Regulation of human interferon signaling by transposon exonization

Abstract

Innate immune signaling is essential for clearing pathogens and damaged cells and must be tightly regulated to avoid excessive inflammation or autoimmunity. Here, we found that the alternative splicing of exons derived from transposable elements is a key mechanism controlling immune signaling in human cells. By analyzing long-read transcriptome datasets, we identified numerous transposon exonization events predicted to generate functional protein variants of immune genes, including the type I interferon receptor IFNAR2. We demonstrated that the transposon-derived isoform of IFNAR2 is more highly expressed than the canonical isoform in almost all tissues and functions as a decoy receptor that potently inhibits interferon signaling, including in cells infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Our findings uncover a primate-specific axis controlling interferon signaling and show how a transposon exonization event can be co-opted for immune regulation.

Keywords: alternative splicing; innate immunity; transposable elements; type I IFN.

Fig. 1.. Human IFNAR2 is constitutively spliced into two main isoforms.

A) Approach used to identify exonized transposable elements (TEs) in immune genes from long-read transcriptome data. B) Count of TE exonization events for each main TE class based on relative orientation to the parent gene. Only TEs that overlapped a protein-coding gene exon for 80% of their length and a splice site for isoforms with TPM ≥ 5 in at least one sample were included. C) Read alignments from human macrophages over IFNAR2-S and IFNAR2-L. D) Long-read RNA-seq read counts comparing IFNAR2-S (orange) and IFNAR2-L (blue) expression in human tissues (GTEx^,). E) Expression levels of IFNAR2-S and IFNAR2-L in matched healthy (lighter shade) and tumor (darker shade) samples in acute myeloid leukemia and diffuse large B-cell lymphoma (TCGA). TM = transmembrane domain. TPM = Transcripts per million.

Fig. 2.. Evolution of the IFNAR2-S isoform and signaling.

A) Primate phylogeny illustrates the evolution of IFNAR2-S ~60–70 mya. B) Schematic reconstruction of IFNAR2-S evolution through the conversion of a syntenic ancestral FAM SINE by a simian primate-specific Alu-Jr. C) Sequence conservation of IFNAR2-S at the nucleotide (nucleotide diversity on top) and amino acid levels (alignment at the bottom). A dot represents a conserved amino acid. D) IFNAR2 exon coverage from NCBI aggregate RNA-seq data for three different simian primates. E) Model of IFNAR2 signaling in primates, where the type I IFN pathway is modulated by the presence of the decoy receptor IFNAR2-S. F). Cellular immune homeostasis can be tuned by the IFNAR2-L : IFNAR2-S ratio.

Fig. 3.. IFNAR2-S functions as a decoy receptor for type I IFN signaling.

A) Cartoon of isoform-specific knockouts (KOs) of the IFNAR2 gene. B) Changes in normalized STAT1 phosphorylation levels as detected by phospho-flow cytometry in wild-type and KO HeLa cell lines upon 30min treatment with increasing doses of IFNβ. Data reflects mean fluorescence intensity (MFI) normalized against untreated cells of the same genotype. C-D) STAT2 and phosphorylated STAT2 (pSTAT2) signal from wild-type and KO A549 cell line. C: ratio of nuclear to cytoplasmic STAT2 signal in cells treated with 1000U/ml of IFNβ normalized to untreated cells (wild-type n=663, IFNAR2-L KO n=377, IFNAR2-S n=574); D: total pSTAT2 signal in untreated cells and cells treated with 10U/ml and 1000U/ml of IFNβ. On the right, immunofluorescence pictures of STAT2 and pSTAT2 from which fluorescence signals were quantified. Pink = (p)STAT2 signal. Scale bar= 50mm. E) RT-qPCR measurements of OASL and ISG15 fold-change induction comparing untreated to 4hrs 10U/ml IFNβ treated HeLa wild-type and KO cell lines. For each genotype, 3 biological replicates (different KO clonal cell lines) were tested and assayed in triplicate. F) RT-qPCR measurements of OASL and ISG15 fold-change induction comparing untreated to 4hrs 10U/ml IFNβ treated wild-type or IFNAR2-S KO cells overexpressing empty vector or IFNAR2-S. G) IFNAR2 isoform expression upon siRNA isoform-specific knockdown. Left: RT-qPCR measurements of IFNAR2-S (orange) and IFNAR2-L (blue) 3 days post transfection of a negative control, anti-IFNAR2-S and anti-IFNAR2-L siRNAs in HeLa cells endogenously expressing an IFNAR2-S-HiBiT tag. Right: Western Blot of IFNAR2-L and NanoBlot of IFNAR2-S 4 days post siRNA transfection confirm isoform specific reduction in protein levels. Blots were turned into black and white and contrast was adjusted using Adobe Photoshop (NanoBlot) or Image Studio v5.5.4 (Western Blot); blots were cropped in Adobe Illustrator H) RT-qPCR measurements of OASL and ISG15 fold-change induction in response to a 4hrs 10U/ml IFNβ treatment in wild-type HeLa cells transfected with negative control, anti-IFNAR2-L, or anti-IFNAR2-S siRNAs. Asterisks (*) indicate p-value < 0.05 between conditions. I) RNA-Seq MA-plot showing differences in 4hrs 10U/ml IFNβ treatment comparing wild-type and IFNAR2-S KO HeLa cells. Significantly differentially induced genes with annotated immune or inflammatory functions are labeled. L) Heatmap of the top 30 most significantly upregulated genes by IFNβ in IFNAR2-S KO cells. Displayed are the log2fold change values from treated vs untreated pairwise comparisons for each HeLa cell line.

