Higher order structure and remodelling of the spliceosomal RNA network

核前mrna剪接由剪接体催化，剪接体由snrnp蛋白U1、U2、U4/U6和U5以及非snrnp蛋白组成。在剪接体组装过程中，一个复杂且高度动态的RNA网络在pre-mRNA和snrna之间形成。我们项目的一个主要目标是使用位点定向UV交联和羟基自由基探测方法确定剪接体活性中心snrna的高阶相互作用。作为一般的实验策略，我们将在剪接体中加入含有多种光敏交联剂或羟基自由基生成试剂Fe(II)- babe （1-(bromoacetamidobenzyl) EDTA）的snRNA或pre-mRNA分子。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。我们还将研究修饰的U2RNA核苷酸在剪接中的作用。作为第二个主要目标，我们计划通过位点定向UV交联方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，而且还可以收集有关剪接体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.