Abstract

Reactive oxygen species can oxidize biological molecules, rendering them inactive or toxic. Oxidized RNA can stall ribosomes during translation, leading to the production of truncated, aggregation-prone nascent polypeptides. Cells have quality control systems that recognize and target oxidatively damaged molecules for repair or degradation. Ribosome collisions that occur due to stalled translation are recognized by a quality control pathway that results in the degradation of the defective mRNA, extraction and degradation of the truncated nascent polypeptide, and recycling of the ribosome subunits for additional rounds of translation. This thesis addresses the quality control of oxidized RNA. It uses two species, Saccharomyces cerevisiae and Chlamydomonas reinhardtii to examine how oxidized RNA is compartmentalized for quality control, and how a photosynthesis protein moonlights as an oxidized RNA quality control factor.

In Chapter 2, with results of immunofluorescence microscopy, genetic analyses, biochemical fractionation, and proteomics, I identify and characterize a previously undiscovered organelle, which we name “oxidized RNA bodies” or ORBs. I show that ORBs are membraneless phase-separated organelles that compartmentalize the translation quality control pathways which handle stalled ribosomes.

In Chapter 3, using immunofluorescence microscopy, genetic analyses, and biochemical fractionation, I present preliminary results on the moonlighting functions of RbcL, the large subunit of Rubisco, in handling oxidized RNA in chloroplasts. I show that a mutant unable to assemble the Rubisco holoenzyme has altered phenotypes related to the moonlighting functions of RbcL and explore the subcellular localization of the protein in various contexts associated with translation.

A powerful approach to understand the significance and management of RNA oxidation could be to find and characterize specialized intracellular compartments dedicated to it. The research contained herein advances the field of RNA quality control by providing a repertoire of candidate components, evidence for biochemical mechanisms that can be tested in future work, and a cytological context for these processes.