binding of aminoglycoside antibiotics to RNA. , 1995, Chemistry & biology.

[31]  A. Furger,et al.  Integrating mRNA Processing with Transcription , 2002, Cell.

[32]  H. Le Hir,et al.  5' exon interactions within the human spliceosome establish a framework for exon junction complex structure and assembly. , 2002, Genes & development.

[33]  Roger E. Moore,et al.  Composition and functional characterization of the yeast spliceosomal penta-snRNP. , 2002, Molecular cell.

[34]  G. Dreyfuss,et al.  Role of the Nonsense-Mediated Decay Factor hUpf3 in the Splicing-Dependent Exon-Exon Junction Complex , 2001, Science.

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

[36]  M. Mann,et al.  Large-scale Proteomic Analysis of the Human Spliceosome References , 2006 .

[37]  Melanie D Ohi,et al.  Characterization of interactions among the Cef1p-Prp19p-associated splicing complex. , 2002, RNA.

[38]  Aaron J. Shatkin,et al.  The ends of the affair: Capping and polyadenylation , 2000, Nature Structural Biology.

[39]  A. Krainer,et al.  Pre-mRNA splicing in the new millennium. , 2001, Current opinion in cell biology.

[40]  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.

[41]  T. Misteli,et al.  A cyclophilin functions in pre‐mRNA splicing , 2002, The EMBO journal.

[42]  W. Tarn,et al.  Functional association of essential splicing factor(s) with PRP19 in a protein complex. , 1994, The EMBO journal.

[43]  H. Le Hir,et al.  The exon–exon junction complex provides a binding platform for factors involved in mRNA export and nonsense‐mediated mRNA decay , 2001, The EMBO journal.

[44]  M B Roth,et al.  SR proteins: a conserved family of pre-mRNA splicing factors. , 1992, Genes & development.

[45]  B. Blencowe,et al.  REF proteins mediate the export of spliced and unspliced mRNAs from the nucleus. , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[46]  Josette Banroques,et al.  Identification by mass spectrometry and functional analysis of novel proteins of the yeast [U4/U6·U5] tri‐snRNP , 1999, The EMBO journal.

[47]  Nikolaus Grigorieff,et al.  Purification and characterization of native spliceosomes suitable for three-dimensional structural analysis. , 2002, RNA.

[48]  B. Kuster,et al.  Functional analysis of the human CDC5L complex and identification of its components by mass spectrometry , 2000, The EMBO journal.

[49]  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.

[50]  E. B. Goodwin,et al.  Nuclear translation: what is the evidence? , 2003, RNA.

[51]  E. Hurt,et al.  The protein Aly links pre-messenger-RNA splicing to nuclear export in metazoans , 2000, Nature.

[52]  Bunpei Nakano,et al.  A CHARACTERIZATION OF INTERACTIONS , 1981 .