Compartmentalization and regulation of snRNP biogenesis in vivo

Cajal小体(CBS)是一种非膜结合的核隔室，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证据表明剪接体SnRNPs，即剪接前mRNA剪接所需的RNA-蛋白质复合体，在CBS中组装。未成熟的SnRNPs独立地从核质运输到CBS进行组装。数学模型预测，由于SnRNP在CB中的浓缩20倍，CBS使SnRNP的组装速度提高了~10倍。为了在活细胞中验证这一预测，我们建议耗尽组织培养细胞中CB特异的蛋白质Colin和其他相关因素；对SnRNP生物发生的动力学分析将确定关键的调控机制。在一个补充的方法中，我们将分析斑马鱼Danio rerio胚胎对Colin的需求。我们首次描述了斑马鱼CB，并表明被吗啉敲除后会导致发育停滞和细胞死亡。这种胚胎表型可能是由于有丝分裂停止，我们在人类组织培养细胞中观察到剪接因子耗尽后的有丝分裂停滞；这表明当前mRNA剪接失败时，物种和系统的细胞都退出了细胞周期。与此一致的是，通过注射纯化的成熟人SnRNPs拯救了斑马鱼Colin变异体，表明Colin是经历快速细胞分裂的胚胎细胞中SnRNP生物发生所必需的。我们将利用人类的SnRNP互补系统和其他检测手段来研究Colin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.