SPLICEOSOME ASSEMBLY AND FUNCTION

从真核细胞的信使核糖核酸前体中去除内含子是由一个大的，复杂的， 以及被称为剪接体的动态分子机器。完整的 组装的剪接体由五个小的核核糖核蛋白组成 颗粒(SnRNPs)，每个含有单个小的核RNA(U1，U2， U4、U5或U6SnRNA)以及许多SnRNP蛋白。在……里面 与多达100种辅助蛋白质合作，SnRNPs 逐步组装到mRNA前体上，然后参与 催化这两个化学步骤的相互作用网络的变化 在剪接反应中，释放剪接的mRNA和切除的内含子， 并准备好这五个SNRNP以供重复使用。一个主要的实验问题是 分析这一复杂的多步骤过程是为了确定 反应中间体。在科纳尔斯卡的带领下，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，以 高效诱导剪接体样SnRNP复合体在体内的组装 哺乳动物的核提取物。这些人工诱导的剪接体样体 复合体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) 在信使核糖核酸剪接反应过程中。使用这种方法，我们 确定了两个新的络合物：U1/U4/U5络合物，它似乎 是U1从5‘剪接位移位的中间体，以及 一种U2/5‘SS复合体，其中U2结合5’外显子的方式与 第二类内含子的EBS2元件与已知的5‘外显子序列结合 作为IBS2。我们建议对这些复合体中的每一个进行表征 在生物化学上，以确定导致形成的事件序列 并开发分析方法来探索这些新的 体内的RNA/RNA配对。这些实验应该会为我们提供新的线索 最初的U4/U6配对在剪接体组装早期解体； U2、U5和U6如何取代U1与5‘剪接位点的结合；如何 活性部位由U2和U6组装而成，以及5‘外显子是如何 在剪接的第一个化学步骤之后被U2和U5保留，以及 在第二步中对齐以进行连接。

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.