Figure 7.

RRC1 and SFPS Regulate Pre-mRNA Splicing by Direct Association with ELF3 Transcript in Vivo.

(A) Schematic diagram indicates the exon–intron structure of ELF3 and the positions of primers used for the RIP assays.

(B) and (C) Relative proportion of the ELF3 spliced (B) and unspliced (C) forms is shown. Total RNA was extracted from Col-0, sfps-2, rrc1-3, and sfps-2 rrc1-3 double mutant seedlings grown under darkness and dark-grown seedling exposed to continuous red light (at 7 μmol m⁻² s⁻¹) for the times indicated. PP2A was used as an internal control. The error bar indicates sem (n = three biological repeats, each repeat includes three technical repeats). Red asterisks indicate significant difference (P < 0.05, based on Student’s t test) from Col-0.

(D) RIP assays show that RRC1 and SFPS associate with ELF3 pre-mRNA in vivo independent of each other. The RNA-protein complex was extracted from the genotypes indicated and immunoprecipitated by anti-GFP/anti-Myc antibodies. The abundance of each gene was quantified by RT-qPCR. The results were normalized to the input of each sample and then normalized to the wild type to calculate the fold-enrichment. Each bar is the mean ± sem (n = 3 independent biological repeats). Red asterisk indicates significant difference (P < 0.05) based on analysis of variance with post hoc Tukey’s honest significant difference test.