Journal: Nucleic Acids Research

Article Title: Body temperature variation controls pre-mRNA processing and transcription of antiviral genes and SARS-CoV-2 replication

doi: 10.1093/nar/gkac513

Figure Lengend Snippet: JAK-STAT signaling is required for the temperature-controlled increase of antiviral genes. (A) The canonical antiviral cascade, also required for defense against SARS-CoV-2, leading to the type I interferon response is shown (created with BioRender.com). Components at all steps of this pathway are activated at 38°C; the expression of all genes shown in color was increased in the RNA-seq experiment in Figure (genes shown in gray were not changed). For RT-qPCR validation, RAW 264.7 cells were incubated at the indicated temperature for 12 h and gene expression was analyzed ( n = 3, mean ± SD). All genes with a predicted increase in expression at 38°C in the RNA-seq data were confirmed by RT-qPCR (validation rate 100%). ( B) RAW 264.7 cells were incubated at the indicated temperature for 12 h and gene expression was analyzed by RT-qPCR. mRNA expression is relative to Hprt ( n = 3, mean ± SD). (C) Elevated temperature is sufficient to induce JAK-STAT signaling. RAW 264.7 cells were incubated at the indicated temperature for 12 h and phosphorylation (activation) of STAT2 was analyzed by western blot ( n = 3, mean ± SD). As positive control, cells were incubated for 16 h with LPS (2.25 μg/ml) ( n = 2, mean ± SD). A representative blot and quantification of three independent experiments are shown. hnRNP-L served as a loading control. (D) Western blot as in panel (C) showing increased expression of Stat2 also at the protein level. A representative blot and quantification relative to hnRNP-L are shown ( n = 3). (E) Western blot as in panel (B) showing increased expression of RIG-I (Ddx58) also at the protein level. A representative blot and quantification relative to hnRNP-L are shown. (F) Inhibition of JAK-STAT signaling abolishes temperature-induced increased expression of antiviral genes and reduces their basal expression. RAW 264.7 cells were incubated in the absence (DMSO) or presence of the JAK inhibitor ruxolitinib for 6 h at 37°C and then at the indicated temperature for 12 h. Expression of the indicated genes was analyzed by RT-qPCR. mRNA expression is relative to Hprt ( n = 3, mean ± SD). Statistical significance was determined by unpaired t -tests and is indicated by asterisks: * P < 0.05; ** P < 0.01; *** P < 0.001; ns = not significant.

Article Snippet: The following primary antibodies were used: rabbit anti-phospho-Stat2 (Tyr690), 1:1000 (#4441, Cell Signaling Technology); rabbit anti-phospho-Stat3 (Tyr705), 1:1000 (#9131, Cell Signaling Technology); mouse anti-RIG-I, 1:1000 (sc-376845, Santa Cruz Biotechnology); rabbit anti-Stat2 (mouse specific), 1:1000 (#4597, Cell Signaling Technology).

Techniques: Expressing, RNA Sequencing, Quantitative RT-PCR, Biomarker Discovery, Incubation, Gene Expression, Phospho-proteomics, Activation Assay, Western Blot, Positive Control, Control, Inhibition

Journal: Nucleic Acids Research

Article Title: Body temperature variation controls pre-mRNA processing and transcription of antiviral genes and SARS-CoV-2 replication

