MASS SPECTROMETRY-DRIVEN DISCOVERY AND CHARACTERIZATION OF NUCLEOSIDE DERIVED-METABOLITES IN C. ELEGANS
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Chemical modifications of nucleosides are increasingly recognized as central regulators of gene expression, stress responses, and host–microbe interactions, yet their metabolic fates and derived small molecules remain largely unexplored. This thesis investigates the metabolic fate of RNA-derived small molecules in C. elegans. This work combines mass spectrometry method development, metabolite discovery, chemical biology, and RNA modification analysis to characterize an uncharted layer of small-molecule diversity shaped by RNA modification chemistry.Chapter 1 focuses on the discovery that maglu#2, an N1-methyladenine gluconucleoside, is a covalent modification in C. elegans RNA. By isolating and enzymatically digesting RNA from C. elegans and its sister-species C. briggsae we demonstrate that this unusual gluconucleoside modification is conserved in some nematodes. Chapter 2 focuses on defining which RNAs are modified by maglu#2 in C. elegans. By developing size-exclusion chromatography as an approach for RNA fractionation and subsequent enzymatic hydrolysis, we demonstrate that maglu#2 is uniquely localized to less abundant small RNA species. In Chapter 3, I examined how microbial diet modulates nucleoside-derived metabolites. Untargeted LC-HRMS profiling of worms raised on Escherichia coli OP50 versus natural bacterial isolates from the CemBio collection revealed extensive diet-dependent remodeling of nucleoside derivatives. This included glucosylated, base-phosphorylated, oxidized, and lipidated nucleosides, as well as unusual cyclic nucleotides, many of which originate from tRNA modifications such as ms2i6A and ms2io6A. These results highlight a previously underappreciated influence of diet on nucleoside metabolism in C. elegans. In Chapter 4 I developed a generalizable data acquisition and analysis approach for the unbiased detection of metabolites sharing specific structural motifs. This label- and derivatization-free workflow leverages correlation analysis between parent ions and characteristic fragment ions or inferred neutral losses allowed systematic identification of metabolites containing selected motifs. The applicability of the approach is demonstrated through the discovery of previously uncharacterized nucleoside-ascaroside conjugates.