Characterization of H1N1 and interferon-β-responsive long non-coding RNAs using single-cell CRISPRi in human lung epithelial and immune cell lines 2260688
Jul 2026· Journal of Immunology· Vol 215· 0 citations
TL;DR
Two novel interferon-responsive human lncRNAs that may play important roles in the regulation of the host interferon response across different cell types are identified.
Abstract
Despite 36,000 long non-coding RNAs (lncRNAs) annotated in the human genome, functional characterization remains limited, and the roles of lncRNAs in the interferon (IFN) response are largely unknown. We previously identified VILMIR as an IFN-stimulated lncRNA that enhances the host IFN response. Through analysis of public bulk RNA-seq datasets, we identified two additional lncRNAs that are consistently upregulated following H1N1 influenza infection and interferon-β (IFN-β) treatment. This study aims to identify immune genes that are perturbed by candidate lncRNA knockdowns (KDs) and explore the relationship between KD efficiency of lncRNA-targeting guide RNAs and the scale of transcriptomic perturbations.
To investigate the roles of these lncRNAs, we used CRISPRi to generate stable KD cell lines in human lung epithelial cells, monocytes, and T-lymphoblasts. These cell lines were treated with IFN-β for 6 hours, and mock versus IFN-treated samples were analyzed using 10x CRISPRi scRNA-seq analysis.
Transcriptome analysis of each lncRNA KD line compared to controls revealed a set of differentially expressed immune genes in response to IFN-β stimulation.
In summary, we have identified two novel interferon-responsive human lncRNAs that may play important roles in the regulation of the host interferon response across different cell types. The results provide mechanistic targets for future studies and serve as preliminary data for future large-scale screens of candidate lncRNAs.
National Institutes of Health Grant R21AI147187
Immune Response Regulation: Molecular Mechanisms (IRM)
Ebola virus (EBOV) causes a highly lethal hemorrhagic fever marked by uncontrolled cytokine release and impaired antiviral defense. EBOV primarily infects human macrophages, where uncontrolled replication reprograms host transcription and fuels hyperinflammation. Long non-coding RNAs (lncRNAs), potent regulators of transcription and immunity, remain largely unexplored in EBOV pathogenesis. We identified EBOV-induced lncRNAs co-regulated with transcription factors (TFs) in primary human macrophages, revealing a novel mechanism by which EBOV rewires host gene expression to promote immune suppression and persistence
Using the highly virulent EBOV (Mayinga variant), we infected primary human macrophages from nine donors. We compared protein-coding and lncRNA transcriptomes of EBOV-infected vs. MOCK-infected macrophages at 24- and 48-hours post-infection. Differentially expressed lncRNAs were correlated with adjacent protein-coding genes to infer cis-regulatory relationships. Functional validation used antisense oligonucleotides, CRISPR interference (CRISPRi), and inducible CRISPR—Cas13d knockdown, followed by qPCR, RNA-seq, and Western blotting
EBOV infection triggered extensive remodeling of macrophage transcriptome. LINC01740 was strongly induced and tightly co-expressed with its neighboring gene, ATF3, a stress-responsive transcription factor known to repress antiviral signaling. LINC01740 knockdown reduced ATF3 expression in macrophages. ATF3 inhibition restored interferon-beta production, enhanced innate immune activation, and induced strong antiviral responses in EBOV-infected macrophages. Our data show that ATF3 impairs IFN-I responses in macrophages, contributing to immune paralysis
Our data revealed LINC01740 as a novel host lncRNA co-opted by EBOV to enhance ATF3 and suppress antiviral responses. Here we show a novel LINC01740—ATF3—IFN-I regulatory axis driving immune evasion and viral persistence, highlighting lncRNAs as potential antiviral therapeutic targets
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Viral Immunology (VIR)
Narasimha Tanuj Gunturu, Anumarla Gopal, Marija Djurkovic-Lopez et al.· Journal of Immunology· 0 citations
Long non-coding RNAs (lncRNAs) can play a major role in modulating innate immune responses by acting as positive or negative regulators. LncRNAs can mediate protein interactions with chromatin and other proteins by serving as scaffolds, decoys, and guides. The expression pattern of most lncRNAs is cell and context specific, making them attractive targets for precise therapeutic intervention. However, only a small number of lncRNAs have been well characterized due to their low abundance, context dependent expression, and complex modes of action. We have previously identified lncRNA VILMIR as an interferon-stimulated gene that regulates host interferon response to viral infection and interferon treatment. In this study, we investigated molecular mechanisms through which VILMIR modulates host immune response.
Using in vitro RNA pulldown assays followed by in vivo RNA immunoprecipitation validation, we identified several VILMIR-interacting proteins, including FUBP1, an RNA-binding protein implicated in transcriptional regulation. Mapping experiments with truncated VILMIR fragments revealed specific regions involved in protein binding.
Our findings suggest that VILMIR modulates transcription of interferon-stimulated genes by forming an RNA-protein complex with FUBP1.
Future studies will focus on elucidating how the VILMIR-FUBP1 complex influences the transcriptional landscape during viral infection and exploring its potential as a therapeutic target to fine-tune antiviral immune responses.
National Institutes of Health Grant R21AI147187
Innate Immune Responses and Host Defense: Molecular Mechanisms (INM)
Nasif Mahmood, Kristen John, Alexandra Istishin et al.· Journal of Immunology· 0 citations
Ribosome Sequencing (RiboSeq) has identified translated open reading frames (ORFs) in “noncoding” elements of the transcriptome, including long noncoding RNAs (lncRNAs) and 5’ untranslated regions (5’UTRs). To date, targeted studies scratch the surface relative to the number of predicted ORFs, and the few high-throughput screens have focused on proliferation in cancer cell lines. To link novel translation events to immune cell function, we performed proliferation and NFkB reporter screens in murine macrophages, linking hundreds of novel ORFs to immune phenotype for the first time.
