Characterization of the mRNP closed-loop structure

描述（由申请人提供）：这些研究的目标是使用带有荧光检测系统（AU-FDS）的分析超速离心新技术，通过检测特定的 GFP 标记蛋白和 mRNA 来分析翻译复合物。 AU 使用各种波长对复合物的大小进行实时分析，以识别生物分子。它提高了复合体实际大小的精确度，并避免了西方和北方二次分析中固有的不精确性和时间密集性。重要的是，AU-FDS 可以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逻辑步骤：识别这些蛋白质复合物的数量、大小和组成。此前，我们使用 AU-FDS 分析结合 Flag 标记的翻译组件（Flag-PAB1 或 RPL25A-Flag）的亲和纯化，从酿酒酵母中鉴定出包含 mRNA、闭环结构组件、eIF4E、eIF4G、PAB1 和 80S 核糖体的 77S 单体翻译复合物。在这些研究的扩展中，我们使用翻译终止因子 Flag-eRF1 的亲和纯化来鉴定四种新型复合物，大小为 21S、28S、38S 和 77S。 21S/28S复合物（约0.5至1.4MDa，取决于其形状）至少包含eRF1、eIF4E、eIF4G1、PAB1、HRP1和mRNA。 21S/28S 复合物中不存在许多常见的应激颗粒蛋白以及 eIF2 和 eIF5 起始因子。我们的数据表明 21S/28S 复合物与假定的闭环 mRNP 结构相似。该模型与最近的建议一致，即 HRP1 在将新从细胞核导入的 mRNA 转变为可翻译形式方面发挥着作用。或者，这些复合物可以是终止后mRNP结构。在这笔资助中，我们将使用 AU-FDS 和质谱的互补技术来鉴定 21S/28S 复合物的所有成分。这些研究将揭示复合物中存在哪些翻译起始成分、翻译终止因子和/或应激颗粒蛋白。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识别这些 Flag-eRF1 复合物在哪一步发挥作用。此外，我们将使用 eIF4E、eIF4G 和 PAB1 中的一些确定的突变来研究假定的闭环 mRNP 的结构和形状。最后，AU-FDS 将使用 PAB1、eIF4E 和 eRF1 的 Flag 标记版本来确定由翻译抑制因子 SBP1、SCD6 和 NPL3 结合的确切翻译复合物。

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.