doi: 10.1093/nar/gkac513

Figure Lengend Snippet: Stat2 alternative splicing coupled to nonsense-mediated decay (NMD) provides a molecular link between elevated temperature and increased de novo transcription of antiviral genes. (A) Delayed increase of antiviral gene expression upon temperature increase. RAW 264.7 cells were incubated at 39°C for the indicated time and RNA was analyzed by RT-qPCR. mRNAs are normalized to 37°C (mean ± SD, n = 3). (B) The temperature-induced increase in antiviral gene expression is reversible. RAW 264.7 cells were incubated for 16 h at 39°C and then transferred to 37°C for the indicated time. RNA was prepared and analyzed by RT-qPCR (mean ± SD, n = 3). mRNAs are normalized to 39°C. (C) RNA stability of antiviral genes is not altered at increased temperature. RAW 264.7 cells were pre-incubated at the indicated temperature for 12 h. ActD was then added and mRNA was quantified 2 and 4 h later. mRNAs are normalized to t = 0 of the respective temperature (mean ± SD, n = 4). ( D) Increased de novo transcription of antiviral genes at increased temperature. RAW 264.7 cells were incubated at 39°C for the indicated time; chromatin-associated RNA was purified and analyzed by RT-qPCR with the forward primer binding to an intronic region (for Irf7 the forward primer binds in an exonic region). mRNA expression is relative to Hprt. (E) Sashimi plot (left) identifying an alternative, NMD-inducing 5′ splice site in Stat2 exon 11, which is used more frequently at 34°C and shows reduced usage at 38°C. Reads from the sashimi plot are quantified (right, bottom). Schematic representation of the NMD-inducing 5′ splice site in Stat2 exon 11 (right, top). Created with BioRender.com. ( F) Radioactive, splicing-sensitive RT-PCR confirming decreased use of the alternative 5′ splice site at warmer temperature and stabilization of the alternative isoform in the presence of the NMD inhibitor CHX. RAW 264.7 cells were incubated at the indicated temperature for 8 h, and then for an additional 4 h in the absence (DMSO) or presence of CHX. RNA was prepared and analyzed by RT-PCR. A representative gel (left) and phosphorimager quantification (right, mean ± SD, n = 3) are shown. ( G) Samples as in panel (F) were analyzed by RT-qPCR. mRNA expression is relative to Hprt. (H) The CLK inhibitor TG003 has a similar effect to the CHX treatment (mean ± SD, n = 3). RAW 264.7 cells were incubated for 6 h in the absence (DMSO) or presence of the CLK inhibitor TG003, and then for an additional 12 h at the indicated temperature. mRNA expression is relative to Hprt (mean ± SD, n = 3). Statistical significance was determined by unpaired t -tests and is indicated by asterisks: * P < 0.05; ** P < 0.01; ns = not significant.

Article Snippet: The following primary antibodies were used: rabbit anti-phospho-Stat2 (Tyr690), 1:1000 (#4441, Cell Signaling Technology); rabbit anti-phospho-Stat3 (Tyr705), 1:1000 (#9131, Cell Signaling Technology); mouse anti-RIG-I, 1:1000 (sc-376845, Santa Cruz Biotechnology); rabbit anti-Stat2 (mouse specific), 1:1000 (#4597, Cell Signaling Technology).

Techniques: Alternative Splicing, Gene Expression, Incubation, Quantitative RT-PCR, Purification, Binding Assay, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: Nucleic Acids Research

Article Title: Body temperature variation controls pre-mRNA processing and transcription of antiviral genes and SARS-CoV-2 replication

doi: 10.1093/nar/gkac513

Figure Lengend Snippet: Deletion or reduced usage of the alternative 5′ splice site of exon 11 in Stat2 increases the expression of antiviral genes. ( A) 3T3 cells were transfected with two different ASOs targeting the alternative 5′ splice site in Stat2 exon 11. Thirty-two hours post-transfection, cells were incubated for 16 h at 37 or 39°C. A scrambled ASO was used as a control. Radioactive, splicing-sensitive RT-PCR confirms decreased use of the alternative 5′ splice site at warmer temperature and in the presence of the ASOs. A representative gel (left) and phosphorimager quantification of two independent experiments performed in triplicates (right, mean ± SD, n = 6) are shown. (B) Radioactive, splicing-sensitive RT-PCR confirms decreased use of the alternative 5′ splice site in the presence of the ASOs in N2a cells. A representative gel (left) and phosphorimager quantification of several independent experiments (right, mean ± SD, Ctrl and scrambled = 6, ASOs = 12) are shown. Empty transfection (Ctrl) and a scrambled ASO were used as a control. ( C) Gene expression levels of the antiviral genes upon ASO treatment in 3T3 cells. Gene expression was investigated by RT-qPCR. mRNAs are normalized to scrambled ASO at 37°C (mean ± SD, n = 6). ( D) Same as in panel (C) for N2a cells (mean ± SD, Ctrl and scrambled = 6, ASOs = 12). (E) Depletion of the alternative 5′ splice site of exon 11 using CRISPR/Cas9 in N2a cells (top: position of the sgRNAs; created with BioRender.com); bottom: a radioactive RT-PCR investigating Stat2 AS in a homozygous clone (g1 + 3C) and a heterozygous clone (g1 + 4D) is shown and quantified (right, mean ± SD, n = 3). (F) Increased expression of Stat2 and other antiviral genes in cells lacking the alternative 5′ splice site. Gene expression was investigated by RT-qPCR. mRNAs are normalized to px458 as nonedited control (mean ± SD, n = 3). Statistical significance was determined by unpaired t -tests and is indicated by asterisks: * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: The following primary antibodies were used: rabbit anti-phospho-Stat2 (Tyr690), 1:1000 (#4441, Cell Signaling Technology); rabbit anti-phospho-Stat3 (Tyr705), 1:1000 (#9131, Cell Signaling Technology); mouse anti-RIG-I, 1:1000 (sc-376845, Santa Cruz Biotechnology); rabbit anti-Stat2 (mouse specific), 1:1000 (#4597, Cell Signaling Technology).

