Degradation of WTAP Blocks Antiviral Responses by Reducing the m6A Levels of IRF3 and IFNAR1 mRNA (2021)
Figure 5

Figure 5. WTAP maintains the protein abundance of IRF3 and IFNAR1 through m6A modification.

A, B Immunoblot analyses to detect the protein expression of critical adaptors in the IFN-I and Jak-STAT signaling pathway in A549 cells after transfection with Ctrl or WTAP-specific siRNAs for 48 hrs. Data are representative of 3 independent biological experiments.

C ELISA quantification of m6A levels in mRNA extracted from shCtrl and shWTAP A549 cells. n = 3 independent biological replicates, and error bars represent standard deviations. Data were compared using Student's t-test.

D, E Schematic representation of the position of m6A motifs within the IRF3 (D) and IFNAR1 (E) transcripts. The sites of the m6A RNA modification element 'DRACH' in IRF3 mRNA 5'-UTRs or IFNAR1 mRNA 3'-UTRs were predicted using RMBase v2.0 (http://rna.sysu.edu.cn/rmbase/). F1/R1 represents the detection site-1 containing three (IRF3) or four (IFNAR1) 'DRACH' elements and F2/R2 represents the detection site-2 containing two (IRF3) or three (IFNAR1) 'DRACH' elements.

F, G Abundance of IRF3 (F) and IFNAR1 (G) transcripts (detection site-1 and -2) among mRNA immunoprecipitated with anti-m6A antibody from shCtrl and shWTAP A549 cells. n = 3 independent biological replicates, and error bars represent standard deviations. Data were compared using Student's t-test.

H Abundance of IRF3 and IFNAR1 transcripts (detection site-1) among mRNAs immunoprecipitated with anti-m6A antibody from A549 cells that were transfected with empty vector (EV) or HA-tagged WTAP plasmid for 24 hrs. n = 4 independent biological replicates, and error bars represent standard deviations. Data were compared using Student's t-test.

I qRT-PCR analyses of the mRNA expression of IRF3 and IFNAR1 in shNC and shWTAP A549 cells. n = 3 independent biological replicates, and error bars represent standard deviations. Data were compared using Student's t-test.

J Semi-qRT-PCR analyses of IRF3 and IFNAR1 mRNA and RNU6 and GAPDH mRNA (loading controls throughout) in the nucleus (Nu) and cytoplasm (Cyto) of A549 cells transfected with Ctrl or WTAP-specific siRNA for 48 hrs. Data are representative of 3 independent biological experiments.

K qRT-PCR analyses of IFNAR1 mRNA expression in shCtrl and shWTAP A549 cells, followed by treatment with Act D (5 μg/mL) for the indicated times. n = 3 independent biological replicates, and error bars represent standard deviations. Data were compared using Student's t-test.

L Immunoblot analyses of IFNAR1 in shCtrl and shWTAP A549 cells, followed by treatment with Act D (5 μg/mL) for the indicated times. Data are representative of 3 independent biological experiments.

M qRT-PCR analyses of the proportion of IRF3 mRNA in polysome fractions from A549 cells transfected with Ctrl or WTAP-specific siRNAs. n = 3 independent biological replicates, and error bars represent standard deviations. Data were compared using Student's t-test.

N, O Wild-type or mutated m6A consensus sequences (A-to-T mutation) of IFNAR1-3' UTR (N) and IRF3-5' UTR (O) were fused with Renilla luciferase reporter in psiCHECKTM-2 vector.

P Relative luciferase activities in 293T cells after transfection with reporter vectors bearing IFNAR1-3' UTR or IRF3-5' UTR with wild-type (WT) or mutated (Mut) m6A sites. Renilla luciferase activity was measured and normalized to firefly luciferase activity. n = 3 independent biological replicates, and error bars represent standard deviations. Data were compared using Student's t-test.

Q Relative luciferase activities of reporter vectors bearing IFNAR1-3' UTR or IRF3-5' UTR with wild-type (WT) m6A sites after cotransfection with empty vector (EV) or HA-tagged WTAP into shWTAP A549 cells. Renilla luciferase activity was measured and normalized to firefly luciferase activity. n = 3 independent biological replicates, and error bars represent standard deviations. Data were compared using Student's t-test.