Volume 51, Issue 2, Pages 174-184 (July 2013) ATP-Dependent roX RNA Remodeling by the Helicase maleless Enables Specific Association of MSL Proteins  Sylvain Maenner, Marisa Müller, Jonathan Fröhlich, Diana Langer, Peter B. Becker  Molecular Cell  Volume 51, Issue 2, Pages 174-184 (July 2013) DOI: 10.1016/j.molcel.2013.06.011 Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 1 The RNA Helicase MLE Binds to roX RNA Containing Conserved Stem Loops (A) Schematic representation of roX1 and roX2 harboring functionally relevant stem-loop structures SLroX1 and SLroX2. Evolutionarily conserved roX-boxes (sequence indicated) are depicted as black bars. (B) Secondary structures of SLroX1 and SLroX2 as predicted by M-fold. The corresponding free energy value (ΔG) is indicated. RoX-boxes are written in red and marked with black bars. (C) MLE binds to roX RNA containing SLroX structures. RNAs were immobilized on amylose beads as shown in Figure S1A. Proteins in a nuclear extract of L2-4 cells were allowed to bind to a 93 or 123 nt fragment containing SLroX1 or SLroX2, respectively (roX1-93 and roX2-123) in the presence of ATP. Experiments were also performed with roX1-93 and roX2-123 derivatives lacking the 5′ (roX1-93Δ5′and roX2-123Δ5′) or the 3′ sequences (roX1-93Δ3′and roX2-123Δ3′) of SLroX1 and SLroX2, respectively. Eluted RNAs were analyzed by PAGE, and association of MLE was monitored by western blotting (IN is input). The percentage of bound MLE was quantified relative to the amount of eluted RNA. Two (roX1) and three (roX2) independent experiments (n) were performed, and values were normalized to MLE's roX1-93 and roX2-123 binding, respectively. SD and extreme highest and lowest values were determined for duplicates and triplicates, respectively. Molecular Cell 2013 51, 174-184DOI: (10.1016/j.molcel.2013.06.011) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 2 ATP Improves MLE’s Binding Strength and Selectivity for a Conserved Stem-Loop Structure MS2 affinity purification and assessment of ATP dependence. RNA-binding proteins were isolated in the presence or absence of ATP as in Figure 1 using the following roX derivatives: (A) SLroX2-containing fragment in sense (roX2-123) and antisense orientation (roX2-123AS); (B) SLroX1-containing fragment in sense (roX1-93) and antisense orientation (roX1-93AS); (C) roX2-123 and the mutant roX2-123mPARK, in which the 3′ region of the stem is mutated; (D) roX2-123 and two mutants, in which four A-U bp in the stem are replaced by G-C bp (roX2-123mGC) or U-A bp (roX2-123mUA), respectively; (E) same as in (D), except that corresponding roX1 derivates were used. Three (A, B, and D) or two (C and E) independent experiments (n) were performed. Values were normalized to MLE’s roX2-123 (A, C, and D) or MLE’s roX1-93 binding (B and E). SD and extreme highest and lowest values were determined for triplicates and duplicates, respectively. Molecular Cell 2013 51, 174-184DOI: (10.1016/j.molcel.2013.06.011) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 3 Experimental Determination of the SLroX2 Secondary Structure (A–D) Enzymatic and chemical probing of roX2. roX2-123 (A) or full-length roX2 (B–D) was subjected to limited digestion with RNases T1, T2, or V1 (A and B), or modification with DMS (A and C) or CMCT (D) followed by primer extension analyses using oligonucleotide O-25 (Table S2) and electrophoretic separation of resulting cDNAs (for details, see the Experimental Procedures). Lanes labeled U, G, C, and A indicate sequencing reactions spiked with respective dideoxynucleotides obtained with the same primer. Contr. corresponds to primer extension analysis on unmodified transcripts. Nucleotides are numbered relative to nucleotide +1 of roX2. (E) Schematic summary of the experimental data shown in (A)–(D). The free energy value (ΔG) of the stem-loop structure (M-fold) is indicated. The box describes the labeling of RNase cleavage sites, modified nucleotides, and their intensities. Molecular Cell 2013 51, 174-184DOI: (10.1016/j.molcel.2013.06.011) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 4 Interaction of MLE with SLroX2 and ATP-Dependent Remodeling (A–C) Footprinting analysis of MLE on full-length roX2 treated with RNase T2 (A), DMS (B), or CMCT (C) in the absence (−) or presence (+) of ATP. cDNAs obtained by primer extension using oligonucleotide O-25 were electrophoretically separated. Lanes labeled with U, G, C, and A apply to the Sanger sequencing ladders obtained with the same primer. Nucleotides are numbered relative to nucleotide +1 of roX2. Nucleotides protected by MLE are indicated by asterisks (A), and circles denote nucleotides that become more accessible to the probes in presence of MLE and ATP (B and C). Intensities of the bands were determined, and the ratio of “signal (+ATP)” to “signal (−ATP)” was