In vivo analysis of NHPX reveals a novel nucleolar localization pathway involving a transient accumulation in splicing speckles

Anthony K. L. Leung, Angus I. Lamond

    Research output: Contribution to journalArticle

    42 Citations (Scopus)
    613 Downloads (Pure)

    Abstract

    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.

    © 2002 Leung et al. This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.jcb.org/misc/terms.shtml). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).

    Original languageEnglish
    Pages (from-to)615-629
    Number of pages15
    JournalJournal of Cell Biology
    Volume157
    Issue number4
    DOIs
    Publication statusPublished - 13 May 2002

    Fingerprint

    Licensure
    Small Nucleolar RNA
    RNA Polymerase I
    Cell Line
    RNA Polymerase II
    HeLa Cells
    Publications
    Proteins
    RNA

    Cite this

    @article{c2221f65581d423b9b3e8cb6df8cdfbf,
    title = "In vivo analysis of NHPX reveals a novel nucleolar localization pathway involving a transient accumulation in splicing speckles",
    abstract = "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. {\circledC} 2002 Leung et al. This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.jcb.org/misc/terms.shtml). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).",
    author = "Leung, {Anthony K. L.} and Lamond, {Angus I.}",
    note = "dc.publisher: Rockefeller University Press dc.description.sponsorship: We thank our colleges in the Lamond laboratory, Dr. Laura Trinkle-Mulcahy for plasmid DsRED-U1A, Dr. Judith Sleeman for the ECFP-SmB cell line, Ursula Ryder for help in RNA manipulation, and other colleagues in the laboratory and Prof. C Proud for helpful discussions. We would also like to thank Dr. T. Kanda for the EGFP-H2B cell line, Dr. K. Collins (University of California, Berkeley, Berkeley, CA) for the htert1787 primary fibroblast cell line, and Dr. B. Chen (Dana-Farber Cancer Institute, Boston, MA) for the affinity-purified anti-NHPX rabbit antiserum, Dr. B. Frenguelli for use of the Zeiss LSM 510 confocal microscope, and S. Shreeman for help in FACS analysis.",
    year = "2002",
    month = "5",
    day = "13",
    doi = "10.1083/jcb.200201120",
    language = "English",
    volume = "157",
    pages = "615--629",
    journal = "Journal of Cell Biology",
    issn = "0021-9525",
    publisher = "Rockefeller University Press",
    number = "4",

    }

    In vivo analysis of NHPX reveals a novel nucleolar localization pathway involving a transient accumulation in splicing speckles. / Leung, Anthony K. L.; Lamond, Angus I.

    In: Journal of Cell Biology, Vol. 157, No. 4, 13.05.2002, p. 615-629.

    Research output: Contribution to journalArticle

    TY - JOUR

    T1 - In vivo analysis of NHPX reveals a novel nucleolar localization pathway involving a transient accumulation in splicing speckles

    AU - Leung, Anthony K. L.

    AU - Lamond, Angus I.

    N1 - dc.publisher: Rockefeller University Press dc.description.sponsorship: We thank our colleges in the Lamond laboratory, Dr. Laura Trinkle-Mulcahy for plasmid DsRED-U1A, Dr. Judith Sleeman for the ECFP-SmB cell line, Ursula Ryder for help in RNA manipulation, and other colleagues in the laboratory and Prof. C Proud for helpful discussions. We would also like to thank Dr. T. Kanda for the EGFP-H2B cell line, Dr. K. Collins (University of California, Berkeley, Berkeley, CA) for the htert1787 primary fibroblast cell line, and Dr. B. Chen (Dana-Farber Cancer Institute, Boston, MA) for the affinity-purified anti-NHPX rabbit antiserum, Dr. B. Frenguelli for use of the Zeiss LSM 510 confocal microscope, and S. Shreeman for help in FACS analysis.

    PY - 2002/5/13

    Y1 - 2002/5/13

    N2 - 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. © 2002 Leung et al. This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.jcb.org/misc/terms.shtml). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).

    AB - 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. © 2002 Leung et al. This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.jcb.org/misc/terms.shtml). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).

    U2 - 10.1083/jcb.200201120

    DO - 10.1083/jcb.200201120

    M3 - Article

    VL - 157

    SP - 615

    EP - 629

    JO - Journal of Cell Biology

    JF - Journal of Cell Biology

    SN - 0021-9525

    IS - 4

    ER -