Volume 39, Issue 3, Pages 385-395 (August 2010) The DEAH Box ATPases Prp16 and Prp43 Cooperate to Proofread 5′ Splice Site Cleavage during Pre-mRNA Splicing  Prakash Koodathingal, Thaddeus Novak, Joseph A. Piccirilli, Jonathan P. Staley  Molecular Cell  Volume 39, Issue 3, Pages 385-395 (August 2010) DOI: 10.1016/j.molcel.2010.07.014 Copyright © 2010 Elsevier Inc. Terms and Conditions

Molecular Cell 2010 39, 385-395DOI: (10.1016/j.molcel.2010.07.014) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 1 An ATP-Dependent Activity Rejects Pre-mRNA Reversibly prior to 5′ Splice Site Cleavage if the Rate of 5′ Splice Site Cleavage Is Slow (A) An ATP-dependent activity competes with 5′ splice site cleavage. U6/sU80 (Sp) spliceosomes stalled in Mg2+ on an ACT1 pre-mRNA substrate were affinity-purified and then either frozen (no incubation) or incubated with the indicated metals and with or without ATP-Mg2+. The migration of the splicing species is indicated on the right. The bottom panel shows the efficiency of 5′ splice site cleavage. Values are represented as means ± standard error of mean (SEM) from three experiments (see also Figure S1). (B) ATP-dependent rejection is reversible. Spliceosomes were stalled and purified as in A and then either frozen (no incubation) or incubated with or without ATP. Spliceosomes incubated with ATP were then washed to remove ATP and incubated further with or without ATP. Dashed lines indicate that samples were not incubated further. (C) In the absence of ATP, U6/sU80 (Sp) spliceosomes catalyzed 5′ splice site cleavage in Mg2+ but with an apparent rate 10-fold lower than that in Mn2+ and 20-fold lower than that in Cd2+ (kMg = 0.010 ± 0.006 min−1; kMn = 0.13 ± 0.02 min−1; kCd = 0.22 ± 0.02 min−1; errors reflect the error in the fit of the data points; see Experimental Procedures). Reactions were as in (A) but without ATP. Error bars represent ± SEM of each data point for three experiments. Molecular Cell 2010 39, 385-395DOI: (10.1016/j.molcel.2010.07.014) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 2 The DEAH Box ATPase Prp16p Rejects Defective Spliceosomes prior to 5′ Splice Site Cleavage (A) 5′ splice site cleavage in U6/sU80 (Sp) spliceosomes is repressed by four different nucleoside triphosphates (NTPs). Reactions were as in Figure 1A, but with Mg2+ only. (B) rPrp16p mutants but not wild-type (WT) permit 5′ splice site cleavage by U6/sU80 (Sp) spliceosomes even in the presence of ATP. Reactions were as in Figure 1A, except that spliceosomes were stalled in yeast extract supplemented with recombinant variants of Prp16p, Prp22p, or Prp43p and then incubated only with Mg2+. The intensity of the middle panel is increased. Values and errors were as in Figure 1A (see also Figure S2). Molecular Cell 2010 39, 385-395DOI: (10.1016/j.molcel.2010.07.014) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 3 Prp16p Can Bind the Spliceosome prior to 5′ Splice Site Cleavage (A) In Cd2+, stalled and affinity purified U6/sU80 (Sp) spliceosomes undergo not only 5′ splice site cleavage but also exon ligation in an ATP- and Prp16p-dependent manner. Reactions were as in Figure 2B but included Cd2+ in the final incubation. (B and C) Prp16p antibodies immunoprecipitate pre-mRNA bound to spliceosomes stalled prior to 5′ splice site cleavage but only if U6 is present (B) and the 5′ splice site conforms to the consensus sequence (C). Spliceosomes were stalled and purified as in Figure 1A, except in (B), lanes 4–6, where U6/sU80 (Sp) was omitted and in (C), lanes 4–6, where the 5′ splice site (5′SS) was mutated (G1C). Top panels show inputs (i) and immunoprecipitates, which were 10% and 40% of a splicing reaction, respectively; bottom panels show quantitation. Values and errors were as in Figure 1A. Molecular Cell 2010 39, 385-395DOI: (10.1016/j.molcel.2010.07.014) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 4 The DEAH Box ATPase Prp43p Mediates Discard of Pre-mRNA Stalled prior to 5′ Splice Site Cleavage (A–D) The spliceosome discards stalled pre-mRNA and this discard requires Prp43p. U6/sU80 (Sp) spliceosomes were assembled on labeled UBC4 pre-mRNA in Mg2+ and in extracts supplemented with wild-type (WT) (A and B) or mutated (Q423E) (C and D) rPrp43p. The stalled spliceosomes were then chased by adding excess unlabeled UBC4 pre-mRNA. The reactions were analyzed by glycerol gradient before (top) and after (bottom) the chase (A and C). Inputs (i) represent 10% of the reaction. The pre-mRNA in each fraction was quantitated (B and D) (see also Figure S3). (E and F) Discarded and unassembled pre-mRNA migrate similarly. (E) Glycerol gradient analysis is shown for pre-mRNA in the absence of extract (top) or in the presence of extract but in the absence of ATP (middle) or with a G1C/U2G double mutation of the 5′ splice site consensus sequence (bottom). (F) Quantitation of E. (G and H) Efficient discard of stalled pre-mRNA is prevented by mutated rPrp16p. (G) Reactions were as in Figures 4A and C, except extracts were supplemented with wild-type (WT) or mutated (K379A) rPrp16p, and reactions were analyzed only after the chase. Molecular Cell 2010 39, 385-395DOI: (10.1016/j.molcel.2010.07.014) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 5 A Collaborative Role for Prp16p and Prp43p in the Fidelity of 5′ Splice Site Cleavage A simplified pathway for splicing is shown, emphasizing the two roles for Prp16p and the role for Prp43p at the stage of 5′ splice site cleavage. Before 5′ splice site cleavage, Prp16p antagonizes splicing by competing with 5′ splice site cleavage and thereby permits rejection of suboptimal pre-mRNA through a kinetic proofreading mechanism. After rejection, a pre-mRNA may return to the 5′ splice site cleavage conformation or discard in a Prp43p-dependent manner. After 5′ splice site cleavage, Prp16p promotes splicing by allowing exon ligation. In a model for kinetic proofreading, the fidelity of 5′ splice site cleavage is enhanced by a competition between 5′ splice site cleavage and the ATP-dependent activity of Prp16p. Specificity for a wild-type substrate would be established if k5′ splice site cleavage (wild-type) > k5′ splice site cleavge (aberrant) and/or if krejection(aberrant) > krejection(wild-type). Our data provide evidence for the former case. Molecular Cell 2010 39, 385-395DOI: (10.1016/j.molcel.2010.07.014) Copyright © 2010 Elsevier Inc. Terms and Conditions