Nuclear Coactivator-62 kDa/Ski-interacting Protein Is a Nuclear Matrix-associated Coactivator That May Couple Vitamin D Receptor-mediated Transcription and RNA Splicing

• Published in: The Journal of biological chemistry Vol. 278; no. 37; pp. 35325 - 35336
• Main Authors: Zhang, Chi, Dowd, Diane R., Staal, Ada, Gu, Chun, Lian, Jane B., van Wijnen, Andre J., Stein, Gary S., MacDonald, Paul N.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 12.09.2003; American Society for Biochemistry and Molecular Biology
• Subjects: Animals; Base Sequence; Calcitriol - pharmacology; Cell Nucleus - metabolism; Chlorocebus aethiops; COS Cells; DNA Primers; HeLa Cells; Humans; Nuclear Proteins - metabolism; Nuclear Receptor Coactivators; Plasmids; Receptors, Calcitriol - metabolism; Recombinant Fusion Proteins - metabolism; Reverse Transcriptase Polymerase Chain Reaction; RNA Splicing - drug effects; RNA, Messenger - drug effects; RNA, Messenger - genetics; Trans-Activators - metabolism; Transcription Factors - metabolism; Transcription, Genetic - drug effects; Transfection
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M305191200

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.