Probing the RNA Structure of a Satellite RNA of Cucumber Mosaic Virus Using SHAPE Method

Abstract

Selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) is a widely used technique for RNA structure analysis using N-methylisatoic anhydride (NMIA) treatment that has been proven to be applicable to different types of RNA templates. In this study, we performed the structural analysis of the viral satellite RNA of cucumber mosaic virus TA-Tb (satCMV TA-Tb) using the SHAPE method. In the preliminary experiment, we optimized the protocol of SHAPE method for analyzing satCMV TA-Tb by determining a suitable quantity of template RNA. This optimization effectively reduced the appearance of a large number of intense bands in the NMIA-untreated sample lane, along with a strong overall background signal that prevented the clear elucidation of the RNA structure. SHAPE analysis indicated the presence of non-structured, single-stranded flexible regions throughout satCMV TA-Tb with prominent flexible stretches located around nucleotide positions 145 to 200. The positions of these flexible regions were particularly consistent with a secondary structure of satCMV TA-Tb predicted by mfold software v.2.3, which consisted of five 5′- and 3′-proximal stem-loops and one internal large multi-branched stem-loop. Sequence alignment and secondary RNA structure prediction of other satCMV sequences that are phylogenetically the same group with satCMV TA-Tb also suggested the presence of 5′- and 3′-proximal stem-loop structures. Our data provide the structural basis for elucidating the mechanism by which satCMV TA-Tb regulates the pathogenicity and replication of its helper virus.