Compartmentalization and regulation of snRNP biogenesis in vivo

snRNP 体内生物发生的区室化和调节

• 批准号: 49251211
• 负责人: Professorin Dr. Karla Neugebauer, Ph.D.
• 金额: --
• 依托单位: Department of Molecular Biophysics and Biochemistry
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2007
• 资助国家: 德国
• 起止时间: 2006-12-31 至 2015-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/49251211?language=en
• 关键词: Compartmentalization; regulation; snRNP; biogenesis; vivo

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.

卡哈尔体是一种非膜结合的核室，含有高浓度的RNA加工机制。在之前的工作中，我们已经提供了直接证据，证明剪接体snRNPs （pre-mRNA剪接所需的rna -蛋白复合物）在CBs中组装。未成熟snRNPs独立地从核质转运到CBs进行组装。数学模型预测，CB将snRNP的组装率提高了约10倍，这是由于CB中snRNP富集了20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的cb特异性蛋白coilin，这是CBs中snRNP浓度和其他相关因素所必需的；snRNP生物发生的动力学分析将确定关键的调控机制。在一种补充的方法中，我们将分析斑马鱼胚胎对肠毒素的需求。我们首次描述了斑马鱼的CB，并表明由morpholino敲低卷曲蛋白导致发育停滞，随后是细胞死亡。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竭；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类snRNPs来挽救斑马鱼卷曲蛋白变形体，这表明在经历快速细胞分裂的胚胎细胞中snRNP的生物发生需要卷曲蛋白。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。