1. An RNA Switch at the 5′ Splice Site Requires ATP and the DEAD Box Protein Prp28p 
Jonathan P Staley, Christine Guthrie 
Molecular Cell 
Volume 3, Issue 1, Pages (January 1999)
DOI: /S (00)

2. Figure 1
A Strategy for Impeding the Switch of U1 for U6: Hyperstabilization of the U1:5′ Splice Site Interaction
(A) The switch of U1 for U6 at the 5′ splice site. U1 forms six base pairs with the actin 5′ splice site of S. cerevisiae. Exons are depicted as shaded rectangles and the intron, as a line.
(B) Extension of the U1:5′ splice site interaction to ten base pairs through 5′ splice site mutations. The boxed base pair was mutated to test for base pairing (Figure 2A).
(C) Extension of the U1:5′ splice site interaction to 12 base pairs through a combination of U1 and 5′ splice site mutations. Boxed bases show the compensatory analyses used to test for base pairing between the 5′ splice site and U1 or U6 (Figures 2B, 4A, 5A, 6).
Molecular Cell 1999 3, 55-64DOI: ( /S (00) )

3. Figure 2
Extension of the U1:5′ Splice Site Interaction In Vivo Confers a Defect in 5′ Splice Site Cleavage
(A) Extension confers a defect that is relieved by high temperature (30°C) or the destabilizing mutation U1-5A. Primer extension of an actin reporter containing a wild-type (lanes 2 and 4) or mutated (lanes 3 and 5) 5′ splice site (Figure 1A and Figure 1B) is shown for cells grown at 16°C or 30°C. Primer extension of the actin reporter containing a wild-type (lanes 6 and 8) or mutated (lanes 7 and 9) 5′ splice site is shown for a U1 wild-type or U1-5A mutant strain (Figure 1B) grown at 16°C. Splicing defects were quantitated in triplicate. Unmarked bands reflect transcription pausing. Lanes 1 and 10: MspI digest of pBR322.
(B) Extending the U6:5′ splice site interaction suppresses the splicing defect conferred by hyperstabilizing the U1:5′ splice site interaction. Growth at various copper concentrations is shown for the indicated U1, U6, and actin 5′ splice site residues (Figure 1C, boxed bases); growth at higher copper reflects higher splicing efficiency.
Molecular Cell 1999 3, 55-64DOI: ( /S (00) )

4. Figure 4
Hyperstabilization of the U1: 5′ Splice Site Interaction In Vitro Traps a Functional Intermediate at Low Temperature
(A) Hyperstabilization in vitro confers a cold-sensitive defect to 5′ splice site cleavage. Actin pre-mRNA containing a wild-type (lanes 1 and 4) or mutated (lanes 2, 3, 5, and 6) 5′ splice site was spliced at 23°C in lengthened U1 mutant extracts (Figure 1C).
(B) Hyperstabilization traps a functional intermediate. A mutated 5′ splice site (lanes 1 and 3), versus a wild-type 5′ splice site (lanes 2 and 4), conferred a defect to 5′ splice site cleavage at 15°C but not at 35°C. An intermediate trapped by hyperstabilizing the U1:5′ splice site interaction at 15°C was converted into an active spliceosome after shifting to 35°C (lane 6) but not while maintaining at 15°C (lane 5); addition of unlabeled competitor before the shift prevented de novo splicing (data not shown).
(C) prp28-1 extracts were also defective in 5′ splice site cleavage at 15°C (cf., lanes 1 and 3); this defect was rescued by rPrp28p (lanes 3–6).
Molecular Cell 1999 3, 55-64DOI: ( /S (00) )

5. Figure 5
Hyperstabilization of the U1:5′ Splice Site Interaction Blocks the Dissociation of U1 and U4
(A) Affinity purification of a spliceosomal intermediate trapped by hyperstabilization. Biotinylated pre-mRNA was incubated with splicing extract at 15°C in the absence (lanes 1–5) or presence (lanes 6–10) of ATP and quenched with nonbiotinylated competitor. Intermediates were affinity purified using streptavidin-agarose beads; snRNAs were detected by primer extension. As a control (ctrl) for the quench, competitor was added with the biotinylated pre-mRNA (lanes 5 and 10). Boxed bases correspond to those in Figure 1C.
(B) Affinity purification of a spliceosomal intermediate trapped by prp28-1. Spliceosomes were assembled at 15°C on biotinylated pre-mRNA with a wild-type 5′ splice site in PRP28 (WT) or prp28-1 (-1) extracts in the absence (lanes 1–3) or presence (lanes 4–6) of ATP. Procedures and controls (lanes 1 and 4), as in (A).
(C) U4 and U6 are base paired in both trapped intermediates. RNA from the affinity-purified intermediates was extracted under native conditions, separated on a native gel, and probed for U4 and U6 by Northern analysis; as a control, half of the sample was heat denatured (lanes 2 and 4).
Molecular Cell 1999 3, 55-64DOI: ( /S (00) )

