The structures of RNA molecules are often important for their function and regulation1,2,3,4,5,6, yet there are no experimental techniques for genome-scale measurement of RNA structure. Here we describe a novel strategy termed parallel analysis of RNA structure (PARS), which is based on deep sequencing fragments of RNAs that were treated with structure-specific enzymes, thus providing simultaneous in vitro profiling of the secondary structure of thousands of RNA species at single nucleotide resolution. We apply PARS to profile the secondary structure of the messenger RNAs (mRNAs) of the budding yeast Saccharomyces cerevisiae and obtain structural profiles for over 3,000 distinct transcripts. Analysis of these profiles reveals several RNA structural properties of yeast transcripts, including the existence of more secondary structure over coding regions compared with untranslated regions, a three-nucleotide periodicity of secondary structure across coding regions and an anti-correlation between the efficiency with which an mRNA is translated and the structure over its translation start site. PARS is readily applicable to other organisms and to profiling RNA structure in diverse conditions, thus enabling studies of the dynamics of secondary structure at a genomic scale.
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We thank D. Herschlag’s group, A. Adler, A. Fire, M. Kay, the Life Technologies SOLiD team, M. Rabani, G. Sherlock and A. Weinberger for assistance and critiques. This work was supported by a National Institutes of Health grant (RO1HG004361). Y.W. is funded by the Agency of Science, Technology and Research of Singapore. H.Y.C. is an Early Career Scientist of the Howard Hughes Medical Institute. E.S. is the incumbent of the Soretta and Henry Shapiro career development chair.
The authors declare no competing financial interests.
This file contains Supplementary Methods and Data, a Supplementary Note, additional references, Supplementary Figures 1-16 with legends and Supplementary Tables 1, 2 and 4 (see separate files for Supplementary Tables 3 and 5). (PDF 1680 kb)
This table lists all genes whose average nucleotide coverage is above 1.0 (Methods). Columns show, for each gene, the annotated transcript length, the total number of sequences mapping to that transcript, the computed load and the type of transcript: mRNA, rRNA, snoRNA, tRNA, snRNA or ncRNA. (XLS 322 kb)
This file contains sub-tables which list the Wilcoxon rank sum test p-value for each GO term when genes were sorted based on their average PARS score in each region of the transcript (5’ UTR, CDS, 3’ UTR). For each group, the FDR correction p-value threshold is indicated, and GO categories having a p-value below this threshold are marked in green. (XLS 44 kb)
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Kertesz, M., Wan, Y., Mazor, E. et al. Genome-wide measurement of RNA secondary structure in yeast. Nature 467, 103–107 (2010). https://doi.org/10.1038/nature09322
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