RUI: Regulation of Secretory Protein mRNA Stability in Heat-Shocked Barley Aleurone Layers

这项研究的长期目标是了解高等植物对环境胁迫反应的细胞生物学。 研究的模式系统是大麦糊粉层的热激反应。 该组织的细胞通常致力于蛋白质分泌;然而，热休克显著地改变了它们的细胞活动。 当组织受到热休克时，分泌蛋白的合成突然停止，但非分泌蛋白的合成继续进行。 这是通过选择性地使编码分泌蛋白质并且在其他方面稳定的信使RNA（mRNA）不稳定来实现的。 热休克还引起细胞内膜区室组织的变化，细胞内膜区室是蛋白质进入分泌途径的位点。 这个隔室，内质网（ER），从堆叠的池状板层排列变成管状形态。 ER上的核糖体密度也减少了约一半。分泌性和非分泌性蛋白质mRNA之间的主要区别是编码氨基末端信号序列的区域，该信号序列指导分泌性蛋白质mRNA的翻译在ER表面发生。 信号识别颗粒（SRP）是一种蛋白质-核酸复合物，它与新生多肽的信号序列相互作用，并将翻译分泌蛋白的核糖体带到ER。 研究发现，热休克可抑制内质网核糖体对接后SRP的释放。“这个项目解决了在热休克过程中选择性地使分泌蛋白mRNA不稳定的机制。 时程实验将研究ER结合或游离池是否发生分泌蛋白mRNA的降解。 已经观察到特定蛋白质与分泌蛋白质mRNA的结合，并且由于热休克而可能增加。 由于这种蛋白质结合在调节mRNA稳定性方面可能是重要的，因此将进行实验以确定RNA中的结合序列。 然后将这些候选序列从分泌蛋白mRNA中删除或添加到正常稳定的转录物中，以确定它们是否赋予热休克不稳定性。 最后，将克隆大麦糊粉层内质网膜上的SRP受体或对接蛋白的基因。 这些克隆将用于生产抗体，以监测热休克对这些蛋白质表达和分布的影响。 植物对环境胁迫的适应性反应对其生存至关重要。 更全面地了解压力对正常细胞生物学的影响以及压力适应的细胞机制对于旨在提高植物生产力和效用的农业努力是重要的。 目前对热休克的研究主要集中在热休克特异蛋白的诱导及其功能上。 这项工作的重点是热休克对正常细胞过程的影响，特别是对正常细胞蛋白质表达的抑制。 了解植物调节基因表达的转录后机制是充分实现基因工程潜力的基础。 该项目对于本科生的参与也具有重要意义。 诺克斯学院在培养学生攻读科学研究生方面有着良好的记录。 此外，该项目还包括一个新颖的教学博士后职位，将更传统的研究博士后经验与本科教学经验相结合。 这种研究与教学相结合的培训是为博士学位的准备量身定制的经验。在小型文理学院谋求教职的科学家。

The long-term goal of this research is to understand the cell biology of higher plants' responses to environmental stress. The model system for investigation is the heat shock response of the aleurone layer of barley grains. The cells of this tissue are normally dedicated to protein secretion; however, heat shock dramatically redirects their cellular activities. The synthesis of secretory proteins is abruptly arrested when the tissue is subjected to heat shock, yet the synthesis of nonsecretory proteins continues. This is accomplished by the selective destabilization of messenger RNAs (mRNAs) that code for the secretory proteins and that are otherwise stable. Heat shock also causes a change in the organization of the intracellular membrane compartment that is the site of entry of proteins into the secretory pathway. This compartment, the endoplasmic reticulum (ER), changes from a stacked cisternal lamellar arrangement to a tubular morphology. Ribosome density on the ER also decreases by about half. The principal distinction between secretory and nonsecretory protein mRNAs is a region encoding an amino terminal signal sequence that directs the translation of secretory protein mRNAs to take place at the ER surface. The signal recognition particle (SRP) is a protein-nucleic acid complex that interacts with the signal sequence of nascent polypeptides and brings the ribosomes that are translating the secretory proteins to the ER. It has been found that heat shock inhibits the release of SRP from the ER following ribosome "docking." This project addresses the question of what mechanisms operate during heat shock to selectively destabilize secretory protein mRNA. Time course experiments will investigate whether degradation of secretory protein mRNA occurs from the ER-bound or free pools. Binding of specific proteins to secretory protein mRNAs has been observed and may be increased as a result of heat shock. Since this protein binding may be of importance in regulating mRNA stability, experiments will be performed to define the binding sequences in the RNA. These candidate sequences will then be either deleted from secretory protein mRNAs or added to normally stable transcripts to determine if they confer heat shock instability. Finally, the genes for the SRP receptor or docking proteins in the ER membrane of barley aleurone will be cloned. The clones will be used to produce antibodies to monitor the effect of heat shock on the expression and distribution of these proteins. A plant's adaptive responses to environmental stress are crucial to its survival. A fuller understanding of the impact of stress on normal cellular biology and of cellular mechanisms for stress adaptation are important to agricultural efforts aimed at enhancing plant productivity and utility. Most studies on heat shock focus on the induction and function of heat shock-specific proteins. This work focuses instead on the impact of heat shock on normal cellular processes, most specifically the suppression of normal cellular protein expression. Understanding the post-transcriptional mechanisms by which plants regulate gene expression is fundamental to fully realizing the potential that genetic engineering offers. This project is also significant for its involvement of undergraduates. Knox College has a strong track record for preparing students for graduate study in the sciences. In addition, this project includes a novel teaching postdoctoral position, combining a more traditional research postdoctoral experience with undergraduate teaching experience. This training in the integration of research and teaching is an experience tailored for the preparation of Ph.D. scientists seeking faculty positions at small liberal arts colleges.