Journal: Genes & Development

Article Title: DUSP11-mediated control of 5′-triphosphate RNA regulates RIG-I sensitivity

doi: 10.1101/gad.340604.120

Figure Lengend Snippet: DUSP11 modulates RIG-I signaling sensitivity to liposomal 5′-triphosphate RNAs. (A) Schematic diagram of the 5′-ppp-RNA transfection assay, and immunoblot analysis of A549 parental wild-type (WT) and DUSP11 knockout (KO) cells transduced with pLenti empty vector (vector), DUSP11-3xFLAG (D11), or DUSP11-3xFLAG-catalytic mutant (D11-CM). A549 WT or DUSP11 knockout cells (12-well) were transfected with 5–10 ng of in vitro transcribed 5′-ppp-RNA for 18 h posttransfection. (B) RT-qPCR analysis of IFNB1 and ISG15 mRNA normalized to GAPDH mRNA in 5′-ppp-RNA transfected in WT and DUSP11 KO cells as in (A). Results are presented relative to mock-transfected WT cells. (C) RT-qPCR analysis of ISG15 mRNA normalized to GAPDH mRNA in DUSP11 KO cells stably expressing empty vector, DUSP11, or DUSP11 catalytic mutant as in A, transfected with 5′-ppp-RNA. Results are presented relative to those of DUSP11-expressing cells (+D11). (D) RT-qPCR analysis of IFNB1 and ISG15 mRNA normalized to GAPDH mRNA in DUSP11 knockout cells transfected with 5′-ppp-RNA pretreated with or without calf intestinal phosphatase (CIP) or in vitro-translated DUSP11-core. Results are presented relative to mock-treated DUSP11 knockout cells. (E) RT-qPCR analysis (left) of ISG15 mRNA normalized to GAPDH mRNA in DUSP11 knockout cells transfected with negative control siRNA (siNC) or siRNA targeting RIG-I (siRIG-I) and subsequently transfected with 5′-ppp-RNA, and immunoblot analysis (right) assessing siRIG-I knockdown efficiency of A549 cells transfected with or without 5′ppp-RNA. Data are derived from n = 3 independent replicates in B–E and are presented as mean ± SEM.

Article Snippet: Mice C57BL/6N Dusp11 tm1a(EUCOMM)Wtsi mice were generated by IVF using frozen sperm (EM: 09991) obtained from the European Mouse Mutant Archive and maintained on the C57BL/6N (Taconic) background.

Techniques: Transfection, Western Blot, Knock-Out, Transduction, Plasmid Preparation, Mutagenesis, In Vitro, Quantitative RT-PCR, Stable Transfection, Expressing, Negative Control, Knockdown, Derivative Assay

Journal: Genes & Development

Article Title: DUSP11-mediated control of 5′-triphosphate RNA regulates RIG-I sensitivity

doi: 10.1101/gad.340604.120

Figure Lengend Snippet: DUSP11 alleviates the interferon response mediated by tumor–fibroblast cell interaction. (A) Schematic diagram of coculture assay (left), and RT-qPCR analysis (right) of IFNB1, MX1, and ISG15 mRNA induction normalized to GAPDH mRNA in MDA-MB-231 breast cancer (BrCa) cells and HFF fibroblast cells either in monoculture or coculture. Cells were cultured for 60–72 h and RNA lysates were collected for RT-qPCR analysis. RT-qPCR results are presented relative to monocultured MDA-MB-231 cells. (B) RT-qPCR analysis (left) of IFNB1 and ISG mRNA transcripts (MX1, ISG15, and IFIT1 mRNA) comparing fold change of cocultured cells silenced of DUSP11 (siD11 coculture) relative to control coculture (siNC coculture), and immunoblot analysis (right) assessing siRNA knockdown of DUSP11 in MDA-MB-231 and HFF cells treated with negative control siRNA (siNC) or siRNA targeting DUSP11 (siD11). (C) RT-qPCR analysis (left) of IFNB1 and MX1 mRNA normalized to GAPDH mRNA in cocultured MDA-MB-231 and HFF cells transduced with pLenti empty vector (vector) or pLenti-DUSP11-3xFLAG (D11), and immunoblot analysis (right) of endogenous and 3xFLAG-tagged DUSP11 in whole-cell lysates. RT-qPCR results are presented relative to those of monocultured MDA-MB-231 vector cells. Data are derived from n = 4 independent replicates in A and B and n = 3 independent replicates in C. In all panels, data are presented as mean ± SEM. (*) P < 0.05 (two-tailed Student's t-test).

