The cellular transcriptome is predominantly composed of RNAs less than 200 nucleotides in length. These small RNAs participate in and regulate numerous biological processes, including translation, transcription, epigenetic reprogramming, apoptosis, cell-wall biosynthesis, natural product biosynthesis, and the cellular immune response. The most abundant small RNA is tRNA, comprising roughly 80% of cellular transcripts. Many classes of small RNAs, however have proven difficult to sequence owing to their high degree of secondary and tertiary structure, presence of epitranscriptomic modifications, and aminoacylation. Although sequencing methods have been developed over the past decade, they possess several drawbacks that make them impractical for studies both clinical and fundamental that require high throughputs and the ability to produce libraries from low quantities of material. In this thesis, I detail the development and application of a high-throughput small RNA sequencing method termed MSR-seq. In Chapter 2, I describe the development of MSR-seq. In Chapter 3, I demonstrate the application of MSR-seq to studying translational regulation during stress response. In Chapter 4, I discuss a study performed to analyze the SARS-CoV-2 packageome of host and viral RNAs, the discover of a chemical treatment that renders several modifications detectable by reverse transcriptase error signatures, and new insights into the function of the host oral microbiome. In Chapter 5, I provide preliminary results on the integration of RNA structural mapping techniques to study the changes in small RNA interactions with other molecular partners. This work demonstrates the power of a new high-throughput small RNA sequencing method and its ability to inform and provide insight into previously intractable areas of biology.
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