Rotavirus RNA chaperone mediates global transcriptome-wide increase in RNA backbone flexibility

Abstract

ABSTRACTDue to genome segmentation, rotaviruses must co-package a set of eleven distinct genomic RNAs. The packaging is mediated by the RNA chaperone NSP2. While the activities of RNA chaperones are well studied with short RNAs, little is known about their global effect on the entire viral transcriptome. Here we used Selective 2′-hydroxyl Acylation Analyzed by Primer Extension and Mutational Profiling (SHAPE-MaP) to systematically examine the secondary structure of the rotavirus transcriptome alone and in the presence of NSP2. Surprisingly, SHAPE-MaP data reveal that despite the well-characterized helix-unwinding activity of NSP2 in vitro, its incubation with rotavirus transcripts does not induce a significant change in the SHAPE reactivities. However, a quantitative analysis of the per nucleotide mutation rate, from which SHAPE reactivities are derived, reveals a global five-fold rate increase in the presence of molar excess of NSP2. Further analysis of the mutation rate in the context of structural classification reveals a larger effect on stems rather than loop elements. Together, these data provide the first experimentally derived secondary structure model of the rotavirus transcriptome and reveal that NSP2 exerts a larger effect on stems, while acting by globally increasing RNA backbone flexibility in a protein concentration-dependent manner.