Regulation of human interferon signaling by transposon exonization

Figures

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  Fig. 1.. Human IFNAR2 is constitutively spliced into two main isoforms. A) Schematic of the approach to identify candidate exonized transposable elements (TEs) in immune genes from long-read RNA sequencing data. B) Count of TE exonization events for each main TE class based on their orientation compared to that of the protein-coding gene they provide the alternative splice site to (sense= same orientation, to the left; antisense = opposite orientation, to the right). 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 included27. C) Read alignments from long-read cDNA sequencing data of human macrophages26 over the short IFNAR2 alternative isoform (IFNAR2-S) and the canonical long IFNAR2 isoform (IFNAR2-L). D) Long-read RNA-seq read counts comparing IFNAR2-S (orange) and IFNAR2-L (blue) expression across normal human tissues (GTEx27). 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 (TCGA33,90). The two tumor types show extreme changes in the relative isoform expression…

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  Fig. 2.. IFNAR2-S functions as a decoy receptor for type I IFN signaling. A) Schematic model of isoform-specific knockouts (KOs) of the IFNAR2 gene (not drawn to scale). IFNAR2-S KO cell lines were generated by deleting the E9-Alu exon (orange); IFNAR2-L KO cell lines were generated by deleting the E9-canonical exon (blue); complete IFNAR2 KO cell lines were generated by deleting exon 7 (E7 KO/IFNAR2 KO). B) Plot shows changes in normalized STAT1 phosphorylation levels as detected by phospho-flow cytometry in wild-type and knockout HeLa cell lines upon 30min treatment with increasing doses of IFNβ. Data shown are mean fluorescence intensity (MFI) normalized against untreated cells of the same genotype. C-D) STAT2 and phosphorylated STAT2 (pSTAT2) signal from wild-type and knockout A549 cell line. On the left, C: violin plots show the 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: histograms show 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…

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  Fig. 3.. 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 shows that both IFNAR2 isoform can bind IFNβ. 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. Target proteins were detected in total cell lysates (pre-pull-down fraction) in all treatment conditions. STAT2 was detected only in IFNAR2-L HaloTag pull-downs, and IFNAR1 in cells treated with IFNβ. Signal was barely detectable in cells treated with IFNα, consistent with the lower affinity of IFNα to IFNAR2. β-actin was not identified in the pull-down fractions. C) Immunofluorescent labeling of IFNAR2-L-HaloTag and IFNAR2-S-HaloTag in untreated conditions (cnt, on the left) and after 30min of IFNβ treatment (100U/ml). Compared to the diffused signal…

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  Fig. 4.. 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 different viruses. SARS-CoV-2 data from (42) and Influenza B data from (43). B) Variants associated with severe COVID-19 located in and near exon E9-Alu as identified in GWAS studies by the COVID-19 Host Genetic Initiative91. C) Bar plot shows the protective effect of IFNβ in the context of SARS-CoV-2 (B.1.617.2) viral infection in wild-type and knockout A549-ACE2 cells. Each bar represents the ratio of viral genome copies 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. SARS-CoV-2 copy number was measured by RT-qPCR. D) Based on three independent SARS-CoV-2 infection experiments, the IFNβ IC50 was calculated for wild-type and IFNAR2-S KO A549-ACE2 cells. Bars show the average and distribution of IC50 values for the two genotypes. 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…

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  Fig. 5.. Evolution of the IFNAR2-S isoform and signaling. A) Primate phylogeny illustrates the evolution of IFNAR2-S at the time of evolution of simian primates ~70–60 mya. B) Schematic reconstruction of IFNAR2-S evolution through the conversion of a syntenic ancestral FAM SINE by a simian primate-specific Alu-Jr Alu. Mutations in the Alu-Jr then allowed for its exonization and the evolution of IFNAR2-S. 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, which binds IFN molecules but cannot initiate a signaling response. F). Cellular immune homeostasis can be tuned by the IFNAR2-L : IFNAR2-S ratio.