---
title: "MDA5 senses disrupted RNA processing: cellular RNAs drive activation during viral infection"
id: "nature-immunology-2-loss-of-cellular-rna-homeostasis-contributes-to-mda5-activation-during-virus"
canonical_url: "https://medichelpline.com/clinical-feed/nature-immunology-2-loss-of-cellular-rna-homeostasis-contributes-to-mda5-activation-during-virus"
content_type: "clinical_feed_article"
specialty: "Infectious Disease"
source_name: "Nature Immunology"
source_url: "https://www.nature.com/articles/s41590-026-02614-3"
published_at: "2026-08-11T10:38:32.000Z"
evidence_level: "Journal Feed"
license: "CC-BY-NC-4.0 / Informational Use"
---
# MDA5 senses disrupted RNA processing: cellular RNAs drive activation during viral infection
## Provenance & Clinical Metadata
- **Canonical URL:** https://medichelpline.com/clinical-feed/nature-immunology-2-loss-of-cellular-rna-homeostasis-contributes-to-mda5-activation-during-virus
- **Specialty:** [Infectious Disease](https://medichelpline.com/clinical-feed/infectious-disease.md)
- **Primary Source:** Nature Immunology
- **Source URL:** [Original Journal Publication](https://www.nature.com/articles/s41590-026-02614-3)
- **Published At:** 2026-08-11T10:38:32.000Z
- **Evidence Rating:** Journal Feed
## Executive GIST (TL;DR)
- MDA5 is an innate cytoplasmic **RNA sensor** that triggers type I interferon responses to viral infection and other pathogens. - The specific endogenous and pathogen-derived RNA agonists for **MDA5** have been incompletely defined. - The authors used single-nucleotide resolution crosslinking and immunoprecipitation (**iCLIP**) to map MDA5 RNA ligands during infection. - During infection with **SARS-CoV-2** or encephalomyocarditis virus (**EMCV**), MDA5 bound predominantly to **cellular RNAs** rather than viral RNAs. - Many MDA5 binding sites mapped to **intronic** regions, were located near **Alu** elements, and were proximal to sequences predicted to form base-paired structures. - Infection increased cytoplasmic levels of aberrant transcripts and intron-containing unspliced RNAs; these species were enriched for MDA5 iCLIP peaks. - Overexpression of the splicing factor **SRSF3** reduced aberrant transcription and prevented MDA5 activation in the experimental system. - The authors propose that **MDA5 surveys RNA processing fidelity** and detects infection by sensing perturbations of post-transcriptional events, particularly **splicing** defects and accumulation of unprocessed RNAs. - This model implies MDA5 activation can reflect loss of cellular RNA homeostasis in addition to direct recognition of pathogen-derived RNA. - Details about experimental design, quantitative outcomes, and full datasets were reported in the source article; specific numeric results and methodological parameters are not fully reproduced here.
## Clinical Analysis & Structured Key Points
Loss of cellular RNA homeostasis contributes to MDA5 activation during virus infection | Nature Immunology Skip to main content Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript. Advertisement Loss of cellular RNA homeostasis contributes to MDA5 activation during virus infection Download PDF Download PDF Abstract MDA5 is an innate immune RNA sensor that senses infection with a range of viruses and other pathogens. MDA5’s RNA agonists are not well defined. Here we used single-nucleotide resolution crosslinking and immunoprecipitation (iCLIP) to study its ligands. Of note, upon infection with SARS-CoV-2 or encephalomyocarditis virus, MDA5 bound overwhelmingly to cellular RNAs. Many binding sites were intronic and proximal to Alu elements and to potentially base-paired structures. Concomitantly, cytoplasmic levels of aberrant transcripts and intron-containing unspliced transcripts increased in infected cells and displayed enrichment of MDA5 iCLIP peaks. Moreover, overexpression of the splicing factor SRSF3 reduced aberrant transcription and abrogated MDA5 activation. Taken together, we propose that MDA5 surveys RNA processing fidelity and can detect infections by sensing perturbations of post-transcriptional events such as splicing. Subjects RIG-I-like receptors Viral infection Main Pathogens are first detected by pattern recognition receptors (PRRs) that activate immune signal transduction pathways and thereby initiate the host response to infection. Canonically, pathogen-derived molecules known as pathogen-associated molecular patterns (PAMPs) activate PRRs 1 . Viral infections are recognized by a class of PRRs that detect