The functions of RNA in cellular biology are diverse. While it has traditionally been thought to be a passive carrier of information from DNA to proteins, the role of RNA has expanded greatly to include other functional classes such as ribozymes, riboswitches, long and short non-coding RNAs1-6.
The regulation of coding and non-coding RNAs occur at transcriptional and posttranscriptional levels. While in some cases this regulation occurs due to sequence specificity of transcripts, in other cases RNA regulation is governed by its structure7. RNA structure has been shown to be involved in the catalytic activity, stability, localization and rate of translation of transcripts in the cell8-11. Understanding what these structural motifs are, how they are organized, and how they might interact with other molecules in the cells, such as other RNAs, proteins and ligands, is critical to our understanding of cellular biology.
Traditionally, the secondary structure of an RNA can be probed in solution, one at a time, using chemicals and enzymes that either cleave or modify single or double stranded regions12-14. The double or single stranded regions are identified by running the cleaved fragments onto a sequencing gel or by reverse transcription followed by capillary electrophoresis. The intensity of the bands on the gels indicates the extent of chemical/enzymatic cleavage15. This method becomes tedious when the structures of long RNAs are of interest. As only a few hundred bases can be resolved at a time, probing the structures of RNAs that are a few kilobases long require running multiple gels. While it is possible to separately clone and probe a region of interest in a long RNA without probing the structure of the full length transcript, doing so risks detecting structural rearrangements that may occur because of the loss in long range contacts. The tedious nature of probing one RNA at a time and the difficulty in probing the structures of long RNAs require the development of a technique that can probe multiple RNA species at a time, independent of their length.
We developed a method, Parallel Analysis of RNA Structure (PARS), to increase the throughput of obtaining experimental RNA structure data independent of RNA size. PARS couples traditional RNA footprinting with high throughput sequencing to identify double and single stranded regions of many RNAs in solution simultaneously. The method provides genome-wide RNA structural information at single nucleotide resolution, greatly expanding the number of RNA secondary structures probed. Here, we describe the PARS protocol in detail. Briefly: RNAs are cleaved by RNase V1 or S1 nuclease independently and the cleavage sites are captured by adaptor ligation followed by high throughput sequencing. The number of sequencing reads mapped to a base in the appropriate library indicates the amount of RNase V1 or S1 nuclease cuts, providing information on whether the base is double or single stranded (Fig1). PARS can be readily applied to probe RNA structural changes that occur under different solution conditions, enhancing our understanding of RNA structure.