title: Analysis of URI nuclear interaction with RPB5 and components of the R2TP/prefoldin-like complex.
abstract: Unconventional prefoldin RPB5 Interactor (URI) was identified as a transcriptional repressor that binds RNA polymerase II (pol II) through interaction with the RPB5/POLR2E subunit. Despite the fact that many other proteins involved in transcription regulation have been shown to interact with URI, its nuclear function still remains elusive. Previous mass spectrometry analyses reported that URI is part of a novel protein complex called R2TP/prefoldin-like complex responsible for the cytoplasmic assembly of RNA polymerase II. We performed a mass spectrometry (MS)-based proteomic analysis to identify nuclear proteins interacting with URI in prostate cells. We identified all the components of the R2TP/prefoldin-like complex as nuclear URI interactors and we showed that URI binds and regulates RPB5 protein stability and transcription. Moreover, we validated the interaction of URI to the P53 and DNA damage-Regulated Gene 1 (PDRG1) and show that PDRG1 protein is also stabilized by URI binding. We present data demonstrating that URI nuclear/cytoplasmic shuttling is affected by compounds that stall pol II on the DNA (α-amanitin and actinomycin-D) and by leptomycin B, an inhibitor of the CRM1 exportin that mediates the nuclear export of pol II subunits. These data suggest that URI, and probably the entire R2TP/prefoldin-like complex is exported from the nucleus through CRM1. Finally we identified putative URI sites of phosphorylation and acetylation and confirmed URI sites of post-transcriptional modification identified in previous large-scale analyses the importance of which is largely unknown. However URI post-transcriptional modification was shown to be essential for URI function and therefore characterization of novel sites of URI modification will be important to the understanding of URI function.
authors.affiliation: Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, New York, New York, United States of America.
authors.forename: Paolo
authors.initials: P
authors.lastname: Mita
authors.forename: Jeffrey N
authors.initials: JN
authors.lastname: Savas
authors.forename: Susan
authors.initials: S
authors.lastname: Ha
authors.forename: Nabil
authors.initials: N
authors.lastname: Djouder
authors.forename: John R
authors.initials: JR
authors.lastname: Yates
authors.forename: Susan K
authors.initials: SK
authors.lastname: Logan
mesh_terms: D021581:Active Transport, Cell Nucleus / Q000502:physiology; D015153:Blotting, Western; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics; D004268:DNA-Binding Proteins / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D005231:Fatty Acids, Unsaturated; D005786:Gene Expression Regulation / Q000235:genetics / Q000502:physiology*; D006801:Humans; D047468:Immunoprecipitation; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D028884:Karyopherins / Q000378:metabolism; D008156:Luciferases; D008297:Male; D013058:Mass Spectrometry; D008856:Microscopy, Fluorescence; D046912:Multiprotein Complexes / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011467:Prostate / Q000378:metabolism*; D040901:Proteomics / Q000379:methods; D012319:RNA Polymerase II / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism; D012097:Repressor Proteins; D000097606:Exportin 1 Protein
chemical_list: D017931:DNA Primers; D004268:DNA-Binding Proteins; D012321:DNA-Directed RNA Polymerases; D005231:Fatty Acids, Unsaturated; D047908:Intracellular Signaling Peptides and Proteins; D028884:Karyopherins; D008156:Luciferases; D046912:Multiprotein Complexes; D012319:RNA Polymerase II; D034741:RNA, Small Interfering; D018160:Receptors, Cytoplasmic and Nuclear; D012097:Repressor Proteins; D000097606:Exportin 1 Protein; C478985:PDRG1 protein, human; C117184:URI1 protein, human; C517511:POLR2E protein, human; C038753:leptomycin B