Adapting RNA interactome capture to explore plant RNA-binding proteins and their RNA targets

Atugala, Dilini M

Adapting RNA interactome capture to explore plant RNA-binding proteins and their RNA targets

dc.contributor.advisor	Muench, Douglas
dc.contributor.author	Atugala, Dilini M
dc.contributor.committeemember	Chua, Gordon
dc.contributor.committeemember	Ro, Dae-Kyun
dc.date	Spring Convocation
dc.date.accessioned	2023-05-11T04:04:19Z
dc.date.embargolift	2023-09-26
dc.date.issued	2022-09-23
dc.description.abstract	RNA-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).
dc.identifier.citation	Atugala, 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 .
dc.identifier.uri	http://hdl.handle.net/1880/116283
dc.identifier.uri	https://dx.doi.org/10.11575/PRISM/dspace/41127
dc.language.iso	English
dc.publisher.faculty	Science
dc.subject.classification	Biological Sciences
dc.title	Adapting RNA interactome capture to explore plant RNA-binding proteins and their RNA targets
dc.type	doctoral thesis
thesis.degree.discipline	Biological Sciences
thesis.degree.grantor	University of Calgary
thesis.degree.name	Doctor of Philosophy (PhD)