Probing RNA Structure With Optimized DMS-MaPseq in Rice.

RNA transcripts form various secondary and tertiary structures that have a wide range of regulatory functions. Several methods have been developed to profile RNA secondary structure in prokaryotes and eukaryotes. These methods, such as dimethyl sulfate (DMS) mutational profiling with high-throughput sequencing (DMS-MaPseq), couple small chemical-mediated RNA modifications with next-generation sequencing. DMS-MaPseq, a powerful method for genome-wide and target-specific RNA secondary structure profiling, has been applied in yeast, mammals, , and , but not in crops. Here, we used DMS-MaPseq to conduct a target-specific and genome-wide profile of RNA secondary structure in rice (). The DMS treatment conditions were optimized for rice leaf and root tissues. To increase the sequencing depth and coverage of low-abundance transcripts in genome-wide DMS-MaPseq, we used streptavidin-biotin depletion to reduce the abundance of highly expressed chloroplast transcripts during library construction. The resulting target-specific and genome-wide rice DMS-MaPseq data were of high quality and reproducibility. Furthermore, we used DMS-MaPseq to profile the RNA secondary structure of an OsmiR399 target region located at 5'UTR of , which participates in rice phosphate homeostasis. An unfolded RNA structure downstream of miRNA target site was observed in predicted RNA secondary structure, reminiscence of the TAM (Target Adjacent nucleotide Motif) involved in mRNA structure-mediated regulation in miRNA cleavage. Our study optimized DMS-MaPseq for probing RNA secondary structure in rice, facilitating the study of RNA structure-mediated regulations in crops.