unusual nucleic acids 2 . A crucial response activated by nucleic acid sensors is the production of type I interferons (T1-IFNs) 3 . These cytokines are essential for protection against all viruses. This system is fine-tuned to differentiate infection from the homeostatic state, to elicit protective immunity followed by return to homeostasis. Excessive or uncontrolled T1-IFN responses fail to protect against infection and lead to long-term damage and disease 4 , 5 . Characterizing the initiation of T1-IFN responses is therefore fundamental to our ability to understand infectious and other diseases, and to design antiviral therapies. Among the PRRs that detect viral infection are the retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) 6 . This family of RNA sensors includes RIG-I, melanoma differentiation-associated protein 5 (MDA5) and laboratory of genetics and physiology 2 (LGP2). RLRs are primarily located in the cytoplasm. All RLRs have a central helicase domain and a carboxy-terminal domain, which together detect unusual, immunostimulatory RNA molecules. MDA5 and RIG-I also contain two tandem amino-terminal caspase activation and recruitment domains (CARDs), which mediate downstream signaling through interaction with the adaptor mitochondrial antiviral-signaling protein (MAVS). Activated MAVS oligomerizes and recruits other factors including the kinase TBK1, that in turn activate IFN regulatory factors (IRFs) and the NF-κB pathway. Ultimately, expression of T1-IFNs and other immune response genes is induced. LGP2 lacks CARDs and modulates MDA5 and RIG-I signaling. RIG-I and MDA5 are activated by different viral infections 6 . For example, influenza A virus (IAV) infection is sensed by RIG-I; conversely, picornaviruses such as encephalomyocarditis virus (EMCV) or rhinovirus are detected by MDA5 7 . Viruses from other families are recognized by both RIG-I and MDA5, including important human pathogens such as members of the Flaviviridae (for example Zika virus, hepatitis C virus), Paramyxoviridae (for example measles virus) and Coronaviridae (for example SARS-CoV) 8 . SARS-CoV-2 infection has been reported to be partially or exclusively sensed by MDA5 (refs. 9 , 10 , 11 , 12 ). Additionally, MDA5 can be activated by infection with non-RNA viruses 13 , 14 , 15 and other pathogens such as Plasmodium sp. 16 , 17 , Mycobacterium tuberculosis 18 or Aspergillus fumigatus 19 . The noteworthy role of MDA5 in the human immune response is highlighted by cases of inherited MDA5 deficiency leading to increased and/or life-threatening susceptibility to viral infections, for example with Rhinovirus 20 , 21 , 22 , 23 . An important aspect of RLR signaling is the ability of these receptors to distinguish immunostimulatory RNAs accumulating in infected cells from the RNA content of cells during homeostasis. RIG-I is activated by RNAs with triphosphate or diphosphate groups at the 5′ end 24 , 25 , 26 . Additional features of RIG-I-stimulatory RNAs include base-pairing and the lack of methylation marks 27 , 28 . Such RNA species do not occur abundantly in cells, as most cellular RNAs are processed at the 5′ end; for example, mRNAs are capped and methylated. In contrast, some viral RNAs such as the genomes of IAV have those features, allowing them to be recognized by RIG-I 29 . As such, RNA sensing by RIG-I can be conceptualized by the paradigm of PAMP detection by PRRs. It is noteworthy that some viruses, including DNA viruses and retroviruses that do not produce viral 5′-(P)PP-RNAs, activate RIG-I indirectly by causing the accumulation of unprocessed and/or mislocalized cellular noncoding RNAs with 5′-PPP moieties 30 , 31 , 32 , 33 . The RNA species that activate MDA5 are less well defined 8 . Early work showed that MDA5 recognizes infection with picornaviruses and the synthetic double-stranded (ds) RNA mimic polyriboinosinic:polyribocytidylic acid (Poly(I:C)) 7 , 34 . In particular, MDA5 is essential for innate immune sensing of long Poly(I:C) 35 . Moreover, purified MDA5 forms multimeric filaments on long strands of dsRNA 36 . RNA viruses with positive-sense genomes, including picornaviruses, replicate by producing a negative-sense copy of their genome that serves as a template for synthesis of progeny positive-sense genomes. Annealing of positive and negative-sense RNAs can form long dsRNA, known as replicative form dsRNA, which was reported to activate MDA5 (refs. 37 , 38 ). Together, these results indicate that, in virally infected cells, MDA5 is