Higher order structure and remodelling of the spliceosomal RNA network

剪接体RNA网络的高阶结构和重塑

• 批准号: 5311507
• 负责人: Professor Dr. Reinhard Lührmann
• 金额: --
• 依托单位: Max-Planck-Institut für Multidisziplinäre Naturwissenschaften
• 依托单位国家: 德国
• 项目类别: Research Units
• 财政年份: 2001
• 资助国家: 德国
• 起止时间: 2000-12-31 至 2006-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/5311507?language=en
• 关键词: Higher; order; structure; remodelling; spliceosomal

Nuclear pre-mRNA splicing is catalysed by the spliceosome, which consists of the snRNPs U1, U2, U4/U6 and U5, and non-snRNP proteins. During spliceosome assembly, a complex and highly dynamic RNA network is formed between the pre-mRNA and snRNAs. One major goal of our project is to identify higher order interactions of the snRNAs in the active centre of the spliceosome using site-directed UV crosslinkung and hydroxyl radical probing approaches. As a general experimental strategy, we will incorporate into spliceosomes snRNA or pre-mRNA molecules that contain either diverse photo-sensitive crosslinkers or the hydroxyl radical generating reagent Fe(II)-BABE (1-(pbromoacetamidobenzyl) EDTA) at defined positions. In addition we will perform structure probing of the RNAs using chemical probes and purified, native spliceosomes. We will also investigate the role of the modified nucleotides of the U2RNA in splicing. As a second major goal, we plan to elucidate by site-directed UV crosslinking approaches snRNA-protein interactions at the catalytic core of the spliceosome. This will allow us not only to identify proteins which are potentially involved in splicing catalysis, but also to gather important information about the remodelling of the spliceosomal RNP structure.

核前mRNA剪接由剪接体催化，剪接体由snRNP U1、U2、U4/U6和U 5以及非snRNP蛋白组成。在剪接体组装过程中，前体mRNA和snRNA之间形成复杂且高度动态的RNA网络。我们的项目的一个主要目标是使用定点UV交联和羟基自由基探测方法来鉴定剪接体活性中心的snRNA的高阶相互作用。作为一般的实验策略，我们将纳入剪接体snRNA或前mRNA分子，含有不同的光敏交联剂或羟基自由基生成试剂Fe（II）-BABE（1-（pbromoacetamidobenzyl）EDTA）在定义的位置。此外，我们将使用化学探针和纯化的天然剪接体对RNA进行结构探测。我们还将研究U2 RNA的修饰核苷酸在剪接中的作用。作为第二个主要目标，我们计划通过定点UV交联方法阐明剪接体催化核心处的snRNA-蛋白质相互作用。这将使我们不仅能够识别潜在参与剪接催化的蛋白质，而且还可以收集有关剪接体RNP结构重塑的重要信息。