Techniques: Expressing, Transfection, Incubation, Control, Reverse Transcription Polymerase Chain Reaction, Gene Expression, Quantitative RT-PCR, CRISPR

Journal: Nucleic Acids Research

Article Title: Body temperature variation controls pre-mRNA processing and transcription of antiviral genes and SARS-CoV-2 replication

doi: 10.1093/nar/gkac513

Figure Lengend Snippet: Schematic representation of the impact of Stat2 on the expression of antiviral genes at higher temperatures. Alternative splicing coupled to NMD decreases Stat2 expression in colder conditions. An increase of Stat2 expression at warmer temperatures together with JAK activity will induce the expression of the antiviral genes. This will then lead to a stronger antiviral immune response and as a consequence a lower viral replication. Created with BioRender.com.

Article Snippet: The following primary antibodies were used: rabbit anti-phospho-Stat2 (Tyr690), 1:1000 (#4441, Cell Signaling Technology); rabbit anti-phospho-Stat3 (Tyr705), 1:1000 (#9131, Cell Signaling Technology); mouse anti-RIG-I, 1:1000 (sc-376845, Santa Cruz Biotechnology); rabbit anti-Stat2 (mouse specific), 1:1000 (#4597, Cell Signaling Technology).

Techniques: Expressing, Alternative Splicing, Activity Assay

Journal: Computational and Structural Biotechnology Journal

Article Title: A five-protein prognostic signature with GBP2 functioning in immune cell infiltration of clear cell renal cell carcinoma

doi: 10.1016/j.csbj.2023.04.015

Figure Lengend Snippet: GBP2 enhances metastasis of ccRCC cells through JAK-STAT signaling. A. Western blotting validation of GBP2 expression in ccRCC cells upon knockdown of GBP2. B. Transwell assays were used to test the migration and invasion abilities upon knockdown of GBP2. * *P < 0.01, * **P < 0.001. Scale bar, 600 µm. C. Western blotting validation of GBP2 expression in ccRCC cells transfected with indicated constructs. D. Transwell assays were used to test the migration and invasion abilities of ACHN and 769-P cells transfected with indicated constructs. * *P < 0.01, * **P < 0.001. Scale bar, 600 µm. E-F. Gene Set Enrichment Analysis revealed that high GBP2 expression was mainly enriched in JAK/STAT signaling based on proteome and transcriptome data from CPTAC (E) and TCGA (F). G. Protein-protein interaction network for GBP2 in ccRCC was constructed using GeneMANIA. Different colors of the network edge indicate the bioinformatics methods applied: physical interaction, predicted, and co-expression. H. The correlation of GBP2 with STAT2/STAT3 at both RNA levels (TCGA) and protein levels (CPTAC) in ccRCC was determined. I-J. GBP2 siRNAs (I) or overexpression vectors (J) were transfected into ccRCC cells. The phosphorylation of STAT family proteins was examined by western blotting. K. Transwell assays were used to test the migration abilities of GBP2-expressing ACHN and 769-P cells treated with JAK inhibitor Ruxolitinib. * * P < 0.01, * ** P < 0.001. n.s, no significant. Scale bar, 600 µm.

Article Snippet: STAT1 (Cell Signaling Technology, Boston), STAT2 (Proteintech, Wuhan), STAT3 (Beyotime Biotechnology, Shanghai), Phospho-STAT1 (Tyr701) (Cell Signaling Technology, Boston), Phospho-STAT2 (Tyr690) (Beyotime Biotechnology, Shanghai), Phospho-STAT3 (Tyr705) (Beyotime Biotechnology, Shanghai).

Techniques: Western Blot, Expressing, Migration, Transfection, Construct, Over Expression