Extended Data Fig. 7: SE stabilizes MTB by preventing 20S-proteasome-mediated degradation in plants.
From: SERRATE drives phase separation behaviours to regulate m6A modification and miRNA biogenesis
a – c, MTB-decay assays. Immunoblot assays showed that different protein stabilities of the plant MTB protein (a) and purified recombinant MTB protein (b) in the presence of the indicated reagents. The statistical analysis (c) of left-over MTB in (b). CHX, cycloheximide, 0.5 mM; MG-132, 50 μM; PYR-41, 50 μM. Only the comparisons with the Col-0 (DMSO) group are shown. P values for se-2 (MG-132) and se-2 (DMSO) vs Col-0 (DMSO) are 0.97 and < 0.0001, respectively. Two-way ANOVA analysis with Tukey’s multiple comparisons test results. See also supplementary table 4 for detailed comparisons. d, e, Y2H (d) and BiFC assays (e) showed interactions between MTB and 20S proteasome subunits, including PAG1, PBE1, and PBE2. In (d), negative controls for Fig. 4h (1:10 serial dilutions) are shown. SD-LT, synthetic defined medium lacking Leu and Trp; -LTHA, lacking Leu, Trp, His and Ade. f, RNA-seq15 analysis showed the expression level of m6A writers in pag1 vs Col-0. Data are mean ± s.d. of three biological replicates. P values for MTA, MTB, FIP37, and SE at pag1-2 vs Col-0 are 1.00, 0.99, 0.99, and 1.00, respectively. Unpaired two-sided t-test. g, Overexpression of MTA in Col-0 and se-1 could promote flowering time whereas overexpression of MTB could only do this in Col-0, but not in the se-1 background. h, MTB-interaction compromised or IDR-depleted SE variants could not complement the developmental defects of se-1. 6As-SE, in which all potential hydrogen donors in C-terminal were mutated except R718; SE-R718A, which has compromised interaction with MTB; SE∆IDR1, which lacks the N-terminal IDR, failed to form spherical co-condensates; LCDFUS-SE∆IDR1, a chimera protein of human FUS’s LCD fused with SE∆IDR1, exhibits a pattern analogous to wild-type SE. Three-week-old plants were imaged. Scale bars, 10 μm (e), 5 cm (g), 1 cm (h). Three independent experiments (a, b) were performed, at least 10 independent colonies and protoplasts for each interaction were tested (d, e), at least ten transgenic plants showed similar phenotype (g, h), and representative images are shown.
