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的反应的细胞生物学。研究的模式系统是大麦籽粒糊粉层的热激响应。这种组织的细胞通常致力于蛋白质的分泌；然而，热休克会戏剧性地改变它们的细胞活动。当组织受到热休克时，分泌性蛋白质的合成突然停止，但非分泌性蛋白质的合成仍在继续。这是通过信使RNA(MRNAs)的选择性失稳实现的，信使RNA编码分泌蛋白，其他方面是稳定的。热休克还会导致细胞内膜隔室的组织发生变化，这是蛋白质进入分泌途径的部位。内质网(ER)从堆叠的脑池板层排列变为管状形态。内质网上的核糖体密度也减少了大约一半。分泌和非分泌蛋白mRNAs之间的主要区别是编码氨基末端信号序列的区域，该序列指导分泌蛋白mRNAs的翻译发生在内质网表面。信号识别颗粒(SRP)是一种蛋白质-核酸复合体，它与新生多肽的信号序列相互作用，将翻译分泌蛋白质的核糖体带到内质网。研究发现，热休克抑制核糖体“对接”后内质网SRP的释放。这个项目解决的问题是，在热休克过程中，什么机制可以选择性地破坏分泌蛋白mRNA的稳定。时间进程实验将调查内质网结合池或游离池是否发生分泌蛋白mRNA的降解。已观察到特定蛋白与分泌蛋白mRNAs的结合，并可能因热休克而增加。由于这种蛋白质结合在调节mRNA稳定性方面可能是重要的，因此将进行实验来定义RNA中的结合序列。然后，这些候选序列将从分泌蛋白mRNAs中删除，或添加到通常稳定的转录本中，以确定它们是否具有热休克不稳定性。最后，大麦糊粉质膜中SRP受体或对接蛋白的基因将被克隆。这些克隆将被用来产生抗体，以监测热休克对这些蛋白质的表达和分布的影响。植物对环境胁迫的适应性反应对其生存至关重要。更充分地了解胁迫对正常细胞生物学的影响和适应胁迫的细胞机制，对于旨在提高植物生产力和实用性的农业努力非常重要。关于热休克的研究大多集中在热休克特异蛋白的诱导和功能上。相反，这项工作侧重于热休克对正常细胞过程的影响，尤其是对正常细胞蛋白表达的抑制。了解植物调控基因表达的转录后机制是充分发挥基因工程潜力的基础。这个项目也很重要，因为它涉及到本科生。诺克斯学院在为学生的科学研究生学习做准备方面有着良好的记录。此外，该项目还包括一个新的博士后教学职位，将更传统的博士后研究经验与本科教学经验相结合。这种研究和教学相结合的培训是为准备在小型文理学院寻找教职的博士科学家量身定做的一种经历。

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.