Mutational analysis of fibrillarin and its mobility in living human cells

Cajal bodies (CBs) are subnuclear organelles that contain components of a n umber of distinct pathways in RNA transcription and RNA processing. CBs hav e been linked to other subnuclear organelles such as nucleoli, but the reas on for the presence of nucleolar proteins such as fibrillarin in CBs remain s uncertain. Here, we use full-length fibrillarin and truncated fibrillarin mutants fused to green fluorescent protein (GFP) to demonstrate that speci fic structural domains of fibrillarin are required for correct intranuclear localization of fibrillarin to nucleoli and CBs. The second spacer domain and carboxy terminal alpha-helix domain in particular appear to target fibr illarin, respectively, to the nucleolar transcription centers and CBs. The presence of the RNP domain seems to be a prerequisite for correct targeting of fibrillarin. Time-lapse confocal microscopy of human cells that stably express fibrillarin-GFP shows that CBs fuse and split, albeit at low freque ncies. Recovered fluorescence of fibrillarin-GFP in nucleoli and CBs after photobleaching indicates that it is highly mobile in both organelles (estim ated diffusion constant similar to0.02 mum(2) s(-1)), and has a significant ly larger mobile fraction in CBs than in nucleoli.