In eukaryotic cells, RNA polymerase II (RNA pol II) transcription and pre-mRNA processing are coordinated events. We have addressed how individual components of the transcription and pre-mRNA processing machinery are organized during mitosis and subsequently recruited into the newly formed daughter nuclei. Interestingly, localization studies of numerous RNA pol II transcription and pre-mRNA processing factors revealed a nonrandom and sequential entry of these factors into daughter nuclei after nuclear envelope/lamina formation. The initiation competent form of RNA pol II and general transcription factors appeared in the daughter nuclei simultaneously, but prior to pre-mRNA processing factors, whereas the elongation competent form of RNA pol II was detected even later. The differential entry of these factors rules out the possibility that they are transported as a unitary complex. Telophase nuclei were competent for transcription and pre-mRNA splicing concomitant with the initial entry of the respective factors. In addition, our results revealed a low turnover rate of transcription and pre-mRNA splicing factors during mitosis. We provide evidence to support a model in which the entry of the RNA pol II gene expression machinery into newly forming daughter nuclei is a staged and ordered process. INTRODUCTIONIn eukaryotic cells, RNA polymerase II (RNA pol II) transcription and pre-mRNA processing are coordinated events that require finely tuned interactions among a large number of proteins (Misteli and Spector, 1999;Maniatis and Reed, 2002;Orphanides and Reinberg, 2002;Proudfoot et al., 2002). One fundamental feature of mammalian cell nuclei is that many components of the RNA synthesis and processing machinery are organized into compartments (Spector, 1993;Lamond and Earnshaw, 1998;Misteli, 2000;Spector, 2001;Hernandez-Verdun et al., 2002;Huang, 2002). The best characterized nuclear compartment is the nucleolus where rRNA synthesis and processing as well as preribosome assembly takes place (Scheer and Hock, 1999;Hernandez-Verdun et al., 2002;Huang, 2002). The nucleus is further divided into other nonmembrane-enclosed compartments, including but not limited to chromosome territories, nuclear speckles, Cajal bodies, promyelocytic leukemia bodies, etc. (Spector, 1993(Spector, , 2001Lamond and Earnshaw, 1998;Gall, 2000). Active transcription sites can be visualized by bromouridine triphosphate (bromo-UTP) incorporation as several thousand foci scattered throughout the nucleus that colocalize with transcription factors, heterogeneous nuclear RNA-associated proteins (hnRNPs), and other RNA processing factors (Iborra et al., 1996;Pombo et al., 2000). However, these sites are not generally coincident with the larger and less abundant "nuclear speckles" where splicing factors are enriched. Mammalian interphase nuclei typically contain 20 -50 nuclear speckles. By electron microscopy, the speckled pattern corresponds to interchromatin granule clusters (IGCs) and perichromatin fibrils (Spector et al., 1983(Spector et al., , 199...

To examine the involvement of interchromatin granule clusters (IGCs) in transcription and pre-mRNA splicing in mammalian cell nuclei, the serine-arginine (SR) protein kinase cdc2-like kinase (Clk)/STY was used as a tool to manipulate IGC integrity in vivo. Both immunofluorescence and transmission electron microscopy analyses of cells overexpressing Clk/STY indicate that IGC components are completely redistributed to a diffuse nuclear localization, leaving no residual structure. Conversely, overexpression of a catalytically inactive mutant, Clk/STY(K190R), causes retention of hypophosphorylated SR proteins in nuclear speckles. Our data suggest that the protein–protein interactions responsible for the clustering of interchromatin granules are disrupted when SR proteins are hyperphosphorylated and stabilized when SR proteins are hypophosphorylated. Interestingly, cells without intact IGCs continue to synthesize nascent transcripts. However, both the accumulation of splicing factors at sites of pre-mRNA synthesis as well as pre-mRNA splicing are dramatically reduced, demonstrating that IGC disassembly perturbs coordination between transcription and pre-mRNA splicing in mammalian cell nuclei.

beta-catenin and plakoglobin are members of the armadillo family of proteins and were first identified as components of intercellular adhering junctions. In the adherens junction beta-catenin and plakoglobin serve to link classical cadherins to the actin-based cytoskeleton. In the desmosome plakoglobin links the desmosomal cadherins, the desmogleins and the desmocollins, to the intermediate filament cytoskeleton. beta-catenin is not a component of the desmosome. Previously we have shown that the central armadillo repeat region of plakoglobin is the site for desmosomal cadherin binding. We hypothesized that the unique amino- and/or carboxyl-terminal ends of beta-catenin may regulate its exclusion from the desmosomal plaque. To test this hypothesis we used chimeras between beta-catenin and plakoglobin to identify domain(s) that modulate association with desmoglein 2. Chimeric constructs, each capable of associating with classical cadherins, were assayed for association with the desmosomal cadherin desmoglein 2. Addition of either the N- or C-terminal tail of beta-catenin to the armadillo repeats of plakoglobin did not interfere with desmoglein 2 association. However, when both beta-catenin amino terminus and carboxyl terminus were added to the plakoglobin armadillo repeats, association with desmoglein 2 was diminished. Removal of the first 26 amino acids from this construct restored association. We show evidence for direct protein-protein interactions between the amino- and carboxyl-terminal tails of beta-catenin and propose that a sequence in the first 26 amino acids of beta-catenin along with its carboxyl-terminal tail decrease its affinity for desmoglein and prevent its inclusion in the desmosome.