activated by long viral dsRNA. However, other findings contradict this view, suggestive of a more complex mechanism. Indeed, we previously demonstrated that higher-order structured RNA produced during infection, which may contain single-stranded RNA and dsRNA, rather than merely dsRNA, activates MDA5 (ref. 39 ). Moreover, studies analyzing the RNAs bound by MDA5 or LGP2 during viral infections suggested that single-stranded sections of viral genomes, rather than dsRNA forms, are detected 40 , 41 , 42 ; however, these studies are constrained by methodological factors, including nonstringent purification of MDA5–RNA complexes, lack of precision in determining MDA5 binding sites, and/or exogenous protein overexpression. Given that MDA5 can sense infection with many different virus families and other pathogens, understanding the nature of MDA5’s RNA agonists is crucial for our comprehension of innate immune responses. Results Dual IP of endogenous MDA5 isolates MDA5-bound RNA Previous studies of RNAs binding to and/or activating MDA5 relied on recombinant or overexpressed protein 40 , 42 , 43 . To identify RNA ligands of endogenous MDA5, we screened cell lines for expression of MDA5 and found that monocytic THP1 cells expressed MDA5 protein at baseline (Extended Data Fig. 1a ). EMCV specifically activates MDA5 (refs. 7 , 44 ) and infects THP1 cells, evident from staining with the J2 monoclonal antibody that detects dsRNA (Extended Data Fig. 1b ), a signature of viral replication 39 , 45 , 46 , and from accumulation of viral RNA (Extended Data Fig. 1c ). Infection induced IFNB1 mRNA and IFN-stimulated genes (ISGs) (Extended Data Fig. 1d ). Transfection of total cellular RNA extracted from EMCV-infected THP1 cells activated IFNB1 promoter-driven luciferase expression in HEK293 reporter cells 47 in an MDA5-dependent manner (Extended Data Fig. 1e ). Together, these data show that MDA5-stimulatory RNA was present in EMCV-infected THP1 cells. To obtain an in-depth view of RNAs binding MDA5 during viral infection, we employed iCLIP 48 . This technique allows deep sequencing of RNAs bound by a protein of interest, with resolution of the nucleotide site(s) where the protein binds, and has been successfully used for other dsRNA binding proteins including Dicer 49 and DDX17 (ref. 50 ). As a control, MDA5 knockout (KO) THP1 cells were generated using CRISPR/Cas9 (Extended Data Fig. 1a,f ). MDA5-KO THP1 did not induce IFNB1 mRNA in response to transfection with total RNA extracted from EMCV-infected HeLa cells, but responded normally to RIG-I stimulation with in vitro transcribed RNA 29 (Extended Data Fig. 1g ). In MDA5-KO cells, T1-IFN responses to EMCV infection were undetectable (Extended Data Fig. 1d ). Furthermore, we surmised that high infectivity levels would be required to detect RNA bound to MDA5; however, EMCV infectivity was low in wild-type (WT) THP1 cells (Extended Data Fig. 1h ). IRF3 signals downstream of multiple nucleic acid sensors, including cGAS, which is required for baseline T1-IFN responses in THP1 cells 51 . Loss of IRF3 may therefore render cells more susceptible to EMCV without impairing MDA5–RNA interactions. Indeed, infectivity levels increased tenfold in IRF3-KO THP1 cells 47 (Extended Data Fig. 1h–k ). We therefore used IRF3-KO THP1 cells to investigate MDA5 ligands generated during EMCV infection. To isolate endogenous MDA5, we raised a panel of monoclonal antibodies. We identified two antibodies that precipitated native and denatured MDA5, termed antibody A (clone 16) and antibody B (clone 22), respectively. To increase the stringency and specificity of MDA5 isolation, we used a dual immunoprecipitation (IP) method. Native MDA5 was first isolated using antibody A, and then eluted with a denaturing solution containing high urea and detergent concentrations. Next, the eluate containing denatured protein was used for a second IP with antibody B, which was specific for the denatured protein (Fig. 1a ). This dual IP successfully pulled down MDA5 from THP1 cells (Fig. 1b ). Silver staining confirmed the specificity of the MDA5 IP (Extended Data Fig. 2a ) Fig. 1: iCLIP isolates RNA bound by endogenous MDA5 in live cells. Full size image a , MDA5 IP was performed on WT THP1 cell lysate using antibody A or IgG control (IP1), or eluted under denaturing conditions and the eluted fraction was used for a second IP with antibody B using the indicated amounts of antibody (IP1 + IP2). Equivalent volumes of unbound fractions from the first and second