title: Proteomics studies of the interactome of RNA polymerase II C-terminal repeated domain.
abstract: BACKGROUND
  Eukaryotic RNA polymerase II contains a C-terminal repeated domain (CTD) consisting of 52 consensus heptad repeats of Y1S2P3T4S5P6S7 that mediate interactions with many cellular proteins to regulate transcription elongation, RNA processing and chromatin structure. A number of CTD-binding proteins have been identified and the crystal structures of several protein-CTD complexes have demonstrated considerable conformational flexibility of the heptad repeats in those interactions. Furthermore, phosphorylation of the CTD at tyrosine, serine and threonine residues can regulate the CTD-protein interactions. Although the interactions of CTD with specific proteins have been elucidated at the atomic level, the capacity and specificity of the CTD-interactome in mammalian cells is not yet determined.
  
  
  RESULTS
  A proteomic study was conducted to examine the mammalian CTD-interactome. We utilized six synthetic peptides each consisting of four consensus CTD-repeats with different combinations of serine and tyrosine phosphorylation as affinity-probes to pull-down nuclear proteins from HeLa cells. The pull-down fractions were then analyzed by MUDPIT mass spectrometry, which identified 100 proteins with the majority from the phospho-CTD pull-downs. Proteins pulled-down by serine-phosphorylated CTD-peptides included those containing the previously defined CTD-interacting domain (CID). Using SILAC mass spectrometry, we showed that the in vivo interaction of RNA polymerase II with the mammalian CID-containing RPRD1B is disrupted by CID mutation. We also showed that the CID from four mammalian proteins interacted with pS2-phosphorylated but not pY1pS2-doubly phosphorylated CTD-peptides. However, we also found proteins that were preferentially pulled-down by pY1pS2- or pY1pS5-doubly phosphorylated CTD-peptides. We prepared an antibody against tyrosine phosphorylated CTD and showed that ionizing radiation (IR) induced a transient increase in CTD tyrosine phosphorylation by immunoblotting. Combining SILAC and IMAC purification of phospho-peptides, we found that IR regulated the phosphorylation at four CTD tyrosine sites in different ways.
  
  
  CONCLUSIONS
  Upon phosphorylation, the 52 repeats of the CTD have the capacity to generate a large number of binding sites for cellular proteins. This study confirms previous findings that serine phosphorylation stimulates whereas tyrosine phosphorylation inhibits the protein-binding activity of the CTD. However, tyrosine phosphorylation of the CTD can also stimulate other CTD-protein interactions. The CTD-peptide affinity pull-down method described here can be adopted to survey the mammalian CTD-interactome in various cell types and under different biological conditions.
authors.affiliation: Moores Cancer Center, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA, 92093, USA. gpineda@ucsd.edu.|Department of Medicine, Division of Hematology-Oncology, University of California, San Diego, George Palade Laboratories Room 256, 9500 Gilman Drive, La Jolla, CA, 92093, USA. gpineda@ucsd.edu.
authors.forename: Gabriel
authors.initials: G
authors.lastname: Pineda
authors.affiliation: Division of Biological Sciences, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA, 92093, USA. z1shen@ucsd.edu.
authors.forename: Zhouxin
authors.initials: Z
authors.lastname: Shen
authors.affiliation: Ludwig Institute for Cancer Research, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA, 92093, USA. cpontede@gmail.com.|Department of Cellular and Molecular Medicine, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA, 92093, USA. cpontede@gmail.com.
authors.forename: Claudio Ponte
authors.initials: CP
authors.lastname: de Albuquerque
authors.affiliation: Moores Cancer Center, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA, 92093, USA. Eduardo.Reynoso@utsouthwestern.edu.|Division of Biological Sciences, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA, 92093, USA. Eduardo.Reynoso@utsouthwestern.edu.
authors.forename: Eduardo
authors.initials: E
authors.lastname: Reynoso
authors.affiliation: Moores Cancer Center, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA, 92093, USA. jec036@gmail.com.|Division of Biological Sciences, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA, 92093, USA. jec036@gmail.com.
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Chen
authors.affiliation: Moores Cancer Center, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA, 92093, USA. cctu010810@ibms.sinica.edu.tw.
authors.forename: Chi-Chiang
authors.initials: CC
authors.lastname: Tu
authors.affiliation: Moores Cancer Center, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA, 92093, USA. wilsontang2@gmail.com.|Division of Biological Sciences, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA, 92093, USA. wilsontang2@gmail.com.
authors.forename: Wingchung
authors.initials: W
authors.lastname: Tang
authors.affiliation: Division of Biological Sciences, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA, 92093, USA. sbriggs@ucsd.edu.
authors.forename: Steve
authors.initials: S
authors.lastname: Briggs
authors.affiliation: Ludwig Institute for Cancer Research, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA, 92093, USA. huzhou@ucsd.edu.|Department of Cellular and Molecular Medicine, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA, 92093, USA. huzhou@ucsd.edu.
authors.forename: Huilin
authors.initials: H
authors.lastname: Zhou
authors.affiliation: Moores Cancer Center, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA, 92093, USA. jywang@ucsd.edu.|Department of Medicine, Division of Hematology-Oncology, University of California, San Diego, George Palade Laboratories Room 256, 9500 Gilman Drive, La Jolla, CA, 92093, USA. jywang@ucsd.edu.|Division of Biological Sciences, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA, 92093, USA. jywang@ucsd.edu.
authors.forename: Jean Y J
authors.initials: JY
authors.lastname: Wang
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D018797:Cell Cycle Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005720:Gamma Rays; D005786:Gene Expression Regulation; D006367:HeLa Cells; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009363:Neoplasm Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010455:Peptides / Q000138:chemical synthesis / Q000378:metabolism*; D010766:Phosphorylation / Q000528:radiation effects; D011485:Protein Binding / Q000528:radiation effects; D054730:Protein Interaction Domains and Motifs; D017433:Protein Structure, Secondary; D040901:Proteomics; D012319:RNA Polymerase II / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D012694:Serine / Q000737:chemistry* / Q000378:metabolism; D015398:Signal Transduction; D014443:Tyrosine / Q000737:chemistry* / Q000378:metabolism
chemical_list: D018797:Cell Cycle Proteins; D009363:Neoplasm Proteins; D010455:Peptides; C571225:RPRD1B protein, human; D014443:Tyrosine; D012694:Serine; D012319:RNA Polymerase II