Identification and comparison of RNA structures in 5’- and 3’-terminal genomic regions across four coronavirus genera by SHAPE analysis

Background: Comprehensive determination of RNA structures in 5’- and 3’-terminal genomic regions across four genetically divergent coronavirus genera and thus their conservation are the first steps toward the identification of their function in coronavirus gene expression. Although these RNA structures have been experimentally analyzed for β coronaviruses, those for α coronaviruses have primarily been analyzed using bioinformatics methods. In addition, information concerning these RNA structures in γ and δ coronaviruses is limited. Methods: To experimentally identify the conserved RNA structures in 5’- and 3’-terminal genomic regions across four coronavirus genera, the chemical probing method selective 2′ hydroxyl acylation analyzed by primer extension (SHAPE) was used. Moreover, on the basis of the determined RNA structures, the potential long-range RNA‒RNA interactions and the predicted 3-dimensional (3D) RNA structures in these genomic regions were identified using RNAcanvas and RNAComposer, respectively. Results: The results obtained were as follows: (i) the overall RNA structures in the 5’- and 3’-terminal genomic regions varied across the four coronavirus genera, as did their 3D structures; (ii) the 3’-terminal RNA structures in the representative γ coronavirus were unique among the four coronavirus genera; (iii) the stem loops (SLs) 1, 2, 4 and 5 in the 5’-termini and the SL2 and potential pseudoknot (PK) structures in the 3’-termini were the conserved RNA structures across the four coronavirus genera; and (iv) potential long-range RNA‒RNA interactions within the 5’- and 3’-terminal genomic regions can be identified. Conclusions: The 5’ and 3’ terminal RNA structures in the represented α, β, γ and δ coronaviruses are analyzed and compared by using the chemical probing method SHAPE. Although different degrees of variations are found in the 5’- and 3’-terminal genomic regions across the four coronavirus genera, conserved RNA structures are identified. The determined RNA structures, the potential long-range RNA‒RNA interactions and the predicted 3D RNA structures within these genomic regions may contribute to the identification of RNA elements important for coronavirus gene expression.