FUNCTIONAL SPLICEOSOME AND EXTRINSIC SPLICING FACTORS

RNA剪接，导致内含子移除的生化过程 从前体RNA出发，是表达的重要一步 对遗传信息的监管。核转录前基因剪接需要 它存在于一种称为剪接体的复杂、动态的结构中 它由小核核糖核蛋白组成。长期的 这项资助的目的是为了了解核预激反应的机制 通过对mRNA剪接的生化和遗传分析 剪接体及其组件。发生了一系列的RNA重排 在剪接体的组装和成熟过程中， 这些重排的驱动力是剪接的ATPase。我们有 广泛地表征了这种依赖于RNA的ATPase，酵母 (酿酒酵母)Prp2蛋白。我们的研究表明Prp2 水解三磷酸腺苷激活第一个剪接体 酯交换反应。最近，我们检测到了几个结构 前信使核糖核酸及U2和U6单链RNA的变化 剪接体激活过程受PRP2的影响。我们假设 受PRP2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, 酯交换反应所需的蛋白质因子(HP)是 也被确认了。现在我们处于一个独特的位置来描述和 调查由一种病毒引起的RNA变化的重要性 剪接ATPase，以及RNA和蛋白质在其中的作用 催化剪接反应。此外，它的特点是 剪接体激活中的Prp2蛋白可以作为一种模型 其他DExH蛋白。 有三个具体目标。(1)我们会继续调查 Prp2激活剪接体过程中发生的RNA变化 和三磷酸腺苷。我们将检验PRP2的作用是形成 剪接的关键RNA相互作用。(二)持续净化 并探讨幽门螺杆菌与血氧的关系。 U6的磷酸盐骨架和金属离子。(3)个人死亡 剪接ATPase显然只与它的“同源”剪接体结合。我们 计划通过搜索RNA和蛋白质来了解这种特异性 与剪接体口袋中的PRP2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.