Fig. 5.

FER is required for pre-mRNA splicing of flowering-related genes in Arabidopsis. a-b Relative mRNA levels of WT and fer-4 mutant at ZT 4. LD, long day condition. ACTIN2 was used as an internal control gene. Error bars indicate the SD of three technical replicates. All experiments were performed at least three times (ns. means no significant; **P < 0.01, Student’s t-test). c Genomic structure of FLC. The gray boxes represent the exons; black lines indicate introns; F, forward primer; R, reverse primer; The primer pairs F1/R1’ and F6’/R6 were used to detect the unspliced RNA for FLC introns 1 and 6, respectively. F1/R1 and F6/R6 were used to detect spliced mRNA in wild-type and fer-4 mutants. ACTIN2 was used as an internal control. Seven-day-old Col-0 and fer-4 seedlings were grown under LD conditions and collected at ZT 12. Splicing efficiency (spliced/unspliced) was calculated. The experiments were performed three times (**P < 0.01, Student’s t-test). d Numbers of differential splicing events between the fer-4 mutant and WT detected by RNA sequencing (RNA-Seq) analysis (n = 3). e Gene Ontology (GO) enrichment of genes with significant splicing changes between fer-4 and WT (n = 3). The black dotted line indicates P = 0.05. The numbers indicated the representative genes involved in the pathway. f Heat maps of differential pre-mRNA splicing of flowering-related genes between fer-4 mutant and wild-type based on the exon inclusion levels detected by RNA-Seq analysis. g Heat maps of differential pre-mRNA splicing of flowering-related genes (MAF1, MAF2 and MAF3) based on the exon inclusion levels from RNA-seq data (n = 3). h Semi-quantitative PCR (semi-qPCR) validation of differential splicing events between fer-4 mutant and Col-0. Seven-day-old Col-0 and fer-4 seedlings were grown under LD conditions and collected at ZT 4. CS indicates constitutive splicing, and AS indicates alternative splicing. Red triangles show the position of primers used by semi-qPCR (Supplemental Table 5). PP2A was used as the internal control. The experiments were independently repeated three times with similar results