Fig. 4.. IFNAR2-S acts as a decoy receptor for the type I IFN signaling.

A) Western blot of IFNβ in lysates of wild-type and knockout HeLa cells either untreated or treated with 1000U/ml IFNβ for 30min. B) Western blots of STAT2, IFNAR1 and β-actin in total lysates and pull-down fractions of IFNAR2 KO HeLa cells overexpressing either the IFNAR2-S or the IFNAR2-L isoform tagged with a C-terminus HaloTag. The HaloTag was used to perform isoform-specific pull-downs to identify interactors of IFNAR2-L and IFNAR2-S independently in untreated cells and cells treated with IFNα for 10min or IFNβ for 5min. β-actin was not identified in the pull-down fractions. Blots were turned into black and white and contrast was adjusted using Image Studio v5.5.4; blots were subsequently cropped in Adobe Illustrator. C) Immunofluorescent labeling of IFNAR2-L-HaloTag and IFNAR2-S-HaloTag in untreated conditions (cnt) and after 30min of IFNβ treatment (100U/ml). Cell lines expressing IFNAR2-L-HaloTag and IFNAR2-S-HaloTag show clear punctuated signal in the cytoplasm and at the cell membrane. Magnification = 40x; Green = wga; membrane staining; Blue = DAPI, nucleus; Red = permeable JFX549 HaloTag ligand. D) Brightfield images of HeLa cells at 4x magnification either untreated or treated with 10U/ml IFNβ for 4 days (left) and matched normalized cell viability measured by crystal violet staining for the same cells. Scale bar = 1000mm. E) Dose-response curves of normalized cell viability measured by CellTiter Glo in response to treatment with increasing doses of IFNβ (0 to 100U/ml). F) Normalized cell viability measured by crystal violet staining of wild-type cells transfected with 5mM negative control, anti-IFNAR2-L, and anti-IFNAR2-S siRNAs and treated with 10U/ml IFNβ for 4 days. G) Normalized cell viability measured by crystal violet staining of wild-type cells transfected with increasing concentration of scrambled, anti-IFNAR2-L, and anti-IFNAR2-S siRNAs and treated with 10U/ml IFNβ for 4 days. H) Cell viability measured by crystal violet staining of wild-type and KO cells treated with 100U/ml of IFNα, IFNβ and IFNγ for 7 days. All treatments were performed in triplicate and depicted as mean and standard error and normalized to untreated controls. Asterisks (*) indicate p-value < 0.05 between conditions.

Fig. 5.. The IFNAR2-L : IFNAR2-S ratio affects cellular responses to viral infection.

A) Box plots show dynamic modulation of IFNAR2 isoform expression levels (TPM) in human cell lines infected with SARS-CoV-2 and Influenza B. Data from () and (). B) Variants associated with severe COVID-19 located in and near exon E9-Alu and associated effect on IFNAR2 relative isoform expression based on splicing quantitative trait loci (sQTL) studies from the eQTL catalogue. C) Bar plot of the protective effect of IFNβ in the context of SARS-CoV-2 (B.1.617.2) infection in wild-type and knockout A549-ACE2 cells. Each bar represents the ratio of viral genome copies (measured by RT-qPCR) in the media of cells that were not pre-treated with IFNβ over viral genome copies in the media of cells that were pre-treated with increasing doses of IFNβ. D) IFNβ IC₅₀ was calculated for wild-type and IFNAR2-S KO A549-ACE2 cells (based on 3 independent SARS-CoV-2 infections). E) Histogram shows the protective effect of IFNβ in the context of dengue virus 2 (DENV-2) viral infection in wild-type and knockout HeLa cells. Protective effect was calculated as the ratio of viral genome load detectable in lysates by RT-qPCR of unstimulated cells over the viral genome load in lysates of cells that were pre-stimulated with 100U/ml of IFNβ. Asterisks (*) indicate p-value < 0.05 between conditions.