Within nucleoplasm, the pre-mRNA substrates and the factors essential for the splicing are most highly concentrated at transcription sites (active genes), which are distributed apparently at random within the three-dimensional space of the nucleus. Thus, genes themselves can be seen as the sites within nuclei that nucleate the assembly of spliceosomes either through rapid binding of splicing factors to nascent RNA or through the coupled assembly of the transcriptional machinery with splicing factors (known as “transcription factory model”). We have developed novel in vivo assays to distinguish between these two models and are extending the assay to address the elusive question of how alternative splicing is controlled during development.

In addition to their nucleoplasmic distribution and function, some splicing factors are concentrated in CBs. Recently, our lab has provided the first strong evidence that CBs are the sites of assembly of complexes required for splicing. These data suggest that the CB may provide a catalytic environment for splicing factor assembly, which might otherwise occur much less efficiently in the nucleoplasm. Moreover, we have established the zebrafish embryo as an excellent system for imaging the nuclear structures before, during and after the onset of zygotic genome transcription. The movie below shows the dynamic behavior of CBs marked with a fluorescently labeled splicing factor in a 32-cell stage embryo; the CBs have dispersed at mitosis, reform during the extremely short (~15 min) interphase and then disperse again. The embryo is amenable to targeted knockdown of key factors in nuclear morphology and gene expression, and we are exploiting this system to understand the structure and function of CBs as well as to investigate the role of splicing in embryonic development.