title: Nuclear coactivator-62 kDa/Ski-interacting protein is a nuclear matrix-associated coactivator that may couple vitamin D receptor-mediated transcription and RNA splicing.
abstract: Nuclear coactivator-62 kDa/Ski-interacting protein (NCoA62/SKIP) is a putative vitamin D receptor (VDR) and nuclear receptor coactivator protein that is unrelated to other VDR coactivators such as those in the steroid receptor coactivator (SRC) family. The mechanism through which NCoA62/SKIP functions in VDR-activated transcription is unknown. In the present study, we identified a nuclear localization sequence in the COOH terminus of NCoA62/SKIP and showed that NCoA62/SKIP was targeted to nuclear matrix subdomains. Chromatin immunoprecipitation studies revealed that endogenous NCoA62/SKIP associated in a 1,25-dihydroxyvitamin D3-dependent manner with VDR target genes in ROS17/2.8 osteosarcoma cells. A cyclic pattern of promoter occupancy by VDR, SRC-1, and NCoA62/SKIP was observed, with NCoA62/SKIP entering these promoter complexes after SRC-1. These studies provide strong support for the proposed role of NCoA62/SKIP as a VDR transcriptional coactivator, and they indicate that key mechanistic differences probably exist between NCoA62/SKIP and SRC coactivators. To explore potential mechanisms, NCoA62/SKIP-interacting proteins were purified from HeLa cell nuclear extracts and identified by mass spectrometry. The identified proteins represent components of the spliceosome as well as other nuclear matrix-associated proteins. Here, we show that a dominant negative inhibitor of NCoA62/SKIP (dnNCoA62/SKIP) interfered with appropriate splicing of transcripts derived from 1,25-dihydroxyvitamin D3-induced expression of a growth hormone minigene cassette. Taken together, these data show that NCoA62/SKIP has properties that are consistent with those of nuclear receptor coactivators and with RNA spliceosome components, thus suggesting a potential role for NCoA62/SKIP in coupling VDR-mediated transcription to RNA splicing.
authors.affiliation: Department of Pharmacology, Case Western Reserve University, Cleveland, Ohio 44106, USA.
authors.forename: Chi
authors.initials: C
authors.lastname: Zhang
authors.forename: Diane R
authors.initials: DR
authors.lastname: Dowd
authors.forename: Ada
authors.initials: A
authors.lastname: Staal
authors.forename: Chun
authors.initials: C
authors.lastname: Gu
authors.forename: Jane B
authors.initials: JB
authors.lastname: Lian
authors.forename: Andre J
authors.initials: AJ
authors.lastname: van Wijnen
authors.forename: Gary S
authors.initials: GS
authors.lastname: Stein
authors.forename: Paul N
authors.initials: PN
authors.lastname: MacDonald
mesh_terms: D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002117:Calcitriol / Q000494:pharmacology; D002467:Cell Nucleus / Q000378:metabolism; D002522:Chlorocebus aethiops; D017931:DNA Primers; D006367:HeLa Cells; D006801:Humans; D009687:Nuclear Proteins / Q000378:metabolism*; D056918:Nuclear Receptor Coactivators; D010957:Plasmids; D012326:RNA Splicing / Q000187:drug effects; D012333:RNA, Messenger / Q000187:drug effects / Q000235:genetics; D018167:Receptors, Calcitriol / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015534:Trans-Activators / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic / Q000187:drug effects; D014162:Transfection
chemical_list: D017931:DNA Primers; D009687:Nuclear Proteins; D056918:Nuclear Receptor Coactivators; D012333:RNA, Messenger; D018167:Receptors, Calcitriol; D011993:Recombinant Fusion Proteins; C111791:SNW1 protein, human; D015534:Trans-Activators; D014157:Transcription Factors; D002117:Calcitriol