Figure 2. PCIF1 N6-methylates 2′-O-methyladenosine in cells.
A. CRISPR-mediated PCIF1 knockout (KO) in HEK293T cells was assessed by anti-PCIF1 immunoblotting. The upper band represents endogenous PCIF1, whereas the lower band is a non-specific band. β-actin, loading control.
B. PCIF1 is required for formation of m6Am in mRNA in cells. m6Am and Am levels in poly(A) RNA was detected by radiolabeling the 5′ nucleotide after decapping. RNA hydrolysates were resolved by 2D-TLC. Representative images are shown from 3 biological replicates. The bar graph on the right represents the mean ± s.e.m of 3 independent experiments. *** p < 0.001, Student’s t-test.
C. m6Am is depleted in PCIF1 KO HEK293T cells as assessed by UHPLC-MS/MS. Each bar represents the mean ± s.e.m of 3 independent experiments. ****: p <0.0001 as assessed by paired t-tests.
D. PCIF1 does not affect the level of internal m6A. 2D-TLC analysis of poly(A) RNA from HEK293T (WT) and PCIF1 KO HEK293T show no effect on the level of m6A.
E. Internal m6A is not affected in PCIF1 KO HEK293T cells as assessed by UHPLC-MS/MS. Each bar represents the mean ± s.e.m of 3 independent experiments. ns: not significant, as assessed by paired t-tests.
F. Wild-type but not a catalytically inactive PCIF1 mutant restores m6Am levels in PCIF1 knockout cells as assessed by 2D-TLC. Each bar represents the mean ± s.e.m of two independent experiments.
G. Wild-type but not catalytically inactive PCIF1 mutant restores m6Am levels in PCIF1 KO cells as assessed by UHPLC-MS/mS. Each bar represents the mean ± s.e.m of two independent experiments. Ns: not significant, * p < 0.05 as assessed by paired t-tests.
H. Western blot analysis demonstrates equivalent expression of wild-type and catalytically inactive FLAG-tagged PCIF1. β-actin, loading control.
I. Overexpression of wild-type but not catalytically inactive PCIF1 increases m6Am levels in HEK293T cells as assessed by 2D-TLC. Upper and left panels show representative images of 3 independent experiments. The bar graph represents the mean ± s.e.m of 3 independent experiments. ns: not significant, ****P ≤ 0.0001, unpaired t-tests.
See also Figure S1.