RPGR ORF15 isoform co-localises with RPGRIP1 at centrioles and interacts with nucleophosmin

Xinhua Shu, A. M. Fry, B. Tulloch, F. D. C. Manson, J. W. Crabb, H. Khanna, A. J. Faragher, A. Lennon, S. He, P. Trojan, A. Giessl, U. Wolfrum, R. Vervoort, A. Swaroop, A. F. Wright

Research output: Contribution to journalArticle

Abstract

The ORF15 isoform of RPGR (RPGRORF15) and RPGR interacting protein 1 (RPGRIP1) are mutated in a variety of retinal dystrophies but their functions are poorly understood. Here, we show that in cultured mammalian cells both RPGRORF15 and RPGRIP1 localize to centrioles. These localizations are resistant to the microtubule destabilizing drug nocodazole and persist throughout the cell cycle. RPGR and RPGRIP1 also co-localize at basal bodies in cells with primary cilia. The C-terminal (C2) domain of RPGRORF15 (ORF15C2) is highly conserved across 13 mammalian species, suggesting that it is a functionally important domain. Using matrix-assisted laser desorption ionization time-of-flight mass spectrometry, we show that this domain interacts with a 40 kDa shuttling protein nucleophosmin (NPM). The RPGRORF15–NPM interaction was confirmed by (i) yeast two-hybrid analyses; (ii) binding of both recombinant and native HeLa cell NPM to RPGRORF15 fusion proteins in vitro; (iii) co-immunoprecipitation of native NPM, RPGRORF15 and RPGRIP1 from bovine retinal extracts and of native HeLa cell NPM and transfected RPGRORF15 from cultured cells and (iv) co-localization of NPM and RPGRORF15 at metaphase centrosomes in cultured cells. NPM is a multifunctional protein chaperone that shuttles between the nucleoli and the cytoplasm and has been associated with licensing of centrosomal division. RPGR and RPGRIP1 join a growing number of centrosomal proteins involved in human disease.
Original languageEnglish
Pages (from-to)1183-1197
JournalHuman Molecular Genetics
Volume14
Issue number9
DOIs
Publication statusPublished - 1 May 2005

Keywords

  • immunoprecipitation
  • yeast one/two-hybrid system
  • mutation
  • cell cycle
  • cattle
  • cultured cells
  • centrioles
  • centrosome
  • fusion proteins
  • cytoplasm
  • hela cells
  • lasers
  • licensure
  • mammals
  • mitotic metaphase
  • microtubules
  • molecular chaperones
  • nocodazole
  • protein isoforms
  • yeasts
  • antibodies
  • cilia
  • retina
  • ionization
  • npm1 gene
  • time-of-flight mass spectometry
  • retinal dystrophies

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    Shu, X., Fry, A. M., Tulloch, B., Manson, F. D. C., Crabb, J. W., Khanna, H., Faragher, A. J., Lennon, A., He, S., Trojan, P., Giessl, A., Wolfrum, U., Vervoort, R., Swaroop, A., & Wright, A. F. (2005). RPGR ORF15 isoform co-localises with RPGRIP1 at centrioles and interacts with nucleophosmin. Human Molecular Genetics, 14(9), 1183-1197. https://doi.org/10.1093/hmg/ddi129