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Abstract
This dissertation is focused on developing an approach for high throughput analysis of RNA from biological systems. Covalent nucleotide modification of the canonical nucleosides (A, U, C, G) is one of the essential features of cellular RNA maturation. Chemical modifications can range from simple isomerization or methylations to more complex chemical additions orchestrated by multiple enzymes. More than 150 chemical modifications have been identified, and transfer RNAs (tRNAs) contain the highest density of these modifications. The similarities among individual tRNA isoacceptors, extensive sample preparation and low throughput of analysis have slowed the characterization of total tRNA pools from organisms beyond the bacterial kingdom. Here, I introduced the reference concept using a set of known standards, which allows one to rapidly characterize tRNA samples both qualitatively and quantitatively. The methods I developed here created a more powerful comparative analysis approach using stable isotope labelled in vitro transcripts, increased speed of data analysis by mass spectral matching and understanding the dynamics of RNA expression in the pathogenic, radioresistant fungi Cryptococcus neoformans when exposed to different environmental stimuli or stresses such as ionizing radiation. The advances made here can be applied to a holistic approach in studying tRNA modifications, expression levels and other cellular processing in a biological system.
