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. 2021 Jun 15;12:3646. doi: 10.1038/s41467-021-23934-y

Fig. 3. TSSC4 contains two conserved domains interacting independently with U5 snRNP and the PRPF19 complex.

Fig. 3

a A schematic representation of TSSC4 deletion mutants. b Co-immunoprecipitation of U5 and PRPF19 complex markers with TSSC4 deletion mutants. Deletion of aa 51–100 abolished interaction with several U5-specific proteins (PRPF6, SNRNP40, CD2BP2, and SNRPB). The same deletion reduced the association of TSSC4 with U5 proteins PRPF8, SNRNP200, and EFTUD2. Deletion of aa 201–250 specifically inhibited association with PRPF19 complex markers (bottom panel). See also Supplementary Fig. 2a, b for TSSC4 mutant localization and co-precipitation of snRNAs. c A schematic representation of TSSC4 homology regions and deletion/substitution mutants. See also Supplementary Fig. 2c for TSSC4 homology alignment. d Co-immunoprecipitation of U5 and PRPF19 complex markers with TSSC4 deletion and substitution mutants. Deletion or mutation of the Hom2 region reduced interaction with U5-specific proteins and deletion/mutation of the Hom4 region inhibited association with the PRPF19 complex. See also Supplementary Fig. 2e for TSSC4 mutant localization. e Simultaneous deletion of conserved domains Hom2 and Hom3 further reduced association of TSSC4 with U5 proteins PRPF8 and EFTUD2. The experiment was performed only once. See also Supplementary Fig. 2e for ΔHom2ΔHom3 mutant localization. f A schematic representation of TSSC4 fragments. g Co-immunoprecipitation of U5 and PRPF19 complex markers with TSSC4 fragments. Expression of the central part of TSSC4 containing Hom2 and Hom3 regions is sufficient for interaction with U5-specific proteins and a weak association with the PRPF19 complex. See also Supplementary Fig. 2f for localization of these constructs. b, d, e, g The positions of molecular weight markers are shown, the markers labeled by asterisk apply only to the GFP samples in the bottom combined GFP panels. Uncropped and unprocessed scans are provided in the Source Data file. b, d, g The experiments were performed three times with similar results.