Switching of OAS1 splicing isoforms mitigates SARS-CoV-2 infection
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Abstract
Background
The rapidly accumulating disease susceptibility information collected from coronavirus disease (COVID-19) patient genomes must be urgently utilized to develop therapeutic interventions for SARS-CoV-2 infection. Chromosome 12q24.13, which encodes the 2’-5’-oligoadenylate synthetase (OAS) family of proteins that sense viral genomic RNAs and trigger an antiviral response, is identified as one of the genomic regions that contains SNPs associated with COVID-19 severity. A high-risk SNP identified at the splice acceptor site of OAS1 exon 6 is known to change the proportions of the various splicing isoforms and the activity of the enzyme.
Methods
We employed in-silico motif search and RNA pull-down assay to define a factor responsible for the OAS1 splicing. Next, we rationally selected a candidate for slicing modulator to modulate this splicing.
Results
We found that inhibition of CDC-like kinase with a small chemical compound induces switching of OAS1 splice isoforms in human lung cells. In this condition, increased resistance to SARS-CoV-2 infection, enhanced RNA degradation, and transcriptional activation of interferon β1, were also observed.
Conclusions
The results indicate the possibility of using chemical splicing modifiers aided by genome-based precision medicine to boost the innate immune response against SARS-CoV-2 infection.
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SciScore for 10.1101/2021.08.23.457314: (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. Cell Line Authentication Contamination: All cells were maintained in an incubator at 37 °C with 5 % CO2, and mycoplasma was confirmed negative in routine polymerase chain reaction tests. Table 2: Resources
Antibodies Sentences Resources Eluted proteins were then analyzed by western blotting with anti-U1-70k mouse monoclonal antibody (9C4.1) ( anti-U1-70ksuggested: None05-1588, Merk Millipore, Burlington, MA, USA) at a dilution of 1:500 for the detection of U1-70k, anti-SR protein (1H4G7) mouse monoclonal antibody (33-9400, Thermo Fisher Scientific, Waltham, MA, USA) at a … SciScore for 10.1101/2021.08.23.457314: (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. Cell Line Authentication Contamination: All cells were maintained in an incubator at 37 °C with 5 % CO2, and mycoplasma was confirmed negative in routine polymerase chain reaction tests. Table 2: Resources
Antibodies Sentences Resources Eluted proteins were then analyzed by western blotting with anti-U1-70k mouse monoclonal antibody (9C4.1) ( anti-U1-70ksuggested: None05-1588, Merk Millipore, Burlington, MA, USA) at a dilution of 1:500 for the detection of U1-70k, anti-SR protein (1H4G7) mouse monoclonal antibody (33-9400, Thermo Fisher Scientific, Waltham, MA, USA) at a dilution of 1:200 for phosphorylated SRSF6, and anti-β-actin ( U1-70k , anti-SR protein ( 1H4G7 )suggested: Noneanti-β-actinsuggested: NoneACTB) mouse monoclonal antibody (Ac-15) (sc-69879, Santa Cruz Biotechnology, Dallas, TX, USA) at a dilution of 1:4,000 for ACTB. sc-69879suggested: (Santa Cruz Biotechnology Cat# sc-69879, RRID:AB_1119529)Experimental Models: Cell Lines Sentences Resources Transcriptome analysis for CaNDY-treated Calu-3 cells: RNAs were extracted using RNeasy Mini kit (QIAGEN, Hilden, Germany) from Calu-3 cells, treated with 10 µM CaNDY or 0.1% DMSO for 18 h, and applied for RNA-Seq analysis. Calu-3suggested: NoneRNA samples were collected from the cells 24 h post-infection (pi), and the virus titers were determined by the 50% tissue culture infectious dose (TCID50) using VeroE6/TMPRSS2 cells at 48 h pi. VeroE6/TMPRSS2suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)Software and Algorithms Sentences Resources RNA-seq reads were mapped to the human genome sequences (GRCh38) using STAR (ver. 2.7.1a, https://github.com/alexdobin/STAR) with ENCODE options, using the Ensembl genome annotation (ver. 102) STARsuggested: (STAR, RRID:SCR_004463)https://github.com/alexdobin/STARsuggested: (Hamilton Microlab STAR Automated Liquid Handling, RRID:SCR_019993)Raw reads were counted with bam files, and TPM values were calculated using RSEM v1.2.31 ( RSEMsuggested: (RSEM, RRID:SCR_013027)For characterizing gene set and transcriptome profiles, we used the Metascape website (https://metascape.org/) and Gene Set Enrichment Analysis (GSEA, https://www.gsea-msigdb.org/gsea/). Metascapesuggested: (Metascape, RRID:SCR_016620)The original RNA-seq data were deposited at the Gene Expression Omnibus (GEO) of National Center for Biotechnology Information (NCBI) with the accession ID GSE174398. Gene Expression Omnibussuggested: (Gene Expression Omnibus (GEO, RRID:SCR_005012)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 did not find any issues relating to the usage of bar graphs.
Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on page 27. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
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- No protocol registration statement was detected.
Results from scite Reference Check: We found no unreliable references.
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