Identification of a Novel Regulatory Element in the Assembly of the Spliceosome

项目总结 前信使核糖核酸剪接调控新生的信使核糖核酸的成熟，这是基因中的一个基本过程 表情。剪接体是一种大的RNA/蛋白质，它促进和调节着前mRNA的剪接 大分子机器。剪接体是通过一个复杂的、多步骤的途径从5个小的 核核糖核蛋白复合体(SnRNPs)，然而调节每一步的分子相互作用仍然是 未知。这项提议的目标是确定一个重要的剪接蛋白Dib1在调控中的作用 剪接体组装。本提案中的工作解决了所提议的模型，即 U4/U6-U5三重SNRNP中的小硫氧还蛋白Dib1限制了Pre-RNP的成功掺入 信使RNA进入剪接体的活性部位，因此起到剪接体的调节作用 集合。本项目正在研究的U4/U6-U5三重SNRNP复合体的蛋白质已经被直接 与人类疾病视网膜色素变性和烧伤-麦基翁综合征有关。因此，理解 Dib1的功能将提供对这些疾病的原因的直接洞察。利用模型生物体， 酿酒酵母的第一个目标将确定Dib1是否是剪接的调控元件 表征一组Dib1突变体对蛋白质稳定性、剪接体组装、剪接的影响 和细胞生长。第二个目标将确定DIb1和Pre之间的时间关系和距离 用单分子荧光技术检测拼接剪接体上的mRNA.成功完成 在这些研究中，将确定剪接体组装中的一个新的调控元件。此外，这项研究将 对Dib1有一个新的理解，它与基因表达的其他方面有关，包括 有丝分裂和转录，但对此知之甚少。

PROJECT SUMMARY Pre-messenger RNA splicing regulates the maturation of a nascent mRNA, an essential process in gene expression. Pre-mRNA splicing is facilitated and regulated by the spliceosome, a large RNA/protein macromolecular machine. The spliceosome is assembled through a complex, multi-step pathway from 5 small nuclear ribonucleoprotein complexes (snRNPS), however the molecular interactions regulating each step is still unknown. The goal of this proposal is to define the roles of an essential splicing protein Dib1 in the regulation of spliceosome assembly. The work in this proposal addresses the proposed model that the presence of a small thioredoxin-like protein Dib1 in the U4/U6-U5 triple snRNP restricts successful incorporation of the pre- messenger RNA into the active site of the spliceosome, therefore acting as a regulator of spliceosome assembly. The proteins of the U4/U6-U5 triple snRNP complex being studied in this project have been directly linked to the human diseases retinitis pigmentosa and Burn-McKeown syndrome. Thus, understanding the function of Dib1 will provide direct insight into the cause of these diseases. Using the model organism, Saccharomyces cerevisiae, the first aim will determine whether Dib1 is a regulatory element of splicing by characterizing the effects of a collection of Dib1 mutants on protein stability, spliceosome assembly, splicing and cell growth. The second aim will determine the temporal relationship and distance between Dib1 and pre- mRNA on the assembling spliceosome using single molecule fluorescence techniques. Successful completion of these studies will identify a novel regulatory element in spliceosome assembly. Moreover, this research will contribute a new understanding of Dib1, which has been linked to other aspects of gene expression including mitosis and transcription, but about which little is known.