Interferon-γ (IFN-γ) is essential for the innate immune response to intracellular bacteria. Noncoding RNAs and RNA-binding proteins (RBPs) need to be further considered in studies of regulation of the IFN-γ-activated signaling pathway in macrophages. In the present study, we found that the microRNA miR-1 promoted IFN-γ-mediated clearance of Listeria monocytogenes in macrophages by indirectly stabilizing the Stat1 messenger RNA through the degradation of the cytoplasmic long noncoding RNA Sros1. Inducible degradation or genetic loss of Sros1 led to enhanced IFN-γ-dependent activation of the innate immune response. Mechanistically, Sros1 blocked the binding of Stat1 mRNA to the RBP CAPRIN1, which stabilized the Stat1 mRNA and, consequently, promoted IFN-γ–STAT1-mediated innate immunity. These observations shed light on the complex RNA–RNA regulatory networks involved in cytokine-initiated innate responses in host–pathogen interactions.
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The data that support the findings of this study are available from the corresponding author upon request. The RNA-seq data from the present study are deposited in the National Center for Biotechnology Information’s Gene Expression Omnibus under accession code GSE127769.
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This work is supported by grants from the National Natural Science Foundation of China (grant no. 81788101), Chinese Academy of Medical Sciences’ Innovation Fund for Medical Sciences (grant no. 2016-12M-1-003) and the National Key Research and Development Program of China (grant no. 2018YFA0507403). We thank H. Lin for technical help.
The authors declare no competing interests.
Peer review information Ioana Visan was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.
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Supplementary Figure 1 Functional screening of microRNAs in the anti−intracellular bacteria responses.
a, Listeria monocytogenes (L.m.) −induced cell death of PMs transfected with antago−NC (antg−NC) or antago−miR−1 (antg−miR−1). The survival of cells was determined 21 h later by 7−AAD uptake. b, qRT−PCR analysis of miR−1 mRNA in sorted mouse cells; results were normalized to U6. HSC, hematopoietic stem cell, NK cell, natural killer cell; macrophage. c, qRT−PCR analysis of mature miR−1 in peritoneal macrophages after intraperitoneal infection with L.m. for the indicated hours. Data were normalized to U6 expression levels. d,h, IFN−γ−induced gene expression level of Stat1 pre−mRNA after either treating with an antg−miR−1 (d) or knockout miR−1(h). e, Immunoblot analysis and quantification analysis of p−STAT1 and STAT1 and β−Actin in control or miR−1−overexpressed PMs after stimulation with IFN−γ for the indicated times. f,g, Expression levels of mature (f) or pre−mRNA transcript (g) of Stat1 in IFN−γ stimulated PMs after prior transfection with miR−1 mimics. Data are representative of three independent experiments (e) or three independent experiments with n = 3 biological replicates (a-h; shown as mean and s.e.m.). Individual data points represent individual biological replicates. *P<0.05, **P<0.01, ***P<0.001, two-tailed unpaired Student’s t−test.
a, Flow cytometry analysis of the percentages of CD49b+NK cells(NK), CD11b+Ly6G+granulocytes, CD11c+MHC−II+DCs and F4/80+CD11b+ macrophages in splenocytes from miR−1+/+ and miR−1−/− mice at postnatal day 15. b, Bacterial load in lung from miR−1 double knockout (miR-1−/−) or littermate (miR-1+/+) − bone marrow transplanted mice after infection for indicated times. n>3 for each genotype. CFU, colony−forming units. c, Hematoxylin−and−eosin staining of lung sections from the indicated mice after infection with L.m. for 36 hr. Scale bars, 20 μm. d, qRT−PCR analysis of Stat1 mRNA in peritoneal lavages from miR−1+/+ and miR−1−/− mice. e, Immunoblot analysis of phosphorylated (p−) or total STAT1 and β−ACTIN in peritoneal lavages from miR−1+/+ and miR−1−/− mice infected with L.m. for 36 hr. f. qRT−PCR analysis of Cxcl9 and Cxcl10 mRNAs in peritoneal lavages from miR-1+/+ and miR-1−/− mice. g. ELISA of IFN−γ and IFN−β in sera. Data are representative of three independent experiments (c,e) or three independent experiments with n = 3 biological replicates (a,d,f,g; shown as mean and s.e.m.). Individual data points represent individual biological replicates. *P<0.05, **P<0.01, ***P<0.001, two-tailed unpaired Student’s t−test.
