Methyltransferase-like 3 Modulates Severe Acute Respiratory Syndrome Coronavirus-2 RNA N6-Methyladenosine Modification and Replication

Abstract

Internal chemical modifications of viral RNA play key roles in the regulation of viral replication and gene expression. Although potential internal modifications have been reported in SARS-CoV-2 RNA, the function of the SARS-CoV-2 N6-methyladenosine (m ⁶ A) modification in the viral life cycle is unclear.

SciScore for 10.1101/2020.10.14.338558: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Institutional Review Board Statement	not detected.
Randomization	not detected.
Blinding	not detected.
Power Analysis	not detected.
Sex as a biological variable	not detected.
Cell Line Authentication	not detected.

Table 2: Resources

Antibodies
Sentences	Resources
The primary antibodies were as follows: anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH; cat. no. 60004-1-lg; Proteintech, Rosemont, IL, USA), mouse monoclonal anti-β-actin (cat. no. sc47778; Santa Cruz Biotechnology, Dallas, TX,	anti-glyceraldehyde 3-phosphate dehydrogenase suggested: None anti-β-actin suggested: (Santa Cruz Biotechnology Cat# sc-47778 HRP, RRID:AB_2714189)
The secondary antibodies including goat anti-mouse IgG and goat anti-rabbit IgG …

SciScore for 10.1101/2020.10.14.338558: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Institutional Review Board Statement	not detected.
Randomization	not detected.
Blinding	not detected.
Power Analysis	not detected.
Sex as a biological variable	not detected.
Cell Line Authentication	not detected.

Table 2: Resources

Antibodies
Sentences	Resources
The primary antibodies were as follows: anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH; cat. no. 60004-1-lg; Proteintech, Rosemont, IL, USA), mouse monoclonal anti-β-actin (cat. no. sc47778; Santa Cruz Biotechnology, Dallas, TX,	anti-glyceraldehyde 3-phosphate dehydrogenase suggested: None anti-β-actin suggested: (Santa Cruz Biotechnology Cat# sc-47778 HRP, RRID:AB_2714189)
The secondary antibodies including goat anti-mouse IgG and goat anti-rabbit IgG (AntiGene Biotech GmbH, Stuttgart, Germany) were incubated for 1 h at a dilution of 1:5000.	anti-mouse IgG suggested: (LSBio (LifeSpan Cat# LS-C69682-5000, RRID:AB_1653096) anti-rabbit IgG suggested: None AntiGene Biotech GmbH , Stuttgart , Germany suggested: None
The lysates were then centrifugated at 16,000 × g for 10 min, and supernatants were subjected to IP with IgG or anti-Flag antibodies overnight.	anti-Flag suggested: None
MeRIP and northern blotting: For MeRIP and northern blotting, 400 μg total RNA from virus-infected Vero-E6 cells was incubated with an anti-m6A antibody (Synaptic Systems, Goettingen, Germany) or an IgG antibody in 300 μL IP buffer (150 mM NaCl, 0.1% NP-40, 10 mM Tris-HCl [pH 7.4]) for 2 h at 4°C.	anti-m6A suggested: None an IgG suggested: (Hangzhou HuaAn Biotechnology Cat# HA1001, RRID:AB_2819166)
Experimental Models: Cell Lines
Sentences	Resources
Virus, cell lines, and cell culture: SARS-CoV-2 was isolated from the bronchoalveolar lavage fluid of a patient (49) and passaged in monkey kidney cells (Vero-E6 cells) for eight generations.	Vero-E6 suggested: None
Vero-E6 cells (American Tissue Culture Collection [ATCC], Manassas, VA, USA; CRL-1586) and HEK293T cell (ATCC; CRL-11268) were cultured in Dulbecco’s modified Eagle’s medium 116 (Gibco, Gaithersburg, MD, USA) containing 10% fetal bovine serum (Gibco) at 37°C with 5% CO2.	HEK293T suggested: ATCC Cat# CRL-11268, RRID:CVCL_1926)
Software and Algorithms
Sentences	Resources
Quantitative reverse transcription polymerase chain reaction (qRT-PCR): Total RNA was extracted from SARS-CoV-2-infected Vero-E6 cells, and reverse transcription was performed using a HiSeript first-strand cDNA synthesis kit (Vazyme Biotech Co, Nanjing, China) according to the manufacturer’s instructions, followed by quantitative PCR with SYBR Green (Yeasen Biotech Co, Shanghai, China) on a CFX Connect Real-Time system (Bio-Rad Laboratories, Hercules, CA, USA).	Bio-Rad Laboratories suggested: (Bio-Rad Laboratories, RRID:SCR_008426)
Statistical analysis: The statistical analysis of qRT-PCR data was performed using two-tail unpaired t-tests in GraphPad Prism Software (GraphPad Software, La Jolla, CA, USA.).	GraphPad Prism suggested: (GraphPad Prism, RRID:SCR_002798) GraphPad suggested: (GraphPad Prism, RRID:SCR_002798)

Results from OddPub: Thank you for sharing your data.

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 found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

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.

Read the original source

Methyltransferase-like 3 Modulates Severe Acute Respiratory Syndrome Coronavirus-2 RNA N6-Methyladenosine Modification and Replication

This article has been Reviewed by the following groups

Listed in

Abstract

Article activity feed