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1 The EMBO Journal Structure of a Dm peptide bound to the OT module Tobias Raisch et al Expanded View Figures A Hs Dm HEAT HEAT MIF4G 9BD 1SHD HEAT HEAT MIF4G 9BD 1SHD M - I B E Time (min) -5BoxB 15 J λ t 1/2 (min) 146 ± ± 3 Relative / R-Luc activity rp BoxB-poly(A) λ-ha GW182 λ-ha GW182 ontrol KD DP2 KD + DP2 E361Q F 18 2 Anti-V5 R-Luc-V5 K Relative / R-Luc activity λ-ha GW182 λ-ha λ 5 6 DP2 E361Q ΔZnF ZnF ED GW G HA- L ΔED D Relative / R-Luc activity osk H HA- M HA- HA-R4 HA-POP2 HA-PA2 HA-PA3 Figure EV1. EV1 The EMBO Journal ª 216 The Authors
2 Tobias Raisch et al Structure of a Dm peptide bound to the OT module The EMBO Journal Figure EV1. Dm interacts with subunits of the deadenylase complexes. A Domain organization of Hs and Dm. consists of -terminal (-), middle (-M), and -terminal regions (-). - contains two HEAT repeat domains (dark and light blue); -M contains a MIF4G domain that also consists of HEAT repeats and a three-helix bundle domain (9BD). - contains another HEAT repeat domain, the superfamily homology domain (SHD). B orthern blot analysis showing the decay of the -5BoxB mra in S2 cells expressing the indicated proteins. The mra half-lives (t 1/2 ) standard deviations calculated from the decay curves of three independent experiments are indicated below the panels. Tethering assay corresponding to the experiment described in Fig 1B but using an reporter that lacks the BoxB hairpins. activity was normalized to R-Luc and set to in cells expressing k-ha. The panel shows mean values standard deviations from three independent experiments. D -tagged was coexpressed with an reporter containing the oskar 3 UTR. R-Luc served as a transfection control. activity was normalized to that of the R-Luc transfection control and set to in cells expressing. The panel shows mean values standard deviations from three independent experiments. E ormalized luciferase activities corresponding to the experiment shown in Fig 3A and B. F Western blot analysis showing the expression of the DP2 mutant (DP2 E361Q) in the experiment described in Fig 3A and B. R-Luc-V5 served as a transfection control. G M Western blot analysis showing the interaction of -tagged Dm (full length) with HA-tagged deadenylase subunits. -tagged firefly luciferase () served as a negative control. Proteins were immunoprecipitated using a polyclonal anti- antibody. s and immunoprecipitates were analyzed by Western blotting using anti- and anti-ha antibodies. For the -tagged proteins, 3% of the inputs and 1% of the immunoprecipitates were loaded, whereas for the HA-tagged proteins, 1% of the input and 3% of the immunoprecipitates were analyzed. In each panel, cell lysates were treated with Rase A prior to immunoprecipitation. Source data are available online for this figure. ª 216 The Authors The EMBO Journal EV2
3 The EMBO Journal Structure of a Dm peptide bound to the OT module Tobias Raisch et al A B D HA-DP1 HA-DP2 HA-ED4 HA-HPat E F G Δ - -Δ HA-ED3 HA-Me31B H - -L - - -L - HA- fragments HA- fragments 1 13 I MBP- - - MBP HA- fragments Figure EV2. Dm interacts with decapping factors and the OT module. A F o-immunoprecipitation assays using -tagged Dm (full length) and HA-tagged decapping factors. Samples were analyzed as described in Fig EV1G M. G I Western blot analysis showing the interaction of -tagged Dm and HA-tagged,, and (either full length or the indicated fragments). Proteins were immunoprecipitated from Rase A-treated cell lysates using anti- antibodies. - served as a negative control. For the detection of tagged proteins, 3% of the input and 1% of the bound fractions were analyzed by Western blotting. For the detection of HA-tagged, 1.5% of the input and % of the bound fractions were analyzed, whereas for HA and HA proteins, 1% of the input and 3% of the immunoprecipitates were analyzed. Source data are available online for this figure. EV3 The EMBO Journal ª 216 The Authors
