TY - BOOK

T1 - Investigation of RNA Structure by High-Throughput SHAPE-Based Probing Methods

AU - Poulsen, Line Dahl

PY - 2015

Y1 - 2015

N2 - RNA exists in cells as dynamic, three dimensional entities, and determination of theirstructure can be an essential step in understanding their function. With the introduction ofnext generation sequencing, it has become possible to study the structure of thousands ofRNAs in a single experiment. A highly successful method to probe RNA structure isSelective 2’-Hydroxyl Acylation analyzed by Primer Extension (SHAPE), however, thismethod is limited by high background rates arising from non-probed molecules and preterminationin the reverse transcription. In this thesis I describe the development of highthroughputSHAPE-based approaches to investigate RNA structure based on novelSHAPE 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 RNAgenome, and the data is used to construct a genome-wide structural map of the virus. Ihave used the data to discover stable structures de novo, including previouslycharacterized structural elements, such as the internal ribosome entry site (IRES), and Ishow that three of the novel structures have been conserved through evolution, indicatingthat 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 pastyears, the interest of studying RNA structure in their native environment has beenincreased, and to allow studying RNA structure inside living cells using the SHAPESelection approach, I introduce a biotinylated probing reagent. This chemical can crosscell membranes and reacts with RNA inside the cells, allowing the structural conformationsto be studied in the context of physiological relevant conditions in living cells. The methodsand results presented in this thesis represent important steps forward in studying RNAstructures with high-throughput technologies, and the selection approach could be key toobtaining high quality sequencing-based probing data in experiments with a highbackground.

AB - RNA exists in cells as dynamic, three dimensional entities, and determination of theirstructure can be an essential step in understanding their function. With the introduction ofnext generation sequencing, it has become possible to study the structure of thousands ofRNAs in a single experiment. A highly successful method to probe RNA structure isSelective 2’-Hydroxyl Acylation analyzed by Primer Extension (SHAPE), however, thismethod is limited by high background rates arising from non-probed molecules and preterminationin the reverse transcription. In this thesis I describe the development of highthroughputSHAPE-based approaches to investigate RNA structure based on novelSHAPE 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 RNAgenome, and the data is used to construct a genome-wide structural map of the virus. Ihave used the data to discover stable structures de novo, including previouslycharacterized structural elements, such as the internal ribosome entry site (IRES), and Ishow that three of the novel structures have been conserved through evolution, indicatingthat 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 pastyears, the interest of studying RNA structure in their native environment has beenincreased, and to allow studying RNA structure inside living cells using the SHAPESelection approach, I introduce a biotinylated probing reagent. This chemical can crosscell membranes and reacts with RNA inside the cells, allowing the structural conformationsto be studied in the context of physiological relevant conditions in living cells. The methodsand results presented in this thesis represent important steps forward in studying RNAstructures with high-throughput technologies, and the selection approach could be key toobtaining high quality sequencing-based probing data in experiments with a highbackground.

UR - https://soeg.kb.dk/permalink/45KBDK_KGL/1pioq0f/alma99122158686605763

M3 - Ph.D. thesis

BT - Investigation of RNA Structure by High-Throughput SHAPE-Based Probing Methods

PB - Department of Biology, Faculty of Science, University of Copenhagen

ER -