SPLICEOSOME ASSEMBLY AND FUNCTION

内含子通过一个大的，复杂的， 和动态分子机器剪接体。完全 剪接体由5个小的核核糖核蛋白组成 每个含有单个小核RNA（U1，U2， U4、U 5或U6 snRNA）以及许多snRNP蛋白。 在 与多达100种辅助蛋白，snRNP， 逐步组装到mRNA前体上，然后参与 不断变化的相互作用网络， 在剪接反应中，释放剪接的mRNA和切除的内含子， 并准备五个snRNP以供重复使用。 一个主要的实验问题， 分析这个复杂的多步骤过程， 反应中间体 在Konarska的领导下，我们使用了 类似剪接位点的寡核苷酸和反义2 '-0-甲基 针对某些snRNA序列的寡核苷酸， 有效地诱导剪接体样snRNP复合物的组装， 哺乳动物核提取物。这些人工诱导的剪接体样 复合物似乎冻结了各种瞬时剪接体， 自然发生的相互作用（snRNP/snRNP，snRNP/底物） 在mRNA剪接反应的过程中。 使用这种方法，我们 发现了两种新的复合物：U1/U4/U 5复合物， 是从5'剪接位点置换U1的中间体，和 U2/5' SS复合物，其中U2以与U2/5' SS复合物几乎相同的方式结合5'外显子。 II组内含子的EBS 2元件结合已知的5 ′外显子序列 IBS2。 我们建议将这些复合物的特征 从生物化学的角度，来确定导致形成的事件顺序， 的复合物，并开发分析，以探索这些新的作用， 体内RNA/RNA配对。 这些实验应该能揭示 最初的U4/U6配对在剪接体组装早期被解除; 与5'剪接位点结合的U1如何被U2、U 5和U6取代; 活性位点由U2和U6组装;以及5'外显子是如何 在剪接的第一化学步骤后由U2和U 5保留，和 在第二步中对齐用于连接。

Introns are removed from eukaryotic mRNA precursors by a large, complex, and dynamic molecular machine known as the spliceosome. The fully assembled spliceosome consists of five small nuclear ribonucleoprotein particles (snRNPs) each containing a single small nuclear RNA (U1, U2, U4, U5, or U6 snRNA) together with a number of snRNP proteins. In collaboration with as many as 100 auxiliary proteins, the snRNPs assemble stepwise onto the mRNA precursor, and then participate in a changing network of interactions that catalyze the two chemical steps of the splicing reaction, release the spliced mRNA and excised intron, and prepare the five snRNPs for reuse. A major experimental problem in analyzing this complicated multistep process has been to identify reaction intermediates. Following the lead of Konarska, we have used oligonucleotides resembling splice sites, and antisense 2'-0-methyl oligonucleotides directed against certain snRNA sequences, to efficiently induce the assembly of spliceosome-like snRNP complexes in mammalian nuclear extracts. These artificially induced spliceosome-like complexes appear to freeze a variety of transient spliceosomal interactions (snRNP/snRNP, snRNP/substrate) that take place naturally over the course of an mRNA splicing reaction. Using this approach, we have identified two novel complexes: a U1/U4/U5 complex that appears to be an intermediate in displacement of U1 from the 5' splice site, and a U2/5' SS complex in which U2 bind the 5' exon in much the same way as the EBS2 element of a Group II intron binds the 5' exon sequence known as IBS2. We propose to characterize each of these complexes biochemically, to identify the sequence of events leading to formation of the complexes, and to develop assays to explore the role of these new RNA/RNA pairings in vivo. The experiments should shed new light on how the initial U4/U6 pairing comes undone early in spliceosome assembly; how U1 binding to the 5' splice site is replaced by U2, U5, and U6; how the active site is assembled from U2 and U6; and how the 5' exon is retained by U2 and U5 after the first chemical step of splicing, and aligned for ligation in the second step.