Coupling of N7-methyltransferase and 3′-5′ exoribonuclease with SARS-CoV-2 polymerase reveals mechanisms for capping and proofreading
Abstract
The capping of mRNA and the proofreading play essential roles in SARS-CoV-2 replication and transcription. Here, we present the cryo-EM structure of the SARS-CoV-2 replication-transcription complex (RTC) in a form identified as Cap(0)-RTC, which couples a co-transcriptional capping complex (CCC) composed of nsp12 NiRAN, nsp9, the bifunctional nsp14 possessing an N-terminal exoribonuclease (ExoN) and a C-terminal N7-methyltransferase (N7-MTase), and nsp10 as a cofactor of nsp14. Nsp9 and nsp12 NiRAN recruit nsp10/nsp14 into the Cap(0)-RTC, forming the N7-CCC to yield cap(0) (^(7Me)GpppA) at 5′ end of pre-mRNA. A dimeric form of Cap(0)-RTC observed by cryo-EM suggests an in trans backtracking mechanism for nsp14 ExoN to facilitate proofreading of the RNA in concert with polymerase nsp12. These results not only provide a structural basis for understanding co-transcriptional modification of SARS-CoV-2 mRNA but also shed light on how replication fidelity in SARS-CoV-2 is maintained.
Additional Information
© 2021 Elsevier Inc. Received 1 April 2021, Revised 20 April 2021, Accepted 19 May 2021, Available online 24 May 2021. This work was supported by the National Natural Science Foundation of China (U20A20135), the National Program on Key Research Project of China (2020YFA0707500 and 2017YFC0840300), Tsinghua University Spring Breeze Fund (2020Z99CFG015), and the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB37030201). Author contributions: Z.R. and Z. Lou conceived the project and designed the experiments. L.Y., Y.Y., M.L., J.G., Y.C.H., L.Z., Y.Z., S.G., Y.Y.H., and R.Z. performed sample purification, and L.Y. prepared for RTC assembly. L.Y., Y.Y., and Y.Z. for sample native electrophoretic mobility shift assays. Y.G., Z. Liu, and T.W. collected cryo-EM data. L.Y., Z. Lou, and Z.R. analyzed the data. Z.R., Z. Lou, L.Y., and L.W.G. wrote the manuscript. All authors discussed the experiments, read, and approved the manuscript. The authors declare no competing interests.Attached Files
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Supplemental Material - 1-s2.0-S0092867421006632-mmc1.pdf
Supplemental Material - 1-s2.0-S0092867421006632-mmc2.mp4
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Additional details
- PMCID
- PMC8142856
- Eprint ID
- 109697
- DOI
- 10.1016/j.cell.2021.05.033
- Resolver ID
- CaltechAUTHORS:20210701-155032256
- U20A20135
- National Natural Science Foundation of China
- 2020YFA0707500
- National Program on Key Research Project of China
- 2017YFC0840300
- National Program on Key Research Project of China
- 2020Z99CFG015
- Tsinghua University
- XDB37030201
- Chinese Academy of Sciences
- Created
-
2021-07-02Created from EPrint's datestamp field
- Updated
-
2021-07-06Created from EPrint's last_modified field
- Caltech groups
- COVID-19