a, qRT−PCR analysis of Stat1 mRNA in BMDMs. The cells were treated with 10 μM ActD for the indicated times. Data were normalized to Gapdh mRNA. b,d, Immunoblot analysis of STAT1 and β−actin in miR−1+/+ or miR−1−/− mice derived BMDMs (b) and in miR−1−overexpressing PMs (d). Cells were treated with 10 μg/ml CHX for the indicated times. Densitometry of STAT1 relative to that of β−actin is shown. c, Stat1 mRNA decay was measured in control or miR−1−overexpressing PMs after IFN−γ treatment. e, Schematic of affinity purification for identification of target mRNAs associated with miR−1. f, Fold changes of Stat1 mRNA in PMs transfected with siRNAs for screening. g, 5′ terminal and 3′ terminal RACE analysis of the full−length Sros1 cloned from PMs. GSPR1−3 and GSPF1−3 indicate the primers used in RACE assays. h, qRT−PCR assay of Sros1 with poly(A)+ or poly(A)− RNA fraction from PMs. i, Relative distributions of RNA populations across each fraction of the sucrose gradient were determined and normalized to the sum of the mRNA signal across all gradient fractions. x axis, fraction number. j, Bioinformatics analysis of Sros1 using Coding Potential Calculator. k, Possible Open Reading Frames (ORFs) was analyzed using ORF−Finder within Sros1 were listed by nucleotide position and amino-acid sequence. Data are representative of three independent experiments (b,d,g,h) or three independent experiments with n=3 biological replicates (a−d,f,i; shown as mean and s.e.m.). Individual data points represent individual biological replicates. *P<0.05, **P<0.01, ***P<0.001, two−tailed unpaired Student’s t−test.
a, Schematic illustration of the deleted region in Sros1−/− mice. b, RT−PCR analysis of genomic region from Sros1−/− and the littermates. c, qRT−PCR analysis of Sros1 in peritoneal lavages. Data are representative of three independent experiments (b) or three independent experiments with n=3 biological replicates (c; shown as mean and s.e.m.). Individual data points represent individual biological replicates. *P<0.05, **P<0.01, ***P<0.001, two−tailed unpaired Student’s t−test.
Flow cytometry analysis of the percentages of CD3−NK1.1+NK cells, CD11b+Ly6G+granulocytes, CD11c+MHC−II+DCs and F4/80+CD11b+ macrophages in splenocytes from Sros1−/− mice and their littermates. Data are representative of three independent experiments with n=3 biological replicates (shown as mean and s.e.m.). Individual data points represent individual biological replicates.
a, qRT−PCR analysis of Sros1 from Sros1−overexpressing or control RAW264.7 cells. b−c, Immunoblot analysis and quantification analysis of total STAT1 and β−actin in Sros1−silenced PMs (b), or Sros1−overexpressing RAW264.7 cells (c) and control cells. Cells were treated with 10 µg/ml CHX for the indicated times. d, Relative distributions of Stat1 mRNA populations across each fraction in the sucrose gradient were determined and normalized to the sum of the mRNA signal across all gradient fractions in Sros1−silenced PMs. x axis, fraction number. e, qRT−PCR analysis of Sros1 and Stat1 mRNA in PMs after L.m. stimulated for indicated times. f. Changes of total STAT1 protein in PMs transfected with siRNAs for functional screening. Ddx17−1 and Ddx17−2 represent different siRNAs targeting to distinct transcripts of Ddx17. g, Immunoblot analysis and quantification analysis of phosphorylated (p−) or total STAT1, total CAPRIN1 or β−Actin in siRNA transfected PMs after treatment with IFN−γ (50 U/ml) for the indicated times. h, qRT−PCR analysis of Sros1 and Stat1 mRNA in siRNA transfected PMs. Data are representative of three independent experiments (b,c,g) or three independent experiments with n=3 biological replicates (a−h; shown as mean and s.e.m.). Individual data points represent individual biological replicates. *P<0.05, **P<0.01,***P<0.001, two−tailed unpaired Student’s t−test.
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Xu, H., Jiang, Y., Xu, X. et al. Inducible degradation of lncRNA Sros1 promotes IFN-γ-mediated activation of innate immune responses by stabilizing Stat1 mRNA. Nat Immunol 20, 1621–1630 (2019) doi:10.1038/s41590-019-0542-7