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| Status |
Public on Oct 21, 2020 |
| Title |
The short- and long-range RNA Interactome of SARS-CoV-2 |
| Organisms |
Homo sapiens; Chlorocebus pygerythrus |
| Experiment type |
Other
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| Summary |
The Coronaviridae are a family of positive- strand RNA viruses that includes SARS-CoV-2, the etiologic agent of the COVID-19 pandemic. Bearing the largest single stranded RNA genome in nature, coronaviruses are critically dependent on long-distance RNA-RNA interactions to regulate the viral transcription and replication pathways. Here we experimentally mapped the in vivo long-range RNA interactome of the full-length SARS-CoV-2 genome and the subgenomic mRNAs. We uncover a network of RNA-RNA interactions spanning tens of thousands of nucleotides that facilitate the unique transcription mode of coronaviruses, and reveal that the viral genome adopts alternative topologies inside cells and undergoes genome cyclization. Moreover, we discover long RNA-bridges between adjacent open reading frames that encircle the programmed ribosomal frame-shifting element, and demonstrate their conservation in vivo for the related MERS-CoV. Finally, the SARS-CoV-2 genome and subgenomic mRNAs engage in different sets of interactions with cellular RNAs. Our findings illuminate RNA-based mechanisms governing replication, discontinuous transcription, and translation of coronaviruses, and will aid future efforts to develop antiviral strategies.
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| Overall design |
We recently developed Crosslinking Of Matched RNAs And Deep Sequencing (COMRADES) for in-depth RNA conformation capture in living cells (Ziv et al., 2018). COMRADES can detect base-paired regions in RNA inside the cell, using a clickable psoralen derivative to specifically crosslink double-stranded RNA, and high throughput sequencing to retrieve the base-pairing information (Figure S1A). Following in vivo crosslinking, the viral RNA is selectively captured, fragmented and subjected to a click-chemistry reaction to add a biotin tag to crosslinked regions. Crosslinked RNA duplexes are then selectively captured using streptavidin affinity purification. Half of the resulting RNA is proximity ligated, following reversal of the crosslink to create chimeric RNA templates for high throughput sequencing. The other half is used as a control, in which reversal of the crosslink precedes the proximity ligation, and accurately represents the background level of non-specific ligation. The coupling of two enrichment steps, first of viral RNA, and second of crosslinked RNA duplexes provides high structural depth for identification of both long- and short-lived conformations. COMRADES can therefore measure (i) the structural diversity of alternative RNA conformations that coexist inside cells; (ii) short-distance, as well as long-distance (over tens of thousands of nucleotides) base-pairing within the same RNA molecule; and (iii) base-pairing between different RNA molecules, such as those of host and viral origin (Kudla et al., 2020; Ziv et al., 2018). Here we applied COMRADES to study the structural diversity of SARS-CoV-2 gRNA and sgmRNAs inside cells. Whereby we have conducted in duplicate - MERS and SARS-CoV-2 gRNA as well as MERS and SARS-CoV-2 sgmRNAs.
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| Contributor(s) |
Ziv O, Price J, Shalamova L, Kamenova T, Goodfellow I, Weber F, Miska EA |
| Citation missing |
Has this study been published? Please login to update or notify GEO. |
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| Submission date |
Jul 17, 2020 |
| Last update date |
Oct 24, 2020 |
| Contact name |
Jonathan Lewis Price |
| Organization name |
University of Cambridge
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| Department |
The Gurdon Inst.
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| Lab |
Miska Lab
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| Street address |
The Gurdon Inst. Tennis Court Road
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| City |
cambridge |
| ZIP/Postal code |
CB2 1QN |
| Country |
United Kingdom |
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| Platforms (2) |
| GPL24676 |
Illumina NovaSeq 6000 (Homo sapiens) |
| GPL28888 |
Illumina NovaSeq 6000 (Chlorocebus pygerythrus) |
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| Samples (16)
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| Relations |
| BioProject |
PRJNA646969 |
| SRA |
SRP272408 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE154662_RAW.tar |
1.8 Gb |
(http)(custom) |
TAR (of TXT) |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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