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.