Fig. 6: CRYs regulates the RNA-binding activity of CIS1 to modulate FLM splicing.

a Diagram illustrating the locations of RNA probes (red lines) and primer pairs (blue bars) used in RNA-EMSA and RIP assays. b RNA-EMSA assay showing that CIS1 binds to the FLM Joint 3 probe but not FLM Joint 2 in vitro and that CIS1 shows the strongest binding affinity to short FLM Joint 3 (FLM Joint 3 s) among the short probes (Intron 2 and FLM Joint 3 s). RNA probes are indicated in (a). c RIP-qPCR assay showing the binding affinity of CIS1 protein to FLM pre-mRNA in vivo. Col-0, cry1 cry2, and cis1-1 (mock) seedlings were grown at 22 °C in LD condition for 7 days and pretreated with darkness (D) for 24 h, treated with MG132 for 2 h, and transferred to blue light (B, 30 μmol m⁻² s⁻¹) for 4 h. RNA fragments (200–400 nt) extracted from seedlings were immunoprecipitated with anti-CIS1 agarose beads (IP). The precipitated RNA was analyzed by RT-qPCR using different primer pairs of FLM pre-mRNA as indicated in (a). The FLM promoter and ACT7 served as negative controls. The level of binding was calculated as the ratio between IP and mock, normalized to that of IPP2 as an internal control; n.d., not detected. Error bars, s.d. of three biological replicates. d Co-localization of CIS1 and U2AF65A proteins in nuclear speckles in N. benthamiana. mCherry served as a negative control. Scale bar = 5 μm. e BiFC assay showing in vivo protein interactions between CIS1 and U2AF65A. CIS1(ΔN)-nYFP and cCFP-U2AF65B were used as negative controls. N. benthamiana leaf epidermal cells were co-infiltrated with the indicated constructs. Scale bar = 20 μm. In b, d, e, three independent experiments were performed with similar results.