2013 Rustbelt RNA Meeting
RNA functions are intimately linked to their ability to fold into complex secondary and tertiary structures. Thus, understanding how these molecules fold is essential to determining how they function. Current methods for investigating RNA structure often use small molecules, enzymes, or ions that cleave or modify the RNA in a solvent accessible manner. While these methods have been invaluable to understanding RNA structure, they can be fairly labor intensive and often focus on short regions of single RNAs. Here we present a new method and data analysis pipeline, called Mod-seq, for assaying the structure of RNAs by high-throughput sequencing. This technique can be utilized both in vivo and in vitro, with any small molecule that modifies RNA and impedes reverse transcriptase. As proof-of-principle, we used dimethyl sulfate (DMS) to probe the in vivo structure of cellular RNAs in Saccharomyces cerevisiae. Mod-seq analysis simultaneously revealed secondary structural information for all four ribosomal RNAs and 32 additional non-coding RNAs. We further show that Mod-seq can be used to detect structural changes in 5.8S and 25S rRNAs in the absence of ribosomal protein L26, correctly identifying its binding site on the ribosome. While this method is applicable to RNAs of any length, its high-throughput nature makes Mod-seq ideal for studying long RNAs and complex RNA mixtures.
Keywords: RNA structure probing, High-throughput sequencing, RNA folding