Fig. 4: SERBP1 and PCIF1 interact and co-catalyze m6Am modifications on mRNA.
a The SERBP1 peptide identified by an MS analysis after an anti-PCIF1 co-immunoprecipitation assay in HEK293T cells. b Co-immunoprecipitation (IP) analysis of SERBP1 binding to PCIF1, using anti-PCIF1. Tissue was collected from the contralateral S1HL on day 21 after SNI or sham surgery in wild-type mouse premicroinjected with AAV-CaMK2α-shPcif1 (to knock down Pcif1 in glutamatergic cells) or a scrambled control. Input, the purified protein control. IB: immunoblotting (n = 10 mice). S1 samples from two mice were pooled to create one sample. c Construction schematic for the mammalian two-hybrid system; the luciferase reporter, with full-length Serbp1 and Gal4, and the VP16 transcription factor with a PCIF1 domain. The binding activity of SERBP1 and different PCIF1 domains (N-WW, d; helical, e; MTase, f; C-terminal, g) at 48 h after their co-transfection into HEK293T cells (n = 3 dishes of cells). h m6Am levels in HT22 cells after 48-hour transfection using: Vehicle (RNase-free water), SERBP1 plasmid with Control (empty vector), SERBP1 with Δ-MTase, SERBP1 with Δ-Helix, or SERBP1 with PCIF1 (n = 4 dishes of cells). i Schematic of CRY2/CIBN optogenetic regulation of the m6Am level through control of PCIF1 and SERBP1 binding activity. Serbp1 and Pcif1 were fused to the Cry2 and Cibn vectors, respectively. j, k Diagram showing Serbp1-Cry2 and Pcif1-Cibn co-transfection and activation of their interaction by blue light (j), and the quantitation of m6Am levels (k) (n = 3 dishes of cells). PBS, transfection of PBS control. The data are represented as mean ± SEM. One-way ANOVA followed with Tukey’s post hoc test was used for (b). One-way ANOVA followed with Dunnett’s post hoc test was used for (d–h and k).
