In vivo analysis of NHPX reveals a novel nucleolar localization pathway involving a transient accumulation in splicing speckles.
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.
Leung
Anthony K L
AK
Wellcome Trust Biocentre, MSI/WTB Complex, University of Dundee, Dundee DD1 5EH, Scotland, UK.
Lamond
Angus I
AI
D001426:Bacterial Proteins / Q000235:genetics; D002451:Cell Compartmentation / Q000235:genetics*; D002466:Cell Nucleolus / Q000378:metabolism* / Q000648:ultrastructure; D020541:Coiled Bodies / Q000235:genetics* / Q000378:metabolism; D004256:DNA Polymerase I / Q000235:genetics; D004257:DNA Polymerase II / Q000235:genetics; D005057:Eukaryotic Cells / Q000166:cytology / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D007399:Interphase / Q000235:genetics; D008164:Luminescent Proteins / Q000235:genetics; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D021381:Protein Transport; D012313:RNA / Q000235:genetics / Q000378:metabolism; D012326:RNA Splicing / Q000235:genetics*; D016601:RNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics; D017411:Ribonucleoproteins, Small Nuclear; D020718:Ribonucleoproteins, Small Nucleolar / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000235:genetics*; D014158:Transcription, Genetic / Q000235:genetics
D001426:Bacterial Proteins; D008164:Luminescent Proteins; C416929:NHP2 protein, human; D009687:Nuclear Proteins; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D017411:Ribonucleoproteins, Small Nuclear; D020718:Ribonucleoproteins, Small Nucleolar; C054562:yellow fluorescent protein, Bacteria; D012313:RNA; D004256:DNA Polymerase I; D004257:DNA Polymerase II