Characterization of the mRNP closed-loop structure

mRNP 闭环结构的表征

• 批准号: 8722662
• 负责人: CLYDE L DENIS
• 金额: $ 0.33万
• 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/8722662
• 关键词: Affinity Chromatography; Animal Model; Binding; Biochemical; Biological; Biological Sciences; Cell Nucleus; Charge; Complex; Coupled; Cytoplasmic Granules; Data; Defect; Detection; Disease; Eukaryota; Event; Fluorescence; Genetic Transcription; Glucose; Goals; Grant; 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 Binding; Protein Biosynthesis; Proteins; Public Health; RNA; RNA Cap-Binding Proteins; Recycling; Regulation; Research; Resort; Ribosomes; Role; Saccharomyces cerevisiae; Scanning; Shapes; Stress; Structural Protein; 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）：这些研究的目的是使用具有荧光检测系统（AU-FDS）的分析超离心新技术，通过检测特定GFP标记的蛋白质和mRNA来分析翻译复合物。Au提供使用各种波长的复合物的大小的实时分析，以识别生物分子。它提供了关于复合体实际大小的更高的精确度，避免了西方和北方二级分析中固有的不精确性和时间密集性。重要的是，AU-FDS允许识别以前未通过标准技术可视化的大分子复合物。最重要的是，虽然其他免疫沉淀研究表明蛋白质相互作用，但AU-FDS研究将这项研究带到了下一个合乎逻辑的步骤：确定这些蛋白质复合物的数量，大小和组成。先前，我们已经使用AU-FDS分析结合Flag标记的翻译组分（Flag-PAB 1或RPL 25 A-Flag）的亲和纯化来从酵母中鉴定S。酿酒酵母是一个77 S单体翻译复合体，含有mRNA、闭环结构组分、eIF 4 E、eIF 4G、PAB 1和80 S核糖体。在这些研究的扩展中，我们使用翻译终止因子Flag-eRF 1的亲和纯化来鉴定四种新的复合物，大小为21 S、28 S、38 S和77 S。21 S/28 S复合物（约0.5至1.4 MDa，取决于其形状）至少含有eRF 1、eIF 4 E、eIF 4G 1、PAB 1、HRP 1和mRNA。许多常见的应激颗粒蛋白和eIF 2和eIF 5起始因子不存在于21 S/28 S复合物中。我们的数据表明，21 S/28 S复合物类似于假定的闭环mRNP结构。这一模型与最近提出的HRP 1在将新从细胞核输入的mRNA转变为可翻译形式中起作用的观点一致。或者，这些复合物可以是终止后mRNP结构。在这项资助中，我们将使用AU-FDS和质谱的互补技术来鉴定21 S/28 S复合物的所有组分。这些研究将告知哪些翻译起始组分、翻译终止因子和/或应激颗粒蛋白存在于复合物中。转录、mRNA输出、翻译起始、终止和P体/应激颗粒形成中的特定突变将用于鉴定这些Flag-eRF 1复合物在哪个步骤起作用。此外，我们还将使用eIF 4 E、eIF 4G和PAB 1中的一些确定的突变来研究推定的闭环mRNP的结构和形状。最后，将使用PAB 1、eIF 4 E和eRF 1的Flag标记版本，通过AU-FDS确定与翻译阻遏物SBP 1、SCD 6和NPL 3结合的确切翻译复合物。