Fig. 7. ULM–UHM binding between SF3b155 and SPF45 promotes splicing of pre-mRNA with short introns.

a SDS-PAGE analysis of the indicated purified recombinant proteins. b GST pull-down assay in RNase A-treated HeLa nuclear extract shows the UHM-dependent binding of GST-SPF45 to SF3b155 but not to U2AF heterodimer. Proteins that associated with these GST-fusion proteins were detected by Western blotting using the indicated antibodies. c Immunoprecipitation of SF3b155 with the indicated GST-fusion proteins shows the competitive binding between U2AF⁶⁵ and SPF45 to SF3b155 via UHM–ULM interactions. The same reaction mixtures in panel a were immunoprecipitated with anti-SF3b155 antibody and the associated proteins were analyzed by Western blotting with antibodies against SF3b155, U2AF⁶⁵, and SPF45. d The graph shows quantification of the bands on Western blots (panel b as the representative blots). U2AF⁶⁵ and GST-SPF45 scanned data were normalized to SF3B155 (U2AF⁶⁵/SF3B155 and GST-SPF45/SF3B155) and plotted. Means ± SEM are given for four independent experiments and two-tailed Student t-test values were calculated (Upper panel: p = 0.0470 for GST vs GST-SPF45 left, p = 0.0322 for GST vs GST-SPF45 right, p = 0.6238 for GST vs GST-SPF45/UHMmt; Lower panel: p = 0.0036 for GST vs GST-SPF45 left, p = 0.0010 for GST vs GST-SPF45 right, p = 0.5023 for GST vs GST-SPF45/UHMmt). *P < 0.05, **P < 0.01, ^n.s.P > 0.05. Source data of all the above panels are provided as a Source Data file.