Role of RNS2 in Ribosome Recycling via Ribophagy

智力上的优点。这个项目的总体目标是确定(1)核糖体RNA衰退与植物自噬分子循环途径之间的联系，以及(2)核糖核酸酶RNS2在这些过程中的作用。核糖体是所有细胞的重要组成部分，是蛋白质合成的机器，以特定的速率被分解和替换。然而，正常核糖体衰退的途径，特别是核糖体RNA(RRNA)衰退的途径还没有被描述。在酵母中的研究发现，在氮饥饿下生长的细胞会诱导一种特殊类型的自噬，称为核噬，这种自噬以核糖体为目标，降解成液泡。这一过程是否发生在植物中并参与正常的rRNA衰变尚不清楚。该项目的初步实验发现，在拟南芥的正常生长条件下，RNS2是rRNA降解所必需的。RNS2是结构性表达的，缺乏RNS2活性的突变体在液泡中积累RNA，rRNA半衰期较长。这些突变体还表现出结构性自噬，这表明在非应激条件下，核糖体降解可能涉及与核吞噬相关的机制。为了实现这个项目的目标，将使用体内标记和显微镜方法分析野生型和rns2突变植物中核糖体RNA和蛋白质的降解途径。该项目将通过在rns2和自噬相关突变体之间产生双重突变体，并通过使用空泡降解抑制剂来确定自噬在核糖体循环中的参与程度。进一步的实验将确定与核糖体衰退有关的细胞室，并将表征在rns2突变植物中明显的分子变化。这项研究的完成将揭示rRNA衰变所需的基本细胞装置，将提供关于植物如何调节这一过程的新知识，并将揭示所涉及的保守酶的生物学作用，这一作用迄今仍不清楚。该项目将为本科生、研究生和博士后实习生提供参与这项研究的所有跨学科领域的机会。除了艾奥瓦州立大学的学生外，还将设立一个实习项目，为大观大学生物学项目的本科生提供研究机会。大观大学是一所四年制本科院校，缺乏研究项目。这项实习计划将包括为期9周的暑期实习，每年夏天将有3名学生接受分子生物学技术的基本培训，并将参与从实验设计到数据解释的科学过程。学生们还将参加由爱荷华州立大学和大观大学教职员工主持的研讨会，在研讨会上，他们将就当前涉及整个社会的科学问题进行正式讨论。这位合作伙伴正在参与一款针对高中生和本科生的视频游戏的开发，游戏中玩家在植物内的三维细胞环境中导航，并完成基于细胞功能和生化反应的任务。来自该项目的研究将作为一种策略被纳入游戏中，以演示细胞组件回收的概念。除了开发对广泛受众有价值的教育工具外，该项目还将为参与其开发的高中生和本科生提供在一个高度跨学科的研究项目中的丰富经验，该项目要求整合研究、编程设计和教育培训。

Intellectual merit. The overall objective of this project is to determine (1) the connection between ribosomal RNA decay and the molecular recycling pathway of autophagy in plants, and (2) the role of the ribonuclease RNS2 in these processes. Ribosomes are essential components of all cells and act as protein synthesis machines that are broken down and replaced at specific rates. The pathways of normal ribosome decay, however, especially that of ribosomal RNA (rRNA) decay, have not been described. Studies in yeast found that cells growing under nitrogen starvation induce a specialized type of autophagy, called ribophagy, which targets ribosomes for degradation to a vacuole for this purpose. Whether this process occurs in plants and is involved in normal rRNA decay are unknown. Preliminary experiments for this project found that RNS2 is required for rRNA degradation under normal growth conditions in Arabidopsis. RNS2 is constitutively expressed, and mutants lacking RNS2 activity accumulate RNA in the vacuole with longer rRNA half-life. These mutants also exhibit constitutive autophagy, which suggests the possible involvement of a ribophagy-related mechanism in ribosome degradation under non-stress conditions. To accomplish the objective of this project, pathways for degradation of ribosomal RNA and proteins will be analyzed using in vivo labeling and microscopy approaches in wild type and rns2 mutant plants. The project will determine the extent to which autophagy is involved in ribosome recycling through generation of double mutants between rns2 and autophagy-related mutants and through use of inhibitors of vacuolar degradation. Further experiments will identify the cellular compartments involved in ribosome decay and will characterize the molecular changes evident in rns2 mutant plants. Completion of this research will reveal the basic cellular apparatus needed for rRNA decay, will provide new knowledge on how plants regulate this process, and will reveal the biological role of the conserved enzymes involved, which so far has remained elusive.Broader impacts. This project will provide opportunities for undergraduate students, graduate students and postdoctoral trainees to participate in all interdisciplinary areas of this research. In addition to Iowa State University students, an internship program will be established to provide research opportunities for undergraduate students in the biology program at Grand View University, a four-year undergraduate institution that lacks research programs. This internship program will consist of a 9-week summer internship in which 3 students each summer will receive basic training in molecular biology techniques and will participate in the scientific process, from experiment design to data interpretation. Students will also participate in a workshop led by Iowa State and Grand View faculty in which they will engage in formal discussions about current scientific issues that concern society as a whole. The co-PI is participating in the development of a video game, aimed at high school and beginning undergraduate students, in which the player navigates a three-dimensional cellular environment within a plant and completes tasks based on cell functions and biochemical reactions. The research from this project will be incorporated into the game play as a strategy to demonstrate concepts of recycling of cellular components. As well as developing an educational tool that will be valuable for a wide range of audiences, this project also will provide the high school and undergraduate students involved in its development with extensive experience in a highly interdisciplinary research project that requiries the integration of research, programming design, and educational training.