RNA helicase regulation of RNA metabolism and gene expression

Owttrim概要*所有有机体都必须能够感知并对环境的变化做出反应。最常见的两种压力是光和温度的变化，这两个因素对光合作用有机体的生存至关重要。为了应对这些压力，细胞必须改变蛋白质的丰度才能生存。蛋白质是从RNA中产生的，这一过程需要将RNA折叠成正确的三维结构。RNA结合蛋白，称为RNA解旋酶，促进所有生物体中的RNA折叠。我们研究的目的是确定生长环境的变化如何调节RNA解旋酶的积累，以及解旋酶如何与RNA相互作用。为了实现这一目标，我们正在使用光合作用模型系统--蓝藻，这是生物燃料和具有医疗活性的化合物的潜在重要来源。蓝藻含有单一的RNA解旋酶，其丰度在20oC时较高，在30oC时较低。我们创造了缺乏功能性RNA解旋酶的突变细胞。这些细胞不能在20摄氏度下生长，并且有正常情况下只在30摄氏度下表达的蛋白质的异常表达。这些结果表明，解旋酶是一种冷应激基因，它意外地在细胞对热应激的反应中发挥作用。我们可以通过向突变细胞返回一个功能性的RNA解旋酶来拯救冷应激细胞。这使我们能够通过移除或改变关键的氨基酸序列来测试RNA解旋酶的哪些区域是发挥功能所必需的。该项目的另一个方面涉及分析将RNA解旋酶丰度保持在30摄氏度以下的机制。我们的研究表明，解旋酶在30℃时被特异性降解。我们正在使用我们的突变解旋酶蛋白来确定这种降解所需的氨基酸序列，并识别导致这种降解的蛋白质。最后，我们希望确定解旋酶与之相互作用的RNA和蛋白质。为了做到这一点，我们在解旋酶中添加了一个特定的氨基酸序列，这是一个蛋白质标签，将允许我们从蓝藻细胞中特异性地纯化标记的解旋酶。然后，我们可以通过对与标记的解旋酶共同纯化的RNA和蛋白质进行测序来确定相互作用的伙伴。综上所述，这些数据将使我们能够制定出解旋酶如何发挥作用的细胞内分子图，并调节蓝藻对温度波动的反应。了解这些过程可能使我们能够设计温度调控的表达平台，产生蛋白质表达受到特定调控的蓝藻，将材料转移到生产所需的商业产品，而不是细胞生长。总体而言，该项目将帮助我们了解RNA解旋酶蛋白在细胞内的表达和功能，使我们能够深入了解如何操纵RNA折叠，以在蓝藻中更有效地产生有益产品。**

Owttrim SUMMARY***All organisms must be able to sense and respond to changes in their environment. Two of the most commonly experienced stresses are changes in light and temperature, factors that are crucial for the survival of photosynthetic organisms. In response to these stresses, cells must alter protein abundance in order to survive. Proteins are produced from RNA in a process that requires the RNA to be folded into the correct 3-D structure. RNA binding proteins, termed RNA helicases, facilitate RNA folding in all organisms. The purpose of our research is to determine how changes in the growth environment regulate RNA helicase accumulation and how helicases interact with RNA. To accomplish this, we are using the photosynthetic model system, cyanobacteria, a potentially important source of biofuels and medically active compounds. Cyanobacteria contain a single RNA helicase whose abundance is elevated at 20oC and low at 30oC. We have created mutant cells that lack a functional RNA helicase. These cells do not grow at 20oC and have aberrant expression of proteins that are normally only expressed at 30oC. These results indicate that the helicase is a cold stress gene that unexpectedly functions in cellular response to heat stress. We can rescue the cold stressed cells by returning a functional RNA helicase to the mutant cells. This allows us to test which areas of the RNA helicase are required for function by removing or altering crucial amino acid sequences. Another aspect of the project involves analysis of the mechanism that keeps RNA helicase abundance low at 30oC. Our research indicates the helicase is specifically degraded at 30oC. We are using our mutant helicase proteins to determine which amino acid sequences are required for this degradation and also identify the proteins causing the degradation. Finally, we wish to identify the RNAs and proteins with which the helicase interacts. To do this, we have added a specific amino acid sequence to the helicase, a protein-tag that will allow us to specifically purify the tagged helicase from cyanobacterial cells. We can then identify interacting partners by sequencing the RNAs and proteins that co-purify with the tagged helicase. Together, the data will allow us to formulate an intracellular molecular picture of how the helicase functions and also regulates cyanobacterial response to temperature fluctuation. Understanding these processes may allow us to design temperature-regulated expression platforms, generating cyanobacteria in which protein expression is specifically regulated, diverting material to production of the desired commercial product and not cell growth. Overall, the project will help us to understand how the RNA helicase protein is expressed and functions within cells, allowing insights into how we can manipulate RNA folding to generate more efficient production of beneficial products in cyanobacteria.**