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| Status |
Public on Apr 27, 2021 |
| Title |
m6A modification in SARS-CoV-2 virus regulates host cell innate immune response |
| Organisms |
Chlorocebus aethiops; Homo sapiens; Severe acute respiratory syndrome coronavirus 2 |
| Experiment type |
Expression profiling by high throughput sequencing
Methylation profiling by high throughput sequencing
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| Summary |
It is urgent and important to understand the relationship of the widespread severe acute respiratory syndrome coronavirus clade 2 (SARS-CoV-2) with host immune response and study the underlying molecular mechanism. RNA modification landscape of SARS-CoV-2 and its functional relevance to host cell innate immune response remain unknown. N6-methylation of adenosine (m6A) in RNA regulates many physiological and disease processes. Here, we investigated m6A modification of SARS-CoV-2 gene in regulating host cell innate immune response. Our data showed that SARS-CoV-2 virus has m6A modification enriched in 3' region of the viral genome. We also found that host cell m6A methyltransferase METTL3 depletion reduced viral load in infected cells, decreased m6A levels in SARS-CoV-2 and host genes, and m6A reduction in viral RNA increased RIG-1 binding and subsequently enhanced downstream innate immune signaling pathway and inflammatory gene expression. METTL3 expression is reduced and inflammatory genes are induced in severe COVID-19 patients. These findings will aid to understand the COVID-19 pathogenesis and help in designing future studies of regulating innate immunity for COVID-19 treatment.
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| Overall design |
For RNA-Seq, total RNAs were extracted from SARS-CoV-2-infected shControl and shMETTL3 Caco-2 cells. Sequencing was performed by Illumina NovaSeq 6000 at the IGM Genomics Center, UCSD. For MeRIP-Seq, supernatant or cellular RNAs were purified from viral infected-Vero or Caco-2 cells. The RNAs were treated with DNaseI, concentrated and quantified. Purified RNA was fragmented to 100–200 nucleotides using Ambion RNA Fragmentation Reagent (AM8740, Life Technologies) and the fragmented RNA purified by ethanol precipitation. An input sample (10% of total fragmented RNA) was reserved. Fragmented RNA was incubated with 10 μl rabbit anti-m6A polyclonal Ab (ab151230, Abcam) in IP binding buffer (10 mM Tris-HCl, 150 mM NaCl, 0.1% NP-40, pH 7.4) for 2 h at 4°C. The mixture was then incubated with 50 μl protein A/G magnetic beads (Thermo Fisher) for 2 h at 4°C, and the beads were collected and washed twice in IP wash buffer (10 mM Tris-HCl, 1 M NaCl, 0.1% NP-40, pH 7.4). Bound RNA was eluted from the beads with m6A elution buffer (10 mM Tris-HCl, 1 M NaCl, 0.1% NP-40, 25 mM m6A, pH 7.4) and extracted with RNA Clean & Concentrator Kits (ZYMO). Input and m6A-containing viral RNA were dissolved in water and either reverse transcribed by iScript cDNA synthesis kit (Bio-Rad) and analyzed by qPCR or processed for library generation using a TruSeq mRNA library preparation kit (Illumina). Sequencing was performed by Illumina NovaSeq 6000 at the IGM Genomics core, UCSD.
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| Contributor(s) |
Li N, Hui H, Rana T |
| Citation(s) |
33961823 |
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| Submission date |
Feb 19, 2021 |
| Last update date |
May 12, 2021 |
| Contact name |
Na Li |
| E-mail(s) |
nal023@health.ucsd.edu
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| Organization name |
University of California, San Diego
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| Department |
Pediatrics
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| Lab |
Tariq Rana's Lab
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| Street address |
9500 Gilman Dr
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| City |
La Jolla |
| State/province |
California |
| ZIP/Postal code |
92037 |
| Country |
USA |
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| Platforms (2) |
| GPL29320 |
Illumina NovaSeq 6000 (Homo sapiens; Severe acute respiratory syndrome coronavirus 2) |
| GPL29742 |
Illumina NovaSeq 6000 (Chlorocebus aethiops; Severe acute respiratory syndrome coronavirus 2) |
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| Samples (16)
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| Relations |
| BioProject |
PRJNA702920 |
| SRA |
SRP307098 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE167075_RAW.tar |
386.6 Mb |
(http)(custom) |
TAR (of BEDGRAPH, BW) |
| GSE167075_shM3_vs_NTC_human_.xlsx |
8.3 Mb |
(ftp)(http) |
XLSX |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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