RNA helicase regulation of RNA metabolism and gene expression

RNA 解旋酶对 R​​NA 代谢和基因表达的调节

• 批准号: RGPIN-2016-05448
• 负责人: Owttrim, George
• 金额: $ 2.4万
• 依托单位: University of Alberta
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2020
• 资助国家: 加拿大
• 起止时间: 2020-01-01 至 2021-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=709686
• 关键词: RNA; helicase; regulation; metabolism; gene

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.

Owttrim总结 所有的生物都必须能够感知和应对环境的变化。两种最常见的压力是光和温度的变化，这两个因素对光合生物的生存至关重要。为了应对这些压力，细胞必须改变蛋白质丰度才能生存。蛋白质是由RNA在一个过程中产生的，该过程需要RNA折叠成正确的3D结构。RNA结合蛋白，称为RNA解旋酶，在所有生物体中促进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，生物燃料和医学活性化合物的潜在重要来源。蓝细菌含有一个单一的RNA解旋酶，其丰度在20 ℃时升高，在30 ℃时降低。我们已经创造了缺乏功能性RNA解旋酶的突变细胞。这些细胞不能在20 ℃下生长，并且异常表达通常仅在30 ℃下表达的蛋白质。这些结果表明，解旋酶是一个冷应激基因，意想不到的功能，在细胞响应热应激。我们可以通过将功能性RNA解旋酶返回突变细胞来拯救冷应激细胞。这使我们能够通过去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功能所必需的。该项目的另一个方面涉及分析在30 ℃下保持RNA解旋酶丰度较低的机制。我们的研究表明，解旋酶在30 ℃下特异性降解。我们正在使用我们的突变体解旋酶蛋白来确定这种降解所需的氨基酸序列，并确定引起降解的蛋白质。最后，我们希望鉴定与解旋酶相互作用的RNA和蛋白质。为了做到这一点，我们已经添加了一个特定的氨基酸序列的解旋酶，一个蛋白质标签，将允许我们专门纯化标记的解旋酶从蓝藻细胞。然后，我们可以通过对与标记的解旋酶共纯化的RNA和蛋白质进行测序来识别相互作用的伴侣。总之，这些数据将使我们能够制定解旋酶如何发挥作用的细胞内分子图像，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节的表达平台，产生蛋白质表达受到特异性调节的蓝细菌，将材料转移到所需商业产品的生产中，而不是细胞生长。总的来说，该项目将帮助我们了解RNA解旋酶蛋白如何在细胞内表达和发挥作用，从而深入了解我们如何操纵RNA折叠以更有效地生产蓝藻中的有益产物。