Figure 4. Act1 RNA binding activity is... | Rockefeller University Press

Figure 4.

$Act1 RNA binding activity is required for IL-17–induced m6A methylation of Act1 5′UTR targets and cap-independent translation. (A) Density plots corresponding to distances of Act1 binding sites on 5′UTR (red) and 3′UTR regions (blue) to the closest annotated m6A sites from the published database (GEO accession no. GSE147489) on gene transcripts. Random distances on each transcript region were used as control (dashed lines). (B) MOC1 cells, Act1 WT, or Act1 ΔSEF1 MEFs were treated with IL-17A, followed by methylated (m6A) RNA immunoprecipitation (MeRIP) and RT-PCR as described in the Materials and methods section. (C) WT Act1 or Act1 ΔSEF1 MEFs with and without IL-17A stimulation were subjected to methylated (m6A) RNA immunoprecipitation (MeRIP) followed by RT-PCR. Graphs show relative levels of indicated mRNAs normalized to Gapdh (mean and SD of three independent plates of cells). (D) WT Act1 or Act1 ΔSEF1 MEFs were transfected with siCTRL, siWTAP, or siWTAP+HA-WTAP. The transfected cells pretreated with rapamycin were treated with IL-17A, followed by western blot analysis. Vehicle control (DMSO) for rapamycin was shown in Fig. S3 C. (E) Act1 WT MEFs were transfected with siCTRL or siWTAP. The transfections of cells pretreated with rapamycin were either left untreated or IL-17A, followed by fractionation and RT-PCR normalized to Gapdh. Graph shows the ratios of mRNAs from translation-active/inactive pools (mean and SD of three independent plates of cells). (F) Wtap-knockdown MEFs with or without restoration of HA-WTAP (HA-WTAP) were analyzed using a bicistronic reporter system as described in Fig. 3 B. The transfected cells pretreated with rapamycin were treated with IL-17 for 24 h, followed by dual luciferase reporter assay. Graphs show firefly and renilla luciferase activity determined by luminescence (mean and SD of three independent plates of cells). Two-tailed t test was performed. All data are representative of three independent experiments. *P < 0.05, **P < 0.01; NS, not significant. Source data are available for this figure: SourceData F4.$

Act1 RNA binding activity is required for IL-17 – induced m ⁶ A methylation of Act1 5′UTR targets and cap-independent translation. (A) Density plots corresponding to distances of Act1 binding sites on 5′UTR (red) and 3′UTR regions (blue) to the closest annotated m⁶A sites from the published database (GEO accession no. GSE147489) on gene transcripts. Random distances on each transcript region were used as control (dashed lines). (B) MOC1 cells, Act1 WT, or Act1 ΔSEF1 MEFs were treated with IL-17A, followed by methylated (m⁶A) RNA immunoprecipitation (MeRIP) and RT-PCR as described in the Materials and methods section. (C) WT Act1 or Act1 ΔSEF1 MEFs with and without IL-17A stimulation were subjected to methylated (m⁶A) RNA immunoprecipitation (MeRIP) followed by RT-PCR. Graphs show relative levels of indicated mRNAs normalized to Gapdh (mean and SD of three independent plates of cells). (D) WT Act1 or Act1 ΔSEF1 MEFs were transfected with siCTRL, siWTAP, or siWTAP+HA-WTAP. The transfected cells pretreated with rapamycin were treated with IL-17A, followed by western blot analysis. Vehicle control (DMSO) for rapamycin was shown in Fig. S3 C. (E) Act1 WT MEFs were transfected with siCTRL or siWTAP. The transfections of cells pretreated with rapamycin were either left untreated or IL-17A, followed by fractionation and RT-PCR normalized to Gapdh. Graph shows the ratios of mRNAs from translation-active/inactive pools (mean and SD of three independent plates of cells). (F) Wtap-knockdown MEFs with or without restoration of HA-WTAP (HA-WTAP) were analyzed using a bicistronic reporter system as described in Fig. 3 B. The transfected cells pretreated with rapamycin were treated with IL-17 for 24 h, followed by dual luciferase reporter assay. Graphs show firefly and renilla luciferase activity determined by luminescence (mean and SD of three independent plates of cells). Two-tailed t test was performed. All data are representative of three independent experiments. *P < 0.05, **P < 0.01; NS, not significant. Source data are available for this figure: SourceData F4.

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