Article Snippet: Mice C57BL/6N Dusp11 tm1a(EUCOMM)Wtsi mice were generated by IVF using frozen sperm (EM: 09991) obtained from the European Mouse Mutant Archive and maintained on the C57BL/6N (Taconic) background.

Techniques: Co-culture Assay, Quantitative RT-PCR, Cell Culture, Control, Western Blot, Knockdown, Negative Control, Transduction, Plasmid Preparation, Derivative Assay, Two Tailed Test

Journal: Genes & Development

Article Title: DUSP11-mediated control of 5′-triphosphate RNA regulates RIG-I sensitivity

doi: 10.1101/gad.340604.120

Figure Lengend Snippet: Reducing DUSP11 increases 7SL RNA 5′-triphosphate levels in tumor-fibroblast extracellular vesicles. (A) Schematic diagram of the coculture conditioned media transfer assay. EV-containing conditioned media was concentrated from coculture media by differential centrifugation and transferred to MDA-MB-231 breast cancer (BrCa) cells silenced for DUSP11 or RIG-I. (B) RT-qPCR analysis of IFNB1 mRNA in siRNA-treated MDA-MB-231 cells incubated in concentrated coculture conditioned media as in A. Results are presented relative to those of untreated mock MDA-MB-231 cells. (C) Schematic diagram of the 5′ end characterization RT-qPCR assay of 7SL RNA isolated from coculture EVs. EV-containing pellets were collected from coculture conditioned media by differential centrifugation. Extracted RNA was treated with DNase I followed by treatment with or without 5′-monophosphate-specific exonuclease Terminator. (D) RT-qPCR analysis of 7SL RNA in EV RNA extracted from siNC or siDUSP11 (siD11) cocultured conditioned media. Results are presented relative to siNC coculture with 5S rRNA as the endogenous control. (E) RT-qPCR analysis of 7SL RNA in EV RNA isolated from siNC or siDUSP11 (siD11) cocultured conditioned media, treated with or without Terminator. Results are presented relative to mock-treated siNC coculture with 5S rRNA as endogenous control. (F) RT-qPCR analysis of 5.8S rRNA in EV RNA isolated from siNC or siDUSP11 (siD11) cocultured conditioned media, treated with or without Terminator. Results are presented relative to mock-treated siNC coculture with 5S rRNA as endogenous control. Data are derived from n = 3 independent replicates in B and D–F. In all panels, data are presented as mean ± SEM. (*) P < 0.05 (two-tailed Student's t-test).

Article Snippet: Mice C57BL/6N Dusp11 tm1a(EUCOMM)Wtsi mice were generated by IVF using frozen sperm (EM: 09991) obtained from the European Mouse Mutant Archive and maintained on the C57BL/6N (Taconic) background.

Techniques: Centrifugation, Quantitative RT-PCR, Incubation, Isolation, Control, Derivative Assay, Two Tailed Test

Journal: Genes & Development

Article Title: DUSP11-mediated control of 5′-triphosphate RNA regulates RIG-I sensitivity

