Nuclear pre-mRNA compartmentalization: Trafficking of released transcriptsto splicing factor reservoirs

In the present study, the spatial organization of intron-containing pre-mRN As of Epstein-Barr virus (EBV) genes relative to location of splicing facto rs is investigated. The intranuclear position of transcriptionally active E BV genes, as well as of nascent transcripts, is found to be random with res pect to the speckled accumulations of splicing factors (SC35 domains) in Na malwa cells, arguing against the concept of the locus-specific organization of mRNA genes with respect to the speckles. Microclusters of splicing fact ors are, however, frequently superimposed on nascent transcript sites. The transcript environment is a dynamic structure consisting of both nascent an d released transcripts, i.e., the track-like transcript environment. Both E BV sequences of the chromosome 1 homologue are usually associated with the track, are transcriptionally active, and exhibit in most cases a polar orie ntation. in contrast to nascent transcripts (in the form of spots), the ass ociation of a post-transcriptional pool of viral pre-mRNA (in the form of t racks) with speckles is not random and is further enhanced in transcription ally silent cells when splicing factors are sequestered in enlarged accumul ations. The transcript environment reflects the intranuclear transport of R NA from the sites of transcription to SC35 domains, as shown by concomitant mapping of DNA, RNA, and splicing factors. No clear vectorial intranuclear trafficking of transcripts from the site of synthesis toward the nuclear e nvelope for export into the cytoplasm is observed. Using Namalwa and Raji c ell Lines, a correlation between the level of viral gene transcription and splicing factor accumulation within the viral transcript environment has be en observed. This supports a concept that the level of transcription can al ter the spatial relationship among intron-containing genes, their transcrip ts, and speckles attributable to various levels of splicing factors recruit ed from splicing factor reservoirs. Electron microscopic in situ hybridizat ion studies reveal that the released transcripts are directed toward reserv oirs of splicing factors organized in clusters of interchromatin granules. Our results point to the bidirectional intranuclear movement of macromolecu lar complexes between intron-containing genes and splicing factor reservoir s: the recruitment of splicing factors to transcription sites and movement of released transcripts from DNA loci to reservoirs of splicing factors.