Single Molecule Flourescence Studies on DExD/H-box Protein:Spliceosome Complexes

描述（由申请人提供）：大多数真核基因含有内含子，在翻译之前必须由剪接体从mrna前体中去除内含子。剪接体是由RNA和蛋白质组成的巨型道尔顿大分子机器。在剪接体的螺旋合成或外显子-外显子连接过程中，ATP不用于化学键的形成。然而，ATP水解是产物形成所必需的结构重排所必需的。这些结构重排产生了明确的、稳定的复合物，这些复合物已在体外分离和研究。目前尚不清楚复合物之间的转换是如何发生的，但有一种假设是必需的DExD/H-box蛋白（Prp2、16、22和43）通过利用ATP水解的能量来破坏蛋白质/RNA或RNA/RNA相互作用，从而启动剪接体的结构重组。鉴于剪接体的~ 3mda大小，传统的生化分析这些酶如何促进剪接是困难的。本课题拟采用单分子荧光（SMF）作为研究DExD/H-box蛋白与剪接体相互作用的新方法。剪接体将组装在prp2缺失酵母细胞提取物衍生的玻璃表面上。由于缺乏Prp2，剪接体将在表面固定化、荧光标记的pre-mRNA底物上组装，但在幼虫形成之前会停滞。Prp酶将使用公开的程序从大肠杆菌中表达和纯化，并使用基因编码的GFP变体或酮生物素进行荧光标记。酶和固定化剪接体之间的相互作用将使用多波长SMF显微镜进行可视化。这些实验将通过明确测试每个DExD/H-box蛋白与剪接体的瞬时结合以及ATP水解和保守蛋白结构域在剪接体结合中的作用，为剪接体催化提供新的见解，这是用集成方法无法实现的。相关性：前mRNA转录物的剪接是基因表达的重要步骤，前mRNA剪接错误与许多癌症相关，包括婴儿脑肿瘤、乳腺癌和视网膜母细胞瘤。了解剪接体如何催化剪接对于理解健康和患病真核细胞中的基因表达至关重要。利用单分子荧光技术阐明DExD/H-box蛋白与剪接体的相互作用将为剪接的化学步骤提供新的见解，这是用系综测量无法实现的。

DESCRIPTION (provided by applicant): Most eukaryotic genes contain introns which must be removed from pre-mRNA by the spliceosome prior to translation. The spliceosome is a mega-Dalton macromolecular machine composed of both RNA and protein components. ATP is not utilized for chemical bond formation during either lariat synthesis or exon-exon ligation by the spliceosome. Nevertheless, ATP hydrolysis is required for structural rearrangements that are essential for product formation. These structural rearrangements result in defined, stable complexes that have been isolated and studied in vitro. It is not known how transitions between complexes occur, but one hypothesis is that essential DExD/H-box proteins (Prp2,16, 22, and 43) initiate structural reorganization of the spliceosome by using the energy of ATP hydrolysis to disrupt protein/RNA or RNA/RNA interactions. Given the ~3 MDa size of the spliceosome, traditional biochemical analysis of how these enzymes facilitate splicing is difficult. In this research proposal, single molecule fluorescence (SMF) will be used as a new method to study interactions between DExD/H-box proteins and the spliceosome. Spliceosomes will be assembled on a derivitized glass surface from Prp2-deficient yeast cell extract. Due to the absence of Prp2, the spliceosomes will assemble on a surface-immobilized, fluorescently-tagged pre-mRNA substrate but will become stalled prior to lariat formation. The Prp enzymes will be expressed and purified from E. coli using published procedures and fluorescently-labeled using either a genetically-encoded GFP variant or keto-biotin. Interactions between the enzymes and the immobilized spliceosomes will be visualized using multi-wavelength SMF microscopy. These experiments will provide new insight into spliceosome catalysis not attainable with ensemble methods by unambiguously testing the proposed transient association of each DExD/H-box protein with the spliceosome and the role of ATP hydrolysis and conserved protein domains in spliceosome binding. RELEVANCE:Splicing of pre-mRNA transcripts is an essential step in gene expression, and errors in pre- mRNA splicing have been correlated with a number of cancers including infant brain tumors, breast cancers, and retinoblastomas. Understanding how the spliceosome catalyzes splicing is critical for understanding gene expression in both healthy and diseased eukaryotic cells. Elucidation of DExD/H-box protein interactions with the spliceosome using single molecule fluorescence will provide new insight into the chemical steps of splicing not possible with ensemble measurements.