1. Volume 62, Issue 6, Pages 943-957 (June 2016)
K63-Ubiquitylation and TRAF6 Pathways Regulate Mammalian P-Body Formation and mRNA Decapping 
Ulas Tenekeci, Michael Poppe, Knut Beuerlein, Christin Buro, Helmut Müller, Hendrik Weiser, Daniela Kettner-Buhrow, Katharina Porada, Doris Newel, Ming Xu, Zhijian J. Chen, Julia Busch, M. Lienhard Schmitz, Michael Kracht 
Molecular Cell 
Volume 62, Issue 6, Pages (June 2016)
DOI: /j.molcel
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2. Molecular Cell 2016 62, 943-957DOI: (10.1016/j.molcel.2016.05.017)
Copyright © 2016 Elsevier Inc. Terms and Conditions

3. Figure 1
K63 Ubiquitin Chains Regulate Gene-Specific mRNA Decay of Prototypical Inflammatory mRNAs
(A) Constitutive mRNA decay of five prototypical inflammatory genes in U2OS cells. Actinomycin D (5 μg/ml) was added for the indicated periods, and RNA was isolated and quantified by RT-qPCR. The graphs show mean relative mRNA levels ± SEM from five independent experiments. Numbers in brackets indicate the average half-lives.
(B) Parental, shUb, and shUb-Ub(K63R) U2OS cells were treated with doxycycline (dox, 1 μg/ml) as indicated. mRNA steady-state levels of the indicated genes were determined by RT-qPCR. The graphs show mean relative mRNA levels ± SEM from two (parental, shUb) or three (shUb-Ub[K63R]) independent experiments.
(C) U2OS cells were treated as described in (B), and actinomycin D decay kinetics of the indicated mRNAs was determined as described in (A). The graphs show mean relative mRNA levels ± SEM from two (shUb) or three (shUb-Ub(K63R) independent experiments. Solid lines indicate the regression curves which were used for calculation of mRNA half-lives from the combined repeated experiments.
(D) The data obtained in (C) were used to calculate the mRNA half-lives of the indicated cells. The graph shows the relative changes in mRNA stability compared to each untreated cell line. IL8, CXCL3, and PTGS2 mRNAs in dox-treated shUb cells were extremely stable and did not decay within the investigated time frame.
(E) Cell extracts of the U2OS cell lines before and after addition of doxycycline as described in (B) were analyzed for expression of the indicated decapping factors, the exonuclease XRN1, and ubiquitylated proteins using the indicated antibodies. The positions of molecular weight markers are indicated.
See also Figure S1.
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4. Figure 2 K63 Ubiquitin Chains Regulate DCP1a Phosphorylation
(A) U2OS cell lines treated with doxycycline as described in the legend of Figure 1. The cells were stimulated with IL-1 (10 ng/ml) for 1 hr as indicated or were left untreated. Subcellular distribution of DCP1a, DCP1a-positive P-bodies and phosphorylation of DCP1a were assessed by indirect immunofluorescence microscopy. The scale bar represents 10 μm; white arrows mark typical P-bodies. Nuclei were stained with Hoechst dye (blue).
(B) The boxplots summarize numbers of DCP1a-positive P-bodies per cell as determined from at least 100 cells per condition. Data were combined from two independent experiments. (C) U2OS cell lines were treated for 2 days (parental, shUb) or 3 days (shUb-Ub(K63R), shUb-Ub(wt)) with doxycycline followed by IL-1 treatment (10 ng/ml) for 30 (K63R, wild-type) or 60 min (parental, shUb) as indicated. Cell extracts were analyzed for phosphorylation or expression of DCP1a, TAK1, and JNK. Anti-actin antibodies were used to control for equal loading. Arrowheads indicate the proteins and their phosphorylated forms.
See also Figure S2.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 3
K63R Ubiquitin Chains Suppress Assembly of Decapping Factors into P-Bodies
U2OS cell lines treated with doxycycline were stimulated with IL-1 (10 ng/ml) for 1 hr as indicated or were left untreated. Colocalization of DCP1a with the decapping factors EDC4 (A and B) or EDC3 (C) in P-body structures was determined by indirect double-immunofluorescence microscopy.
(A) Representative images for the DCP1a/EDC4 experiments are shown. The scale bar represents 10 μm.
(B and C) The box plots summarize numbers of double positive P-bodies per cell as determined from at least 170 cells per condition. Shown are quantifications from one out of two independent experiments which yielded comparable results.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 4
Expression of K63R Ubiquitin Prevents the Interactions of Decapping Factors in Degradation-Competent P-Bodies
Immuno-PLAs were used to determine the interaction of endogenous decapping factors and their subcellular localization in shUb-Ub(wt) and shUb-Ub(K63R) cells.