doi: 10.1101/gad.340604.120

Figure Lengend Snippet: DUSP11 catalytic activity promotes RNA virus replication. (A) VSV viral titer of negative control siRNA (siNC), siRNA targeting DUSP11 (siD11), or RIG-I (siRIG-I)-treated NHDF cells determined by plaque assay (left), and representative image of plaque assay of cells at 48 h postinfection (hpi) (right). Cells treated with siRNA were infected with WT VSV at MOI of 0.25 PFU/cell and virus supernatant was collected 48 hpi for plaque assay analysis. (B) M51R VSV viral titer of siNC or siRIG-I-treated A549 WT and DUSP11 KO cells determined by plaque assay (left), and representative image of plaque assay of cells at 40 hpi (right). Cells treated with siRNA were infected with M51R VSV at MOI of 0.05 PFU/cell. (C) M51R VSV viral titer of siNC or siRIG-I-treated SVEC4-10 WT and DUSP11 KO cells determined by plaque assay. Cells were infected with M51R VSV at MOI of 0.1 PFU/cell for 24 h. (D) SINV viral titer of siNC or siRIG-I-treated A549 WT and DUSP11 KO cells determined by plaque assay. Cells treated with siRNA were infected with SINV at MOI of 0.05 PFU/cell for 24 h. (E) M51R VSV viral titer of A549 DUSP11 knockout cells reconstituted with empty vector (vector), DUSP11 (DUSP11) or catalytic mutant DUSP11 (D11-CM) as in Figure 1A determined by plaque assay (left), and representative image of plaque assay of cells at 36 hpi (right). Cells were infected with M51R VSV at MOI of 0.05 PFU/cell and virus supernatant was collected 12, 24, and 36 hpi for plaque assay analysis. Data are derived from n = 4 independent replicates in A, B, and D), and n = 3 independent replicates in C and E. In all panels, data are presented as mean ± SEM. (*) P < 0.05; (**) P < 0.01 (two-tailed Student's t-test).

Article Snippet: Mice C57BL/6N Dusp11 tm1a(EUCOMM)Wtsi mice were generated by IVF using frozen sperm (EM: 09991) obtained from the European Mouse Mutant Archive and maintained on the C57BL/6N (Taconic) background.

Techniques: Activity Assay, Virus, Negative Control, Plaque Assay, Infection, Knock-Out, Plasmid Preparation, Mutagenesis, Derivative Assay, Two Tailed Test

Journal: Genes & Development

Article Title: DUSP11-mediated control of 5′-triphosphate RNA regulates RIG-I sensitivity

doi: 10.1101/gad.340604.120

Figure Lengend Snippet: DUSP11 promotes RNA virus replication by dephosphorylating viral RNA PAMPs. (A) Schematic diagram of the VSV leader and trailer RNA 5′ end characterization assay. RNA was extracted from A549 or HEK293 WT and DUSP11 KO cells infected with VSV at MOI of five PFU/cell for 24 h and treated with or without 5′-monophosphate-dependent exonuclease XRN1. Purified RNA was then subject to Northern blot analysis. (B) Northern blot analysis on VSV leader, trailer and small RNAs purified from A549 WT/DUSP11 KO cells infected with WT VSV. (C) Northern blot analysis on VSV leader, trailer, and small RNAs purified from HEK293 WT/DUSP11 KO cells infected with WT VSV. (D) Graphical representation of the relative band density percentage ratio (+XRN1/−XRN1) of VSV leader and trailer RNA in A549 WT and DUSP11 KO cells as determined by Northern blot analysis in B. (E) Graphical representation of the relative band density percentage ratio (+XRN1/−XRN1) of VSV leader and trailer RNA in HEK293 WT and DUSP11 KO cells as determined by Northern blot analysis in C. (F) Graphical representation of the relative band density percentage ratio (+XRN1/−XRN1) of 5.8s rRNA in A549 WT and DUSP11 KO cells as determined by Northern blot analysis in B. Data are derived from n = 4 independent replicates for D and F and n = 3 independent replicates for E. In all panels, data are presented as mean ± SEM.

Article Snippet: Mice C57BL/6N Dusp11 tm1a(EUCOMM)Wtsi mice were generated by IVF using frozen sperm (EM: 09991) obtained from the European Mouse Mutant Archive and maintained on the C57BL/6N (Taconic) background.

