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

DExD/H-box 蛋白：剪接体复合物的单分子荧光研究

• 批准号: 7222280
• 负责人: Aaron Andrew Hoskins
• 金额: $ 2.47万
• 依托单位: BRANDEIS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-02-01 至 2007-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7222280
• 关键词: ATP Hydrolysis; Binding; Biochemical; Biotin; Boxing; Brain Neoplasms; Catalysis; Cell Extracts; Chemicals; Complex; DNA Sequence Rearrangement; Deletion Mutagenesis; Enzymes; Escherichia coli; Eukaryotic Cell; Exons; Fluorescence; Fluorescence Microscopy; Gene Expression; Genes; Glass; Green Fluorescent Proteins; Heterogeneous Nuclear RNA; Imagery; Immobilization; Immobilized Enzymes; In Vitro; Infant; Introns; Label; Ligation; Malignant Neoplasms; Measurement; Methods; Mutagenesis; Numbers; Oligonucleotides; Play; Procedures; Proteins; Publishing; RNA; RNA Splicing; Research Proposals; Retinoblastoma; Role; Saccharomyces cerevisiae; Spliceosomes; Surface; Tertiary Protein Structure; Testing; Transcript; Translations; Variant; Yeasts; analog; chemical bond; dalton; insight; mRNA Precursor; malignant breast neoplasm; mutant; research study; single molecule; size; yeast protein

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.

描述（由申请人提供）：大多数真核基因含有内含子，在翻译前必须通过剪接体从前mRNA中去除内含子。剪接体是由RNA和蛋白质组成的巨分子机器。在套索合成或剪接体的外显子-外显子连接过程中，ATP不用于化学键的形成。然而，ATP水解是必需的结构重排，是必不可少的产品形成。这些结构重排导致已在体外分离和研究的确定的、稳定的复合物。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盒蛋白（Prp 2，16，22和43）通过使用ATP水解的能量破坏蛋白质/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~3 MDa大小，这些酶如何促进剪接的传统生物化学分析是困难的。在这项研究计划中，单分子荧光（SMF）将被用作一种新的方法来研究DExD/H-box蛋白和剪接体之间的相互作用。剪接体将在Prp 2缺陷型酵母细胞提取物衍生的玻璃表面上组装。由于Prp 2的缺乏，剪接体将在表面固定的荧光标记的前mRNA底物上组装，但在脂质体形成之前将变得停滞。Prp酶将从E.使用公开的方法将其转化至大肠杆菌中，并使用遗传编码的GFP变体或酮基生物素进行荧光标记。将使用多波长SMF显微镜观察酶和固定化剪接体之间的相互作用。这些实验将提供新的见解剪接体催化无法实现的合奏方法，通过明确测试拟议的瞬时协会的每个DExD/H盒蛋白与剪接体和作用的ATP水解和保守的蛋白质结构域的剪接体结合。相关性：前体mRNA转录物的剪接是基因表达的重要步骤，前体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了解健康和患病真核细胞中的基因表达至关重要。使用单分子荧光阐明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，而这是集合测量不可能实现的。