IPs were collected. Cell lysate (input), IP and unbound fractions were analyzed by western blot for MDA5. b , MDA5-WT or MDA5-KO THP1 cells were infected with EMCV (MOI = 5) or left uninfected for 22 hr. Cells were lysed, and MDA5 dual IP with antibody A then B was performed. Cell lysates, unbound fractions and IP fractions were analyzed by western blot for MDA5. Actin was used as a loading control. The asterisk indicates a nonspecific band. c , Lysates from mock-infected or EMCV-infected (MOI = 10) WT THP1 cells, with or without UV crosslinking at 150 mJ cm −2 , were treated with high or low concentrations of RNase A. MDA5 dual IP was performed (using Ab A and pooled ascites fluids from a selection of hybridoma clones including Ab B), followed by RNA radiolabeling using PNK enzyme, or no PNK as a negative control. Samples were resolved by SDS-PAGE, transferred to nitrocellulose membranes and exposed to radiofilm. d , e , Lysates from UV-crosslinked, EMCV-infected (MOI = 10) WT THP1 cells were treated with increasing concentrations of RNase A, followed by MDA5 dual IP (using Ab A and pooled ascites fluids from a selection of hybridoma clones including Ab B) and RNA radiolabeling. Samples were resolved by SDS–PAGE, transferred to nitrocellulose and exposed to radiofilm ( d ). High or low molecular weight (MW) sections of membrane from ( d ) were cut as indicated, and RNA extracted by proteinase K digestion, followed by TBE Urea gel electrophoresis and radioblot ( e ). f , Diagram of the MDA5 iCLIP method. SMI, size-matched input; input material run alongside IP sample and excised from membrane at same MW range. UMI, five bases added at random to allow removal of PCR duplicates during data processing. NGS, next-generation sequencing. All data are representative of at least two independent experiments. Source data Next, we performed MDA5 dual IP on lysates from UV-crosslinked cells, and radioactively labeled the bound RNA using polynucleotide kinase (PNK). Gel electrophoresis and radioblot analysis showed a high molecular weight (MW) smear migrating more slowly than the MW of MDA5 (135 kDa), which was dependent on both UV crosslinking and PNK treatment (Fig. 1c ). The intensity of this smear was enhanced by virus infection, indicating increased RNA binding to MDA5 in infected cells (Fig. 1c and Extended Data Fig. 2b ). RNase A treatment reduced the smear, presumably due to the bound RNA being partially degraded to smaller oligonucleotides protected by the protein (Fig. 1c,d ). Extraction and proteinase K digestion of either high MW or low MW sections from the membrane produced RNA of varying lengths, with intermediate and low RNase A concentrations yielding RNA of more than 100 nt (Fig. 1e ). We selected the low RNase A treatment condition to capture MDA5-bound RNA in the 100–500-nt-length range, which is suitable for iCLIP and sequencing 48 . We then applied our dual IP approach to a modified iCLIP protocol 48 (Fig. 1f ). An adaptor for reverse transcription was added to RNA at the 3′ end, and the RNA was radiolabelled at the 5′ end, followed by isolation of RNA by gel electrophoresis and radioblot, where the total sample above the MW of MDA5 was isolated (Extended Data Fig. 2d ). An aliquot of pre-IP input material was processed alongside the IP samples and served as a ‘size-matched input’ (SMI) control for comparison to IP samples 52 . Next, the RNA was reverse transcribed into cDNA using primers containing a sample-specific barcode for multiplexing and a unique molecular identifier (UMI) for identification of PCR duplicates. Of note, reverse transcription is halted at the RNA–protein crosslink site, which allows identification of nucleotide positions bound by MDA5. The cDNA was purified and size-selected by electrophoresis, circularized and cleaved to produce cDNA containing forward and reverse PCR sites for amplification and sequencing (Fig. 1f ). MDA5 binds host-derived RNA during EMCV infection We employed our endogenous MDA5 iCLIP method to identify RNAs specifically bound by MDA5 during virus infection (Extended Data Fig. 2c,d ). IRF3-KO THP1 cells were left uninfected or were infected with EMCV. As a negative control for IP specificity, MDA5-KO THP1 cells were used. EMCV infectivity was lower in MDA5-KO cells compared to IRF3-KO cells (henceforth referred to as MDA5-WT) (Extended Data Fig. 1i–k ), likely due to baseline T1-IFN production via cGAS-IRF3 (ref. 51 ), priming cells against infection. Nevertheless, these cells lacked MDA5 and