Fig. 7: CWC15 promotes phosphorylation of SE.
From: The spliceosome-associated protein CWC15 promotes miRNA biogenesis in Arabidopsis
a Interaction between CWC15 and PRP4KA detected by BiFC. Paired cYFP- and nYFP-fusion proteins were co-expressed in leaves of N. benthamiana. Green color indicates the BiFC signal detected by a confocal microscopy at 48 h after infiltration. Scale bar = 10 μm. Three independent experiments yielded consistent results. b, c Co-IP between CWC15 and PRP4KA. 2HA-CWC15 was co-expressed with FM-PRP4KA or GFP−2FLAG in N. benthamiana leaves, respectively. IPs were performed using anti-FLAG antibodies or and anti-HA antibodies. 2HA-CWC15, FM-PRP4KA and GFP-2FLAG were detected by western blot. Two independent experiments yielded consistent results. d Phosphorylation status of endogenous SE in amiRCWC15 and Col-0. SE was detected by SDS-PAGE and phosphor-tag gel in parallel using anti-SE antibodies. e Phosphorylation levels of SE in amiRCWC15 relative to Col. Phosphorylated SE proteins were compared with total SE proteins (set as 1) in Col and amiRCWC15, respectively. Error bars indicate SD from two biological replicates (n = 2) and data are presented as mean values +/- SD. Asterisks indicate significantly reduced phosphorylation compared to Col-0 plants based on P-value (two-tailed unpaired t-test; P < 0.01): 0.002787. f The proposed action model of CWC15 in miRNA biogenesis. CWC15 binds the MIR promoters to facilitate the recruitment of Pol II, which may depend on the function of CDC5. CWC15 interacts with SE and HYL1 to enhance pri-miRNA processing. Moreover, CWC15 interacts with PRP4KA and the 20 S proteosome to promote phosphorylation-dependent SE degradation. Source data are provided as a Source Data file.
