Characterization of Co-Transcriptional RNA Structure Evolution Using Shape-Seq

RNA is a versatile molecule that is capable of both storing information and adopting structures that serve specific biological functions. Our understanding of the relationship between RNA structure and function is dependent on our ability to interrogate these structures, however, current approaches to RNA structure determination are limited because they focus on full-length RNA and do not provide information about intermediate folding events that are frequently critical to the function of the RNA. The ability to characterize RNA folding pathways is of particular importance because RNAs not only fold co-transcriptionally, but also frequently perform specific functions, such as termination, antitermination and splicing, co-transcriptionally. We have developed a novel technique that can map the dynamic folding pathways that RNAs undergo co-transcriptionally using SHAPE-seq, a high-throughput method that combines chemical probing of RNA structure with next generation sequencing to characterize the structures of thousands of RNAs in a mixture. Co-transcriptional SHAPE-seq allows us to observe the maturation of RNAs into a final structure by characterizing the structure of each length of RNA as it is transcribed. We have used co-transcriptional SHAPE-seq to uncover novel insights into the mechanism through which the B. cereus crcB fluoride riboswitch regulates transcription by observing the RNA folding events that lead to transcription termination or antitermination at nucleotide resolution.