4 Tobias Raisch et al Structure of a Dm peptide bound to the OT module The EMBO Journal A Alignment Drosophila ED D.melanogaster D.anannassae D.erecta D.grimshawi D.mojavensis D.sechellia D.simulans D.virilis D.willistoni D.yakuba D.pseudoobscura ITPVTPDPSTSAQSTHFPFLADS AATASLLMQRQYHYHLLL-QQQQQLAMAQHQLALAA LTPLTPEP-TAQTTTHFPLLADS AAAATLLLQRQYHYHLLLHQQQQQLALAQHQLALAA LTPLTPDPSTAAQTTHFPFLADS AAAATLLLQRQYHYHLLLQQQQQQLALAQHQLALAA LTPEHS----TMSSTQFPLLTDVQL TAALLLQRQYQYHLLIQHHQQQIALAQHQLALAA LTPE-P----TMSSTQFPLLTDSQVM----ASAASTLFLQRQYQYHLLLQQQQQQLALAKHQLALAA ITPVTPDPSTSAQSTHFSFLAD AATASLLLQRQYHYHLLL-QQQQQLAMAQHQLALAA ITPVTPDPSTSAQSTHFSFLAD AATASILLQRQYHYHLLL-QQQQQLAMAQHQLALAA LTPE-P----TMSSTQFPLLTDSHV--MA-STAASTLLMQRQYQYHLLLQQQQQQLAFAQHQLALAA LTPESPAPFGSQAATQFPLLTDAV-AMA-ATAATLLFQRQYHYHLLIQQQQQQLALAQHQLALAA LTPLTPDPTTAAQTTHFPFLADS AAAATLLLQRQYHYHLLLQQQQQQLALAQHQLALAA -TPLTPEPPQYGSQTQFPLLTGSMMMMGLHTGQMLLHQHYQYHLIIQQQQQQLALAHHLALAA D.melanogaster 19 D.anannassae 12 D.erecta 132 D.grimshawi 128 D.mojavensis 114 D.sechellia 132 D.simulans 113 D.virilis 115 D.willistoni 136 D.yakuba 132 D.pseudoobscura132 α1 α2 β1 SAAAASA--SHQQTDEIARSLKIFAQVTTGAAEAAGSMQDVMQEFATGYASDDLGRMSYGSAPPQVQM SAAAVV--HQKDEIARSLKIFAQLTTGASEAHGSMQDVMQEFAAGYVSDDIRFHYGSAPPQAMT SAAAASA--HQQKDEIARSLKIFAQVTTGAAEAVGTVQDVMQEFATGYASDDLSRFSYGSAPPQAQT SAAAA----HHQKDEIAHSLKMFALLTAADASSDAMHDFAAGYVSDDFRF-----ATPQ SAAAA----HQQKDEIAHSLKMFALLTATDSASTQDAMQEFASGYVSDELFQY-----Q SAAAASA--SHQQTDEIARSLKIFAQVTTSAAEAAGSMQDVMQDFATGYASDDLGRMSYGSAQPQAQT SAAAASA--SHQQTDEIARSLKIFAQVTTGAAEVAGSMQDVMQDFATGYASDDPGRMSYGSALPQAQT SAAAA----HHQEDEIAHSLKMFAFLTAADTGSQDAMQDFAAGYVSDEFFQY-----HS SAAAAAATHQQKDEIARSLKIFAQLTTTAEHAAQDVMQEFALGYVSDDLRFQ------SVQS SAAAASA--HQQKDEIARSLKIFAQVTTGAAEAAGSVQDVMQEFATGYASDDLSRFAYPPQPQT SAAASGV--HQQKDEIARSLKVFAQLTSAPAEVPQDVMQEFASGYASDDLFAYSPAP----S 1BM 3BM OT module binding region (BR) D.melanogaster 177 D.anannassae 188 D.erecta 2 D.grimshawi 189 D.mojavensis 175 D.sechellia 2 D.simulans 181 D.virilis 176 D.willistoni 2 D.yakuba 2 D.pseudoobscura196 PPQQQHQQQQGLHLPLGRPAQLQTGGLMPI----PLATHWL--YREHLVWR--MSYIPAA PPPP---PPPSLQAPLGHTSSPQASPVLMPVSVPAPLTAHWLS--YREQLVWR--HMSYMPTA PPPSQQQQQQGLHLPLGRTAQLHSGALMS----PLATHWL--YREQLVWR--MSYMPAV PQQPQQQLLRQQFPSATTTVAGSAAAGVLPS---PLAATQ-LLW-EHLVWRSMSMAYMP TVTPQSSPLPRQYP--AGSTASATAAVSFVPP---SLAAAHWLFYREHLVWR--SMSYVPL QPQQQHQQQQGLHLPLGRPAQLQVGGLMPI----PLATHWL--YREHLVWR--MSYVPAA QPQQQHQQQQGLHLPLGRPAQLQVGGLMPM----PLATHWL--YREHLVWR--MSYVPAA SVAPQSSQLPSQVPISSAGTGGATAEGATFPS---SLAAAHWILSYREHLHVWRTMSMSYMPV PQLQQRHLTPTFTAASGSAGAMAAATAAFPM-----TTAWLY--YREQLSVWR--MSYMPAA PPPS--QQQQGMHLPLGRSPVQLQSGALMS----PLATHWL--YREQLVWR--MSYMPAA PAQLHRLPAAGAAAHPP QVVPA--PAPLSARWLQ--YREQLVWR--SMAYMPI B D Relative / R-Luc activity BoxB λ-ha: Anti-HA Relative / R-Luc levels E BR BR Hs 3 Hs 3 Hs 3 Hs 3 -SHD MBP-Hs 3 -Dm BR pulldowns Figure EV3. ª 216 The Authors The EMBO Journal EV4
