Intermolecular base-pairing interactions, a unique topology and exoribonuclease-resistant noncoding RNAs drive formation of viral chimeric RNAs in plants.

In plants, exoribonuclease-resistant RNAs (xrRNAs) are produced by many viruses. Whereas xrRNAs contribute to the pathogenicity of these viruses, the role of xrRNAs in the virus infectious cycle remains elusive. Here, we show that xrRNAs produced by a benyvirus (a multipartite RNA virus with four genomic segments) in plants are involved in the formation of monocistronic coat protein (CP)-encoding chimeric RNAs. Naturally occurring chimeric RNAs, we discovered, are composed of 5'-end of RNA 2 and 3'-end of either RNA 3 or RNA 4 bearing conservative exoribonuclease-resistant 'coremin' region. Using computational tools and site-directed mutagenesis, we show that de novo formation of chimeric RNAs requires intermolecular base-pairing interaction between 'coremin' and 3'-proximal part of the CP gene of RNA 2 as well as a stem-loop structure immediately adjacent to the CP gene. Moreover, knockdown of the expression of the XRN4 gene, encoding 5'→3' exoribonuclease, inhibits biogenesis of both xrRNAs and chimeric RNAs. Our findings suggest a novel mechanism involving a unique tropology of the intermolecular base-pairing complex between xrRNAs and RNA2 to promote formation of chimeric RNAs in plants. XrRNAs, essential for chimeric RNA biogenesis, are generated through the action of cytoplasmic Xrn 4 5'→3' exoribonuclease conserved in all plant species.