Higher order structure and remodelling of the spliceosomal RNA network

剪接体是由剪接体催化的，剪接体由SnRNPs U1、U2、U4/U6和U5以及非SnRNP蛋白组成。在剪接体组装过程中，在前mRNA和SnRNAs之间形成了一个复杂的、高度动态的RNA网络。我们项目的一个主要目标是使用定点紫外光交联法和羟基自由基探针法确定剪接体活性中心的SnRNAs的高阶相互作用。作为一般的实验策略，我们将在剪接体中掺入含有不同光敏交联剂或羟基自由基生成剂Fe(II)-Babe(1-(对溴乙酰氨基苯甲基)EDTA)的SnRNA或Pre-mRNA分子。此外，我们还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U2RNA的修饰核苷酸在剪接中的作用。作为第二个主要目标，我们计划通过定点紫外光交联法阐明剪接体催化核心的SnRNA-蛋白质相互作用。这将使我们不仅能够识别可能参与剪接催化的蛋白质，而且还可以收集关于剪接体RNP结构重塑的重要信息。

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.