The SARS-CoV-2 nucleocapsid protein preferentially binds long and structured RNAs
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Abstract
The SARS-CoV-2 nucleocapsid protein (NCAP) functions in viral RNA genome packaging, virion assembly, RNA synthesis and translation, and regulation of host immune response. RNA-binding is central to these processes. Little is known how NCAP selects its binding partners in the myriad of host and viral RNAs. To address this fundamental question, we employed electrophoresis mobility shift and competition assays to compare NCAP binding to RNAs that are of SARS-CoV-2 vs. non-SARS-CoV-2, long vs. short, and structured vs. unstructured. We found that although NCAP can bind all RNAs tested, it primarily binds structured RNAs, and their association suppresses strong interaction with single-stranded RNAs. NCAP prefers long RNAs, especially those containing multiple structures separated by single-stranded linkers that presumably offer conformational flexibility. Additionally, all three major regions of NCAP bind RNA, including the low complexity domain and dimerization domain that promote formation of NCAP oligomers, amyloid fibrils and liquid-liquid phase separation. Combining these observations, we propose that NCAP-NCAP interactions that mediate higher-order structures during packaging also drive recognition of the genomic RNA and call this mechanism recognition-by-packaging. This study provides a biochemical basis for understanding the complex NCAP-RNA interactions in the viral life cycle and a broad range of similar biological processes.
HIGHLIGHTS
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NCAP primarily binds structured RNAs.
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NCAP prefers multiple RNA structures separated by single-stranded linkers.
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NCAP favors binding to long RNAs.
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SciScore for 10.1101/2021.12.25.474155: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics not detected. Sex as a biological variable not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Table 2: Resources
Recombinant DNA Sentences Resources In vitro transcription and purification: The sequences of S1hp, S1.5hp, and S2hp were cloned from a gBlock containing the 5’ 1,000 nt of SARS-CoV-2 genome into the pUC19 vector using restriction sites EcoRI and KpnI. pUC19suggested: RRID:Addgene_50005)Briefly, the coding sequences for NCAP and its truncations with N-terminal 6xHis-SUMO tag were cloned into the pEC-28a vector. pEC-28asuggested: NoneSoftware and Algorithms Sentences Resources The gel images were obtained using an Amersham™ Typhoon™ scanner (Cytiva) and … SciScore for 10.1101/2021.12.25.474155: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics not detected. Sex as a biological variable not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Table 2: Resources
Recombinant DNA Sentences Resources In vitro transcription and purification: The sequences of S1hp, S1.5hp, and S2hp were cloned from a gBlock containing the 5’ 1,000 nt of SARS-CoV-2 genome into the pUC19 vector using restriction sites EcoRI and KpnI. pUC19suggested: RRID:Addgene_50005)Briefly, the coding sequences for NCAP and its truncations with N-terminal 6xHis-SUMO tag were cloned into the pEC-28a vector. pEC-28asuggested: NoneSoftware and Algorithms Sentences Resources The gel images were obtained using an Amersham™ Typhoon™ scanner (Cytiva) and were quantified using Quantity One (BIO-RAD, version 4.6.9) or ImageJ. Quantity Onesuggested: (Quantity One 1-D Analysis Software, RRID:SCR_014280)ImageJsuggested: (ImageJ, RRID:SCR_003070)The data were fitted and plotted using Prism (GraphPad, version 8). Prismsuggested: (PRISM, RRID:SCR_005375)GraphPadsuggested: (GraphPad Prism, RRID:SCR_002798)Statistical analyses: Unpaired Student’s t-tests with two tails were performed in Excel (Microsoft). Excelsuggested: NoneResults from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: We did not find any issues relating to colormaps.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
- Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
- No protocol registration statement was detected.
Results from scite Reference Check: We found no unreliable references.
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