calculated. The fold difference is indicated on each band analyzed. (D) Schematic representation of experimental data shown in (A)–(C) on the predicted SLroX2 secondary structure in absence or in presence of ATP. See box for details on labeling. MLE binding sites on the stem-loop structure in absence of ATP are labeled on the left. Enhanced accessibility of putative base-paired nucleotides in the presence of MLE and ATP suggests the formation of an alternative secondary structure. In the model, SLroX2 is converted into two small hairpins (ASL1 and ASL2), which are formed by pairing of bases formerly located in the 5′ and 3′ stem of SLroX2. Molecular Cell 2013 51, 174-184DOI: (10.1016/j.molcel.2013.06.011) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 5 Interaction of MLE with SL8 and ATP-Dependent Remodeling (A) Footprinting analysis of MLE on full-length roX2 treated with DMS in the absence (−) or presence (+) of ATP as in Figure 4B. For analysis of SL8, primer extension was performed using oligonucleotide O-22. Circles denote nucleotides that become more accessible to DMS. Quantification was performed as described above. Orange circles denote 2- to 99-fold increase in signal intensity (+ATP/−ATP), and red circles indicate a >100-fold increased signal. (B) Schematic representation of experimental data shown in (A) on the SL8 secondary structure as predicted by M-fold. Molecular Cell 2013 51, 174-184DOI: (10.1016/j.molcel.2013.06.011) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 6 Probing the Conformation of roX2 In Vitro and In Vivo by Oligonucleotide Pull-Down (A) Probing roX2-123 secondary structures in vitro. Biotinylated probes (P0–P8) were used to capture the original stem-loop structure (left) or the modeled alternative structure (right), and recovered RNA was analyzed by PAGE. The cartoon illustrates the position on roX2-123, to which the probes hybridize (for details, see text and Figure S6). (B) Probing roX2-123 secondary structures in vitro in presence of MLE. Complexes of roX2-123 and purified MLE or MLEGET were formed in the absence or presence of ATP. Probes P6 and P8 were used as in (A) to capture the original stem-loop structure and the alternative secondary, respectively. Recovery of roX2-123 with probe P6 without ATP was set to 1. Data are represented as mean ± SD. (C) Probing the conformation of roX2 in vivo. The same probes as in (A) were used to capture roX2-123 secondary structures in crosslinked chromatin from L2-4 cells. Eluted RNA was subjected to quantitative reverse-transcription PCR (RTqPCR). Values were normalized to GAPDH mRNA (see the Experimental Procedures). Data are represented as mean ± SD. The red line indicates the threshold of background. Molecular Cell 2013 51, 174-184DOI: (10.1016/j.molcel.2013.06.011) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 7 Remodeling of SLroX2 by MLE Generates a Binding Site for MSL2 (A–C) MS2 affinity purification shows ATP-dependent enrichment of MLE and MSL2 with roX2 (A) and roX2-123 (B and C). (A and B) RNA-binding proteins were retrieved from L2-4 nuclear extract in the presence or absence of ATP with the indicated RNAs. (C) Interaction of purified recombinant MLE and MSL2 with roX2-123 was assayed by MS2 affinity purification. Eluted RNA was analyzed by PAGE, and copurifying proteins were monitored by western blotting using specific MLE and MSL2 antibodies, respectively (IN is input). (D and E) ATPase activity regulates complex formation of MLE and roX2-123. Shown is EMSA with roX2-123 in the presence and absence of ATP and 25–500 nM recombinant MLE (D) or MLEGET (E). Black bar indicates retarded RNA:protein complexes. (F and G) Shown is formation of higher-order complexes of MLE, MSL2, and roX2-123. EMSA was performed with roX2-123 (F) and roX2-123AS (G) in the presence and absence of ATP and constant concentration of recombinant MLE or MLEGET (10 nM) and increasing amounts of MSL2 (10–50 nM). (H) Interaction of MSL2 and MLE is mediated by RNA and stimulated by ATP. Protein-RNA complexes were formed with purified recombinant MLE, MSL2, and roX2-123 (F) or roX2-123AS (G) in the presence or absence of ATP. Following immunoprecipitation using an MLE antibody, elution fractions were analyzed for their RNA content by PAGE and for copurifying MSL2 by western blotting. INPUT and IP fractions are labeled. (I) Shown is schematic representation of the potential function of the RNA helicase MLE suggested in this work. MLE specifically recognizes the SLroX2 stem-loop structure and remodels it in an ATP-dependent fashion. The affinity of MLE for the remodeled RNA is enhanced, which in turn may provide a platform for the recruitment of MSL2. For details, see text. Molecular Cell 2013 51, 174-184DOI: (10.1016/j.molcel.2013.06.011) Copyright © 2013 Elsevier Inc. Terms and Conditions