RUI: Normal Cellular Process During Heat Shock: Secretory Protein mRNA Stability

RUI：热休克期间的正常细胞过程：分泌蛋白 mRNA 稳定性

• 批准号: 9507328
• 负责人: Mark Brodl
• 金额: $ 18.5万
• 依托单位: Knox College
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-08-01 至 1998-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9507328&HistoricalAwards=false
• 关键词: RUI; Normal; Cellular; Process; During

MCB-9507318 Brodl To study the cell biology of higher plants' responses to environmental stress, a model system for investigation is the heat shock response of barley aleurone cells. Normally aleurone cells are dedicated to protein secretion; however, heat shock dramatically redirects their cellular activities. Heat shock abruptly arrests secretory protein synthesis, yet nonsecretory protein synthesis continues. This is accomplished by the selective destabilization of otherwise stable secretory protein mRNAs. Heat shock also causes the loss of the stacked cistemal ER lamellae upon which secretory protein mRNAs are translated; only short, single tubular ER remain. Ribosome density decreases by 50%. This may provide the discriminatory mechanism or selectively stopping the synthesis of secretory proteins; nonseeretory protein mRNAs are translated by "free" ribosomes. The principal distinction between secretory and nonsecretory protein mRNAs is a region encoding an amino terminal signal sequence that directs secretory protein mRNA translation to take place at the ER surface. This proposal investigates the mechanisms that operate during heat shock to selectively destabilize secretory protein mRNA. There are two central objectives in this work: 1) To determine whether the signal sequence is an important factor in selectively targeting secretory protein mRNA for destabilization. Using PCR the coding region for the signal sequence (ss) of the secretory protein a-amylase will be fused in-frame to the coding region of b-glucuronidase (GUS). Constructs will also be made that include either or both the 5' and 3' untranslated regions (UTRs) of a-amyase such that they flank GUS or ss-GUS. Transcription will be driven by the tetracycline- regulated Top l0 promoter. The constructs will be microprojectile bombarded into intact aleurone layers. The levels of the GUS reporter transcript will be monitored by GUS selected PCR amplification at set intervals following tetrac ycline-induced transcription arrest. 2) To understand something of the fate of secretory protein mRNAs upon heat shock. For this objective two types of experiments will be performed: a) The levels of secretory protein mRNAs in free and ER-bound polysomes before and at selected intervals during heat shock will be monitored in order to learn whether secretory protein mRNAs remain attached to the ER during heat shock. b) UV cross-linking experiments with radiolabeled a-amylase mRNA (including the UTRs) and cell lysates will be performed to investigate whether heat shock induces the binding of RNA binding proteins that may play a role in the destabilization of secretory protein mRNAs. %%% Plants respond to higher-than-normal, non-lethal temperatures by synthesizing heat shock proteins and by transiently shutting down the synthesis of other proteins necessary for normal growth and development. My laboratory works to understand how the shut down is controlled. Our model system for study is aleurone, an outer layer of cells surrounding the cereal grain whose function is to start the breakdown of starch reserves during germination. If the aleurone layer is briefly exposed to higher-than-normal temperature (heat shock), the synthesis of proteins involved this breakdown, including the starch-digesting enzyme, amylase, is shut down. The synthesis of other proteins, those not released (secreted) from the aleurone, continues unabated. Early experiments have determined the cause of this selective cessation of protein synthesis. Messenger RNAs (mRNAs) (transcripts of genetic information encoded by DNA) encoding the secreted proteins are destabilized, but nonsecretory protein mRNAs remain stable. To determine why, focus is brought to bear on another difference between mRNAs for secretory and non-secretory proteins. Secretory proteins frequently have additional amino acids tacked onto one end which are cleaved from the mature protein, but function to target the prote in to the secretory system and are encoded by their mRNA. This sequence of amino acids is called the "signal sequence". Does the signal sequence contribute to the instability of mRNAs encoding secreted proteins? To answer that question, genetic engineering will be used to fuse the signal sequence coded by amylase mRNA to the mRNA encoding an easily assayed (reporter) protein (b-glucuronidase) that is not normally secreted. The stability of the reporter protein mRNA with and without the signal sequence will be monitored during heat shock. If the hypothesis that the signal sequence confers sensitivity to heat is correct, then the proteins which cause destabilization by binding to the signal sequence-containing mRNA during heat shock will be identified. These experiments to deepen our understanding of the plant cellular and genetic response to environmental extremes are of importance to agriculture in both temperate and tropical climates. They are also important because they reveal part of the basic mechanism of cellular adaptation to changes in temperature. ***

