Characterization of the mRNP closed-loop structure

mRNP 闭环结构的表征

• 批准号: 9333556
• 负责人: CLYDE L DENIS
• 金额: $ 2.17万
• 依托单位: UNIVERSITY OF NEW HAMPSHIRE
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-06-15 至 2017-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9333556
• 关键词: Affinity Chromatography; Animal Model; Binding; Binding Proteins; Biochemical; Biological; Biological Sciences; Cell Nucleus; Charge; Complex; Coupled; Cytoplasmic Granules; Data; Defect; Detection; Disease; Eukaryota; Event; Fluorescence; Genetic Transcription; Glucose; Goals; Grant; Health; Immunoprecipitation; Individual; Initiator Codon; Investigation; Light; Link; Macromolecular Complexes; Mass Chromatography; Mass Spectrum Analysis; Messenger RNA; Methionine; Modeling; Mutation; Nature; Nuclear; Peptide Initiation Factors; Play; Poly(A) Tail; Poly(A)-Binding Protein I; Process; Production; Protein Biosynthesis; Proteins; Public Health; RNA; RNA Cap-Binding Proteins; Recycling; Regulation; Research; Resort; Ribosomes; Role; Saccharomyces cerevisiae; Scanning; Shapes; Stress; Structural Genes; Structure; Study models; Sucrose; Suggestion; System; Systems Analysis; Techniques; Terminator Codon; Time; Training; Transfer RNA; Translation Initiation; Translational Repression; Translations; Yeasts; analytical ultracentrifugation; biological systems; graduate student; mRNA Export; macromolecular assembly; messenger ribonucleoprotein; novel; protein complex; protein expression; termination factor; tool; undergraduate student

DESCRIPTION (provided by applicant): The goal of these studies is to use the novel technique of analytical ultracentrifugation with fluorescence detection system (AU-FDS) to analyze translation complexes by detecting specific GFP- tagged proteins and mRNA. AU provides real-time analysis of the size of complexes using various wavelengths to identify biological molecules. It provides enhanced precision as to the actual size of complexes and avoids the inexactitude and the time-intensive nature inherent in secondary Western and Northern analysis. Importantly, AU-FDS allows the identification of macromolecular complexes that have not previously been visualized by standard techniques. Most critically, while other immunoprecipitation studies indicate that proteins interact, AU-FDS studies take this research to the next logical step: identifying the number, size, and composition of these protein complexes. Previously, we have used AU-FDS analysis coupled with an affinity purification of a Flag- tagged translational component (Flag-PAB1 or RPL25A-Flag) to identify from the yeast S. cerevisiae a 77S monosomal translation complex that contained mRNA, the closed-loop structural components, eIF4E, eIF4G, PAB1, and the 80S ribosome. In expansion of these studies we have used affinity purification of translation termination factor, Flag-eRF1, to identif four novel complexes, 21S, 28S, 38S, and 77S in size. The 21S/28S complexes (about 0.5 to 1.4 MDa depending on their shape) contained at least eRF1, eIF4E, eIF4G1, PAB1, HRP1, and mRNA. Many common stress granule proteins and eIF2 and eIF5 initiation factors were not present in the 21S/28S complexes. Our data suggest that the 21S/28S complexes are similar to the putative closed-loop mRNP structure. This model is consonant with the recent suggestion that HRP1 plays a role in transitioning mRNA, newly imported from the nucleus, into a translatable form. Alternatively, these complexes could be post- termination mRNP structures. In this grant we shall use the complementary techniques of AU-FDS and mass spectrometry to identify all of the components of the 21S/28S complexes. These studies will inform as to which translation initiation components, translation termination factors, and/or stress granule proteins are present in the complexes. Specific mutations in transcription, mRNA export, translation initiation, termination, and P body/stress granule formation will be used to identify at which step these Flag- eRF1 complexes are functioning. In addition, we will use a number of defined mutations in eIF4E, eIF4G, and PAB1 to investigate the structure and shape of the putative closed-loop mRNPs. Finally, the exact translational complexes bound by translational repressors, SBP1, SCD6, and NPL3, will be determined by AU-FDS using Flag-tagged versions of PAB1, eIF4E, and eRF1.

描述（由申请人提供）：这些研究的目的是通过荧光检测系统（AU-FDS）使用新的分析超速离心技术来通过检测特定的GFP标记的蛋白质和mRNA来分析翻译复合物。 AU使用各种波长来鉴定生物分子，对复合物的大小进行实时分析。它为复合物的实际大小提供了增强的精度，并避免了中学西方和北方分析中固有的不符合性和耗时的性质。重要的是，AU-FDS允许鉴定大分子复合物，这些复合物以前尚未通过标准技术可视化。最关键的是，尽管其他免疫沉淀研究表明蛋白质相互作用，但Au-FDS研究将这项研究取得了下一个逻辑步骤：确定这些蛋白质复合物的数量，大小和组成。 以前，我们已经使用了AU-FDS分析，并与标记标记的翻译成分（FLAG-PAB1或RPL25A-FLAG）的亲和力纯化从酵母菌S. cerevisiae a 77S单子体翻译复合体中识别，其中包含mRNA，其中包含mRNA，封闭式组件，eif4e，eif4e，eif4g，pab1，pab1和80s and the 80s和80s，andys ob1和80s and the nocer-loop结构组件。在这些研究的扩展中，我们使用了翻译终止因子Flag-erf1的亲和力纯化来识别四个新型复合物，21s，28s，38s和77s的大小。 21s/28s复合物（约0.5至1.4 MDA取决于其形状）至少包含ERF1，EIF4E，EIF4G1，PAB1，HRP1和mRNA。在21s/28s复合物中，不存在许多常见的应激颗粒蛋白和EIF2和EIF5的起始因子。我们的数据表明，21S/28S复合物与推定的闭环MRNP结构相似。该模型与最近的建议相吻合，即HRP1在转变从核中新进口的mRNA起作用，从核中导入到可翻译形式。或者，这些复合物可以是终止MRNP结构的。 在这笔赠款中，我们将使用AU-FDS和质谱的互补技术来识别21S/28S复合体的所有组件。这些研究将告知哪种翻译起始成分，翻译终止因子和/或应力颗粒蛋白存在于复合物中。转录，mRNA输出，翻译起始，终止和P身体/应力颗粒的形成中的特定突变将用于确定这些标志ERF1复合物在哪个步骤中起作用。此外，我们将在EIF4E，EIF4G和PAB1中使用许多定义的突变来研究推定的闭环mRNP的结构和形状。最后，使用PAB1，EIF4E和ERF1的FLAG标签版本确定，由翻译阻遏物SBP1，SCD6和NPL3绑定的精确翻译复合物将由AU-FDS确定。