RiboSeq reads from 21 immune cell datasets were collected and processed. Input datasets included macrophages, dendritic cells, CD4/8 T cells, and B cells, with both resting and activated samples. Novel translation events were called against a custom transcriptome. RiboSeq predicted ORFs were validated using mass spectrometry data from immune cells and MHC I peptidomics. 3,000 high confidence novel ORFs on lncRNAs and in 5’UTRs were knocked out in a CRISPR dropout screen and a Tlr2-NFkB sorting screen. A top hit from each screen was characterized in follow-up studies.
Over 20,000 novel translation events were predicted across all cell types (adj p < 0.01), and hundreds were found to have supporting proteomics data (adj p < 0.01). In follow-up CRISPR screens 200 novel ORFs impacted cell proliferation, notably ORFs in the 5’UTR of Tlr1, Tlr2, and Irf2. 15 novel ORFs were implicated in Tlr2-NFkB signaling. We followed up on two hits which were derived from retroviral envelope proteins, one acting as a secreted cytokine and one as a transmembrane protein.
We have generated a resource of novel translation events in immune cells and demonstrated that 100s of novel ORFs play a role in macrophage biology. Of the predicted ORFs, 8 were found to be derived from retroviral proteins, two of which were top hits in our proliferation and NFkB screens, indicating novel repurposing of retroviral elements.
NIH NIAID F31 Grant
Innate Immune Responses and Host Defense: Cellular Mechanisms (INC)
Eric Malekos, Susan Carpenter, Zach Rugee et al.· Journal of Immunology· 0 citations
Findings indicate that RP3-340N1.2 is aberrantly expressed in LUAD and may participate in tumor-associated cellular behaviors through a miR-4650-5p/SHC1-related regulatory mechanism.
Fang Chen, Yan Yan, Wenting Yang et al.· PLoS ONE· 0 citations
Ebola virus (EBOV) infection causes severe hemorrhagic fever marked by dysregulated cytokine production, impaired antiviral defenses, and multi-organ failure. Macrophages are primary targets of EBOV, and viral replication profoundly alters macrophage transcriptional programs, driving hyperinflammation. Although long non-coding RNAs (lncRNAs) are increasingly recognized as regulators of immunity and viral pathogenesis, their roles in EBOV infection remain poorly understood. We performed comprehensive transcriptomic profiling of primary human monocyte-derived macrophages infected with the highly pathogenic EBOV Mayinga variant. Infection triggered extensive remodeling of both coding and non-coding transcriptomes, including hundreds of differentially expressed lncRNAs. Functional analysis of neighboring protein-coding genes of EBOV-induced lncRNAs (EVILs) revealed enrichment of pathways linked to cytokine signaling, transcriptional regulation, and cell signaling, all of which are central to Ebola virus disease (EVD) pathogenesis. Among the most strongly induced EVILs, LINC01740 and its neighboring protein-coding gene, Activating Transcription Factor 3 (ATF3), were significantly upregulated. Antisense oligonucleotide-mediated inhibition of LINC01740 reduced ATF3 mRNA and protein levels. CRISPR/Cas13d-mediated knockdown of ATF3 restored type I interferon (IFN-I) signaling and antiviral gene expression in EBOV-infected macrophages. Mechanistically, ATF3 functions as a negative regulator of IFN-I and type I interferon-stimulated gene expression, thereby suppressing antiviral immune responses in EBOV-infected macrophages. Together, these findings identify a previously unrecognized LINC01740-ATF3-IFN-I regulatory axis that EBOV exploits to promote immune suppression and viral replication.
O. Shtanko, Tanuj Gunturu, Anumarla Gopal et al.· bioRxiv· 0 citations
Long noncoding RNAs (lncRNAs) are emerging as critical regulators of tumor initiation and progression through transcriptional and posttranscriptional mechanisms. UPK1A antisense RNA 1 (UPK1A-AS1), a cancer‐associated lncRNA, has been reported to participate in oncogenic processes; however, its overall landscape across human malignancies and its biological role in therapy resistance remain poorly understood. Given the increasing importance of identifying functional lncRNAs with prognostic and therapeutic potential, this study presents a comprehensive multiomics characterization of UPK1A-AS1 and its experimental validation in hepatocellular carcinoma (HCC). We integrated datasets from The Cancer Genome Atlas (TCGA), the Genotype‐Tissue Expression Project (GTEx), the cancer immunology data engine (CIDE), and the cBioPortal for cancer genomics (cBioPortal) to systematically assess its expression pattern, genomic alterations, clinical significance, and immunological associations. Our analyses revealed that UPK1A-AS1 is significantly upregulated in multiple tumor types, with copy‐number amplification as the predominant genomic alteration driving its overexpression. Elevated UPK1A-AS1 expression was correlated with advanced disease stage, poor differentiation, immune exclusion, and unfavorable prognosis, supporting its potential as a cancer type‐dependent biomarker. In parallel, functional studies demonstrated that hypoxia transcriptionally induces UPK1A-AS1 in HCC, where it promotes sorafenib resistance by suppressing apoptosis. Silencing UPK1A-AS1 restored apoptotic and enhanced sorafenib efficacy both in vitro and in vivo. Collectively, our findings suggest that UPK1A-AS1 is a hypoxia‐inducible oncogenic lncRNA that plays dual roles in cancer, with cancer type‐dependent associations with progression and immune modulation across malignancies and mechanistically mediating hypoxia‐associated drug resistance in HCC.
Ze-Kai Li, Min Luo, Shu-Sen Fang et al.· Analytical Cellular Patholog...· 0 citations
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