My thesis focuses on the investigation and characterization of RNA modifications. RNA is post-transcriptionally modified on the nucleoside bases and/or on the ribose-phosphate backbone. RNA modifications are conserved from prokaryotes to eukaryotes. Each modification has its own functions in vivo. Efficient and quantitative detection methods are necessary for the elucidation of the functions of specific RNA modifications. Some methods are useful for the detection of a specific modification, while other methods can detect multiple RNA modifications simultaneously. In the chapters 2 and 3 of my thesis, I describe the methods that I developed for the sensitive and quantitative detection of pseudouridine (Ψ) and glycosylated queuosine (Q). Chapter 4 is about the usage of small RNA sequencing methods (MSR-seq) for the investigation of tRNA modification response under stress conditions. Chapter 2 describes the development of a sensitive and quantitative method called “CLAP” for Ψ detection. This PCR-based method takes advantages of the CMC reaction with Ψ which leaves a stop signature during reverse transcription. This method is useful for sensitive quantification of the modification fraction of target Ψ sites in low abundant long non-coding RNAs and mRNAs. CLAP has the potential of being adapted for the studies of other RNA modifications. Chapter 3 describes an acid denaturing gel based Northern blot method for the quantitative detection of glycosylated Q modifications. This method combines the acid denaturing gel and Northern blot. Non-radioactive Northern blot probes are used for detection of Q-modified tRNAs. The acid denaturing gel based Northern blot is useful for measuring the glycosylated Q modification levels in tRNAs. By combining the acid denaturing gel based Northern blot and 3-(Acrylamido)phenylboronic acid (APB) gel based Northern blot, we investigated the Q modification and glycosylation kinetics in tRNAs in three human cell lines, HEK293T, HeLa, and MCF7. Chapter 4 describes a study of using small RNA sequencing method to study tRNA abundance and modification response under different stress conditions including heat shock, H2O2, and NaAsO2. We identified new stress responses by m3C modifications in tRNAs and a tRNA-based regulation of translational elongation, and a relationship between tRNA modification and tRNA fragment biogenesis. In conclusion, efficient and simple detection methods are often required for studying the functional roles of RNA modifications. The development of quantitative methods of Ψand glycosylated Q will help the elucidation of the diverse functions of Ψ in different RNA species and uncover the mysterious functions of glycosylated Q modifications in tRNAs.




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