Fig. 5

Knockdown of ac4C modification mediated by NAT10 expedites the ITGB5 mRNA degradation. (A) qRT-PCR analysis showing the specified mRNA level in PANC-1 cells expressing NAT10 siRNA (siNAT10) or control siRNA (siCtrl) for 72 h. (B) The half-life of ITGB5 mRNA in PANC-1 and MIA PaCa-2 cells transfected with siCtrl or siNAT10. PDAC cells were treated with actinomycin D (Act.D, 5 µg/mL) and collected at the indicated time for qRT-PCR analysis. (C) PANC-1 cells were transfected with indicated shRNAs. Polysome profiling was performed using 5–50% sucrose gradient fractionation and the absorption of the gradient at 260 nm was recorded. Representative images are shown. (D) Quantification of ITGB5 mRNA in 11 gradient fractions using qRT-PCR, from three replicates of polysome profiling. (E) Schematic illustration of the CRISPR-dCasRx method to engineer ac4C writer NAT10. Effective small guide RNA (sgRNA) targeting the CDS in the ITGB5 mRNA. sgRNA (a) and (b) were targeting the potential ac4C positive cytosine (red). (F) NAT10-KD PANC-1 cells were transfected with CRISPR/dCasRx protein fused with NAT10 or acetylase-dead NAT10 G641E (NAT10-dead). Immunoblotting showing the dCasRx fusion protein. (G) Subcellular localization of dCasRx-NAT10 and dCasRx-NAT10-dead fusion protein in NAT10-KD PANC-1 cells. Scale bar: 20 μm. (H) acRIP analysis followed by qRT-PCR, showing the ac4C levels on endogenous ITGB5 mRNA in NAT10-KD PANC-1 (upper) and MIA PaCa-2 (lower) cells, which were transfected with indicated dCasRx fusion proteins and sgRNA (a), sgRNA (b), or control (con) sgRNA. (I) Relative ITGB5 mRNA expression in NAT10-KD PDAC cells transfected with indicated dCasRx fusion proteins and sgRNA (a). (J) ITGB5 mRNA decay in NAT10-KD PDAC cells transfected with indicated dCasRx fusion proteins and sgRNA (a). (K) Immunoblotting images showing endogenous ITGB5 protein expression when PDAC cells were treated with indicated dCasRx proteins and sgRNAs for 48 h. *p < 0.05, **p < 0.01, ***p < 0.001. (A), Student’s t-test. (B) and (I), One-way ANOVA. (H) and (J), Two-way ANOVA