Single cell RNA-seq uncovers the nuclear decoy lincRNA PIRAT as a regulator of systemic monocyte immunity during COVID-19

  1. Marina Aznaourova
  2. Nils Schmerer
  3. Harshavardhan Janga
  4. Zhenhua Zhang
  5. Kim Pauck
  6. Judith Hoppe
  7. Sarah M Volkers
  8. Daniel Wendisch
  9. Philipp Georg
  10. Margrit Gündisch
  11. Elisabeth Mack
  12. Chrysanthi Skevaki
  13. Christian Keller
  14. Christian Bauer
  15. Wilhelm Bertrams
  16. Andrea Nist
  17. Thorsten Stiewe
  18. Achim D Gruber
  19. Clemens Ruppert
  20. Yang Li
  21. Holger Garn
  22. Leif E Sander
  23. Bernd Schmeck
  24. Leon N Schulte

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Abstract

The systemic immune response to viral infection is shaped by master transcription factors such as NFκB or PU.1. Although long non-coding RNAs (lncRNAs) have been suggested as important regulators of transcription factor activity, their contributions to the systemic immunopathologies observed during SARS-CoV-2 infection have remained unknown. Here, we employed a targeted single-cell RNA-seq approach to reveal lncRNAs differentially expressed in blood leukocytes during severe COVID-19. Our results uncover the lncRNA PIRAT as a major PU.1 feedback-regulator in monocytes, governing the production of the alarmins S100A8/A9 – key drivers of COVID-19 pathogenesis. Knockout and transgene expression, combined with chromatin-occupancy profiling characterized PIRAT as a nuclear decoy RNA, diverting the PU.1 transcription factor from alarmin promoters to dead-end pseudogenes in naïve monocytes. NFκB-dependent PIRAT down-regulation during COVID-19 consequently releases a transcriptional brake, fueling alarmin production. Our results suggest a major role of nuclear noncoding RNA circuits in systemic antiviral responses to SARS-CoV-2 in humans.

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    SciScore for 10.1101/2021.11.05.467458: (What is this?)

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

    Table 1: Rigor

    EthicsIRB: The BioInflame study was approved by the ethics committee of the Charité - Universitätsmedizin Berlin (EA2/030/09) and the University Medical Center Marburg (55/17).
    Consent: All blood donors were at least 18 years of age and provided written informed consent for use of their blood samples for scientific purposes.
    Sex as a biological variableImmunosuppressed, pregnant and HIV-positive patients were excluded from the study.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    1 C1 + A7 antibody or FLAG antibody (Table S8), as described by Tawk et al. (45) and added to the diluted lysate, followed by rotation at 4 °C over-night.
    FLAG
    suggested: None
    Cells were washed twice with BD Pharmingen Stain Buffer (Cat. No. 554656) and labelled cell suspensions were pooled and incubated with oligo-labelled AbSeq antibodies directed against CD206 (Cat. No. 940068), CD163 (Cat. No. 940058) and HLA-DR (Cat. No. 940010) for 30 minutes on ice.
    CD206
    suggested: (BD Biosciences Cat# 940068, RRID:AB_2875959)
    CD163
    suggested: (BD Biosciences Cat# 940058, RRID:AB_2875949)
    HLA-DR
    suggested: (BD Biosciences Cat# 940010, RRID:AB_2875901)
    Experimental Models: Cell Lines
    SentencesResources
    THP1 and Hek293T cells were purchased from ATCC and cultured in RPMI 1640 (Thermo Fisher), 10 % FCS, 1% penicillin/streptomycin solution (Thermo Fisher).
    THP1
    suggested: None
    Lentiviral transduction: HEK293T cells were co-transfected with lentiviral vector, pseudotyping- and helper-plasmid (pVSVG and psPAX2) using lipofectamine 2000 (Thermo Fisher).
    HEK293T
    suggested: None
    Recombinant DNA
    SentencesResources
    Control cells were generated using a pX458 vector with scrambled gRNA.
    pX458
    suggested: RRID:Addgene_101731)
    Lentiviral transduction: HEK293T cells were co-transfected with lentiviral vector, pseudotyping- and helper-plasmid (pVSVG and psPAX2) using lipofectamine 2000 (Thermo Fisher).
    pVSVG
    suggested: RRID:Addgene_85140)
    psPAX2
    suggested: RRID:Addgene_12260)
    Software and Algorithms
    SentencesResources
    After pre-processing of BD Rhapsody scRNA-seq data, read counts were loaded into the R (v3.6.3) environment and further analyzed using the Seurat package (v3.1.4).
    Seurat
    suggested: (SEURAT, RRID:SCR_007322)
    S1A) were obtained through NCBI Sequence Read Archive (datasets ERR030888-ERR030903)
    NCBI Sequence Read Archive
    suggested: (NCBI Sequence Read Archive (SRA, RRID:SCR_004891)
    Reads in fastq-format were quality-trimmed and mapped to the human GRCh38 reference (GENCODE), using the CLC genomics workbench.
    GENCODE
    suggested: (GENCODE, RRID:SCR_014966)
    Hierarchical clustering was done using Cluster 3.0 (Eisen lab).
    Cluster
    suggested: (Cluster, RRID:SCR_013505)
    For pathway enrichment analysis and induced network analysis ConsensusPathDB (48) was used.
    ConsensusPathDB
    suggested: (ConsensusPathDB, RRID:SCR_002231)
    Other plots were generated using GraphPad Prism, Excel or BoxPlotR (http://shiny.chemgrid.org/boxplotr/).
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    Excel
    suggested: None
    BoxPlotR
    suggested: (BoxPlotR, RRID:SCR_015629)
    Sequence conservation was determined using NCBI BLASTN and the major species reference genome, respectively.
    BLASTN
    suggested: (BLASTN, RRID:SCR_001598)
    BLAST hits with ≥ 20 complementary nucleotides located within a genomic range of max.
    BLAST
    suggested: (BLASTX, RRID:SCR_001653)

    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: 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.

    About SciScore

    SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

  2. Version published to 10.1101/2021.11.05.467458 on bioRxiv