Figure 4.

Splicing of RPS30A pre-mRNAs through alternative pre-spliceosome assembly pathways. (A) Experimental design.Binding of labeledU1 and U2 was visualized with red and green excitation. The intron of each pre-mRNA molecule was labeled with 8 dye molecules on average; intron release was detected as loss of blue-excited fluorescence. (B) Fraction of intron fluorescent spots lostfrom a single field of view at various times after adding WCE with ATP (red; N = 923) or without ATP (black; N = 1272). The open red symbol is a photobleaching-independent estimate (average N = 474 ± 12 spot per field of view after 35 min; see Experimental Procedures(C)Time records of intron fluorescence loss in WCE+ATP. Top, an example of fluorescence loss that occurs in a single step, presumably due to splicing.Bottom, fluorescence loss in multiple steps, presumably due to photobleaching.Insets: Gallery of the images (1.34 × 1.34 μm) corresponding to each time point. (D)Rastergram summarizing the presence of fluorescent U1 (red) or U2 (green) snRNPs, or both (yellow), onindividual RPS30A pre-mRNA molecules.Only pre-mRNAs that were observed to loseintron fluorescence in a large step, presumably due to the intron departure,were selected for analysis (N = 54).Records are ordered by the time interval in which intron departure occurred (shading).(E)Time courses of intron departure (open symbols;from CoSMoS experiment) and the second step of splicing (filledsymbols;from ensemble experiment). The red arrow indicates an apparent lag in the intron departure detected in the CoSMoS experiments (see text). (F) Genesis of the U1•U2•pre-mRNA complex that most closely preceded intron departure from each pre-mRNA molecule in (D). Fractions of complexes formed through the U1→U2 (red) or U2→U1 (green) pathways are indicated.(G) Summary of the pathways observed in this experiment.Catalysis of splicing and subsequent intron release can occur through pre-spliceosomes assembled by both U1→U2 and U2→U1 branches.