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.