Figure 5
p.E415G compromises recognition of the downstream 5′ splice site by U1-70K.
(a,b) RT-PCR of E15E16 (wt and p.E415G) and E16E17 (wt and p.E415G) minigenes in HeLa cells. (c) Schematic structures of MS2-attached wild-type (wt) and p.E415G substrates used for isolation of early spliceosome complex (left). Immunoblotting (IB) of purified spliceosome complex assembled on the indicated substrates (right). β-glo-MS2 is a control construct carrying MS2-attached human β-globin exon 1-intron 1-exon 235. A representative gel image of three independent experiments is shown. Signal intensities of U1-70K bound to wt-MS2 are on average 2.19-fold (SD = 1.21, n = 3, p < 0.05 by Student’s t-test) higher than those bound to p.E415G-MS2.
