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

描述(申请人提供)：大多数真核基因包含内含子，在翻译前必须由剪接体将其从前-mRNA中移除。剪接体是一个由RNA和蛋白质组成的百万道尔顿大分子机器。在套索合成或剪接体的外显子-外显子连接过程中，ATP不被用于化学键的形成。然而，对产物形成至关重要的结构重排需要三磷酸腺苷的水解。这些结构重排导致确定的，稳定的络合物，已被分离和研究在体外。目前尚不清楚复合体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相互作用来启动剪接体的结构重组。考虑到剪接体的~3MDA大小，传统的生化分析这些酶是如何促进剪接的是困难的。在本研究方案中，单分子荧光(SMF)将作为一种新的方法来研究DExD/H-box蛋白与剪接体的相互作用。剪接体将由Prp2缺乏的酵母细胞提取物组装在衍生的玻璃表面上。由于缺少Prp2，剪接体将在表面固定的、荧光标记的前-mRNA底物上组装，但在套索形成之前将停滞不前。PRP酶将使用已公布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。酶和固定的剪接体之间的相互作用将用多波长SMF显微镜可视化。这些实验将为剪接体催化提供新的见解，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水解和保守的蛋白质结构域在剪接体结合中的作用，为系综法无法获得的剪接体催化提供新的见解。相关性：前信使核糖核酸转录本的剪接是基因表达的重要步骤，前信使核糖核酸剪接错误与许多癌症有关，包括婴儿脑瘤、乳腺癌和视网膜母细胞瘤。了解剪接体是如何催化剪接的，对于了解健康和疾病真核细胞中的基因表达至关重要。用单分子荧光法阐明DExD/H-box蛋白与剪接体的相互作用将为我们提供新的视角，了解剪接的化学步骤，这是整体测量不可能实现的。

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.