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Salivary gland polytene cells in the dipteran Chironomus tentans provide exceptional experimental possibilities to analyze processing of specific pre-mRNAs in intact eukaryotic cell nuclei. Here we give a brief account of how these experimental advantages can be exploited to analyze the splicing process in vivo. In multi-intron pre-mRNAs, spliceosomes assemble and splicing is initiated cotranscriptionally for all introns. Intron excision may, however, occur mainly co- transcriptionally or mainly posttranscriptionally depending on the position of each intron in relation to the remaining transcription time and intron-specific efficiencies of excision. As measured for the U2 snRNP and an SR protein, 10-15% of the spliceosomal components are bound to pre-mRNA at active gene loci at a given moment, while the majority of the spliceosomal components are present in the nucleoplasm. A continuous redistribution of the spliceosomal components takes place in the nucleus as a result of a close coupling between transcription and spliceosomal assembly.

We have previously reported that EBNA-5, one of the Epstein-Barr virus-encoded proteins, accumulates in the nuclear bodies containing PML, the promyelocytic leukemia associated protein. In this study, we examine the intranuclear distribution of SV40 large T-antigen (SV40T), the p53 tumor suppressor protein (p53), and PML in a conditionally immortalized cell line, IDH4. In IDH4 cells, the expression of SV40T is regulated by a dexamethasone (Dex)-driven promoter. Withdrawal of Dex results in down-regulation of SV40T and growth arrest, whereas addition of Dex to the growth-arrested cells results in up-regulation of SV40T and proliferation. In proliferating IDH4 cells, SV40T is concentrated in nuclear dots that are also positive for p53. Many of these dots are juxtaposed to PML positive structures but do not colocalize with them. After removal of Dex, SV40T-p53 dots gradually disappear, while the PML structures remain. Induction of SV40T in nonproliferating IDH4 cells causes a coordinated redistribution of SV40T and p53. The immunostaining for SV40T and p53 is first weak, then strong with a homogeneous distribution, and 3-4 days later becomes dot-like again. This reappearance of SV40T-p53 dots coincides with the recovery of proliferation in restimulated IDH4 cells. Also, the p53 pattern correlates with the SV40T pattern with regard to both morphology and intensity during both suppression and induction of SV40T. Taken together, our data suggest that (i) the level of p53 is coregulated with the level of SV40T in a dose-dependent fashion; (ii) the formation of SV40T-p53 nuclear dots correlates with the transformed phenotype; (iii) the SV40T-p53 dots localize preferentially to the neighborhood of PML bodies which are already present in normal cells.

POM121 is an integral membrane protein specifically localized in the pore membrane domain of the nuclear envelope. We have investigated the intracellular distribution of rat POM121 heterologously overexpressed in monkey COS cells by immunofluorescence and fluorescence digital imaging microscopy. At low levels of expression overexpressed POM121 was distributed in the nuclear envelope in a punctate fashion, partially overlapping with the distribution of nuclear pores. At high levels of expression, however, the overexpressed protein accumulated in intranuclear bodies. These bodies represent a novel subnuclear structure, displaying a defined cylindrical structure and a distinct localization at or adjacent to the inner nuclear membrane. The C-terminal portion of POM121, which contains a pentapeptide repeat domain common to a subfamily of related nucleoporins, was sufficient to mediate targeting to the nuclear envelope as well as formation of intranuclear bodies.

During skeletal muscle development three intermediate filament proteins are expressed: nestin, vimentin, and desmin. Vimentin and desmin belong to the class III intermediate filaments and are closely related to each other, whereas nestin is a more distantly related, class VI, intermediate filament. It was previously observed by conventional immunocytochemistry that the intracellular patterns of nestin, desmin, and vimentin appeared indistinguishable, despite nestin's more distant evolutionary relationship. We here extend this analysis by applying three-dimensional fluorescence digital imaging microscopy to compare the intracellular distribution of nestin with that of desmin, vimentin, actin, and tubulin in G6 human fetal skeletal muscle cells. We show that in vitro differentiation of G6 cells can produce an intermediate filament expression pattern similar to that observed during myogenesis in vivo, i.e., downregulation of vimentin but not of nestin and desmin during myotube maturation. The image analysis demonstrated that the degree of overlap between nestin and desmin/vimentin was very extensive in myoblasts and in multinucleate myotubes in all regions of the cells. In contrast, nestin did not colocalize with tubulin or actin in G6 myoblasts. In particular, nestin immunoreactivity was not detected at the microtubule-organizing center, and it was only sparsely observed at the cell periphery where actin stress fibers were seen. Our data lend further support to the notion that nestin interacts very closely with the two more distantly related class III intermediate filament proteins desmin and vimentin in the entire muscle cell, before and after myotube formation. A comparison of conserved amino acid residues in the different IFs suggest that charged amino acid residues in the alpha-helical rod domain may play a role in the interaction.

The intranuclear distribution of a new antigen (F78) associated with U snRNPs (small nuclear RNA-protein complexes) was compared with that of the RNP and Sm protein antigens previously identified on individual snRNP particles. Human and rat cells were double stained with human autoantisera and mouse monoclonal antibodies. The binding of the human and mouse antibodies was detected with secondary antibodies conjugated with fluorescein and rhodamine, respectively. The resulting immunofluorescence patterns were compared by digital image analysis. The F78, RNP, and Sm antigens show speckled fluorescence patterns which overlap to a great extent. The F78 pattern, however, also contains two classes of structural elements not present in the RNP pattern. Furthermore, during mitosis expression of the F78 antigen is completely suppressed from early prophase to telophase, while the RNP and Sm antigens are found evenly distributed throughout the cytoplasm of the dividing cells.

A monoclonal antibody (aRB1C1) raised against an Rb fusion protein detects a limited number (4-10) of relatively large intranuclear foci in an EBV-immortalized cord blood cell line (IB4). These domains also bind an anti-EBNA-5 monoclonal antibody. The Rb antibody reactive sites also co-localize with the SV40 T antigen in transformed monkey cells (COS). The nuclear structures stained by aRB1C1 and EBNA-5 antibodies are distinct from the structures detected with antibodies against centromeric proteins and certain snRNP epitopes. EBNA-5/Rb-positive domains do not selectively react with antibodies against the La antigen known to associate with the small EBV-encoded nuclear RNA species designated as the EBERs.