RNA structural transitions are important in the function and regulation of

RNA structural transitions are important in the function and regulation of RNAs. roles that RNA structures play in the cell. RNA structures can influence each step BCX 1470 in the life cycle of a genefrom 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 BCX 1470 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 BCX 1470 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 Rabbit polyclonal to Ataxin3. 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) (Fig. 1A). 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. Figure 1 Measuring RNA melting temperatures by deep sequencing RESULTS Parallel analysis of RNA structures with temperature elevation To carry out PARTE, we first defined conditions that allowed comparable results at different temperatures. RNA footprinting with RNase V1 of the well known, structured domains of the Tetrahymena ribozyme (Guo et al., 2004) revealed that RNase V1 retains its double-stranded specificity up to 75C, and comparable footprinting results are obtained at different temperatures by correspondingly shorter incubation times with the enzyme to maintain single-hit kinetics (Fig. S1). Optimized conditions defined by these experiments were then used to probe RNA folding at different temperatures genome-wide. Next, we extracted total RNA from log-phase growth culture of yeast and performed polyA selection to enrich for mRNAs. Control RNAs, including domains of the ribozyme and human long non-coding RNAs HOTAIR and HOTTIP were added into the reactions (Rinn et al., 2007; Wang et al., 2011). The RNA pool was folded in vitro at 23 and 30C, and the 30C pool i s split and shifted to 30, 37, 55, or 75C for five m inutes. The RNA samples were then subjected to RNase V1 treatment with single hit kinetics, and the resulting fragments were cloned for deep sequencing on the SOLiD platform as previously described (Kertesz et al., 2010) (Fig. 1A). We performed two biological replicates for each temperature, yielding ten PARTE experiments in total. We generated over 3 million deep sequencing reads for each sample, and mapped the reads to the yeast transcriptome to identify the cleavage.