RNA Architecture Underlies Discontinuous Transcription and Evolution of Coronavirus

Coronaviruses employ discontinuous transcription to produce canonical subgenomic RNAs (sgRNAs) essential for gene expression, as well as non-canonical sgRNAs with unclear function. Nevertheless, the mechanisms regulating the balance between canonical and non-canonical sgRNAs throughout the viral replication cycle remain unknown. We conducted an integrated analysis on 234 transcriptome and 12 RNA-RNA interactome samples across various coronavirus genera. RNA-RNA interactions were identified between TRS-B and TRS-L flanking regions in the same genomic direction, correlating with the formation of canonical junctions. Non-canonical junctions frequently span short or long genomic distance, with short-range junctions generally mediated by reverse complementary stem-loops and coinciding with genomic deletion regions. Conserved long-range non-canonical sgRNAs were identified across different coronaviruses, and these sgRNAs harbor ORF10 or an evolving gene to suppress antiviral innate immune responses. This work establishes a structural framework that enables an understanding of the regulatory mechanism behind discontinuous transcription and the evolutionary pathway of coronaviruses.