The NHPX protein is a nucleolar factor that binds directly to a conserved RNA target sequence found in nucleolar box C/D snoRNAs and in U4 snRNA. Using enhanced yellow fluorescent protein (EYFP)– and enhanced cyan fluorescent protein–NHPX fusions, we show here that NHPX is specifically accumulated in both nucleoli and Cajal bodies (CBs) in vivo. The fusion proteins display identical localization patterns and RNA binding specificities to the endogenous NHPX. Analysis of a HeLa cell line stably expressing EYFP–NHPX showed that the nucleolar accumulation of NHPX was preceded by its transient accumulation in splicing speckles. Only newly expressed NHPX accumulated in speckles, and the nucleolar pool of NHPX did not interchange with the pool in speckles, consistent with a unidirectional pathway. The transient accumulation of NHPX in speckles prior to nucleoli was observed in multiple cell lines, including primary cells that lack CBs. Inhibitor studies indicated that progression of newly expressed NHPX from speckles to nucleoli was dependent on RNA polymerase II transcription, but not on RNA polymerase I activity. The data show a specific temporal pathway involving the sequential and directed accumulation of NHPX in distinct subnuclear compartments, and define a novel mechanism for nucleolar localization.
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13 May 2002
Article|
May 13 2002
In vivo analysis of NHPX reveals a novel nucleolar localization pathway involving a transient accumulation in splicing speckles
Anthony K.L. Leung,
Anthony K.L. Leung
Wellcome Trust Biocentre, MSI/WTB Complex, University of Dundee, Dundee DD1 5EH, Scotland, UK
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Angus I. Lamond
Angus I. Lamond
Wellcome Trust Biocentre, MSI/WTB Complex, University of Dundee, Dundee DD1 5EH, Scotland, UK
Search for other works by this author on:
Anthony K.L. Leung
Wellcome Trust Biocentre, MSI/WTB Complex, University of Dundee, Dundee DD1 5EH, Scotland, UK
Angus I. Lamond
Wellcome Trust Biocentre, MSI/WTB Complex, University of Dundee, Dundee DD1 5EH, Scotland, UK
Address correspondence to Angus I. Lamond, Wellcome Trust Biocentre, MSI/WTB Complex, University of Dundee, Dundee DD1 5EH, Scotland, UK. Tel.: 44-1382-345473. Fax: 44-1382-345695. E-mail: [email protected]
*
Abbreviations used in this paper: CB, Cajal body; ECFP, enhanced cyan fluorescent protein; EYFP, enhanced yellow fluorescent protein; FIB, fibrillarin; FLIP, fluorescence loss in photobleaching; GFP, green fluorescent protein; NOR, nucleolar organizing regions; PEG, polyethene glycol; rDNA, ribosomal DNA; snoRNA, small nucleolar RNA.
Received:
January 28 2002
Revision Received:
March 20 2002
Accepted:
March 22 2002
Online ISSN: 1540-8140
Print ISSN: 0021-9525
The Rockefeller University Press
2002
J Cell Biol (2002) 157 (4): 615–629.
Article history
Received:
January 28 2002
Revision Received:
March 20 2002
Accepted:
March 22 2002
Citation
Anthony K.L. Leung, Angus I. Lamond; In vivo analysis of NHPX reveals a novel nucleolar localization pathway involving a transient accumulation in splicing speckles . J Cell Biol 13 May 2002; 157 (4): 615–629. doi: https://doi.org/10.1083/jcb.200201120
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