Nuclear pre-mRNA Compartmentalization: Trafficking of Released Transcripts to Splicing Factor Reservoirs

Abstract

In the present study, the spatial organization of intron-containing pre-mRNAs of Epstein-Barr virus (EBV) genes relative to location of splicing factors is investigated. The intranuclear position of transcriptionally active EBV genes, as well as of nascent transcripts, is found to be random with respect to the speckled accumulations of splicing factors (SC35 domains) in Namalwa cells, arguing against the concept of the locus-specific organization of mRNA genes with respect to the speckles. Microclusters of splicing factors are, however, frequently superimposed on nascent transcript sites. The transcript environment is a dynamic structure consisting of both nascent and released transcripts, i.e., the track-like transcript environment. Both EBV sequences of the chromosome 1 homologue are usually associated with the track, are transcriptionally active, and exhibit in most cases a polar orientation. In contrast to nascent transcripts (in the form of spots), the association of a post-transcriptional pool of viral pre-mRNA (in the form of tracks) with speckles is not random and is further enhanced in transcriptionally silent cells when splicing factors are sequestered in enlarged accumulations. The transcript environment reflects the intranuclear transport of RNA 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 envelope for export into the cytoplasm is observed. Using Namalwa and Raji cell lines, a correlation between the level of viral gene transcription and splicing factor accumulation within the viral transcript environment has been observed. This supports a concept that the level of transcription can alter the spatial relationship among intron-containing genes, their transcripts, and speckles attributable to various levels of splicing factors recruited from splicing factor reservoirs. Electron microscopic in situ hybridization studies reveal that the released transcripts are directed toward reservoirs of splicing factors organized in clusters of interchromatin granules. Our results point to the bidirectional intranuclear movement of macromolecular complexes between intron-containing genes and splicing factor reservoirs: the recruitment of splicing factors to transcription sites and movement of released transcripts from DNA loci to reservoirs of splicing factors. INTRODUCTIONPrevious results have demonstrated that spliceosome formation and/or splicing can be co-transcriptional (Beyer and Osheim, 1988;LeMaire and Thummel, 1990;Tennyson et al., 1995). In addition, recent results show that transcription and splicing are coupled through interactions of certain factors participating in both of these processes. A large macromolecular complex called "transcriptosome" or "mRNA factory," containing both transcription and splicing factors, is formed (Corden and Patturajan, 1997;McCracken et al., 1997;Steinmetz, 1997). Polymerase II (pol II) transcriptional...

Prespliceosomal Assembly on Microinjected Precursor mRNA Takes Place in Nuclear Speckles

“…In a parallel experiment, HeLa cells were incubated with 50 g/ml ␣-amanitin, 50 g/ml 5,6-dichloro-1-␤-d-ribofuranosylbenzimidazole, or 4 g/ml actinomycin D for 3 h and then microinjected with premRNA. Movement to enlarged/rounded up SC35 domains, which is typical for transcriptionally inhibited cells (Sinclair and Brasch, 1978;Spector et al, 1991;Melčák et al, 2000), was not abolished (our unpublished results). This indicated that the movement of microinjected RNAs to the speckles was independent of the ongoing nuclear transcription.…”

“…A concomitant dual mechanism for the intranuclear movement of endogenous (pre)-mRNAs and splicing factors was proposed (Melčák et al, 2000). Apparently, for most genes expressed at low level, splicing factors are recruited from the splicing factor reservoirs to the sites of transcription and splicing is cotranscriptional.…”

“…In some cell types, they occupy as much as 20% of the nuclear volume. At the electron microscope level, they consist of morphologically well-defined interchromatin granule clusters and of domains of perichromatin fibrils, some of which are believed to represent precursor-mRNAs (premRNAs) (Fakan and Puvion, 1980;Spector et al, 1983;Puvion et al, 1984;Spector, 1990;Fakan, 1994;Raška, 1995;Melčák et al, 2000).…”

“…Primary transcripts of certain genes as well as the spliced RNAs are mapped at sites of active transcription and outside of speckles (Zhang et al, 1994;Smith et al, 1999). On the other hand, pre-mRNAs from some other genes are shown to be associated with nuclear speckles (Xing et al, 1993(Xing et al, , 1995Huang and Spector, 1996;Ishov et al, 1997;Smith et al, 1999;Snaar et al, 1999;Johnson et al, 2000;Melčák et al, 2000). It thus remains unclear as to whether the nuclear speckles reflect localized accumulations of active splicing factors and whether the nucleus is compartmentalized with respect to splicing.…”

“…This RNA movement persists in transcriptionally inhibited cells. The movement of endogenous pre-mRNA to the speckles has been demonstrated in both transcriptionally active and silenced cells (Xing et al, 1995;Dirks et al, 1997;Ishov et al, 1997;Smith et al, 1999;Snaar et al, 1999;Johnson et al, 2000;Melčák et al, 2000). In addition, it has been shown that microinjected intron-containing RNA is within the cell nucleus processed into functional mRNA (Graessmann and Graessmann, 1982) and that several microinjected RNAs transcribed by RNA polymerase II and III localized at specific nuclear sites at which their corresponding endogenous counterparts were present in the steady-state distribution as shown by in situ hybridization (Jacobson et al, 1995(Jacobson et al, , 1997 Jacobson and Pederson, 1998a,b;Pederson, 1999).…”