6. Figure 6
By Native Gels, Hyperstabilization of the U1:5′ Splice Site Interaction Traps the Prespliceosome, as Does prp28-1
Spliceosomal intermediates were assembled at 15°C on 32P-labeled actin pre-mRNA containing a wild-type 5′ splice site (lane 2) or a mutated 5′ splice site (lane 1; Figure 1C). Intermediates were also assembled at 15°C on pre-mRNA in wild-type extract (lanes 3 and 4) or prp28-1 mutant extract (lanes 5 and 6); rPrp28p was added at 1 μM (lanes 4 and 6). The intermediates were treated with heparin and separated on native gels. By Northern, the poorly resolved complexes U1·U2 and U2·U4/U6·U5 (lanes 1 and 2) were clearly distinguished; the intermediate trapped in prp28-1 extract (lane 5) contained only U2 (data not shown).
Molecular Cell 1999 3, 55-64DOI: ( /S (00) )

7. Figure 3
Hyperstabilizing the U1:5′ Splice Site Interaction Exacerbates prp28-1, Weakening the Interaction Suppresses prp28-1
(A) Exacerbation of the prp28-1 splicing defect. Primer extension of the actin reporter containing a wild-type (lanes 1 and 3) or mutated (lanes 2 and 4) 5′ splice site (Figure 1B) in a wild-type (lanes 1 and 2) or mutant (lanes 3 and 4) strain at 30°C.
(B) Suppression of the prp28-1 cold-sensitive growth defect. Growth is shown for the wild-type or mutant strain containing wild-type U1, the mutant 4U, or the mutant 2A,10A (in addition to the chromosomal copy of U1). The schematic shows disruption of actin U1:5′ splice site base pairing by the mutant 4U, which suppresses prp28-1 at 18°C, and extension of base pairing by the mutant 2A,10A, which is synthetically lethal with prp28-1 at 22°C.
Molecular Cell 1999 3, 55-64DOI: ( /S (00) )

8. Figure 7 The Switch of U1 for U6 Requires Prp28p and ATP
(A) The Prp28 requirement. An intermediate with a hyperstabilized U1:5′ splice site interaction was trapped at 15°C and shifted to 35°C, as in Figure 4B. Spliceosome assembly was monitored by affinity purification of intermediates and primer extension of bound snRNAs, as in Figure 5A. The levels of U1, U4, and U6 bound to pre-mRNA were quantitated relative to U5 and normalized to the levels before the shift. Bars 1–3 show levels before the shift for extract immunodepleted of Prp28p; the data for mock-depleted extract before the shift are equivalent (data not shown). The snRNA levels after the shift are depicted for mock-depleted extract (bars 4–6) and immunodepleted extract (bars 7–9). Wild-type rPrp28p (bars 10–12), or the ATPase mutant K227E (bars 13–15), were added before the shift. Error bars reflect variation in duplicate experiments.
(B) The ATP requirement. After trapping an intermediate with a hyperstabilized U1:5′ splice site interaction at 15°C (bars 1–3), the splicing reaction was incubated with 1.5 mM glucose to deplete ATP and then shifted to 35°C to test for formation of active spliceosomes (bars 4–9; see schematic). ATP was added before the shift to test for rescue (bars 7–9).
Molecular Cell 1999 3, 55-64DOI: ( /S (00) )

9. Figure 8 Prp28p and ATP Exchange U1 for U6 at the 5′ Splice Site
In the simplest model, Prp28p utilizes ATP to unwind the U1:5′ splice site interaction. Alternatively, Prp28p could destabilize this interaction by displacing a stabilizing protein, such as yU1C (Tang et al. 1997). Finally, since U6 appears to play an active role in displacing U1 (Figure 2B), Prp28p could act to facilitate this role for U6.
Molecular Cell 1999 3, 55-64DOI: ( /S (00) )