5 The EMBO Journal Structure of a Dm peptide bound to the OT module Tobias Raisch et al Figure EV3. Sequence alignment of the ED regions of the indicated Drosophila species. A The residues conserved in all of the aligned sequences are shown with a red background, and the residues with > % similarity are highlighted with a salmon background. The residues interacting with and are indicated by magenta and cyan diamonds, respectively, including main-chain and minor side-chain contacts. The residues mutated in this study are indicated by asterisks colored in blue (mutations that disrupt OT module binding) and in orange (I123M mutation for the incorporation of selenomethionine as an anomalous scatterer). B Tethering assay using the -5BoxB reporter in S2 cells expressing the indicated k-ha-tagged ED fragments. A plasmid-expressing R-Luc mra was used as a transfection control. The activities were normalized to those of the R-Luc transfection control and set to in the presence of the k-ha. The panel shows mean values standard deviations from three independent experiments. Western blot analysis showing the expression of the k-ha-tagged proteins used in the experiment described in (B). served as a transfection control. D An experiment similar to that described in (B) was performed using an reporter that lacks the BoxB hairpins. The panel shows mean values standard deviations from three independent experiments. E pull-down assay showing that the MBP-tagged 3 IM peptide does not compete with the -tagged BR for binding to the purified OT module. Source data are available online for this figure. Figure EV4. omparison of OT module conformations and details of Dm BR binding. A Superposition of the mutant OT module (apo form) crystallized in space group P2 1 (blue; chains A, B, ), and the wild-type OT module crystallized in space group P (red; PDB code 4D; Boland et al, 213). B Superposition of the mutant OT module structure obtained in the absence (blue, chains A, B, ) and presence of the Dm BR (orange; chains A, B, ). The BR peptide is shown in red. Superposition of the two OT module complexes from the asymmetric unit of the crystals obtained in the presence of the Dm BR peptide. omplex 1 (chains A, B, ) is colored in orange and complex 2 (chains E, F, G) in green. The BR peptide is shown in red and dark green. D rystal packing of the OT module mutant bound to the Dm BR peptide. E Orientation of helix a23 in the wild type (PDB code 4D; Boland et al, 213) and mutant OT module apo complexes. olors are as in (A). The black lines indicate the change in the relative orientation of the helix axes. F Orientation of the helix a23 in the mutant OT module complex as compared to the orientation in the complex of the SHD with the human 1 IM (PDB code 4QO; Bhandari et al, 214). G Alternative view of the 1BM binding pocket centered on Dm F13. Selected residues of and of the BR peptide are shown as gray and red sticks, respectively. Residues mutated in this study are underlined. H Alternative view of the 3BM binding pocket centered on Dm F152. Selected residues of and of the BR peptide are shown as cyan and red sticks, respectively. Residues mutated in this study are underlined. I Additional close-up of the 3BM binding site emphasizing the role of K737 with hydrogen bonds as dashed green lines. Residues mutated in this study are underlined. EV5 The EMBO Journal ª 216 The Authors
