Journal: Proceedings of the National Academy of Sciences of the United States of America
Article Title: A CRISPR-Cas9 knockout screening identifies IRF2 as a key driver of OAS3/RNase L-mediated RNA decay during viral infection
doi: 10.1073/pnas.2412725121
Figure Lengend Snippet: IRF2 promotes OAS3 expression in unstressed cells. ( A and B ) The levels of OAS3, IRF2, RNase L, and GAPDH were analyzed in the indicated U2OS ( A ) or A549 ( B ) cell lines by western blot. ( C ) The OAS3 mRNA levels were monitored by RT-qPCR in indicated U2OS ( Left ) or A549 ( Right ) cell lines. Mean values ± SD (n = 3). **** P < 0.0001 (two-tailed t test). ( D ) The levels of OAS3 were analyzed by western blot using indicated antibodies in A549 IRF2 KO cells expressing either wild-type IRF2 or DNA binding mutant IRF2K78R. ( E and F ) Total RNAs were isolated from A549 WT or IRF2 KO cells expressing wild-type IRF2, IRF2K78R, or OAS3 and monitored for integrity by a bioanalyzer after transfection with poly(I:C) (10 ng/mL, 4 h). The red arrows indicated ribosomal RNA cleavage products. ( G ) IgG and IRF2 ChIP-sequencing in A549 cells. Analysis of ChIP-sequencing data focuses on the promoter region of OAS3. (H) IRF2 ChIP was performed in A549 WT and IRF2 KO cells. IRF2 binding on the OAS3 promoter was determined by qPCR. Mean values ± SD. *** P < 0.001 (two-tailed t test). ( I ) Schematic of pGL3-OAS3-luciferase constructs harboring OAS3 WT promotor region (−1 to −900 bp upstream of transcription start site [TSS]) and IRF2 binding motif region deleted (Δ−1 to −200 bp). ( J ) The relative luciferase activity (ratio of Firefly:Renilla) was measured in U2OS cells transiently transfected with the empty pGL3 and pGL3 vector harboring either WT and Δ200 bp OAS3 promoter region. ( K ) The relative luciferase activity was monitored U2OS WT or IRF2 KO cells 24 h following transfection with the pGL3-OAS3 vector.
Article Snippet: The OAS3 luciferase reporter plasmid was generated by cloning the OAS3 promoter region (TSS −1 to −900) into pGL3 basic luciferase reporter (Addgene #128046).
Techniques: Expressing, Western Blot, Quantitative RT-PCR, Two Tailed Test, Binding Assay, Mutagenesis, Isolation, Transfection, ChIP-sequencing, Luciferase, Construct, Activity Assay, Plasmid Preparation
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![IRF2 promotes OAS3 expression in unstressed cells. ( A and B ) The levels of OAS3, IRF2, RNase L, and GAPDH were analyzed in the indicated U2OS ( A ) or A549 ( B ) cell lines by western blot. ( C ) The OAS3 mRNA levels were monitored by RT-qPCR in indicated U2OS ( Left ) or A549 ( Right ) cell lines. Mean values ± SD (n = 3). **** P < 0.0001 (two-tailed t test). ( D ) The levels of OAS3 were analyzed by western blot using indicated antibodies in A549 IRF2 KO cells expressing either wild-type IRF2 or DNA binding mutant IRF2K78R. ( E and F ) Total RNAs were isolated from A549 WT or IRF2 KO cells expressing wild-type IRF2, IRF2K78R, or OAS3 and monitored for integrity by a bioanalyzer after transfection with poly(I:C) (10 ng/mL, 4 h). The red arrows indicated ribosomal RNA cleavage products. ( G ) IgG and IRF2 ChIP-sequencing in A549 cells. Analysis of ChIP-sequencing data focuses on the promoter region of OAS3. (H) IRF2 ChIP was performed in A549 WT and IRF2 KO cells. IRF2 binding on the OAS3 promoter was determined by qPCR. Mean values ± SD. *** P < 0.001 (two-tailed t test). ( I ) Schematic of <t>pGL3-OAS3-luciferase</t> constructs harboring OAS3 WT promotor region (−1 to −900 bp upstream of transcription start site [TSS]) and IRF2 binding motif region deleted (Δ−1 to −200 bp). ( J ) The relative luciferase activity (ratio of Firefly:Renilla) was measured in U2OS cells transiently transfected with the empty pGL3 and pGL3 vector harboring either WT and Δ200 bp OAS3 promoter region. ( K ) The relative luciferase activity was monitored U2OS WT or IRF2 KO cells 24 h following transfection with the pGL3-OAS3 vector.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_1408/pmc11551408/pmc11551408__pnas.2412725121fig02.jpg)