PhD defense: Line Dahl Poulsen

Investigation of RNA structure by high-throughput SHAPE-based probing methods

Supervisisor: Jeppe Vinther, Associate Professor, Department of Biology

Exam Committee
Dr. Andrew K. Tuplin, University of Leeds
Associate Professor Birte Vester, University of Southern Denmark
Associate Professor Jan Christiansen, Department of Biology

Abstract
RNA exists in cells as dynamic, three dimensional entities, and determination of their structure can be an essential step in understanding their function. With the introduction of next generation sequencing, it has become possible to study the structure of thousands of RNAs in a single experiment. A highly successful method to probe RNA structure is Selective 2’-Hydroxyl Acylation analyzed by Primer Extension (SHAPE), however, this method is limited by high background rates arising from non-probed molecules and pre-termination in the reverse transcription. In this thesis I describe the development of high-throughput SHAPE-based approaches to investigate RNA structure based on novel SHAPE reagents that permit selection of full-length cDNAs. The SHAPE Selection (SHAPES) method is applied to the foot-and-mouth disease virus (FMDV) plus strand RNA genome, and the data is used to construct a genome-wide structural map of the virus. I have used the data to discover stable structures de novo, including previously characterized structural elements, such as the internal ribosome entry site (IRES), and I show that three of the novel structures have been conserved through evolution, indicating that they are functional. The SHAPES method is further applied to the hepatitis C virus (HCV), where the data is used to refine known and predicted structures. Over the past years, the interest of studying RNA structure in their native environment has been increased, and to allow studying RNA structure inside living cells using the SHAPE Selection approach, I introduce a biotinylated probing reagent. This chemical can cross cell membranes and reacts with RNA inside the cells, allowing the structural conformations to be studied in the context of physiological relevant conditions in living cells. The methods and results presented in this thesis represent important steps forward in studying RNA structures with high-throughput technologies, and the selection approach could be key to obtaining high quality sequencing-based probing data in experiments with a high background.