为了研究高等植物对环境胁迫的反应的细胞生物学，研究大麦糊粉细胞的热激反应是一个模型系统。正常情况下，糊粉细胞致力于蛋白质的分泌；然而，热休克显著地改变了它们的细胞活动。热休克会突然阻止分泌性蛋白质合成，但非分泌性蛋白质合成仍在继续。这是通过选择性地使其他稳定的分泌蛋白mRNAs失稳来实现的。热休克还导致堆积的胞质内质网片层的丢失，分泌蛋白mRNAs在其上被翻译；只剩下短的、单管状的内质网。核糖体密度下降50%。这可能提供区别机制或选择性地阻止分泌蛋白的合成；非分泌性蛋白mRNAs是由“自由”核糖体翻译的。分泌型和非分泌型蛋白mRNAs之间的主要区别是编码氨基末端信号序列的区域，该序列指导分泌型蛋白mRNAs在内质网表面进行翻译。这项建议研究了在热休克过程中选择性破坏分泌蛋白mRNA稳定的机制。这项工作有两个中心目标：1)确定信号序列是否是选择性靶向分泌蛋白mRNA以使其失稳的重要因素。利用聚合酶链式反应将分泌性蛋白a-淀粉酶的信号序列的编码区(Ss)框内融合到b-葡萄糖苷酸酶的编码区(GUS)。还将构建包括a-淀粉酶的5‘和3’非翻译区(UTRs)之一或两者的构建体，使得它们位于GUS或ss-GU的两侧。转录将由四环素调控的Top l0启动子驱动。这些结构将被微弹轰击成完整的糊粉层。在四环素诱导转录停止后，GUS选择的PCR扩增将以设定的间隔监测GUS报告转录本的水平。2)了解分泌蛋白mRNAs在热休克时的命运。为此，将进行两种类型的实验：a)在热休克之前和在选定的时间间隔，将监测游离和内质网结合的多聚体中分泌蛋白mRNAs的水平，以了解在热休克期间分泌蛋白mRNAs是否仍然附着在内质网上。B)用放射性标记的α-淀粉酶mRNA(包括UTRs)和细胞裂解产物进行UV交联实验，以研究热休克是否诱导RNA结合蛋白的结合，这可能在分泌蛋白mRNAs的失稳中发挥作用。%植物对高于正常的非致死温度的反应是合成热休克蛋白，并暂时停止正常生长和发育所需的其他蛋白质的合成。我的实验室致力于了解关闭是如何控制的。我们研究的模型系统是糊粉，这是谷粒周围的一层外层细胞，其功能是在发芽期间启动淀粉储备的分解。如果糊粉层短暂地暴露在高于正常温度的环境中(热休克)，参与这种分解的蛋白质的合成，包括淀粉消化酶，淀粉酶，就会停止。没有从糊粉中释放(分泌)的其他蛋白质的合成继续有增无减。早期的实验已经确定了这种选择性停止蛋白质合成的原因。编码分泌蛋白的信使RNAs(MRNAs)(由DNA编码的遗传信息的转录本)不稳定，但非分泌蛋白mRNAs保持稳定。为了确定原因，重点放在分泌蛋白和非分泌蛋白的mRNAs之间的另一个差异上。分泌蛋白的一端通常有附加的氨基酸，这些氨基酸是从成熟蛋白上切割出来的，但它的功能是将蛋白定位到分泌系统中，并由它们的mRNA编码。这种氨基酸序列被称为“信号序列”。信号序列是否导致编码分泌蛋白的mRNAs的不稳定性？为了回答这个问题，基因工程将被用来将由淀粉酶mRNA编码的信号序列与编码一种容易检测的(报告)蛋白质(b-葡萄糖醛酸酶)的mRNA融合在一起，这种蛋白质通常不会被分泌。在热休克过程中，有或没有信号序列的报告蛋白mRNA的稳定性将被监测。如果信号序列赋予热敏感性的假设是正确的，那么在热休克过程中，通过与含有信号序列的mRNA结合而导致不稳定的蛋白质将被识别出来。这些实验加深了我们对植物对极端环境的细胞和遗传反应的理解，对温带和热带气候下的农业都很重要。它们也很重要，因为它们揭示了细胞适应温度变化的基本机制的一部分。***