Determination of in vivo RNA structure in low-abundance transcripts.

Abstract

RNA structure plays important roles in diverse biological processes. However, the structures of all but the few most abundant RNAs are presently unknown in vivo. Here we introduce DMS/SHAPE-LMPCR to query the in vivo structures of low-abundance transcripts. DMS/SHAPE-LMPCR achieves attomole sensitivity, a 100,000-fold improvement over conventional methods. We probe the structure of low-abundance U12 small nuclear RNA (snRNA) in Arabidopsis thaliana and provide in vivo evidence supporting our derived phylogenetic structure. Interestingly, in contrast to mammalian U12 snRNAs, the loop of the SLIIb in U12 snRNA is variable among plant species, and DMS/SHAPE-LMPCR determines it to be unstructured. We reveal the effects of proteins on 25S rRNA, 5.8S rRNA and U12 snRNA structure, illustrating the critical importance of mapping RNA structure in vivo. Our universally applicable method opens the door to identifying and exploring the specific structure-function relationships of the multitude of low-abundance RNAs that prevail in living cells.