Alternative splicing is a key factor contributing to genetic diversity and evolution. Intron retention, one form of alternative splicing, is common in plants but rare in higher eukaryotes, because messenger RNAs with retained introns are subject to cellular restriction at the level of cytoplasmic export and expression. Often, retention of internal introns restricts the export of these mRNAs and makes them the targets for degradation by the cellular nonsense-mediated decay machinery if they contain premature stop codons. In fact, many of the database entries for complementary DNAs with retained introns represent them as artefacts that would not affect the proteome. Retroviruses are important model systems in studies of regulation of RNAs with retained introns, because their genomic and mRNAs contain one or more unspliced introns. For example, Mason-Pfizer monkey virus overcomes cellular restrictions by using a cis-acting RNA element known as the constitutive transport element (CTE). The CTE interacts directly with the Tap protein (also known as nuclear RNA export factor 1, encoded by NXF1), which is thought to be a principal export receptor for cellular mRNA, leading to the hypothesis that cellular mRNAs with retained introns use cellular CTE equivalents to overcome restrictions to their expression. Here we show that the Tap gene contains a functional CTE in its alternatively spliced intron 10. Tap mRNA containing this intron is exported to the cytoplasm and is present in polyribosomes. A small Tap protein is encoded by this mRNA and can be detected in human and monkey cells. Our results indicate that Tap regulates expression of its own intron-containing RNA through a CTE-mediated mechanism. Thus, CTEs are likely to be important elements that facilitate efficient expression of mammalian mRNAs with retained introns.

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Adenovirus protein VII is the major protein component of the viral nucleoprotein core. It is highly basic, and an estimated 1070 copies associate with each viral genome, forming a tightly condensed DNA-protein complex. We have investigated DNA condensation, transcriptional repression, and specific protein binding by protein VII. Xenopus oocytes were microinjected with mRNA encoding HA-tagged protein VII and prepared for visualization of lampbrush chromosomes. Immunostaining revealed that protein VII associated in a uniform manner across entire chromosomes. Furthermore, the chromosomes were significantly condensed and transcriptionally silenced, as judged by the dramatic disappearance of transcription loops characteristic of lampbrush chromosomes. During infection, the protein VII-DNA complex may be the initial substrate for transcriptional activation by cellular factors and the viral E1A protein. To investigate this possibility, mRNAs encoding E1A and protein VII were comicroinjected into Xenopus oocytes. Interestingly, whereas E1A did not associate with chromosomes in the absence of protein VII, expression of both proteins together resulted in significant association of E1A with lampbrush chromosomes. Binding studies with proteins produced in bacteria or human cells or by in vitro translation showed that E1A and protein VII can interact in vitro. Structure-function analysis revealed that an N-terminal region of E1A is responsible for binding to protein VII. These studies define the in vivo functions of protein VII in DNA binding, condensation, and transcriptional repression and indicate a role in E1A-mediated transcriptional activation of viral genes.The adenovirus nucleoprotein core consists of doublestranded genomic DNA, three highly basic viral proteins VII, V, and (mu), as well as protein IVa2 and the 55-kDa terminal protein (1,8,33,42,(52)(53)(54)61). Protein VII is the major protein component of the core, with an estimated 1,070 copies present per virion (20). Along with , it is bound noncovalently to the DNA in a sequence-independent manner (2, 6, 36, 55). Protein V contacts the DNA as well and also acts as a bridge between protein VII and the outer capsid (19,55). Protein IVa2 makes sequence-specific contacts with the viral DNA packaging sequence and is thought to play a role in DNA packaging (64). Salt-extracted preparations of the core contain only DNA and protein VII, suggesting that this protein is the most tightly DNA bound of all core proteins (60). Similarly, Sarkosyl preparations of the core contain predominantly DNA and protein VII (8).Structural features of the DNA-protein complex within the adenovirus capsid and during infection remain largely unknown. DNA within the capsid is in a highly compact configuration, and electron microscopy studies of purified viral core reveal structures reminiscent of beads on a string, or higherorder chromatin compaction, depending on the method of preparation (8,18,45,49,50,60,63). Nuclease digestion of core preparations results in discrete populations of prote...

Adenovirus protein VII is the major component of the viral nucleoprotein core. It is a highly basic nonspecific DNA-binding protein that condenses viral DNA inside the capsid. We have investigated the fate and function of protein VII during infection. "Input" protein VII persisted in the nucleus throughout early phase and the beginning of DNA replication. Chromatin immunoprecipitation revealed that input protein VII remained associated with viral DNA during this period. Two cellular proteins, SET and pp32, also associated with viral DNA during early phase. They are components of two multiprotein complexes, the SET and INHAT complexes, implicated in chromatin-related activities. Protein VII associated with SET and pp32 in vitro and distinct domains of protein VII were responsible for binding to the two proteins. Interestingly, protein VII was found in novel nuclear dot structures as visualized by immunofluorescence. The dots likely represent individual infectious genomes in association with protein VII. They appeared within 30 min after infection and localized in the nucleus with a peak of intensity between 4 and 10 h postinfection. After this, their intensity decreased and they disappeared between 16 and 24 h postinfection. Interestingly, disappearance of the dots required ongoing RNA synthesis but not DNA synthesis. Taken together these data indicate that protein VII has an ongoing role during early phase and the beginning of DNA replication.The adenovirus nucleoprotein core consists of doublestranded genomic DNA, the highly basic viral proteins VII, V, and (mu), as well as protein IVa2 and the 55-kDa terminal protein (1,5,19,25,31,33,34,42). Protein VII is the major protein component of the core with an estimated 1,070 copies present per virion (9) and is primarily responsible for establishing viral chromatin structure. It can potently condense DNA in vitro and in vivo and also repress transcription (3,6,21,35). This is consistent with the highly condensed configuration of viral chromatin found within the virion. When delivered to the nucleus, the chromatin is silent prior to stimulation by the viral transcriptional activator E1A (13).We and others have reported that protein VII from infectious viral particles enters the nucleus along with viral DNA and remains associated with it, suggesting that the protein VII-DNA complex is the substrate for transcriptional activation by E1A during early phase (8,15,18,21). Moreover we found that protein VII can associate with E1A protein in vitro (21).In this report we have studied the fate and function of protein VII during the early phase of infection. We demonstrate that virus-derived "input" protein VII persists in the nucleus throughout early phase and the beginning of viral DNA replication, suggesting that it has an ongoing role in gene regulation and perhaps DNA replication. During this period protein VII is found in discrete nuclear dot structures and viral DNA continues to associate with protein VII.We have also investigated the association of protein VII with cel...