Nat. Methods doi:10.1038/nmeth.4057 (2016)

Credit: NATURE METHODS

One method for genome-wide probing of RNA structures in cells combines the use of dimethyl sulfate (DMS), which reacts with unpaired adenine and cytosine residues, with deep sequencing. However, this approach is unable to detect RNA species with low expression or to reliably distinguish heterogeneous RNA populations. To address this limitation, Zubradt et al. devised a modified approach called DMS-MaPseq, which uses DMS-mediated RNA modification with RT-PCR performed by a high-fidelity thermostable group II intron reverse transcriptase (TGIRT) combined with next-generation sequencing. DMS-MaPseq reduced the signal-to-noise ratio and produced reproducible results between samples. This method enabled the authors to probe the RNA structure of oskar and gurken in Drosophila ovaries and to observe low-abundance mRNA species such as human FXR2. It also allowed discernment of distinct RNA structures in a heterogeneous population, such as a single-nucleotide polymorphism in MRPS21 that produced a local RNA structural shift. Finally, this method enabled the comparison of the premature and mature mRNA structures of the yeast ribosomal protein RPL14A. Overall, the DMS-MaPseq approach offers an improved method that expands the analysis of diverse RNA secondary structures with the potential to provide more insight into RNA function.