SARS‐CoV‐2, SARS‐CoV, and MERS‐CoV encode circular RNAs of spliceosome‐independent origin

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.

New insights on circular RNAs and their potential applications as biomarkers, therapeutic agents, and preventive vaccines in viral infections: with a glance at SARS-CoV-2

“…It is reported that two circRNAs are a significant component of coronavirus transcriptome because of having ORF3a and M and containing strong IRES signals. 121 It has also been observed that SARS-CoV-2, like the majority viruses, can change the expression of host circRNAs. Consequently, 42 of the total 6,118 circRNAs were remarkably dysregulated, of which 17 circRNAs were upregulated and 25 were downregulated upon SARS-CoV-2 infection at various phases.…”

Circular RNAs as emerging regulators in COVID-19 pathogenesis and progression

“…Yang S et al. ( 17 ) predicted 351, 224, and 2764 circRNAs derived from SARS-CoV-2, SARS-CoV, and Middle East respiratory syndrome coronavirus, respectively. Moreover, 75 potential SARS-CoV-2 circRNAs were identified from RNA samples extracted from SARS-CoV-2-infected Vero E6 cells.…”

“…In addition, a study by Yang S et al. ( 17 , 98 ) found that abundant and diverse circRNAs are generated by SARS-CoV-2, SARS-CoV and MERS-CoV and represent a novel type of circRNA that differs from circRNAs encoded by DNA genomes, and forward splice junctions (FSJs) representing noncanonical “splicing” events were detected in these circRNA genome sequences. Furthermore, the study also reported the existence of alternative back-splicing events in SARS-CoV-2 circRNAs that share either 5’ or 3’ breakpoints.…”

“…CircRNAs are resistant to RNase R degradation and have a longer half-life, and their expression patterns are affected by viral infection. Several studies have demonstrated that circRNAs are abundant in the circulatory system of COVID-19 patients and may be reliable biomarkers of disease progression or prognosis ( 15 – 17 ). A study by Wu Y et al.…”

Circular RNAs Represent a Novel Class of Human Cytomegalovirus Transcripts

“…Interestingly, the most studied DNA viruses related to circRNAs are tumor viruses, including the Epstein Barr virus (EBV) ( 13 – 16 ), Kaposi Sarcoma herpesvirus (KSHV) ( 14 , 17 – 19 ), and human papillomaviruses (HPVs) ( 20 ). We recently reported that circRNAs are an important component of the transcriptome encoded by RNA viruses of the betacoronavirus genus of Coronaviridae, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), SARS-CoV, and Middle East respiratory syndrome (MERS-CoV) ( 21 ).…”