A DEAD Protein that Activates Intron Self-Splicing without Unwinding RNA  Amanda Solem, Nora Zingler, Anna Marie Pyle  Molecular Cell  Volume 24, Issue 4, Pages 611-617 (November 2006) DOI: 10.1016/j.molcel.2006.10.032 Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 1 Protein-Mediated Splicing (A) Mss116-, Cyt-19-, and Ded1-promoted splicing of ai5γ. Reactions were performed with 600 nM protein, 1 mM ATP, and 8 mM MgCl2 at 30°C; time points, 0, 15, 30, 60, and 120 min. Control-lane time points, 0 and 120 min. Self-splicing products are provided as a marker. I-Lar, intron lariat; P, precursor; I-Lin, a population containing linear and/or broken lariat intron RNA; LE, ligated exons; 3E, 3′ exon; and 5E, 5′ exon. (B) Splicing with 600 nM Mss116 in the presence of ATP, AMPPNP, and ADP. Time points, 0 and 120 min. (C) 600 nM NS3, Dbp8, and the Dbp8/Esf2 complex cannot facilitate splicing. Time points, 0 and 120 min. Molecular Cell 2006 24, 611-617DOI: (10.1016/j.molcel.2006.10.032) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 2 Structure and ATPase Activity of Mss116 and Mutant Variants (A) Schematic of Mss116 architecture. Mss116 contains a core of two RecA folds (yellow and brown) and an arginine-rich CTD (red) linked by a predicted α helix (Jpred, Cuff and Barton, 2000; green, upper panel). Conserved motifs of the RecA core domains are shown in the lower panel. Mutated amino acids are indicated in red. (B) ATPase activities. Rates of ATP hydrolysis were normalized to the RNA-stimulated wild-type activity. Error bars represent standard deviation from three experiments. In this experiment, the final ATP concentration was 10 μM, which results in hydrolysis under highly sensitive but nonsaturating conditions. Note that specific activities for ATP hydrolysis were determined under standard conditions of ATP saturation (1 mM, see Figure S2). Molecular Cell 2006 24, 611-617DOI: (10.1016/j.molcel.2006.10.032) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 3 RNA and ATP Binding by Mss116 Variants (A) Protein-bound RNA is retained on the nitrocellulose membrane, while the remaining free RNA is bound to the nylon membrane. The percentage of bound RNA is indicated below. At a concentration of 250 nM, WT and mutant proteins exhibit comparable RNA affinities. (B) ATP binding was detected by UV crosslinking α-32P[ATP] to the protein, followed by SDS-PAGE. Mss116 WT protein, the SAT→AAA mutant, and the K158A mutant show comparable ATP-binding efficiencies, while the K158R mutant displays a severe ATP-binding defect. The K158 mutations cause slightly different effects on Mss116 function than previously reported for other helicases (Sung and Stratton, 1996 and references therein; Tanner et al., 2003). However, inspection of the tightly packed active site of the Vasa helicase (a similar RNA-specific DEAD protein [Sengoku et al., 2006]) suggests that there might be significant steric defects for a K→R mutation in motif I, potentially resulting in the ATP-binding defect observed here. The K158A mutant appears to retain sufficient interactions with ATP to maintain binding. Molecular Cell 2006 24, 611-617DOI: (10.1016/j.molcel.2006.10.032) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 4 Unwinding and Splicing Activity of Mss116 and Mutant Variants (A) Unwinding of a 12 bp or, when specified, 18 bp duplex with a 3′ or 5′ss overhang or a blunt end in the presence of 100 nM Mss116 WT and mutant proteins, 0.1 nM RNA, and 30 mM KCl. Time points, 0 and 30 min. A control without protein is provided to demonstrate duplex stability under these conditions. D, heat-denatured control indicating position of labeled single strand. (B) Unwinding of a 12 bp duplex with a 3′ss overhang in the presence of 250 nM Mss116 WT and mutant proteins, 0.5 nM RNA, and 100 mM KCl. Time points, 0 and 60 min. Protein concentration and monovalent salt were varied over a wide range in an effort to detect trace helicase activity by the mutant proteins. (C) Splicing of ai5γ in the presence of 250 nM Mss116 WT and mutant proteins. Time points, 0 and 120 min. After 2 hr, the lariat fraction in the reaction mixture is 72% (WT), 36% (SAT/AAA), and 16% (K158A). The K158R mutant shows no splicing activity. Note that these values are only a qualitative measure of activity. Molecular Cell 2006 24, 611-617DOI: (10.1016/j.molcel.2006.10.032) Copyright © 2006 Elsevier Inc. Terms and Conditions