Extended Data Fig. 10: The interaction between GRP20 and spliceosome in the condensates depending on HDR.
a, The spliceosome U5 subunits identified by IP-MS. b, The quantification of YFP signals in the nucleus of tobacco leaves in Fig. 8b. The fraction of YFP intensity to DAPI intensity for a single nucleus was estimated. The data were presented as mean ± SD from 26 or 27 nuclei for each transient experiment indicated in each column. PC: positive control. c, Co-IP of GRP20 and Prp18 from Arabidopsis. GRP20-YFP and Prp18-FLAG fusion proteins driven by the CaMV-35S promoter were co-expressed in Arabidopsis. Seedlings from such transgenic plants were used to extract nuclear proteins for immunoprecipitation using GFP antibody-conjugated agarose beads. The pre-IP and IP samples were analyzed by immunoblotting using antibodies against FLAG and GFP, separately. Arabidopsis co-expressing GFP and Prp18-FLAG driven by the 35 S promoter was used as a negative control. The pre-IP sample using an antibody against Histone 3 was the loading control. d, The band intensity of Prp18-GST in His pull-down experiments for GRP20, GRP20ΔHDR and HDR in Fig. 8c. His-sumo is a negative control. Nearly none of Prp18 was captured by GRP20ΔHDR and 40% of Prp18 was captured by HDR, compared to full-length GRP20. The experiments are conducted by three independent replicates with similar results. The data were presented as mean ± SEM. e, The fluorescent intensity of Prp18-RFP and GRP20-YFP estimated in the drawing line across the nucleus (left to right distance shown in the X-axis) in Fig. 8d by Image J. The data suggested that Prp18-RFP and GRP2-YFP are co-localized in the nuclei and have stronger co-localization in three condensates in the nucleus indicated by black arrows. f, The condensate formation in the nucleus showing interactions between GRP20 and Prp18 by BiFC assay. The MMD1 and JMJ16 were used as a control that do not form condensates under interaction. The yellow arrows indicate condensates in the tobacco nuclei when GRP20-YFPn interacts with Prp18-YFPc. The fluorescent intensity of YFP after interactions in the right panel was estimated by drawing red lines across the nucleus (left to right distance shown in the X-axis) in the left panel. YFP signals represented positive interaction. Two obvious peaks indicated by black arrows were observed in the interaction nucleus of GRP20 and Prp18 suggesting two condensates, however, none of peaks (condensates) were observed in positive interacting nucleus. Two-sided Student’s t test is used for statistics in 10b and 10d.
