Cell biology: the emergence of the transcriptosome
The use of increasingly sophisticated methods for visualizing subcellular structures and for localizing individual proteins within them, has given rise to a picture of nuclear interactions remarkably similar to that suggested by the largely biochemical experiments described above. Considerable attention has focused on nuclear structures called speckles, which appear to correspond to interchromatin granule clusters that had initially been observed with the electron microscope (for review, see Spector 1993). Speckles, of which there are 20-40 in a typical mammalian nucleus, have been visualized by immunofluorescent staining with antibodies against splicing factors, frequently SR proteins. It now seems clear that speckles represent storage sites, or perhaps sites of assembly or recycling of splicing complexes, rather than sites of active splicing (for review, see Singer and Green 1997; Misteli and Spector 1998). Both hyperphosphorylated RNAP II0 (Bregman et al. 1995) and polyadenylation factors (Schul et al. 1998) can also be observed associated with speckles, although apparently at the periphery. Transcriptional activation seems to result in a redistribution of factors from the speckles, and indeed RNAP II0 (Bregman et al. 1995) and the SR protein ASF/SF2 (Misteli et al. 1997) appear to migrate from speckles to sites of transcription. In cells expressing an RNAP II with a truncated CTD, however, relocalization of splicing factors to transcription sites does not occur (Misteli and Spector 1999), in keeping with the biochemistry describing interactions between the CTD and splicing factors. Thus the localization and dynamics of transcription and processing factors within the nucleus is consistent with the functional interactions observed in vitro.
Combining biochemistry with cell biology, Mintz et al. (1999) recently described a purification protocol for speckles. Although it was not possible to quantitate purification, EM visualization was consistent with a significant enrichment. The preparation contained RNAP II0 and was significantly enriched in splicing factors, especially SR proteins. A number of previously unknown proteins were also identified, and one, of 137 kD, seemed especially interesting. The protein is the apparent mammalian homolog of a recently described yeast splicing factor, Rse1 (Caspary et al. 1999), but shows similarity across its entire length to a polyadenylation factor (CPSF-160) and to a protein implicated in DNA repair (UV-DDB). Although the significance of these similarities is currently unknown, all three proteins are conserved from yeast to human and suggest potentially interesting relationships between these processes.
The above studies have provided evidence that RNAP II0 can colocalize with certain RNA processing factors, supporting the view that transcription and processing are indeed linked physically and functionally in the nuclei of mammalian cells. But this idea has recently been significantly extended by studies employing X. laevis oocytes. Gall and colleagues have for many years analyzed nuclear organization by studying the oocyte germinal vesicle, taking advantage of the large size of the organelle. In a recent study (Gall et al. 1999), they provided evidence for a remarkable convergence of the transcriptional and processing machineries. Specifically, by following the localization and movement of numerous required factors, it was shown that not only RNAP II and other components of the transcription, splicing and polyadenylation machineries, but also RNAP I and III and related factors accumulate initially following transport into the nucleus in structures known as Cajal (or coiled) bodies. Here it seems that factors needed for RNAP I-, II-, and III-mediated synthesis associate into massive holocomplexes, or transcriptosomes, and are subsequently transported to sites of RNA synthesis. In the case of RNAP II, "pol II transcriptosomes" are exported from Cajal bodies as the previously described B snurposomes, which consist of multiple pol II transcriptosomes and are likely identical to the interchromatin granule clusters, or speckles, characterized in somatic cells. The B snurposomes would then provide a reservoir of pol II transcriptosomes to active genes, and these particles in turn contain all the factors necessary for complete synthesis of the mature mRNA. Supporting the idea that processing factors are indeed associated with RNAP II throughout transcription, staining of highly active lampbrush chromosomes with antibodies directed against both splicing and polyadenylation factors revealed uniform staining along the chromosome's entire length, coincident with RNAP II staining (Gall et al. 1999). All in all, this picture resembles rather closely that emerging from the biochemical studies.
The idea of a multifunctional pol II transcriptosome provides a neat mechanism for insuring efficient and accurate processing of RNAP II-generated transcripts. But it is not without some conceptual challenges. For example, how would such a massive structure translocate along the DNA? Might we want to reconsider the idea that the DNA is mobile, with the transciptosome being stationary? Most genes have multiple intervening sequences, and considerable data from some twenty years ago indicates that introns are not necessarily removed in a 5'-to-3' order, and in some cases not until transcription is complete. How might the transcriptosome deal with this? One possibility is that the transcriptosome doesn't necessarily contain (a) complete spliceosome(s), but perhaps only factors necessary to define splice sites, or intron boundaries, and for committing the RNA to subsequent splicing. And what about regulation? Are there gene-specific transcriptosomes, or are regulatory factors not part of the transcriptosome? This is an especially vexing problem when considering regulated processing, where it is thought that changes in the relative concentrations of certain essential factors (e.g., SR proteins) can contribute to changing patterns of alternative processing. Despite these questions, the concept of the transcriptosome provides an exciting new way to think about how genes are expressed in the cell nucleus.