1. Volume 60, Issue 3, Pages 487-499 (November 2015)
Structure of the RNA Helicase MLE Reveals the Molecular Mechanisms for Uridine Specificity and RNA-ATP Coupling 
J. Rajan Prabu, Marisa Müller, Andreas W. Thomae, Steffen Schüssler, Fabien Bonneau, Peter B. Becker, Elena Conti 
Molecular Cell 
Volume 60, Issue 3, Pages (November 2015)
DOI: /j.molcel
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2. Molecular Cell 2015 60, 487-499DOI: (10.1016/j.molcel.2015.10.011)
Copyright © 2015 Elsevier Inc. Terms and Conditions

3. Figure 1
The Stable Core of the DExH Helicase MLE Has Unusual RNA-Binding and Unwinding Properties
(A) Domain organization of D. melanogaster MLE. The portion of the molecule included in the crystal structure (MLEcore) is shown in colors (from dsRBD2 in pink to the L3 linker region in gray), with the individual domains labeled. The regions of the molecule not included in the crystal structure are in white.
(B) EMSAs carried out with ss17 with an A10 or U10 3′ extension. Both substrates were labeled at the 5′ end with 32P and incubated with identical increasing concentrations of MLEcore protein (5, 10, and 20 nM). The two distinct species could be due to one or two molecules of MLE per RNA substrate.
(C) On the left, EMSA with a blunt-ended RNA substrate ds17 (ss17 annealed with the complementary sequence css17). The substrate was incubated with either MLEcore (residues 105–1158) or MLEcore-ΔRB (residues 257–1158) at identical increasing concentrations (5, 10, and 20 nM). On the right is a Coomassie-stained SDS-PAGE of the protein samples used in the EMSA.
(D) Unwinding activity of MLEcore and MLEcore-ΔRB. RNA duplexes (ds17) with or without a U10 overhang were incubated with the same concentration of proteins (10 nM) over an increasing amount of time (indicated) and separated by native PAGE.
(E) RNase protection patterns of MLEcore. A single-stranded (C∗U)28C RNA internally labeled with 32P at the uridine α-phosphates was incubated with proteins and nucleotides as indicated and treated with RNase A/T1, and the reaction products were analyzed by denaturing PAGE. The size of the protected fragments is indicated on the left.
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4. Figure 2
Overall Structure of MLEcore: An Assembly of Five RNA-Binding Domains and Extended Regions
(A) Crystal structure of MLEcore in two orientations related by a 90° rotation around a vertical axis. The structure is shown in either cartoon representation (top panels) or surface representation (bottom panels). The domains are colored as in Figure 1A and labeled. RNA is in black, with the 5′ end (nucleotide 1) and the 3′ end (nucleotide 10) indicated. Distinctive structural elements discussed in the text are indicated. This and all other structure figures were generated with the PyMOL Molecular Graphics System (version 1.2, Schrödinger).
(B) Close-up view of the interactions among the β-hairpin of the DExH domain, the OB-like fold, and dsRBD2.
(C) Model of a dsRNA (gray) bound to MLE dsRBD2. The model was obtained by superposing the structure of RHA dsRBD2 bound to a dsRNA (Fu and Yuan, 2013) onto the dsRBD2 domain in MLEcore. For clarity, only the dsRBD2 (pink) and RecA2 (orange) domains of MLEcore are shown.
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5. Figure 3 The RNA-Binding Path through MLEcore
(A) Schematics showing the protein-RNA interactions in the MLEcore-U10-ADP-AlF4 structure. Polar contacts are highlighted with dotted lines, and stacking contacts are shown with thick gray lines. Interacting residues are shown with ellipses and circles, depending on whether they use the side chain or the main chain to mediate the RNA-binding contact.
(B–F) Zoomed-in panels relative to the overall structure shown in the center. The nucleotides and discussed MLE residues are labeled. Dotted lines indicate polar contacts.
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6. Figure 4 Effect of Structure-Based Mutations
(A and B) Residues targeted for mutagenesis colored per domain (according to Figure 1A) and in the context of the structure.
(C) EMSAs carried out with 25 nM U20 ssRNA substrate and MLE mutants at increasing concentrations (5, 10, and 20 nM). The presence of two gel-shifted bands likely represents distinct complexes with either one or two molecules of MLE bound to the RNA substrate.
(D) EMSAs carried out with 25 nM ds17 dsRNA substrate and MLE mutants at increasing concentrations (0.25, 2, and 5 μM).
(E) RIP assays performed with GFP-tagged MLEFL-WT and indicated mutants. The abundance of roX2 RNA was quantified in qRT-PCR using amplicons located in stem loop 2 and stem loop 7 of roX2. Relative roX2 enrichment (IP/input) of mutants compared to WT is presented. Data are represented as mean ± SD. n is the number of experiments.
(F) Representative confocal images of stable S2 cell lines expressing WT and mutant MLEFL-GFP proteins. Immunostaining against GFP, MLE, and MSL2 and DNA counterstaining with DAPI are shown. The percentage of cells with X chromosome territory-localized MLEFL-GFP (GFP column) and eMLE (MLE column) is indicated. n is the number of cells analyzed. Scale bar is 5 μm.
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7. Figure 5
Coupling the Presence or Absence of the Nucleotide γ-Phosphate to RNA
(A) Structure (upper panel) and schematics (lower panel) of interactions in the structure of S. cerevisiae Prp43 bound to ADP (He et al., 2010; Walbott et al., 2010). Helicase motifs are shown in different colors and indicated.
(B) Structure (upper panel) and schematics (lower panel) of interactions in the structure of D. melanogaster MLE bound to RNA and ADP-AlF4.
(C) Unwinding activity of MLEcore and MLEcore-ΔHook. RNA duplexes (ds17) with a U10 overhang were incubated with the same concentration of proteins (10 nM) over an increasing amount of time (indicated) and separated by native PAGE.
(D) EMSAs carried out with 25 nM ss17-U10 ssRNA substrate and MLE mutants at increasing concentrations (5, 10, and 20 nM).
Molecular Cell  , DOI: ( /j.molcel )
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