Fig 1: TSSC4 associates specifically with U5 snRNP and PRPF19 protein.a Characterization of proteins co-precipitating with TSSC4-GFP and TSSC4-FLAG by SILAC-IP. Primarily U5-specific proteins (yellow), Sm proteins (green), chaperones involved in U5 biogenesis (blue) co-purified with TSSC4. The ratio M/L shows a SILAC ratio of medium labeled proteins co-precipitated with tagged TSSC4 (M) and control immunoprecipitation from unlabeled cells (L). The experiment was performed once with TSSC4-GFP and once with TSSC4-FLAG. b TSSC4 interacts with proteins specific for U5 snRNP and the PRPF19 complex. TSSC4-GFP immunoprecipitation followed by western blotting and detection of proteins specific for U5 (PRPF8, SNRNP200, EFTUD2, and PRPF6—top panel and DDX23 and TXNL4A—bottom panel), U2 (SNRPA1) and U4/U6 (PRPF31 and SART3) snRNPs and PRPF19 complex (PRPF19 and CDC5L). SNRPB is one of the Sm proteins common to all snRNPs, and AAR2 is a PRPF8 chaperone. The positions of molecular weight markers are shown, the marker labeled by asterisk applies only to the GFP sample in the bottom combined GFP panels. See also Supplementary Fig. 1 for TSSC4-FLAG immunoprecipitation. c TSSC4 precipitates specifically U5 snRNA. TSSC4-GFP immunoprecipitation followed by RNA isolation and detection of co-purified RNAs by silver staining (top panel) and Northern blotting with a probe against U5 snRNA (bottom panel). Immunoprecipitation of Sm proteins with Y12 antibody served as a positive control and GFP as a negative control. d TSSC4 association with U5-specific and PRPF19 proteins is RNA independent. TSSC4-GFP immunoprecipitation from RNase A treated or untreated cell lysates was followed by detection of U5-specific proteins and PRPF19. The positions of molecular weight markers are shown, the marker labeled by asterisk applies only to the GFP sample in the bottom combined GFP panel. See Supplementary Fig. 1c for control of the RNase treatment. b, c The experiments were performed three times or d two times with similar results. b–d Uncropped and unprocessed scans are provided in the Source Data file.
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