Structural Studies of RNA Elements in Pre-mRNA Splicing

描述(申请人提供)：本申请的重点是建立前mRNA分支位点的结构和功能之间的关系，该RNA形成包含用于前mRNA剪接的第一次攻击的亲核体。我们工作的长期目标是了解真核剪接装置的RNA和蛋白质组件是如何组装的，定义剪接位点，并催化从前mRNA分子中移除非编码序列(内含子)。剪接是真核RNA成熟过程中不可或缺的一步，选择性剪接位点的选择是发育阶段和组织特异性蛋白质多样性的来源。由于剪接错误是某些癌症和神经退行性疾病的基础，因此了解剪接过程具有生物医学意义。真核细胞核的剪接体是由5个可回收的小核RNA和大量蛋白质动态组装而成的，而在某些原核生物和真核细胞器中发现的第II类内含子是由6个二级结构域组成的单个RNA分子。这两个剪接系统通过相同的化学机制和立体化学去除内含子，并具有一定的序列相似性，但只有第二类内含子可以在没有蛋白质的情况下进行体外剪接反应。因此，这两个系统很可能具有共同的进化祖先，并且RNA在剪接体反应中也起着主要的催化作用。结构性信息方面的差距具有有限的可比性。其具体目的是：1)确定单个RNA-RNA相互作用对剪接体分支位点结构的贡献及其对剪接活性的影响；以及2)探索II组自剪接内含子的前mRNA分支位点的结构，以便将其与剪接体的对应物进行比较。溶液中的结构研究将通过同核和异核核磁共振进行；各种突变和修饰对剪接活性的影响将通过对体外剪接试验产物形成的扰动来评估。许多创新的核磁共振技术将被用来帮助确定高分辨率溶液的结构模型和RNA结构域的取向。拟议的实验将为了解真核细胞剪接活性的起源和分子基础提供新的见解。

DESCRIPTION (provided by applicant): The focus of this application is to establish the relationship between structure and function of the pre-mRNA branch site, the RNA formation that contains the attacking nucleophile for the first of pre-mRNA splicing. The long-range objective of our work is to understand how RNA and protein components of the eukaryotic splicing apparatus assemble, define splice sites, and catalyze the removal of noncoding sequences (introns) from pre-mRNA molecules. Splicing is an integral step in the maturation of eukaryotic RNAs, and alternative splice site selection is a source of developmental stage- and tissue-specific protein diversity. Since errors in splicing are the basis of certain cancers and neurodegenerative diseases, an understanding of the splicing process is of biomedical importance. The spliceosome of eukaryotic nuclei is a dynamic assembly of five recyclable small nuclear (sn)RNAs and numerous proteins, whereas the Group II intron, found in certain prokaryotes and eukaryotic organelles, is a single RNA molecule comprising six secondary structural domains. The two splicing systems remove introns via the identical chemical mechanism and stereochemistry, and share certain sequence similarities, but only the Group II intron can carry out the splicing reaction in vitro in the absence of proteins. It is therefore likely that the two systems share evolutionary ancestry and that RNA also plays a major catalytic role in the spliceosomal reaction. Gaps in structural information have limited comparison. The Specific Aims are: 1) determine the contributions of individual RNA-RNA interactions to the spliceosomal branch site structure and their implications on splicing activity; and 2) probe the structure of the pre-mRNA branch site of the Group II self-splicing intron in order to compare it with its spliceosomal counterpart. Structural studies in solution will be carried out by homonuclear and heteronuclear NMR; the effect of various mutations and modifications on splicing activity will be assessed by perturbations to product formation of in vitro splicing assays. A number of innovative NMR techniques will be exploited to assist in determination of high resolution solution structural models and orientation of RNA domains. The proposed experiments will provide new insights into the ancestry and the molecular basis of splicing activity in eukaryotic cells.