Mutational analysis of fibrillarin and its mobility in living human cells.

Cajal bodies (CBs) are subnuclear organelles that contain components of a number of distinct pathways in RNA transcription and RNA processing. CBs have been linked to other subnuclear organelles such as nucleoli, but the reason for the presence of nucleolar proteins such as fibrillarin in CBs remains uncertain. Here, we use full-length fibrillarin and truncated fibrillarin mutants fused to green fluorescent protein (GFP) to demonstrate that specific 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 fibrillarin, 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 frequencies. Recovered fluorescence of fibrillarin-GFP in nucleoli and CBs after photobleaching indicates that it is highly mobile in both organelles (estimated diffusion constant approximately 0.02 microm(2) s(-1)), and has a significantly larger mobile fraction in CBs than in nucleoli.

Synthesis, processing, and transport of RNA within the three-dimensional context of the cell nucleus.

Fluorescence in situ hybridization and immunocytochemical techniques have contributed significantly to our current understanding of how transcription, RNA processing, and RNA transport are spatially and temporally organized in the cell nucleus. New technologies enabling the visualization of nuclear components in living cells specifically advanced our knowledge of the dynamic aspects of these nuclear processes. The picture that emerges from the work reviewed here shows that the positioning of genes within the three-dimensional nuclear space is of crucial importance, not only for its expression, but also for the efficient processing of its transcripts. Splicing factors are recruited from speckles to sites of active transcription, which can be present within, at the periphery, or at a relatively large distance from speckles. Furthermore, results are discussed showing that transcripts are exported by means of random diffusion.

RNA polymerase II localizes at sites of human cytomegalovirus immediate-early RNA synthesis and processing.

Pre-mRNA synthesis in eukaryotic cells is preceded by the formation of a transcription initiation complex and binding of unphosphorylated RNA polymerase II (Pol II) at the promoter region of a gene. Transcription initiation and elongation are accompanied by the hyperphosphorylation of the carboxy-terminal domain (CTD) of Pol II large subunit. Recent biochemical studies provided evidence that RNA processing factors, including those required for splicing, associate with hyperphosphorylated CTDs forming "transcription factories." To directly visualize the existence of such factories, we simultaneously detected human cytomegalovirus immediate-early (IE) DNA and RNA with splicing factors and Pol II in rat 9G cells inducible for IE gene expression. Combined in situ hybridization and immunocytochemistry revealed that, after induction, both splicing factors and Pol II are present at the sites of IE mRNA synthesis and of IE mRNA processing that extend from the transcribing gene. Noninduced cells revealed no such associations. When IE mRNA-synthesizing cells were treated with a transcription inhibitor, these associations disappeared within 30 min. Our results show that the association of Pol II and splicing factors with IE DNA is dependent on its transcriptional activity and furthermore suggest that splicing factors are still associated with Pol II during active splicing.

Dynamics of RNA polymerase II localization during the cell cycle.

Mitosis is characterized by condensation of chromatin, cessation of RNA transcription, and redistribution of nuclear proteins. We investigated the distribution of the hypo- and hyperphosphorylated forms of RNA polymerase II in mitotic cells from different cell lines by immunofluorescence. In interphase cells, the hyperphosphorylated RNA polymerase II (Pol IIO) is present in speckles and diffusely throughout the nucleoplasm. In prophase, when speckles disappear, Pol IIO concentrates at the surface of chromosomes and, in addition, localizes in small spots throughout the cytoplasm. The association of Pol IIO with the surface of chromosomes is visible until the chromosomes start to decondense during late anaphase/early telophase. In telophase cells, Pol IIO is absent in newly formed nuclei but present in the cytoplasm, while Pol IIO disappears nearly completely in late telophase cells. In early G1 cells, when cell nuclei increase in size, Pol IIO becomes present in the nucleus, first in small spots and later diffusely and in speckles. The hypophosphorylated form of RNA polymerase II (Pol IIA) is nearly absent in mitotic cells suggesting that Pol IIA is hyperphosphorylated at the onset of mitosis. Because Pol IIO, unlike Pol IIA, cannot assemble in transcription preinitiation complexes, the conversion of Pol IIA to Pol IIO and the lining of chromosomes with Pol IIO might be underlying a mechanism by which mitotic cells repress their transcriptional activity.