Role of RNS2 in Ribosome Recycling via Ribophagy

知识价值。本项目的总体目标是确定(1)植物核糖体RNA衰变与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基本组成部分，作为蛋白质合成机器，以特定的速率分解和替换。然而，正常核糖体衰变的途径，特别是核糖体RNA （rRNA）的衰变，尚未被描述。对酵母的研究发现，在氮饥饿下生长的细胞会诱导一种特殊类型的自噬，称为核糖体自噬，它以核糖体为目标，将其降解为液泡。这个过程是否发生在植物中，是否参与正常的rRNA衰变尚不清楚。本课题前期实验发现，在拟南芥正常生长条件下，RNS2是rRNA降解所必需的。RNS2是组成性表达的，缺乏RNS2活性的突变体在液泡中积累RNA， rRNA半衰期更长。这些突变体也表现出组成性自噬，这表明在非应激条件下核糖体降解可能涉及与核糖体自噬相关的机制。为了完成这个项目的目标，我们将使用活体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突变体以及通过使用液泡降解抑制剂来确定自噬参与核糖体循环的程度。进一步的实验将确定参与核糖体衰变的细胞区室，并将表征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，将为植物如何调节这一过程提供新的知识，并将揭示迄今为止仍然难以捉摸的所涉及的保守酶的生物学作用。更广泛的影响。本项目将为本科生、研究生和博士后学员提供参与本研究所有跨学科领域的机会。除了爱荷华州立大学的学生外，还将设立实习项目，为大观大学生物专业的本科生提供研究机会。大观大学是一所缺乏研究项目的四年制本科院校。该实习计划将包括为期9周的暑期实习，每年暑期将有3名学生接受分子生物学技术的基础培训，并参与从实验设计到数据解释的科学过程。学生们还将参加一个由爱荷华州立大学和大观大学教师领导的研讨会，在这个研讨会上，他们将对当前与整个社会有关的科学问题进行正式讨论。这名联合研究人员正在参与一款电子游戏的开发，该游戏的目标是高中生和本科生，在游戏中，玩家在植物内部的三维细胞环境中导航，并根据细胞功能和生化反应完成任务。这个项目的研究将作为一种策略融入到游戏中，以展示蜂窝组件回收的概念。除了开发一种对广大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高度跨学科研究项目的丰富经验，该项目需要将研究、程序设计和教育培训结合起来。

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.