Compartmentalization and regulation of snRNP biogenesis in vivo

Cajal小体（CB）是一种非膜结合的核区室，含有高浓度的RNA加工机制。在以前的工作中，我们已经提供了直接的证据，剪接体snRNP，RNA-蛋白质复合物所需的前mRNA剪接，组装在CB。未成熟的snRNP独立地从核质运输到CB进行组装。数学建模预测CB将snRNP组装的速率提高约10倍，这是由于CB中snRNP的20倍富集。为了在活细胞中测试这种预测，我们建议耗尽CB特异性蛋白卷曲蛋白的组织培养细胞，这是CB中snRNP浓度所需的，以及其他相关因素; snRNP生物合成的动力学分析将确定关键的调控机制。在一个互补的方法，我们将分析的斑马鱼，Danio rerio胚胎卷曲蛋白的需求。我们描述了斑马鱼CB的第一次，并表明线圈蛋白敲低吗啉导致发育停滞，随后细胞死亡。这种胚胎表型可能是由于有丝分裂停滞，这是我们在人类组织培养细胞中观察到的剪接因子耗尽;这表明当前体mRNA剪接失败时，两个物种和系统的细胞都退出了细胞周期。与此相一致，斑马鱼卷曲蛋白morphant是通过注射纯化的成熟人snRNP来拯救的，表明卷曲蛋白是经历快速细胞分裂的胚胎细胞中snRNP生物合成所需的。我们将使用人类snRNP互补系统和其他检测方法来研究卷曲蛋白在斑马鱼中的重要作用。

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.