A virus‐derived microRNA targets immune response genes during SARS‐CoV‐2 infection
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This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/5514037.
In this preprint, Singh et al. identify two novel small RNA transcripts produced during SARS-COV-2 infection. The authors go on to demonstrate Dicer-mediated miR-7a production, gene-specific targeting of interferon stimulated genes (ISGs) during the course of infection, and use bioinformatic analyses from clinical isolates to demonstrate these miR transcripts are not a cell culture artifact.
Methodology is well-described and conclusions are well justified. The manuscript's one limitation is a loss of function study in which the authors mutate the endogenous MiR-7a sequences and evaluating whether they see decreases in ISG expression or diminished virulence of the mutant strain. Still, the …
This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/5514037.
In this preprint, Singh et al. identify two novel small RNA transcripts produced during SARS-COV-2 infection. The authors go on to demonstrate Dicer-mediated miR-7a production, gene-specific targeting of interferon stimulated genes (ISGs) during the course of infection, and use bioinformatic analyses from clinical isolates to demonstrate these miR transcripts are not a cell culture artifact.
Methodology is well-described and conclusions are well justified. The manuscript's one limitation is a loss of function study in which the authors mutate the endogenous MiR-7a sequences and evaluating whether they see decreases in ISG expression or diminished virulence of the mutant strain. Still, the present work demonstrates a novel and compelling discovery that will contribute to our emerging understanding of host-pathogen interactions during SARS-CoV-2 infection.
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SciScore for 10.1101/2021.09.09.459577: (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 Size selected RNA was used to prepare libraries following previously described methodology (Barucci et al, 2020), which included the ligation of 3’end and 5’end adaptors each having four randomized nucleotides to minimize ligation biases. Blinding not detected. Power Analysis not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Cells were incubated with antibodies recognizing the spike protein of SARS-CoV-2 (anti-S2 H2 162, a kind gift from Dr. Hugo Mouquet, Institut Pasteur, Paris, France) for 30 minutes at 4°C, and then with secondary antibodies (anti-human-Alexa Fluor-647) for 30 … SciScore for 10.1101/2021.09.09.459577: (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 Size selected RNA was used to prepare libraries following previously described methodology (Barucci et al, 2020), which included the ligation of 3’end and 5’end adaptors each having four randomized nucleotides to minimize ligation biases. Blinding not detected. Power Analysis not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Cells were incubated with antibodies recognizing the spike protein of SARS-CoV-2 (anti-S2 H2 162, a kind gift from Dr. Hugo Mouquet, Institut Pasteur, Paris, France) for 30 minutes at 4°C, and then with secondary antibodies (anti-human-Alexa Fluor-647) for 30 minutes at 4°C. anti-S2suggested: Noneanti-human-Alexasuggested: NoneFrom the total lysate (~0.5 mg/ml), 10 % lysate was saved as input and IP was performed using an anti-pan-AGO antibody (clone 2A8, MABE56 Sigma-Aldrich) and an anti-FLAG M2 antibody (F3165, Sigma-Aldrich) was used for control IPs. anti-pan-AGOsuggested: (Z. Mourelatos Lab - University of Pennsylvania Cat# pan-AGO (7G1-1, RRID:AB_2827163)anti-FLAGsuggested: (Sigma-Aldrich Cat# F3165, RRID:AB_259529)Experimental Models: Cell Lines Sentences Resources A549-ACE2, Caco-2 and Vero E6 cells were cultured in high-glucose DMEM media (Gibco) supplemented with 10% Fetal Bovine Serum (FBS; Sigma) and 1% penicillin-streptomycin (P/S; Gibco). Vero E6suggested: NoneCells were then lysed using TRIzol LS reagent (Thermo Fischer Scientific) and RNA was extracted following the manufacturer’s instructions or lysed in RIPA buffer (Thermo Fischer Scientific) for western blot analysis. siRNA-mediated knockdown: A549-ACE2 cells were transfected using Lipofectamine RNAiMax (Life Technologies) with 10 nM of control (#4390843, Ambion) or DICER1 siRNAs (#4390824, Ambion), following the manufacturer’s instructions. A549-ACE2suggested: NoneImmunoprecipitation: A549-ACE2 and Caco-2 cells, infected and not, were lysed in FA buffer as described above. Caco-2suggested: CLS Cat# 300137/p1665_CaCo-2, RRID:CVCL_0025)Software and Algorithms Sentences Resources Cells were acquired on an Attune NxT Flow Cytometer (Thermo Fisher) and data analyzed with FlowJo software. FlowJosuggested: (FlowJo, RRID:SCR_008520)Reads were aligned on the Homo sapiens genome (Build version GRCh38, NCBI) using Hisat2 (Kim et al, 2015) Hisat2suggested: (HISAT2, RRID:SCR_015530)After alignment, reads mapping to annotated protein-coding genes were counted using featureCounts (version 2.0.1). featureCountssuggested: (featureCounts, RRID:SCR_012919)Counted reads for protein-coding genes were used for differential expression analysis using the R/Bioconductor package DESeq2 (Love et al, 2014) (version 1.26.0). R/Bioconductorsuggested: NoneDESeq2suggested: (DESeq, RRID:SCR_000154)The selected 18-26-nucleotide reads were aligned on the SARS-CoV-2 genome (assembly Jan.2020/NC_045512v2, UCSC) or on the Homo sapiens genome (Build version GRCh38, NCBI) using Bowtie2 (Langmead & Salzberg, 2012; Li et al, 2009) v.2.4.2 with the following parameters: -L 6 -i S,1,0.8 -N 0 Bowtie2suggested: (Bowtie 2, RRID:SCR_016368)First, a bed file with the coordinates of all the putative 22-nucleotide RNAs encoded by the SARS-CoV-2 genome was created and used to extract their sequences using bedtools. bedtoolssuggested: (BEDTools, RRID:SCR_006646)To compute the size distribution around a specific region, reads mapping to a specific region of the genome were extracted using samtools and size distribution was computed using bioawk. samtoolssuggested: (SAMTOOLS, RRID:SCR_002105)2 complementary sites on human 3’UTR: The sequences of the 3’UTR of human genes have been retrieved using the Ensembl BioMart (database Ensembl Genes 101 – Human genes (GRCh38.p13)). Ensembl BioMartsuggested: NoneResults from OddPub: Thank you for sharing your data.
Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:One limitation of this study is that we could not evaluate whether the presence and abundance of the CoV2-miR-7a.2 correlates with disease progression or with impaired ISGs activation in patients with severe COVID-19 disease outcome. Thus, future studies will address the relevance of the CoV2-miR-7a in the progression of the COVID-19 disease. Furthermore, the recent evolution of the CoV2-miR-7a sequence in the SARS-CoV-2 genome may facilitate the development of specific therapeutic approaches to potentially target and dampen the virulence of SARS-CoV-2 infection in COVID-19 patients.
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.
Results from scite Reference Check: We found no unreliable references.
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