MECHANISMS OF PRE-MRNA SPLICING IN HIGHER EUKARYOTES

我们研究的长期目标是了解这些机制。 高等真核生物中的前信使核糖核酸剪接。后生动物中的大多数前mRNA是 高度复杂，包含多个内含子，必须精确地 为了产生有功能的信使核糖核酸而切除。拼接缺陷是 导致许多遗传性疾病，而剪接是关键的一步 在几乎所有病毒的生命周期中。因此，阐明 剪接机制是一个至关重要的问题。这 研究建议的重点是实现对 哺乳动物剪接体组装的早期步骤。这些步骤是 尤其重要的是，负责识别 每个剪接点，并用于将适当的配对聚集在一起 5‘和3’剪接位点的组成和交替 剪接的前mRNAs在这个时候工作。剪接体组装是 由不依赖于ATP的E复合体的形成引发，随后 ATP依赖的A复合体的形成。在以前的工作中，这些 对络合物进行了纯化，发现其含有几种关键成分 剪接机械，包括U1和U2 SnRNP，非SnRNP 剪接因子U2AF和SR剪接蛋白家族。这个 本提案的第一个目标的总体目标是了解每个 这些组件相互作用，并与特定序列相互作用 在E和E的组装过程中不同时间前mRNA中的元件 一种复合体。第二个具体目标是重组U2 snRNP 使用重组U2SnRNP的野生型和突变型衍生物的颗粒 相关蛋白质。然后，重组的SnRNP将被添加回 耗尽U2 SnRNP的剪接提取物以确定 U2的每个SnRNP组件在两者中的特定功能 剪接体组装和剪接反应的催化步骤。 在第三个具体目标中，制定了一个工作模式，以 构造性地解释剪接位点选择的分子基础 拼接的前信使RNA。该模型的关键特征之一是外显子 包含一个或多个指定为外显子的特定序列元件 促进相邻5‘或3’剪接位点的使用的“选择器”。 外显子选择子被认为是通过差异来发挥作用的 与SR剪接蛋白家族成员的相互作用。另一个 该模型的中心特征是将三个不同的因素捆绑在一起 前信使核糖核酸、外显子选择子、5‘和3’剪接中的元件 外显子两侧的位点，相互促进彼此结合到前- MRNA.模型的核心方面将通过组合进行测试 鉴定白纹伊蚊外显子内SR蛋白的结合部位 功能E复合体和确定这些结合位点是否影响 拼接-位置选择。此外，使用纯化因子的研究将 以确定与外显子结合的因子 选择子和侧翼的5‘和3’剪接位点相互影响 有约束力的。

The long term objective of our research is to understand the mechanisms of pre-mRNA splicing in higher eukaryotes. Most pre-mRNAs in metazoa are highly complex, containing multiple introns that must be precisely excised in order to generate functional mRNA. Splicing defects are responsible for many genetic diseases, and splicing is a critical step in the life cycle of virtually all viruses. Thus, elucidating the mechanisms of splicing is a problem of fundamental importance. This research proposal is focused on achieving a detailed understanding of the early steps in mammalian spliceosome assembly. These steps are especially important because the mechanisms responsible for identifying each of the splice sites and for bringing together the appropriate pairs of 5' and 3' splice sites in both constitutively and alternatively spliced pre-mRNAs operate at this time. Spliceosome assembly is initiated by the ATP-independent formation of the E complex followed by formation of the ATP-dependent A complex. In previous work these complexes were purified and found to contain several key components of the splicing machinery, including U1 and U2 snRNPs, the non-snRNP splicing factor U2AF, and the SR family of splicing proteins. The general goal of the first aim of this proposal is to understand how each these components interact with one another and with specific sequence elements in the pre-mRNA at distinct times during assembly of the E and A complexes. The second specific aim is to reconstitute the U2 snRNP particle using wild-type and mutant derivatives of recombinant U2 snRNP- associated proteins. The reconstituted snRNP will then be added back to splicing extracts depleted of U2 snRNP in order to determine the specific functions of each of the U2 snRNP components in both spliceosome assembly and the catalytic steps of the splicing reaction. In the third specific aim, a working model has been formulated to explain the molecular basis for splice-site selection in constitutively spliced pre-mRNAs. One of the key features of the model is that exons contain one or more specific sequence elements, designated "exonic selectors", that promote the use of the adjacent 5' or 3' splice site. The exonic selectors are proposed to function via differential interactions with members of the SR family of splicing proteins. Another central feature of the model is that factors bound to three distinct elements in the pre-mRNA, the exonic selector, and the 5' and 3' splice sites flanking an exon, reciprocally promote each other binding to pre- mRNA. The central aspects of the model will be tested by a combination of identifying the binding sites of SR proteins within the exons in functional E complex and determining whether these binding sites affect splice-site selection. In addition, studies used purified factors will be carried out to determine whether factors that bind to exonic selectors and the flanking 5' and 3' splice sites influence one anothers binding.