1. Volume 54, Issue 4, Pages 573-585 (May 2014)
MOV10 Is a 5′ to 3′ RNA Helicase Contributing to UPF1 mRNA Target Degradation by Translocation along 3′ UTRs 
Lea H. Gregersen, Markus Schueler, Mathias Munschauer, Guido Mastrobuoni, Wei Chen, Stefan Kempa, Christoph Dieterich, Markus Landthaler 
Molecular Cell 
Volume 54, Issue 4, Pages (May 2014)
DOI: /j.molcel
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2. Molecular Cell 2014 54, 573-585DOI: (10.1016/j.molcel.2014.03.017)
Copyright © 2014 Elsevier Inc. Terms and Conditions

3. Figure 1 MOV10 Has 5′ to 3′ Directional Unwinding Activity In Vitro
(A) MOV10 contains an N-terminal cysteine/histidine-rich domain (CH domain) and a C-terminal helicase core consisting of two RecA-like domains 1A and 2A. Amino acids in motif I or II, which were mutated to create helicase-deficient MOV10 mutants, are indicated by boxes.
(B) Analysis of UV-crosslinked MOV10-RNA complexes using SDS-PAGE and western analysis. HEK293 cells stably expressing FLAG/HA-tagged MOV10 WT, K530A, or D645N were labeled with 4SU and UV-crosslinked as indicated, followed by anti-FLAG affinity purification. Crosslinked RNA was radiolabeled for detection. IPs were separated by SDS-PAGE, transferred to nitrocellulose membrane, exposed to a phosphorimager, and subjected to anti-HA western analysis.
(C–E) Time-dependent in vitro unwinding assays for MOV10 WT, K530A, and D645N proteins. RNA duplexes containing 5′ overhangs were incubated with purified MOV10 WT (C), K530A (D), or D645N (E) protein in the presence of ATP. Reactions were stopped at 0, 1, 2.5, 5, 10, 15, 20, and 30 min after addition of ATP, and the RNA was separated on nondenaturing Tris-borate-EDTA (TBE) gels followed by visualization using a phosphorimager. Incubation with BSA for 30 min served as a negative control, while 30 s incubation at 95°C served as a positive control.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 2
RNA-Binding Patterns of MOV10 Helicase Mutants Indicate Impaired Directional Translocation along RNA
(A) Correlation coefficients of T-C transition counts per position between individual MOV10 PAR-CLIP libraries.
(B) Venn diagram displaying the overlap of mRNAs containing the top 5,000 consensus clusters from MOV10 WT, K530A, and D645N PAR-CLIPs.
(C). Bar plot depicting the percentage of T-C changes located in different transcript regions.
(D) Relative position of T-C transitions of MOV10 WT, K530A, and D645N throughout 3′ UTRs. y axis shows density of the median relative coverage over all 3′ UTRs.
(E) Browser view of T-C transitions found in the ELAVL1 3′ UTR.
(F) Cumulative fraction plot of distances between T-C transitions in top 5,000 clusters of all PAR-CLIP libraries relative to the K530A library 1.
(G) Density plot of lengths of the top 5,000 MOV10 WT, K530A, and D645N clusters. The median cluster lengths are indicated above the graph.
(H) Average centered PhastCons conservation scores around MOV10 WT, K530A, and D645N 3′ UTR crosslinking sites. For comparison, conservation scores were computed for sites shifted 100 nt relative to the crosslinking site.
(I) Positional accessibility around MOV10 WT, K530A, and D645N crosslinking sites calculated using mfold. Positional accessibility of shuffled regions is shown in gray.
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5. Figure 3 MOV10 Binds to LINE-1 Elements
(A) FPKM of reads as well as their corresponding shuffled sequences on positive strand mapping to LINE-1 elements (GenBank accession number: M ).
(B) Density of MOV10 T-C transitions across M based on reads mapping to positive strand.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 4 MOV10 Is Associated with UPF1
(A) Plot of log2-transformed SILAC ratios from the crossover label-swap experiments to identify RNA-bound proteins in the proximity of MOV10 (RNA-dependent interactions). The pie chart indicates the fraction of proteins with enriched SILAC ratios, which were annotated or experimentally verified as RNA-binding (Baltz et al., 2012). Lines indicate the cut-off of 1.5.
