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Pre-mRNAs are synthesized and processed in the cell nucleus. Accurate pre-mRNA processing is a prerequisite for export of mature mRNAs to the cytoplasm and comprises several coand posttranscriptional modifications, like Y-capping, adenosine methylation, 3/cleavage and poly(A) addition, and intron removal during splicing. These activities must take place at the site of transcription or during intranuclear transport between the gene and the nuclear envelope. PremRNA processing requires many proteins and small nuclear RNAs (snRNAs), most of which are concentrated in a number of well-defined nuclear substructures, e.g., interchromatin granule clusters, perichromatin fibrils and coiled bodies. The function of these nuclear substructures is not fully understood, but must somehow be related to pre-mRNA processing. Evidently, knowledge of the dynamic interplay between sites of pre-mRNA synthesis, sites containing factors for pre-mRNA processing, and (routes of) intranuclear RNA transport is important for understanding nuclear pre-mRNA metabolism. The finding that only a fraction of the nuclear pre-mRNA population is eventually exported as mature mRNA to the cytoplasm suggests that nuclear (pre-)mRNA metabolism plays an important role in the control of gene expression. 2. Transcription of pre-mRNA genes

Publisher Summary This chapter discusses transcription by RNA polymerase II and nuclear architecture. Nuclear transcription is carried out by three different DNA-dependent RNA polymerases, termed as RNA polymerase I, II, and III (RPI, RPII, RPIII). It is found that anti-RPII antibodies may not discriminate between RPII molecules that are engaged in transcription and those that are inactive. It is suggested that the RPII-antigen distribution may be an overestimation of the number of RPII transcription sites. It is observed that antibodies directed against DNA–RNA hybrids label specific structures in the nucleoplasm and the nucleolus. These structures correspond largely to structures that become labeled by [ 3 H]uridine. It is observed that RPII transcription sites are distributed throughout the nucleus, without any preference for either the nuclear interior or the periphery. It is found that actively transcribed genes are located predominantly at the periphery of domains of condensed chromatin. It is suggested that intron- and exon-specific probes can be used to study the localization of successive steps in RNA processing and transport of pre-mRNA and mature RNA from the site of synthesis to the nuclear envelope.

This overview describes the spatial distribution of several enzymatic machineries and functions in the interphase nucleus. Three general observations can be made. First, many components of the different nuclear machineries are distributed in the nucleus in a characteristic way for each component. They are often found concentrated in specific domains. Second, nuclear machineries for the synthesis and processing of RNA and DNA are associated with an insoluble nuclear structure, called nuclear matrix. Evidently, handling of DNA and RNA is done by immobilized enzyme systems. Finally, the nucleus seems to be divided in two major compartments. One is occupied by compact chromosomes, the other compartment is the space between the chromosomes. In the latter, transcription takes place at the surface of chromosomal domains and it houses the splicing machinery. The relevance of nuclear organization for efficient gene expression is discussed.

Transcription and replication are, like many other nuclear functions and components, concentrated in nuclear domains. Transcription domains and replication domains may play an important role in the coordination of gene expression and gene duplication in S-phase. We have investigated the spatial relationship between transcription and replication in S-phase nuclei after fluorescent labelling of nascent RNA and nascent DNA, using confocal immunofluorescence microscopy. Permeabilized human bladder carcinoma cells were labelled with 5-bromouridine 5′-triphosphate and digoxigenin-11-deoxyuridine 5′-triphosphate to visualize sites of RNA synthesis and DNA synthesis, respectively. Transcription by RNA polymerase II was localized in several hundreds of domains scattered throughout the nucleoplasm in all stages of S-phase. This distribution resembled that of nascent DNA in early S-phase. In contrast, replication patterns in late S-phase consisted of fewer, larger replication domains. In double-labelling experiments we found that transcription domains did not colocalize with replication domains in late S-phase nuclei. This is in agreement with the notion that late replicating DNA is generally not actively transcribed. Also in early S-phase nuclei, transcription domains and replication domains did not colocalize. We conclude that nuclear domains exist, large enough to be resolved by light microscopy, that are characterized by a high activity of either transcription or replication, but never both at the same time. This probably means that as soon as the DNA in a nuclear domain is being replicated, transcription of that DNA essentially stops until replication in the entire domain is completed.

The artificial UTP-analogue 5-bromouridine 5'-triphosphate (BrUTP) has been used to label pre-mRNA in vitro and in vivo [1,2]. We have investigated the effect of bromouridine (BrU) in pre-mRNA on the efficiency of splicing. An adenovirus major late II construct was used to prepare four different transcripts, each containing a different amount of BrU. These four transcripts were tested in an in vitro splicing assay. We found that splicing is strongly inhibited if all uridines (U) in the transcript were substituted for BrU. Splicing was restored to some extent if 50% of the Us were replaced by BrU. The splicing efficiency returned to an almost normal level if only 1 our of every 10 Us was substituted for BrU. This demonstrates that only a pre-mRNA containing a small amount of BrU can be spliced normally in vitro. Furthermore, these results strongly suggest that some Us in the adenoviral transcript, probably those at the splice sites, cannot be replaced by BrU and are therefore critical in the splicing reaction.

The cell nucleus is highly organized. Many nuclear functions are localized in discrete domains, suggesting that compartmentalization is an important aspect of the regulation and coordination of nuclear functions. We investigated the subnuclear distribution of the glucocorticoid receptor, a hormone-dependent transcription factor. By immunofluorescent labeling and confocal microscopy we found that after stimulation with the agonist dexamethasone the glucocorticoid receptor is concentrated in 1,000-2,000 clusters in the nucleoplasm. This distribution was observed in several cell types and with three different antibodies against the glucocorticoid receptor. A similar subnuclear distribution of glucocorticoid receptors was found after treatment of cells with the antagonist RU486, suggesting that the association of the glucocorticoid receptor in clusters does not require transformation of the receptor to a state that is able to activate transcription. By dual labeling we found that most dexamethasone-induced receptor clusters do not colocalize with sites of pre-mRNA synthesis. We also show that RNA polymerase II is localized in a large number of clusters in the nucleus. Glucocorticoid receptor clusters did not significantly colocalize with these RNA polymerase II clusters or with domains containing the splicing factor SC-35. Taken together, these results suggest that most clustered glucocorticoid receptor molecules are not directly involved in activation of transcription.