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荧光检测系统（AU-FDS）分析超离心的新技术，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提供实时分析的大小配合物使用各种波长来识别生物分子。它为复合体的实际大小提供了更高的精度，并避免了次级西部和北部分析固有的不精确性和时间密集性。重要的是，AU-FDS允许鉴定以前没有通过标准技术可视化的大分子复合物。最关键的是，当其他免疫沉淀研究表明蛋白质相互作用时，AU-FDS研究将这项研究带入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。在此之前，我们使用AU-FDS分析结合Flag标记的翻译成分（Flag-PAB1或RPL25A-Flag）的亲和纯化，从酵母酿酒酵母中鉴定出含有mRNA、闭环结构成分、eIF4E、eIF4G、PAB1和80S核糖体的77S单体翻译复合体。在这些研究的扩展中，我们使用了翻译终止因子Flag-eRF1的亲和纯化，鉴定了四个新的复合物，大小分别为21S， 28S， 38S和77S。21S/28S复合物（根据其形状约为0.5至1.4 MDa）至少含有eRF1， eIF4E, eIF4G1, PAB1， HRP1和mRNA。许多常见的应激颗粒蛋白和eIF2和eIF5起始因子不存在于21S/28S复合物中。我们的数据表明，21S/28S复合物与假定的闭环mRNP结构相似。这一模型与最近的研究一致，即HRP1在将新从细胞核导入的mRNA转化为可翻译形式的过程中起作用。或者，这些复合物可能是终止后的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21S/28S配合物的所有成分。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确定这些Flag- eRF1复合物在哪个步骤起作用。此外，我们将使用eIF4E、eIF4G和PAB1中许多已定义的突变来研究假定的闭环mRNPs的结构和形状。最后，AU-FDS将使用flag标记版本的PAB1、eIF4E和eRF1来确定与翻译抑制因子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.