Abstract
Circular RNAs (circRNAs) are a newly recognized component of the transcriptome with critical roles in autoimmune diseases and viral pathogenesis. To address the importance of circRNA in RNA viral transcriptome, we systematically identified and characterized circRNAs encoded by the RNA genomes of betacoronaviruses using both bioinformatical and experimental approaches. We predicted 351, 224, and 2764 circRNAs derived from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), SARS-CoV, and Middle East respiratory syndrome coronavirus, respectively. We experimentally identified 75 potential SARS-CoV-2 circRNAs from RNA samples extracted from SARS-CoV-2-infected Vero E6 cells. A systematic comparison of viral and host circRNA features, including abundance, strand preference, length distribution, circular exon numbers, and breakpoint sequences, demonstrated that coronavirus-derived circRNAs had a spliceosome-independent origin. We further showed that back-splice junctions (BSJs) captured by inverse reverse-transcription polymerase chain reaction have different level of resistance to RNase R. Through northern blotting with a BSJ-spanning probe targeting N gene, we identified three RNase R-resistant bands that represent SARS-CoV-2 circRNAs that are detected cytoplasmic by single-molecule and amplified fluorescence in situ hybridization assays. Lastly, analyses of 169 sequenced BSJs showed that both back-splice and forward-splice junctions were flanked by homologous and reverse complementary sequences, including but not limited to the canonical transcriptional regulatory sequences. Our findings highlight circRNAs as an important component of the coronavirus transcriptome, offer important evaluation of bioinformatic tools in the analysis of circRNAs from an RNA genome, and shed light on the mechanism of discontinuous RNA synthesis.
Keywords: RNA biology; SARS-CoV-2; circular RNA; coronavirus; virology.
【저자키워드】 SARS-CoV-2, coronavirus, Virology., RNA Biology, circular RNA, 【초록키워드】 Transcriptome, Virology, SARS-CoV, viral pathogenesis, severe acute respiratory syndrome Coronavirus, RNA, Autoimmune disease, polymerase chain reaction, Features, Middle East respiratory syndrome Coronavirus, Fluorescence, VERO E6 cells, N gene, Autoimmune diseases, distribution, homologous, Critical, mechanism, In situ hybridization, betacoronaviruses, RNase, Analysis, complementary, Middle East, Vero E6, viral transcriptome, Chain Reaction, not limited, acute respiratory syndrome, acute respiratory syndrome coronavirus, acute respiratory syndrome coronavirus 2, approaches, RNA synthesis, RNA samples, abundance, transcriptional regulatory sequences, RNA genome, respiratory syndrome coronavirus, offer, infected Vero E6 cells, probe, Host, highlight, junction, RNA genomes, polymerase chain, breakpoint, amplified, predicted, sequenced, assays, characterized, cytoplasmic, demonstrated, analysis, canonical, betacoronavirus, RNA sample, Circular, 【제목키워드】 SARS-CoV, MERS-CoV, RNA,