Muench, DouglasAtugala, Dilini M2023-05-112022-09-23Atugala, D. M. (2022). Adapting RNA interactome capture to explore plant RNA-binding proteins and their RNA targets (Doctoral thesis). University of Calgary, Calgary, Canada). Retrieved from https://prism.ucalgary.ca .http://hdl.handle.net/1880/116283RNA-binding proteins (RBPs) function in every step of RNA metabolism, from synthesis todecay. Thus, the global identification of RBPs and their RNA targets provide important insight intopost transcriptional gene regulation during organismal development and in response toenvironmental cues. To better understand the repertoire of RBPs in Arabidopsis, an RNAinteractome capture (RIC) approach was applied to root and leaf tissues, and to suspensionculture cells. RIC identified 416, 611 and 110 proteins comprising the root, leaf and cell culturemRNA-binding proteomes (RBPomes), respectively. Similar to other RNA interactome studiesthere were a large number of ‘moonlighting’ RBPs (up to 46%) that had activities not related toRNA biology, but rather to processes that included intermediate metabolic pathways,photosynthesis, cytoskeleton, and membrane transporters. These results highlight the overlapbetween interactome studies and reports RBPs that have not been identified in other Arabidopsisstudies. Additionally, the root RIC results provide insight into the RBPome of a tissue that has notbeen reported previously.To understand the responses of cellular RBPs to an abiotic stress, we applied RIC coupledwith stable isotope dimethyl labeling-mass spectrometry to Arabidopsis roots treated withnaphthenic acid fraction compounds (NAFCs) isolated from oil sands tailings pond water. NAFCtreatment resulted in a remodeling of the cellular RBPome, identifying 205 RBPs that increasedin their association with RNA. In addition to RBPsrelated to RNA biology processes, classical stressresponsive proteins were detected and included metabolic enzymes involved in carbohydratemetabolism, suggesting that these RBPs may govern metabolic changes during stress responses.Lastly, an approach was developed and tested for the identification of in vivo target RNAsof RBPs under stringent, denaturing extraction conditions. This immunoprecipitation approachidentified over 300 mRNA targets of a specific RBP (AtGRP7). This approach has the potential tobe used as a simplified option to identify RNA targets over other more advanced techniques suchas crosslinking and immunoprecipitation (CLIP).EnglishBiological Sciencesdoctoral thesis