Compartmentalization and regulation of snRNP biogenesis in vivo

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Cajal bodies (CBs) are non-membrane bound nuclear compartments that contain high concentrations of RNA processing machineries. In previous work, we have provided direct evidence that spliceosomal snRNPs, RNA-protein complexes required for pre-mRNA splicing, assemble in CBs. Immature snRNPs traffick independently from the nucleoplasm to CBs for assembly. Mathematical modelling predicted that CBs enhance the rate of snRNP assembly by ~10-fold, due to the 20-fold enrichment of snRNPs in the CB. To test this prediction in living cells, we propose to deplete tissue culture cells of the CB-specific protein coilin, which is required for snRNP concentration in CBs, and other relevant factors; analysis of the dynamics of snRNP biogenesis will identify key regulatory mechanisms. In a complementary approach, we will analyze the requirement for coilin in embryos of the zebrafish, Danio rerio. We describe the zebrafish CB for the first time and show that coilin knockdown by morpholino causes developmental arrest followed by cell death. This embryonic phenotype may be due to mitotic arrest, which we observed in human tissue culture cells upon depletion of splicing factors; this would suggest that cells of both species and systems exit the cell cycle when pre-mRNA splicing fails. Consistent with this, the zebrafish coilin morphant is rescued by injection of purified mature human snRNPs, showing that coilin is required for snRNP biogenesis in embryonic cells undergoing rapid cell divisions. We will pursue the essential role of coilin in zebrafish, using the human snRNP complementation system and other assays.