6 Tobias Raisch et al Structure of a Dm peptide bound to the OT module The EMBO Journal A B L19 α22 Wild-type OT module Mutant OT module OT module mutant apo OT module mutant bound to Dm D hain hain D hain H hain G hain B hain F hain A hain E hains A, B,, D hains E, F, G, H E α22 F α22 L18 L18 α21 29 α21 L19 α22 α23 L19 α22 α23 Wild-type OT module Mutant OT module SHD- Hs 1 IM Mutant OT module G 1BM I123 H 3BM V148 I β5 G156 I1895 L1962 α5 α4 H1949 L127 F13 α3 L1946 F1876 L1889 V188 α2 Y157 β5 M149 F7 K737 F152 Y2 β4 Y6 F6 Y9 β2 β1 M4 M145 M3 K737 F152 A153 1 T154 Figure EV4. ª 216 The Authors The EMBO Journal EV6
7 The EMBO Journal Structure of a Dm peptide bound to the OT module Tobias Raisch et al Figure EV5. Validation of the sequence assignment of the BR peptide bound to the OT module and activity of BR mutants. A D Anomalous difference Fourier map (black mesh) calculated at a resolution of 7.5 Å and contoured at the 4. r level. Data (available as source data) were collected at the Selenium K-edge peak wavelength from a crystal containing selenomethionine-substituted Dm BR peptide (I123M mutant). The panels show closeup views of the binding sites of the 1BM (A, B) and the 3BM (, D) in the same orientations as in Fig 6. Residues mutated in this study are underlined. E G Western blot analysis showing the interaction of -tagged Dm (wild type or ) with HA-tagged,, and. -tagged firefly luciferase () served as a negative control. Proteins were immunoprecipitated using a polyclonal anti- antibody. s and immunoprecipitates were analyzed by Western blotting as described in Fig EV1G M. H A tethering assay using the -5BoxB reporter and the indicated k-ha-tagged proteins was performed in S2 as described in Fig 1B. The panel shows mean values standard deviations from three independent experiments. I Tethering assays in human HEK293T cells, using a b-globin reporter containing 6 binding sites (6xbs) for the MS2 protein and MS2-HA-tagged Hs 2 (wild type or the indicated variants, 2 DIM and 2 IM-to-BR). In the 2 IM-to-BR protein, the IM was replaced by the Dm BR. A plasmid expressing an mra lacking MS2 binding sites (ontrol) served as a transfection control. The b-globin-6xbs mra levels were normalized to those of the control mra and set to in the presence of MS2-HA. The panel shows mean values standard deviations from three independent experiments. J orthern blot of representative RA samples corresponding to the experiment shown in (I). K Western blot analysis showing the expression of the MS2-tagged proteins used in the experiment shown in (I) and (J). V5-MBP served as a transfection control. L o-immunoprecipitation assay in human HEK293T cells showing the interaction of V5-SBP-tagged 2 (wild type or the indicated variants) with endogenous and. V5-SBP-tagged -MBP served as a negative control. Source data are available online for this figure. EV7 The EMBO Journal ª 216 The Authors
8 Tobias Raisch et al Structure of a Dm peptide bound to the OT module The EMBO Journal A 1BM F13 SeMet123 B A124 SeMet123 L127 9 F13 V188 α2 L127 L1962 3BM A124 Y157 T154 L1946 F1876 H1949 α4 α3 D T154 Y157 K737 1 α5 β5 F152 1 E F152 SeMet149 Y2 SeMet145 HA E F G HA- 5 6 I J K Relative 6xbs RA levels Figure EV5. 2 ΔIM IM-to-BR ontrol 6xbs 2 ΔIM IM-to-BR Anti-HA V5-MBP 95 2 ΔIM HA- IM-to-BR 5 6 L MS2-HA- MS2-HA- MS2-HA- V5-SBP- Anti-V5 Anti- Anti- 95 H Relative / R-Luc activity -5BoxB-poly(A) MBP 2 ΔIM IM-to-BR -MBP 2 ΔIM SBP-pull down λ-ha IM-to-BR ª 216 The Authors The EMBO Journal EV8
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