(A) A representative image series is shown. Red spots mark positive PLA signals for DCP1a:EDC4 interactions. To determine PLA complexes that colocalized with P-bodies, cells were stained simultaneously with an anti-EDC4 antibody (green spots). PLA assays omitting the primary DCP1a antibody (upper second right panel) or the primary EDC4 antibody (lower second right panel) of each PLA antibody pair were used as negative controls. Yellow spots highlighted by yellow arrows indicate DCP1a:EDC4 complexes present within P-bodies, as shown exemplarily in the right panel. Green spots indicate EDC4-positive P-bodies lacking an interaction of DCP1 with either EDC4, XRN1, or EDC3. The scale bar represents 10 μm.
(B) Relative changes in EDC4-positive P-bodies, numbers of PLA spots, and merges representing degradation-competent P-bodies were calculated from the four DCP1a:EDC4 experiments. Values from untreated cells were set as 100%. Each bar represents the relative mean ± SEM of at least 300 analyzed cells.
(C) (Left) Boxplots show PLA spot distribution as determined from four (DCP1a:EDC4) or two (DCP1a:XRN1, DCP1a:EDC3) independent experiments. Numbers 1–10 refer to the experimental conditions as shown in columns one and two of the table. (Right) The table summarizes the total number of analyzed cells, the mean number of PLA spots/cell and the relative changes across the treatment conditions.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 5 Identification of DCP1a as Ubiquitylated Protein
(A) HEK293IL-1R cells were transfected with expression vectors encoding hexahistidine-tagged ubiquitin (HIS-Ubiquitin) or empty vector. In addition, cells were transfected to express a constitutively active TAK1-TAB1 fusion protein (HA-TAK1kdTAB1ad) composed of the kinase (kd) domain of TAK1 and the activation domain (ad) of TAB1 and GFP-DCP1a. Lysates were prepared in denaturing buffer, and ubiquitylated proteins were isolated by affinity purification on Ni2+-NTA beads. Lysates were analyzed by immunoblotting for the presence of GFP-DCP1a, or phosphorylation of GFP-DCP1a and JNK as shown. Anti-tubulin antibodies were used to control for equal protein loading. On the same blots, the simultaneous modification of GFP-DCP1a by ubiquitylation (as seen by the multiple upshifted bands) and phosphorylation was revealed by immunoblotting. The positions of molecular weight marker proteins are indicated. In (B)–(E), cells were transfected with the indicated combinations of expression constructs. Ubiquitylation of epitope-tagged DCP1a was determined as in (A), and proteins or their phosphorylated/ubiquitylated forms were detected by immunoblotting using the indicated antibodies. (B and C) Cells were transfected to express GFP-DCP1a together with empty vector or wild-type ubiquitin or versions mutated at lysines K29, K48, or K63 as indicated (B) or inactive (S315A) or phospho-mimetic (S315D) mutants of GFP-DCP1a (C). (D) Cells were transfected to express MYC-DCP1a, or mutants in which the C-terminal lysines (K R), the residues known to be required for trimerization (L551R, I555S, F561R, L565S, MT3), or the entire C terminus was mutated (Δ ). (E) HEK293IL-1R cells were transfected with expression vectors encoding histidine-tagged ubiquitin or empty vector. In addition, cells were transfected to express GFP-DCP1a or FLAG-tagged TRAF2 or TRAF6. (F) HEK293IL-1R cells were stimulated with IL-1 for 1 hr or were left untreated. Ubiquitylated proteins were isolated from nondenaturing (left panel) or denaturing lysates (right panel) with an anti-ubiquitin antibody, and the presence of polyubiquitylated DCP1a (black arrowheads) or ubiquitylated proteins was detected by immunoblotting with anti-DCP1a (upper panels) or anti-ubiquitin antibodies (lower panels). IgG immunoprecipitations served as controls; the position of the IgG heavy chain (IgG hc) is indicated. (G) HEK293IL-1R cells were transfected with expression vectors encoding histidine-tagged ubiquitin or FLAG-TRAF6. One part of the lysate was directly analyzed by immunoblotting (lower panels), while the remaining fraction was used to purify ubiquitylated proteins via the His tag (upper panels). The antibodies and ubiquitylated proteins are shown. (H) An identical experiment was performed in HEK293 cell lines stably transfected with pX459 (vector) or pX459 containing a single guide (sg) RNA directed against TRAF6 facilitating CRISPR/CAS9-mediated knockdown of TRAF6.
See also Figures S3 and S4.
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 6
Identification of TRAF6 as DCP1a Interactor and Regulator of DCP1 Phosphorylation and P-Body Formation
(A) HEK293IL-1R cells were transfected with expression vectors encoding histidine-tagged ubiquitin or empty vector. In addition, cells were transfected to express GFP-DCP1a or FLAG-tagged TRAF2 or TRAF6. Lysates were prepared and immunoprecipitations were performed with control antibodies (IgG) or anti-FLAG antibodies. Coimmunoprecipitation of GFP-DCP1a with TRAF6 or TRAF2 in the presence or absence of wild-type ubiquitin was analyzed by immunoblotting.
