Differential stability of RNA structures in the transcriptome corresponds to the diverse roles that RNA structures play in the cell. RNA structures can influence each step in the life cycle of a gene—from transcription, to pre-mRNA splicing, RNA transport, translation, and RNA decay (Wan et al., 2011
). However, it is difficult to identify functional structural elements in the transcriptome because practically every RNA has the propensity to fold into extensive RNA structures. In addition to whether a base is paired, the stability of base pairing impacts the biological function of RNAs in important ways (Ringner and Krogh, 2005
). Some RNAs, such as ribozymes and structural RNA scaffolds(Guo et al., 2004
; Wang and Chang, 2011
), form stable secondary and tertiary structures; other RNAs, such as RNA thermometers and riboswitches, undergo structural rearrangements at specific temperatures or in the presence of ligands, respectively, to mediate gene regulation (Breaker, 2010
; Chowdhury et al., 2006
). As such, differential RNA stability is one way to distinguish diverse RNA structures and to identify functionally important elements in the transcriptome. While RNA folding energies are difficult to predict computationally because of contributions from complex tertiary RNA structures and ligand interactions(Wilkinson et al., 2005
), RNA folding energies have been experimentally probed by measuring RNA Tm via several methods(Luoma et al., 1980
; Rinnenthal et al., 2010
; Wilkinson et al., 2005
). Tm is defined as the temperature at which half of the molecules of a double-stranded species become single-stranded. RNA structures of low Tm are more dynamic and exhibit lower energetic cost to unwind and access; conversely, RNA structures of high Tm are relatively more stable and demand higher energetic cost to unfold.
We recently reported genome-wide RNA structure data for the yeast transcriptome by coupling RNA footprinting, using RNase V1 and S1 nuclease, to high throughput sequencing (termed Parallel Analysis of RNA Structures, or PARS) (Kertesz et al., 2010
). However, the relative stabilities of these structures and their influence on cellular biology remain unanswered. Inspired by the precedent of Tm measurement via RNA footprinting (e.g. SHAPE(Wilkinson et al., 2005
), here we directly measure the melting temperature at single nucleotide resolution across the yeast transcriptome. We coupled RNA footprinting using RNase V1 to high throughput sequencing to probe for double stranded regions across 5 temperatures, from 23 to 75 Degrees Celsius (°C) (). This approach, termed Parallel Analysis of RNA structures with Temperature Elevation (PARTE), revealed the energetic landscape of the transcriptome and its multiple roles in post-transcriptional regulation.
Measuring RNA melting temperatures by deep sequencing