Using SHAPE-Seq as a tool to understand RNA structure / function relationships
Loughrey, David Alan
RNA is a key player in many cellular processes. In both eukaryotes and prokaryotes, RNAs regulate gene expression by affecting transcription, translation, protein function, and RNA degradation through various types of RNA-RNA or RNA-protein interactions. The functionality of RNA in the cell is rooted in its structure. Intricate three-dimensional structural forms contribute to the myriad function of RNAs, such as blocking or allowing cellular processes and helping to create highly specific binding sites for proteins, other cellular RNAs, and specific metabolites. However, a detailed mechanistic understanding of the RNA structure / function relationship is still lacking. SHAPE-Seq has the potential to resolve the RNA structure / function relationship on a scale necessitated by the omics-level questions of today. This technique combines chemical probing with next-generation sequencing to provide quantitative, single-nucleotide resolution information of RNA structure in a high-throughput multiplexed fashion. The flexibility and accuracy of SHAPE-Seq, as part of the toolbox of powerful related next generation sequencing methods, has the ability to provide us with a reference point to both validate computational RNA structural prediction algorithms and observe RNA structures as they exist and interact in their natural environment. Here, we present a systematic analysis, optimization and extension of the SHAPE-Seq technique in order to realize its full potential in being part of this biological characterization repertoire. We introduce an extension to the technique that accounts for multiple reverse transcriptions sites which opens the door for the secondary structure of RNAs longer than one kilobase, such as RNA viruses, ribosomal RNAs and long noncoding RNAs, to be determined for the first time. Additionally, we use in-cell SHAPE-Seq to uncover mechanistic details for important small bacterial RNA networks and develop the first cellular nucleotide-resolution picture of how the structures of the RNAs mediate these protein-dependent interactions. We anticipate that our work with the SHAPE-Seq technology will provide the scientific community with a broad yet precise set of tools, aimed at providing unprecedented characterization of cellular RNA regulatory mechanisms. This will hopefully serve to be illuminating in offering deeper insight into a whole manifest of wide-ranging biological questions.
Chemical engineering; Next Generation Sequencing; RNA; SHAPE-Seq; Small Bacterial RNAs; Structomics; Structural Engineering; Biology; Biomedical engineering
Delisa, Matthew; Ke, Ailong
Ph. D., Chemical Engineering
Doctor of Philosophy
dissertation or thesis