Commitment to Splice Site Pairing Coincides with A Complex Formation Sharlene R Lim, Klemens J Hertel  Molecular Cell  Volume 15, Issue 3, Pages 477-483 (August 2004) DOI: 10.1016/j.molcel.2004.06.025 Copyright © 2004 Cell Press Terms and Conditions

Figure 1 dsx/βg Splicing (A) Schematic of dsx/βg pre-mRNA containing competing proximal and distal 3′ splice sites. White boxes represent β-globin exon sequences. The gray box represents the dsx exon sequence. The hairpin loop structure represents the enhancer activated by the addition of MS2:RS fusion proteins. Horizontal lines between boxes represent introns. “X” indicates the absence of a 5′ splice site. (B) Representative autoradiogram showing dsx/βg splicing in the presence (lanes 1–6) or absence (lanes 7–12) of MS2:RS9G8. Identities of spliced and unspliced mRNAs are indicated on the right. A log scale graph of the proximal-to-distal product ratios is indicated below the autoradiogram. The x axis corresponds to lanes in the splicing gel shown above. Error bars indicate the standard deviation of at least three independent experiments. The average proximal-to-distal product ratio is shown below each corresponding lane. (C) Quantitation of the total percentage of splicing in the presence (dashed line) or absence (solid line) of MS2:RS9G8. (D) Comparison of dsx/βg splicing complex formation in the presence (lanes 1–5) or absence (lanes 6–10) of MS2:RS9G8 by native polyacrylamide gel electrophoresis. E complex comigrates with the nonspecific H complex. Molecular Cell 2004 15, 477-483DOI: (10.1016/j.molcel.2004.06.025) Copyright © 2004 Cell Press Terms and Conditions

Figure 2 Kinetic Trap Assay to Test for Splice Site Pairing Commitment at E Complex (A) Diagram of the kinetic trap protocol to test for splice site pairing commitment. The dsx/βg substrate is preincubated to enrich for E or A complex, either in the absence (left side, pathway A) or presence (right side, pathway B) of MS2:RS9G8. Splicing is then reinitiated by the addition of a chase reaction. In samples preincubated in the absence of fusion protein, MS2:RS9G8 is added concurrent with the chase addition. Expected spliced products are indicated below. (B) Enrichment of E complex. Dsx/βg pre-mRNA was preincubated in extract depleted of ATP prior to native agarose gel electrophoresis. (C) Chase reaction of Dsx/βg pre-mRNA enriched for E complex. Splicing was reinitiated by addition of ATP and creatine phosphate. The presence or absence of ATP, MS2:RS9G8, and competitor RNA is indicated above each lane. “Preincubation” refers to the reaction conditions during the preincubation, and “chase” refers to changes in reaction conditions during the final incubation. Error bars indicate the standard deviation of at least four independent experiments. The average proximal-to-distal product ratio is shown below each corresponding lane. Molecular Cell 2004 15, 477-483DOI: (10.1016/j.molcel.2004.06.025) Copyright © 2004 Cell Press Terms and Conditions

Figure 3 Kinetic Trap Assay to Test for Splice Site Pairing Commitment at A Complex (A) Enrichment of A complex. Dsx/βg pre-mRNA was preincubated in extract depleted of ATP for 30 min at 30°C. ATP and creatine phosphate were then added, followed by additional incubation at 30°C for 5 or 10 min before native polyacrylamide gel electrophoresis. (B) Chase reaction of Dsx/βg pre-mRNA enriched for A complex. The presence or absence of ATP, MS2:RS9G8, and competitor RNA is indicated above each lane. “Preincubation” refers to the reaction conditions during the preincubation, “A complex enrichment” refers to changes in reaction conditions during the second preincubation at 30°C, and “chase” refers to changes in reaction conditions during the final incubation. Error bars indicate the standard deviation of at least three independent experiments. The average proximal-to-distal product ratio is shown below each corresponding lane. Molecular Cell 2004 15, 477-483DOI: (10.1016/j.molcel.2004.06.025) Copyright © 2004 Cell Press Terms and Conditions

Figure 4 Commitment to Splicing and Splice Site Pairing Are Separate Steps during Spliceosomal Assembly (A and B) In the first level of commitment (A), pre-mRNA substrates become committed to the general splicing pathway with formation of E complex. During the second level of commitment (B), specific splice site pairing is established with the formation of A complex. Molecular Cell 2004 15, 477-483DOI: (10.1016/j.molcel.2004.06.025) Copyright © 2004 Cell Press Terms and Conditions