MECHANISMS OF PRE-MRNA SPLICING IN HIGHER EUKARYOTES

拟议工作的长期目标是实现详细的 了解Pre-mRNA剪接的机制。拼接起到了 在高等真核生物中大多数前mRNAs的成熟过程中起着重要作用， 而选择性剪接涉及到调节一种 基因的数量。大多数前mRNA是高度复杂的，包含多个 大小从65到100,000个核苷酸的内含子。因此， 阐明了准确切割每个内含子的机制， 而不删除必要的蛋白质编码信息是基本的 生物学上的重要性。拟议研究的主要重点是 剪接体的组装、结构和功能。尽管有很大一部分 之前的工作的重点是识别参与其中的小RNA 剪接，对He的蛋白质成分知之甚少 剪接体。在拟议的研究中，中间剪接复合体 在体外剪接反应中组装的组件将被提纯并 特色化的。最近建立的一种适用于大规模生产的两步法 将使用剪接体的纯化来分离每个剪接体 复合体，并且复合体的RNA和蛋白质成分将是 已确认身份。在含有突变的前mRNA上组装的复合体 在反应的特定步骤以及中间体上的片段拼接 体外剪接过程中产生的剪接复合体 反应，将被检验。这些研究应该会导致 稳定与Pre-mRNA相互作用的剪接因子的鉴定 并参与剪接反应的不同步骤。此外 ，对剪接早期产生的中间复合体进行分析 反应应该导致确定起作用的因素 在建立剪接位点选择模式中的重要作用。在……里面 在随后的研究中，将针对特定蛋白质产生抗体。 它们似乎在拼接过程中起着核心作用。然后抗体就会 用于克隆编码这些因子的基因，并进一步定义它们的 在剪接反应中起作用。最后，对其进行了结构分析 剪接体将使用电子显微镜进行观察。这些研究 将使用电子显微镜对剪接体进行分析。 这些研究应该提供关于空间安排的独特信息 剪接体中的前信使核糖核酸和剪接成分。

The long-term objective of the proposed work is to achieve a detailed understanding of the mechanisms of pre-mRNA splicing. Splicing plays a essential role in the maturation of most pre-mRNAs in higher eukaryotes, and alternative splicing is involved in regulating the expression of a number of genes. Most pre-mRNAs are highly complex, containing multiple introns that range in size from 65 to 100,000 nucleotides. Thus, elucidating the mechanisms by which each intron is accurately excised, without deleting essential protein coding information is of fundamental biological importance. The main focus of the proposed research is the assembly, structure and function of the spliceosome. Although a great deal of prior work has focused on the identification of the snRNAs involved in splicing, relatively little is known about the protein components of he spliceosome. In the proposed studies, intermediate splicing complexes assembled during the in vitro splicing reaction will be purified and characterized. A two-step method recently established for the large-scale purification of the spliceosome will be employed to isolate each splicing complex, and the RNA and protein components of the complexes will be identified. Complexes assembled on pre-mRNAs containing mutations that block splicing at a specific step of the reaction, as well as intermediate splicing complexes generated during the course of the in vitro splicing reaction ,will be examined. These studies should lead to the identification of splicing factors that interact stably with the pre-mRNA and are involved in different steps of he splicing reaction. In addition ,analysis of intermediate complexes generated very early in the splicing reaction should lead to the identification of factors that play an important role in establishing the pattern of splice-site selection. In subsequent studies, antibodies will be raised against specific proteins that appear to play a central role in splicing. The antibodies will then be used to clone the genes encoding these factors and further defined their function in the splicing reaction. Finally, structural analysis of the spliceosome will be carried out using electron microscopy. These studies analysis of the spliceosome will be carried out using electron microscopy. These studies should provide unique information on the spatial arrangement of pre-mRNA and splicing components within the spliceosome.