Figure 1. PCIF1 N6-methylates 2′-O-methyladenosine in vitro in an m⁷G cap-dependent manner.

A. Schematic of PCIF1 indicating the position of predicted functional domains. The location of the sites of mutations used in the study are shown. The catalytic domain includes a four amino acid motif, NPPF, which is predicted to be essential for mediating methylation (Iyer et al., 2016). The location of the site guide RNAs (gRNAs; 5′- CGGUUGAAAGACUCCCGUGG-3′ and 5′- ACUUAACAUAUCCUGCGGGG-3′) used in Figure 2 are indicated.

B. Oligonucleotide sequences used in methyltransferase assays.

C. PCIF1 methylates m⁷G-ppp-Am-N₂₀ RNA. GST-PCIF1 (50 nM), but not the catalytically inactive mutants APPA or SPPG efficiently converts m⁷G-ppp-Am (4 μM) to m⁷G-ppp-m⁶Am as assessed by UHPLC-MS/MS. Under the same conditions (SAM, 160 μM, 10 min), PCIF1 does not convert any of the 5 internal adenosines to m⁶A. Each bar represents the mean ± s.e.m of 3 independent experiments. n.s: not significant, ***: p < 0.001, as assessed by unpaired Student’s t-tests.

D. PCIF1 methylates cap-adjacent adenosine regardless of 2′-O-ribose methylation. GST-PCIF1, but not the APPA or SPPG PCIF1 mutants efficiently converts m⁷G-ppp-A-N₂₀ (4 μM) to m⁷G-ppp-m⁶A-N₂₀. Assays were performed as in C. Each bar represents the mean ± s.e.m of 3 independent experiments. ***: p < 0.001, as assessed by unpaired t-tests.

E. PCIF1 enzyme kinetics. m⁷G-ppp-Am-N₂₀ (at indicated concentration) was incubated with GST-PCIF1 (20 nM) for the indicated times in the presence of 1.33 μM ³H-SAM and 10 μM SAM. Methylation was determined by the presence of ³H in the RNA, as assessed by scintillation counting. Each point represents the mean ± s.e.m of 3 independent experiments.

F. Michaelis-Menten kinetics of PCIF1 methylytransferase activity toward m⁷G-ppp-Am and m⁷G-ppp-A. Each point represents the mean ± s.e.m of 3 independent experiments.

G. PCIF1 activity depends on the presence of the m⁷G cap. m⁷G-ppp-Am-N₂₀ or ppp-Am-N₂₀ (4 μM) was incubated with GST-PCIF1 as in C. PCIF1 converted Am to m⁶Am specifically in the m⁷G capped RNA. Each bar represents the mean ± s.e.m of 3 independent experiments. ***: p < 0.001, as assessed by unpaired t-tests.

H. PCIF1 directly binds the m⁷G cap. Anti-FLAG immunoblotting was used to detect binding of 3xFLAG-PCIF1 from HeLa cell extracts to m⁷GTP-conjugated beads. The beads were eluted with m⁷G-ppp-A or G-ppp-A. eIF4E and eIF4G were used to control for binding to m⁷G.