Fig. 6: Increased PCIF1 is responsible for gain of m⁶Am at Maf1 mRNA in the S1HL after nerve injury.

a Flowchart of the screening for downstream targets of PCIF1 by m⁶Am-Seq and RNC-Seq. SNI+shPcif1 indicates PCIF1 downregulation in S1HL of SNI mice via injection of AAV-shPcif1. b Distribution of m⁶Am peaks across mRNA segments of S1HL from three groups. c Motif analysis of m⁶Am peaks of mRNAs’ genomic sequences. The minus sign in X axis, upstream genomic nucleotides. d Venn diagram analysis revealed 436 genes consistently regulated by PCIF1 and SERBP1, based on integrated analysis of three complementary datasets: (i) genes with SNI-induced m⁶Am increases that were reversed by PCIF1 knockdown (m⁶Am-Exo-Seq), (ii) genes with increased SERBP1 binding post-SNI (SERBP1-RIP-Seq), and (iii) genes that exhibited reduced translation efficiency following SNI (RNC-Seq). e Gene ontology (GO) analysis of biological processes for the 436 genes. f Heatmap visualization of p-values and fold changes for the 436 genes. g Top 10 genes with most significance among 436 genes. h The representative image of m⁶Am peaks on Maf1 gene in S1HL. i Maf1 m⁶Am in S1HL on day 14 post-SNI measured via RNA immunoprecipitation (RIP)-PCR with four PCR primer pairs. The first two pairs include the RNA cap site (n = 8 mice). The forward F, and reverse R arrows represent paired PCR primers. j, k Maf1 m⁶Am (j) and the binding level of PCIF1 to Maf1 mRNA (k) in S1HL of SNI or Sham Pcif1^fl/fl mice after preinjection of AAV-CaMK2α-Cre (Cre) or AAV-CaMK2α-mCherry (Ctrl) into S1HL (n = 10 mice). Maf1 m⁶Am level (l) and PCIF1 binding to Maf1 mRNA (m) on day 21 after injection of AAV-DIO-Pcif1 or AAV-DIO-mCherry into S1HL of naïve CaMK2α-Cre mice (n = 10 mice). For (i–m), data are represented as mean ± SEM. S1 samples from two mice were pooled to create one sample (j–m). Student’s unpaired t-test (two-tailed) was used for panels (i, j, k). One-way ANOVA followed with Tukey’s post hoc test was used for (l and m).