Mapping the Tertiary Structure of RNAs using SHAPE-JuMP

Function is closely related to form in biological systems, and one of the more recent advancements in structure-function relationships is in our rapidly expanding understanding of the tertiary structure of RNA. The ability of an RNA to fold back on itself and form tertiary interactions affects catalysis, protein synthesis, and assembly of ribonucleoprotein complexes.1,2 Here, we utilize SHAPE-JuMP (selective 2’-hydroxyl acylation analyzed by primer extension with juxtaposed merged pairs) to examine the tertiary structure of RNAs. SHAPE-JuMP is a technology developed in the Weeks laboratory. It uses an RNA crosslinker trans-bis-isatoic anhydride (TBIA), reverse transcription, sequencing, and computational analysis to reveal where RNA interactions occur in three-dimensional space. We show that reverse transcriptase C8 can successfully locate cross-links in RNase P, mapping RNase P’s tertiary structure using SHAPE-JuMP. We also reveal that the C8 polymerase can successfully reverse transcribe the random-primed 16S subunit of E. coli ribosomal RNA. The 16S rRNA subunit is 1533 nucleotides in length, which suggests that performing SHAPE-JuMP experiments on longer RNAs with the C8 polymerase is feasible, and may ultimately reveal their three-dimensional structures. When fully realized, this project will serve as a model system for measuring and understanding the spatial dynamics of RNA.Bachelor of Scienc

Toward a next-generation atlas of RNA secondary structure

RNA structure plays a crucial role in gene maturation, regulation and function. Determining the form and frequency of RNA folds is essential for a better understanding of how RNA exerts its functions. Low-throughput studies have focused on RNA primary sequences and expression levels, but with an emphasis on relatively small numbers of transcripts. However, with the recent advent of high-throughput technologies, it is realistic to begin analyzing RNA secondary structures on a genomewide scale. Here, we review genome-wide RNA secondary structure profiles as well as advances in computational structure predictions. We further discuss the novel characteristics of RNA secondary structure across messenger RNAs. Probing RNA secondary structure by high-throughput sequencing will enable us to build atlases of RNA secondary structures, an important step in helping us to understand the versatility of RNA functions in diverse cellular processes