FUNCTIONAL SPLICEOSOME AND EXTRINSIC SPLICING FACTORS

RNA剪接，导致内含子移除的生化过程 来自前体RNA，是表达的重要步骤， 基因信息的调控。 核前mRNA剪接需要 ATP和发生在一个复杂的，动态的结构称为剪接体 其由小的核核糖核蛋白组成。 长期 这项资助的目的是了解核预- mRNA剪接通过生物化学和遗传分析， 剪接体及其组分。 一系列的RNA重排发生 在剪接体的组装和成熟过程中， 这些重排的驱动力是剪接ATP酶。我们有 广泛表征这种RNA依赖性ATP酶，酵母 （酿酒酵母）Prp 2蛋白。 我们的研究表明Prp 2 水解ATP以激活剪接体， 酯交换反应 最近，我们发现了几个结构性的 前体mRNA和U2和U6 snRNA的变化是 PRP 2介导的剪接体激活过程。 我们假设 受PRP 2和ATP影响的RNA核苷酸可能是 参与构建剪接的催化中心。 此外，本发明还提供了一种方法， 酯交换反应所需的蛋白质因子（HP）为 也被识别。 现在我们处于一个独特的位置来描述和 研究RNA变化的意义，这些变化是由一种 剪接ATP酶，以及RNA和蛋白质的参与 催化剪接反应。 此外， Prp 2蛋白在剪接体激活中可作为一种模型， 其他DexH蛋白。 有三个具体目标。(1)我们将继续调查 Prp 2激活剪接体过程中发生的RNA变化 和ATP。 我们将检验PRP 2的作用是形成 关键的RNA相互作用。 (2)我们将继续净化 探讨HP、氧与HP蛋白表达的关系。 U6的磷酸盐骨架和金属离子。 (3)个体DEAH 剪接ATP酶显然只与其“同源”剪接体结合。 我们 我计划通过寻找RNA和蛋白质来了解这种特异性 与剪接体口袋中的PRP 2相互作用。

RNA splicing, the biochemical process leading to the removal of introns from precursor RNA, is an important step in the expression and regulation of genetic information. Nuclear pre-mRNA splicing requires ATP and occurs in a complex, dynamic structure called the spliceosome which is composed of small nuclear ribonucleoproteins. The long-term objective of this grant is to understand the mechanism of nuclear pre- mRNA splicing through biochemical and genetic analysis of the spliceosome and its components. A series of RNA rearrangements occur during the assembly and maturation of the spliceosome, and one of the driving forces for these rearrangement is the splicing ATPases. We have extensively characterized such an RNA-dependent ATPase, the yeast (Saccharomyces cerevisiae) Prp2 protein. Our studies suggest that Prp2 hydrolyzes ATP to activate the spliceosome for the first transesterification reaction. Recently, we detected several structural changes in the pre-mRNA and the U2 and U6 snRNAs as results of the spliceosome activation process medicated by PRP2. We hypothesize that the RNA nucleotides that are affected by PRP2 and ATP are likely to be involved in building up the catalytic center for splicing. In addition, a protein factor (HP) required for the transesterification reaction was also identified. Now we are in a unique position to describe and to investigate the significance of the RNA changes that are caused by a splicing ATPase, as well as the involvement of RNA and protein in catalyzing the splicing reaction. Furthermore, the characteristics of the Prp2 protein in spliceosome activation may serve as a model for other DExH proteins. There are three specific aims. (1) We will continue to investigate the RNA changes that occur during the activation of the spliceosome by Prp2 and ATP. We will test the hypothesis that the role of PRP2 is to form critical RNA interactions for splicing. (2) We will continue to purify the HP protein and to investigate the relationship among HP, oxygen in the phosphate backbone of U6, and metal ions. (3) Individual DEAH splicing ATPase apparently only binds to its "cognate" spliceosome. We plan to understand this specificity by searching for RNA and proteins that interact with PRP2 in the spliceosomal pocket.