thus served as a control for MDA5 IP specificity. Sequencing reads were processed and mapped to combined human and virus genomes to simultaneously identify both viral and host sequences, followed by removal of PCR duplicates and extraction of crosslink sites (Extended Data Fig. 3a ). We obtained 10 4 –10 6 mapped, deduplicated reads per sample with four independent biological repeats (Extended Data Fig. 3b,c ). Principal- component analysis (PCA) demonstrated that replicate samples separated according to treatment and genotype (Extended Data Fig. 3d ). To our surprise, we found that the majority (>99%) of RNA sequences detected in iCLIP samples mapped to the human rather than the viral genome (Fig. 2a ). There was no enrichment of EMCV RNA in the MDA5 IP compared to the RNA input control samples. We next identified MDA5-RNA crosslink sites, herein referred to as MDA5 binding sites, using PureCLIP 53 that compares input and IP sequencing reads. MDA5 binding sites were only identified in host RNA, and not in viral RNA. The distribution of MDA5 binding sites between intergenic regions, introns, untranslated regions and coding sequences was similar between the samples, and more than half of all binding sites were in introns (Fig. 2b ). Given that MDA5 has been previously suggested to bind repetitive RNA 43 , we determined the proportion of binding sites present in repetitive regions. Around 35% of binding sites were located within repetitive regions, which included SINEs ( Alu , mammalian-wide interspersed repeat (MIR)), LINEs (L1 and L2) and other repeat categories (Fig. 2c ). We then extended our analysis to 101-nt regions spanning binding sites (50 nt upstream and downstream of binding site) and found that ~65–71% of these regions overlapped with repetitive elements, indicating that a majority of RNAs bound by MDA5 contain repetitive elements. Fig. 2: MDA5 binds cellular RNAs during EMCV infection. Full size image a , Percentage of RNA sequences in RNA input or MDA5 iCLIP samples mapping to the host or EMCV genome. b , Genomic classification of single-nucleotide MDA5 binding sites detected by iCLIP. c , Proportion of single-nucleotide MDA5 binding sites, or 101-nt regions (50 nt upstream and downstream of single-nucleotide binding site) that are located within repetitive regions of the human genome. d–f , Sequences consisting of 50 nt upstream and downstream of each MDA5 binding site (total length 101 nt) from MDA5-WT EMCV-infected samples were analyzed for de novo RNA motifs using MEME-ChIP. Specific motifs enriched in MDA5-WT-EMCV-infected samples compared to MDA5-WT uninfected samples, and associated E -values (adjusted P value multiplied by the number of motifs in the input file) are shown. Localization of the motifs discovered by MEME-ChIP relative to the crosslink site (position 0, black arrow) ( e ). The proportion of binding sites containing Poly(A) and Poly(U) motifs in MDA5-WT EMCV-infected samples when analyzed with either MEME-ChIP or HOMER algorithms as above ( f ). g , Proportion of motifs in MDA5 binding sites from EMCV-infected cells. h–j , Motifs enriched in MDA5-WT uninfected samples compared to the MDA5-KO-EMCV-infected negative control were analyzed as in c–e . M = A or C; W = A or U. Proportion of motifs in MDA5 binding sites from uninfected cells ( j ). Data points in a represent four independent biological repeats, and bars/error bars represent the mean and s.d. Analyses in b–j were conducted on combined data from four independent biological repeats. Source data We then employed HOMER 54 and MEME-ChIP 55 to identify motifs enriched in MDA5-bound RNA. Based on MDA5’s footprint on dsRNA of 14–15 nt (ref. 56 ) and the notion of MDA5 filament formation 36 , we focused on the 101-nt-length regions containing MDA5 binding sites centrally. Both algorithms identified an enrichment of Poly(A) and Poly(U) sequences in MDA5 binding sequences in EMCV-infected cells (Fig. 2d and Extended Data Fig. 4a ). These Poly(A)/(U) motifs were positioned near, but not directly overlapping, the MDA5 crosslinked nucleotide (Fig. 2e ). Between 30% and 50% of all MDA5 binding sequences contained either a Poly(A) or a Poly(U) motif, depending on the control and motif algorithm applied (Fig. 2f,g ). Of note, these motifs were enriched compared to uninfected, MDA5-sufficient cells and to infected, MDA5-deficient cells, indicative of specificity. Additionally, motifs that contained GGUU and AACC seque
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