(B) Cells were transfected to express MYC-DCP1a or the indicated mutants. In addition, wild-type FLAG-TRAF6 or a catalytically inactive mutant of TRAF6 (FLAG-TRAF6C70A) was cotransfected. Comparable amounts of FLAG-TRAF6 or FLAG-TRAF6C70A were immunoprecipitated with anti-FLAG antibodies. Immune complexes were analyzed by anti-FLAG, anti-MYC, and anti P-DCP1a antibodies to measure equal recovery of FLAG-TRAF6 and the coprecipitation of DCP1a or P-DCP1a by immunoblotting. The upper panel of each antibody hybridization corresponds to FLAG-TRAF6 (wt) and the lower panel to FLAG-TRAF6C70A (C70A) immunoprecipitations.
(C) Cells were transfected to express increasing amounts of FLAG-TRAF6 together with MYC-DCP1 or empty vector encoding the MYC epitope only (MYC). Two hours before cell lysis, JNK kinase activity was blocked by addition of the inhibitor SP Phosphorylation and expression of ectopically expressed DCP1a, the JNK substrate c-JUN, and TRAF6 were analyzed by immunoblotting of cell extracts using the indicated antibodies.
(D–F) HEK293IL-1R cells were transfected with empty pSuper (vector) or pSiRPG encoding a shRNA against TRAF6. After 3 days’ selection in puromycin (3 μg/ml), cells were stimulated with IL-1 for 1 hr or were left untreated, and cell extracts were analyzed for phosphorylation and expression of p65 NF-κB and IκBα (D), or for phosphorylation and expression of DCP1a, JNK, c-JUN, and TRAF6 (E). (F) Extracts from the same conditions were analyzed for expression of P-body components, TRAF2 and TRAF6. The graphs summarize the individual values and the mean expression levels ± SEM of proteins from three independent experiments.
(G) P-bodies positive for DCP1a, EDC4, and XRN1 were determined by triple-immunofluorescence in vector- or pSiRPG-shTRAF6-transfected cells. P-bodies containing comparable amounts of all three proteins appear as white merged spots, green P-bodies contain primarily DCP1a, and yellow P-bodies indicate partial reduction of EDC4. The scale bar represents 10 μm. The knockdown efficiency of TRAF6 in one of the experiments was determined in parallel by immunoblotting (right panel).
See also Figures S5 and S6.
Molecular Cell  , DOI: ( /j.molcel )
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9. Figure 7
Mutation of the Six C-Terminal Lysines of DCP1a Suppresses Decapping Activity and Assembly of the Decapping Complex and Alters P-Body Formation
(A) HEK293IL-1R cells were transfected to express GFP, GFP-DCP1a, GFP-DCP1aK R, or GFP-DCP1aΔ GFP or GFP-tagged proteins were precipitated with GFP-beads, and the complexes were incubated with 32P-cap-labeled luciferase mRNA (32P-luc. mRNA). Reaction mixtures were separated by thin-layer chromatography, and the hydrolyzed 32P-m7-GDP product of the decapping reaction was detected by autoradiography. In parallel, the purified protein complexes and lysates were analyzed by immunoblotting for comparable protein expression and immunoprecipitation efficiency.
(B) Quantification of decapping activity associated with GFP-DCP1a wild-type or with GFP-DCP1a mutants. The data were normalized to the amounts of DCP1a in the GFP-beads fractions. Shown are individual and mean values relative to the activity of GFP-DCP1a wild-type ± SEM obtained from three independent experiments.
(C) GFP-bead precipitation experiments were performed as in (A). Lysates and precipitates were analyzed for expression and coprecipitation of the endogenous decapping factors (EDC4, EDC3, DCP2) or of the exonuclease XRN1 by specific antibodies. Comparable enrichment of GFP-DCP1a wild-type or its mutants was validated with anti-GFP antibodies.
(D) Quantification of relative amounts of endogenous decapping factors and XRN1 which associate with GFP-DCP1a or its mutants. Binding of cofactors was normalized to the amount of precipitated GFP-DCP1a. Shown are individual and mean values ± SEM from four independent experiments.
(E) GFP, GFP-DCP1a wild-type, or its mutants were expressed in HEK293IL-1R cells and analyzed by fluorescence microscopy for number and size of processing bodies. Representative merged images are shown; colors indicate GFP fluorescence (green), Hoechst staining (blue), and XRN1 or EDC4 immunofluorescence (red). The scale bar represents 10 μm.
(F) The graphs show boxplots of pairwise comparisons of GFP-DCP1a (wt) with GFP-DCP1aK R. P-body numbers were counted from EDC4 and XRN1 immunofluorescence experiments and represent 81 (wt) or 141 (K R) cells, respectively. From the same experiments, the size of 100 bodies was calculated from separate measurements of the diameters of GFP-DCP1a-, EDC4-, or XRN1-containing P-body structures for each cell. All differences between the four pairwise comparisons are significant (p < 0.001).
See also Figure S7.
Molecular Cell  , DOI: ( /j.molcel )
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