Ribosomes play an essential role in protein manufacturing in the cell, and are made up of ribosomal RNA (rRNA) and proteins. While scientists understand a great deal about how ribosomes are created, surprisingly little is known about how they are broken down at the end of their useful life. Understanding ribosome turnover is important, because each cell invests a lot of energy and resources to maintain a sufficient number of ribosomes to keep up with its protein production demands.

As described in a recent publication in the journal Autophagy, a collaboration between Iowa State University’s Loomis Professor of Plant Physiology Dr. Diane Bassham, Associate Professor of Biochemistry Dr. Gustavo MacIntosh, and their research groups resulted in the discovery that eukaryotic cells may be using a process called autophagy to break down ribosomes. In autophagy, a compartment (called an autophagosome) is created by autophagy-related proteins (ATGs) around the cargo slated for destruction, and the autophagosome with its cargo is trafficked from the cytoplasm of the cell to an organelle called a vacuole (in plant cells) or a lysosome (in animal cells). Fusion with the vacuole or lysosome results in the cargo being deposited inside the organelle with the enzymes required for its destruction. One such enzyme, called RNS2, is from the RNase T2 family of ribonucleases (enzymes that break down RNA into smaller components).

Hypothesizing that the process of autophagy may play a role in breakdown of ribosomes, the research team developed a method to measure ribosomal RNA accumulation in the vacuole of mutant plant (Arabidopsis thaliana) cells lacking the RNS2 ribonuclease. The mutant is called rns2-2. The researchers used confocal microscopy to look for evidence of autophagy activation and the accumulation of autophagosomes. Fluorescent labeling of an ATG protein on the surface of autophagosomes provided evidence of an increased number of autophagosomes (indicated by small, fluorescent blue dots in the image) in the rns2-2 mutant when compared to normal, wild type (WT) Arabidopsis thaliana plant cells. This result allowed the research team to further hypothesize that autophagy activation in the rns2-2 mutant was compensation for the plant cells’ inability to degrade rRNA with the vacuolar ribonuclease RNS2.

The research team also found evidence of the involvement of more than one autophagy pathway in the breakdown of ribosomes. As described in their publication, mutations in an autophagy gene (ATG5) blocked the activity of the autophagy pathway and prevented accumulation of rRNA in the vacuole. But, mutations in a different autophagy gene (ATG9), did not prevent accumulation of the rRNA in the vacuole, suggesting the pathway used by ATG5 and ATG9 to deliver rRNA to the vacuole may be different. As Dr. Bassham notes, “Our results shed light on the mechanisms by which ribosomal components are recycled and in turn, on the way in which ribosome number and quality are controlled.”  Dr. Bassham credits NSF MCB support to “allow Dr. Gustavo MacIntosh and I to reinforce and expand our collaboration by establishing a group consisting of a post-doctoral researcher, several graduate students, and undergraduate students to work together in the analysis of the relationship between autophagy and RNA degradation, allowing progress that would have not been possible had our research groups continued to work independently.”

When asked about the broader impacts of her research, Dr. Bassham responded:

“A major impact of our project has been training students in research. In addition to training several graduate students and a post-doc who worked full time on the research project, ten undergraduate students participated in laboratory research in our summer internship program, including several from the primarily undergraduate institution Grand View University in Des Moines, IA, who would otherwise not have the opportunity to gain research experience. A second outcome is the continued development, headed by Iowa State University Professor Eve Wurtele, of an educational video game called Meta!Blast that is used to teach cell biology to undergraduate and high school students. In the game, the player navigates a three-dimensional cellular environment within a plant and completes tasks based on cell functions and biochemical reactions. Interactivity aids retention and understanding of concepts and the use of multimedia allows us to reach diverse populations of students. The process of autophagy was incorporated into the game as a result of this project.”

This work is funded by the Division of Molecular and Cellular Biosciences, Award #MCB-1051818.