Fig 2: Effects of FBW2 overexpression or loss of function in silencing mutants restores high-molecular-weight AGO1 complexes(A) Representative pictures of 27-day-old plants of Col-0, hyl1-2, hen1-6, ago1-27, and their crosses with fbw2-4 or 35S:3HA-FBW2 (FBW2OE) as indicated (based on two biological replicates; see also Figure S3A). Right: quantitative analysis of the experiment represented on the left, with n > 12 plants per genotype. °p < 0.05 and °°°p < 0.001 (Student’s t test) compared with Col-0. *p < 0.05, **p < 0.01, and ***p < 0.001 (Student’s t test) compared with the corresponding single mutant.(B and C) Western blot of protein extracts from the same seedlings as indicated in Figure S3A (for biological replicates see Figures 5 and S3–S5). CB staining was used as loading control. AGO1 signal was quantified by ImageJ, normalized to the corresponding CB. Numbers below the panel indicate relative to the corresponding mutants (hyl1-2, hen1-6 and ago1-27, respectively) set at 1.0. @ indicates hybridization with the corresponding antibodies.(D and E) Gel filtration analysis of AGO1-based RISC complexes in Col-0, hyl1-2, hy1-2 fbw2-4, and fbw2-4 13-day-old seedlings (a biological replicate is shown in Figure S4). Proteins of known molecular weight are shown on top of the blot. CB staining was used as loading control and “@” indicates hybridization with the AGO1 antibody. (E) Shown are protein and sRNA analysis of the input fraction prior to gel filtration. Methylene blue (MB) staining of the membrane was used as loading control.(F) sRNA analysis from even fractions, spanning the same range (from the GF of [D]). For this analysis, 10µg of the RNA per lane was loaded. The “@” symbol indicates hybridization with the indicated oligonucleotide probes.

Fig 3: Loss of FBW2 modifies AGO1 loading in hyl1-2(A) Western blot of total protein extracts from 2-week-old seedlings from Col-0, hyl1-2, hy1-2 fbw2-4, and fbw2-4 mutants. Two biological replicates (1 and 2) are shown. CB staining and ACTIN were used as loading controls, and @ indicates hybridization with the corresponding antibodies. The arrow indicates the HYL1 protein band, and * indicates aspecific cross-reacting bands.(B) Denaturing polyacrylamide gel of sRNA from immunoprecipitated AGO1 (based on two biological replicates). RNAs from the same protein extracts shown in (A) were indiscriminately labeled by replacing their 5' phosphate with a radioactive one using polynucleotide kinase (PNK). An oligo corresponding to the siR255 serves as control for sRNA size.(C and D) Deep-sequencing analyses of total and AGO1-IP sRNA (performed on three biological replicates). (C) Relative abundance of miRNA in AGO1 IP samples with significant differential expression in single and double mutants compared with WT (Col-0) and single mutants; the relative abundance is expressed as a heatmap (see Key at the bottom), with the samples being compared indicated below each heatmap (*Q value #0.05, **Q value #0.01, and **Q value #0.001). (D) Boxplot representing the abundance of reads (in reads per million, RPM) mapping to eight different features of the Arabidopsis genome TAIR 10 in AGO1 IP samples. These include the following, from left to right: cDNA; mature miRNA; siRNA precursors dependent on Pol4; ribosomal RNAs (rRNAs); small nuclear and small nucleolar RNA (snRNA and snoRNA); TAS precursors; transposable elements (TEs); and tRNA-derived sRNA (tRNA).

Fig 4: Distribution of AS events with significant changes in AS profiles in the se-1, hyl1-2 and dcl1-7 mutants (A) in total, (B) within first introns, (C) within first introns at the 5′ splice sites.

Fig 5: RH6/RH8/RH12 is required for D-body formation.(A) Representative images and fluorescence recovery curves of D-body components. Leaf epidermal cells of N. benthamiana (inoculated with DCL1-YFP/RFP-SE for 72 hours) and root tip cells (elongation region) of 1-week-old A. thaliana (pHYL1::HYL1-YFP) plants were analyzed. The region of interest is highlighted with a red circle. Scale bars, 2 µm. The intensity was normalized against the average prebleach fluorescence. n = 3 for each group. The green line indicates the half-recovery time. (B) Whole-plant images of 6-week-old plants. Scale bars, 0.5 cm. (C) Detection of DCL1 subcellular localization with anti-DCL1 antibodies. Left: One set of representative images. Right: The quantification analysis. The numbers 0 to 3 represent the number of DCL1 granules in each nucleus. In total, 150 to 200 nuclei were analyzed for each genotype. Scale bars, 1 µm. (D and E) Subcellular localization of HYL-YFP (D) and YFP-SE (E) in 3-week-old A. thaliana plants. Fifteen root tip cells (elongation region) for HYL-YFP and 115 to 125 leaf epidermal cells for YFP-SE were analyzed for each genotype. Scale bars, 5 µm. Photo credit: Ningkun Liu, Institute of Zoology, Chinese Academy of Sciences.