Chemical probing methods—in which folded RNAs are modified by chemoselective reagents and analyzed by reverse transcription—are standard approaches for in vitro RNA secondary structure determination. However, reagents such as dimethyl sulfate (DMS), which methylates adenosine and cytosine bases in unpaired RNA regions, have seen limited application in cells and organisms. Ding et al. now report a method for genome-wide RNA secondary structure analysis in vivo. Using 'structure-seq', which combines in vivo DMS modification with deep sequencing, the authors profiled Arabidopsis thaliana seedlings and obtained single-nucleotide resolution modification data on over 10,000 transcripts. Analysis of the DMS modification profiles of the mRNA transcriptome revealed cases where RNA secondary structure may have a direct role in regulating gene expression. For instance, enhanced DMS modification profiles upstream of start codons correlate with high ribosome association and translational efficiency. In contrast, mRNA regions upstream of alternative polyadenylation or alternative splice sites showed lower DMS modification frequency. Interestingly, mRNAs coding for stress response proteins displayed evidence for greater structural flexibility than those involved in housekeeping roles, a property that may be functionally important for environmentally responsive RNAs. In addition to providing an approach for probing RNA structure under physiological conditions, structure-seq also highlights practical gaps between in vitro, in vivo and in silico RNA structural prediction methods that merit the community's attention.
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Sheppard, T. Folding in the wild. Nat Chem Biol 10, 87 (2014). https://doi.org/10.1038/nchembio.1445