RNA Folding Energy of Long-Range Genomic Interactions Regulates Discontinuous Transcription in SARS-CoV-2

Ross, Stephen J., Ye, Chengjin, Moxon, Simon ORCID: https://orcid.org/0000-0003-4644-1816, Muhlberger, Elke, Martinez-Sobrido, Luis and Cifuentes, Daniel (2026) RNA Folding Energy of Long-Range Genomic Interactions Regulates Discontinuous Transcription in SARS-CoV-2. Viruses, 18 (6). ISSN 1999-4915

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Abstract

Coronaviruses use discontinuous transcription to generate subgenomic RNAs (sgRNAs) that encode structural and accessory proteins. However, the factors regulating sgRNA abundance in SARS-CoV-2 remain unclear. Here, we combined strand-specific RNA sequencing, RNA–RNA interaction mapping, prediction of RNA folding energies, and targeted mutagenesis to define the regulation of (–) sgRNA synthesis in SARS-CoV-2 infection. We demonstrated that the relative (–) sgRNA abundance across viral genes is stable throughout infection and largely correlates with corresponding (+) sgmRNA levels. Through meta-analysis of published SPLASH data, we found that the frequency of long-range interactions between the 5′ genomic transcription regulatory sequence TRS-Leader and downstream TRS-Body sequences correlates with sgRNA abundance. Notably, the folding energy (ΔG) of these duplexes quantitatively predicts (–) sgRNA transcript levels. Mutations in non-coding regulatory regions that altered the ΔG resulted in corresponding changes in (–) sgRNA expression, suggesting a causal role for TRS duplex stability in transcriptional regulation. Analysis of naturally occurring mutations near regulatory sites further suggests that modulation of duplex stability may also serve as an evolutionary mechanism to fine-tune viral gene expression. Together, our findings identify the pairing stability of TRS-Leader:TRS-Body as a determinant of discontinuous transcription and reveal how RNA pairing potential contributes to the regulation of (–) sgRNA synthesis in SARS-CoV-2.

Item Type:	Article
Additional Information:	Data Availability Statement: Sequencing files will be deposited in Genbank. All Illumina sequencing data for this manuscript is deposited under the accession number GSE295329. Link to data: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE295329 (accessed on 21 April 2026). All SPLASH data used for analysis can be found with the accession number GSE164565 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE164565 (accessed on 28 September 2022)).
Uncontrolled Keywords:	sars-cov-2,mhv,coronavirus,discontinuous transcription,rna-rna interactions,folding energy
Faculty \ School:	Faculty of Science > School of Biological Sciences
Depositing User:	LivePure Connector
Date Deposited:	16 Jun 2026 09:26
Last Modified:	18 Jun 2026 21:01
URI:	https://ueaeprints.uea.ac.uk/id/eprint/103408
DOI:	10.3390/v18060620

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