Probing in vivo RNA Structure With Optimized DMS-MaPseq in Rice.

Abstract

RNA transcripts form various secondary and tertiary structures that have a wide range of regulatory functions. Several methods have been developed to profile in vivo 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, Drosophila, and Arabidopsis thaliana, but not in crops. Here, we used DMS-MaPseq to conduct a target-specific and genome-wide profile of in vivo RNA secondary structure in rice (Oryza sativa). 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 in vivo RNA secondary structure of an OsmiR399 target region located at 5′UTR of OsPHO2, which participates in rice phosphate homeostasis. An unfolded RNA structure downstream of miRNA target site was observed in predicted in vivo 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 in vivo RNA secondary structure in rice, facilitating the study of RNA structure-mediated regulations in crops.