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.
