Fig. 4: SPF45 binds to all introns while U2AF⁶⁵ cannot bind to short introns.

a Cellular formaldehyde crosslinking and immunoprecipitation experiments shows SPF45 binding to all the indicated introns. Mini genes containing these four introns were individually co-transfected into HEK293 cells with a plasmid expressing Flag-SPF45 protein. The Flag-SPF45 was immunoprecipitated after formaldehyde crosslinking and then co-precipitated pre-mRNAs were quantified by RT–qPCR using specific primers (see the schematic mini gene below). Means ± SEM are given for three independent experiments and two-tailed Student t-test values were calculated (p = 0.0353 for AdML vs HNRNPH1 intron, p = 0.0083 for AdML vs EML3 intron, p = 0.0638 for AdML vs MUS81 intron). *P < 0.05, ***P < 0.0005, ^n.s. P > 0.05. b Cellular CLIP experiments shows strong U2AF⁶⁵ binding to control AdML pre-mRNA but not much to the three indicated short introns. Mini genes containing these four introns were individually co-transfected into HEK293 cells and irradiated with UV light. The lysates were immunoprecipitated with anti-U2AF⁶⁵ and anti-SF3b155 antibodies and then immunoprecipitated RNAs were quantified by RT–qPCR using specific primers (see the schematic mini gene below). Means ± SEM are given for three independent experiments and two-tailed Student t-test values were calculated (p = 0.0034 for AdML vs HNRNPH1 intron, p = 0.0066 for AdML vs EML3 intron, p = 0.0020 for AdML vs MUS81 intron). **P < 0.01. Source data of the above graphs are provided as a Source Data file.