(B) Plot of log2-transformed SILAC ratios from the two label-swap experiments for the identification of RNA-independent MOV10 interaction partners.
(C) Plot of log2-transformed SILAC ratios from the two label-swap experiments for the identification of RNA-independent UPF1 interaction partners.
(D) Proteins identified with high unique peptide counts in the UPF1 IP. x axis shows proteins ranked accordingly to the number of unique peptides detected for that given protein.
(E) Western analysis of MOV10, AGO2, and UPF1 coIPs. Cells stably expressing FLAG/HA-MOV10, -AGO2, or -UPF1 were transiently transfected with MYC-AGO2, -MOV10, -MOV10 K530A, -UPF1, -UPF1 K498A, or -TNRC6B expression constructs as indicated. Input and anti-FLAG IPs were subjected to western analysis using antibodies against the HA tag and the MYC tag. Asterisks indicate unspecific bands.
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7. Figure 5
MOV10 Mutants and UPF1 Bind to Similar Sites in 3′ UTRs, and MOV10 Binding Is Independent of Translation
(A) Percentage of T-C transitions in different transcript regions of UPF1 PAR-CLIPs from untreated (UPF1 1 and 2) and puromycin-treated (UPF1 puro) cells.
(B and C) Relative distribution of T-C transitions over 3′ UTRs (B) and CDSs (C) of UPF1 PAR-CLIPs from untreated and puromycin-treated cells.
(D) Percentage of T-C transitions in different transcript regions of MOV10 PAR-CLIPs from untreated (MOV10 WT 1 and 2) and puromycin-treated (MOV10 puro) cells.
(E and F) Relative distribution of T-C transitions over 3′ UTRs (E) and CDSs (F) of MOV10 PAR-CLIPs from untreated and puromycin-treated cells.
(G) Venn diagram displaying the overlap between all UPF1-bound mRNAs and MOV10 mRNA targets containing the top 5,000 PAR-CLIP clusters.
(H) Density distribution of distances between UPF1 and MOV10 T-C transition positions. Data are shown for one representative PAR-CLIP library each.
(I) Density distribution of distances between T-C transition positions and the stop codon. Data are shown for one representative PAR-CLIP library each.
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 6
MOV10 Depletion Leads to Increased mRNA Half-Lives of NMD-Targeted β-Globin Reporter Transcript
(A) Schematic representation of tetracycline-responsive β-globin reporters containing either a wild-type β-globin gene (βWT) or the corresponding PTC-containing β-globin (β39) used to monitor mRNA decay (Lykke-Andersen and Wagner, 2005). A wild-type β-globin reporter with an extended 3′ UTR from GAPDH (β-UAC-GAP) was cotransfected together with the tetracycline-responsive reporters and used as an internal control.
(B and C) Northern blot analysis of β-globin constructs showing mRNA decay of βWT (B) and β39 (C) in mock-, MOV10-, or UPF1-depleted cells. Ratios indicated in red represent levels of βWT relative to β-UAC-GAP and are plotted in log2 scale in the graph on the right. Error bars represent SD from two replicate experiments.
Molecular Cell  , DOI: ( /j.molcel )
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9. Figure 7
MOV10 Depletion Leads to Increased mRNA Half-Life of Selected Transcripts Bound by MOV10 and UPF1
(A) Changes in mRNA half-lives upon MOV10 knockdown. MOV10-bound mRNAs were divided into top 25%, intermediate 50%, and bottom 25% target sets based on their number of T-C changes normalized to 3′ UTR length and the expression level of the respective mRNA (FPKM value). p values are calculated by the one-sided Wilcoxon signed-rank test.
(B) mRNA half-lives measured by high-throughput sequencing for transcripts used in qRT-PCR validation.
(C) Decay of CBX6, CDKN1B, HOXA9, WEE1, MID1IP1, and MYC mRNAs as well as the known UPF1-regulated PANK2 mRNA after Actinomycin D treatment as measured by qRT-PCR. mRNA levels are shown relative to RPL18A and normalized to the 0 hr time point, with ±SD from three technical replicates.
(D) Decay of WEE1 and MYC mRNAs in HEK293 cells (293) and HEK293 cells stably expressing FLAG/HA-MOV10 K530A (K530A) with and without depletion of endogenous MOV10. Data are displayed as in (C).
Molecular Cell  , DOI: ( /j.molcel )
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