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。 拼接起着 在高等真核生物中大多数前体mRNA的成熟中起重要作用， 而选择性剪接参与调节 基因的数量 大多数前体mRNA是高度复杂的，含有多个 内含子大小范围从65到100，000个核苷酸。 因此，在本发明中， 阐明了精确切除每个内含子的机制， 而不删除必要的蛋白质编码信息， 生物学重要性。 拟议研究的主要重点是 剪接体的组装、结构和功能。 虽然很多 先前的工作集中在识别参与的snRNAs， 剪接，相对较少的是知道的蛋白质组成的he 剪接体 在拟议的研究中， 在体外剪接反应期间组装的蛋白质将被纯化， 表征了 最近建立的两步法用于大规模的 将使用剪接体的纯化来分离每个剪接 复合物，并且复合物的RNA和蛋白质组分将是 鉴定 在含有突变的前mRNA上组装的复合物， 在反应的特定步骤阻断剪接，以及中间体 在体外剪接过程中产生的剪接复合物 反应，将被检查。 这些研究将导致 与前mRNA稳定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 ，分析在剪接中非常早期产生的中间复合物 反应应导致确定的因素发挥作用， 在建立剪接位点选择模式中的重要作用。 在 随后的研究，抗体将针对特定的蛋白质 在拼接中起着重要作用。 抗体就会 用于克隆编码这些因子的基因，并进一步确定它们的结构。 在剪接反应中起作用。 最后，结构分析 剪接体将使用电子显微镜进行。 这些研究 剪接体的分析将使用电子显微镜进行。 这些研究应提供关于空间布局的独特信息 前体mRNA和剪接体中的剪接成分。

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.