Techniques: Virus, Infection, Purification, Northern Blot, Derivative Assay

Journal: Genes & Development

Article Title: DUSP11-mediated control of 5′-triphosphate RNA regulates RIG-I sensitivity

doi: 10.1101/gad.340604.120

Figure Lengend Snippet: Mice lacking DUSP11 display an enhanced signature of interferon signaling. Transcriptomic analysis was performed on liver samples collected from age/sex pair-matched DUSP11-deficient (n = 6 mice: n = 3 male, n = 3 female) versus wild-type mice (n = 6 mice: n = 3 male, n = 3 female). Data were generated from two experiments: untreated (n = 3 mice: n = 2 male, n = 1 female) and mock-treated (n = 3 mice: n = 1 male, n = 2 female) conditions. Combined data were modeled controlling for sex and experiment-treatment (based on Supplemental Fig. S8A) using DESeq2. (A) Volcano plot of differentially expressed genes between DUSP11-deficient mice versus wild-type mice. (B) Heat map of differentially expressed genes up-regulated between DUSP11-deficient mice versus wild-type mice. (C) Analysis of gene ontology (GO) biological process terms enriched for genes up-regulated between DUSP11-deficient versus wild-type mice. (D) Schematic diagram of the VSV 5′-ppp PAMP RNA in vivo transfection assay and VSV infection assay. DUSP11-deficient (DUSP11def) and wild-type (WT) mice were either treated with in vitro transcribed VSV 5′-ppp PAMP RNA (intraperitoneal injection, 15 μg) or infected with WT VSV (intravenous tail vain injection, 2 × 106 PFU). Sera and tissue samples were harvested 6 h posttreatment. (E) ELISA analysis of sera IFN-α levels from VSV 5′-ppp PAMP RNA or mock-treated mice. Sera were collected from age/sex pair-matched DUSP11-deficient (n = 3 mice: n = 1 male, n = 2 female for mock, and n = 5 mice: n = 3 male, n = 2 female for VSV 5′-ppp PAMP RNA-treated) and wild-type (n = 3 mice: n = 1 male, n = 2 female for mock, and n = 5 mice: n = 3 male, n = 2 female for VSV 5′-ppp PAMP RNA-treated) mice. (F) RT-qPCR analysis of IFNA4 and IFNB1 mRNA normalized to GAPDH mRNA in spleen RNA from VSV 5′-ppp PAMP RNA-treated mice. RT-qPCR results are presented relative to those of mock-treated mice. Spleen samples were collected from age/sex pair-matched DUSP11-deficient (n = 6 mice: n = 4 male, n = 2 female) and wild-type (n = 6 mice: n = 4 male, n = 2 female) mice. (G) RT-qPCR analysis of VSV transcript normalized to GAPDH mRNA in spleen RNA from VSV-infected mice. RT-qPCR results are presented relative to those of mock-treated uninfected mice. Spleen samples were collected from age/sex pair-matched DUSP11-deficient (n = 8 mice: n = 3 male, n = 5 female) and wild-type (n = 8 mice: n = 3 male, n = 5 female) mice. Data are presented as mean ± SEM.

Article Snippet: Mice C57BL/6N Dusp11 tm1a(EUCOMM)Wtsi mice were generated by IVF using frozen sperm (EM: 09991) obtained from the European Mouse Mutant Archive and maintained on the C57BL/6N (Taconic) background.

Techniques: Generated, In Vivo, Transfection, Infection, In Vitro, Injection, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR

Journal: Genes & Development

Article Title: DUSP11-mediated control of 5′-triphosphate RNA regulates RIG-I sensitivity

doi: 10.1101/gad.340604.120

Figure Lengend Snippet: Model for DUSP11-mediated regulation of RIG-I signaling. The RNA triphosphatase DUSP11 dephosphorylates endogenous host and exogenous viral 5′-triphosphate RNAs and reduces the sensitivity of the RIG-I signaling response to these RNAs. The higher proportion of 5′-triphosphate RNAs in the absence of DUSP11 results in aberrant RIG-I sensing and increased interferon signaling.

Article Snippet: Mice C57BL/6N Dusp11 tm1a(EUCOMM)Wtsi mice were generated by IVF using frozen sperm (EM: 09991) obtained from the European Mouse Mutant Archive and maintained on the C57BL/6N (Taconic) background.

Techniques: