Autophagy- and RNS2-mediated mechanism of ribosomal RNA degradation in Arabidopsis thaliana

Morriss, Stephanie
Major Professor
Gustavo C. MacIntosh
Committee Member
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Biochemistry, Biophysics and Molecular Biology

Ribosomal RNA degradation is necessary to remove nonfunctional rRNA and maintain homeostasis through degradation of aging or normal rRNA. While pathways for surveillance of nonfunctional rRNA have been characterized, mechanisms of degradation of normal rRNA are not yet well understood. Ribosomal RNA, the majority of cellular RNA, forms a massive resource sink. These studies examine the mechanism of degradation of rRNA in Arabidopsis and the effects of disruption of this process through mutation of key genes.

Ribosomal RNA in many organisms, including yeast, zebrafish, Tetrahymena and Arabidopsis, is degraded by the RNase T2 family of enzymes, a family highly conserved throughout eukaryotes. Mutation of the Arabidopsis RNase T2, RNS2, results in increased rRNA half-life and constitutive autophagy. An autophagy-dependent mechanism of ribosomal degradation, known as ribophagy, has been shown in yeast but not plants. Here, I report the effects of mutation of autophagy genes on rRNA accumulation in Arabidopsis. I show that RNA accumulates in rns2-2 and autophagy mutants atg9-4 and atg5-1, indicating that RNS2 and the autophagy machinery are necessary to maintain normal rRNA levels in Arabidopsis. This accumulation is vacuolar in rns2-2 but not atg5-1. These evidence support an autophagy-dependent rRNA degradation by RNase T2 in plants.

To further examine the effects of mutation in this rRNA degradation pathway, I performed two transcriptomic studies. My initial analysis of WT and rns2-2 in seedlings revealed differentially expressed genes (DEG) related to carbon flux pathways and cell wall modifying processes. Alteration of carbon flux in the mutant was verified using metabolite analysis, while the impact on cell wall was studied using cell wall component analysis and phenotypic data which revealed that rns2-2 plants are larger due to an increase in water content and cell elongation. I next performed an expanded transcriptomic analysis including WT, rns2-2, atg9-4, and rns2-2atg9-4 in adult plants and found cell wall and oxidative stress associated DEG. Further analysis indicated a role for reactive oxygen species signaling constitutive autophagy in rns2-2 plants. These results support existence of a ribophagy-like mechanism in Arabidopsis and dissect the importance of rRNA degradation in maintenance of cellular homeostasis in plants.