Distinct modes of recruitment of the CCR 4– NOT complex by Drosophila and vertebrate Nanos
2016; Springer Nature; Volume: 35; Issue: 9 Linguagem: Inglês
10.15252/embj.201593634
ISSN1460-2075
AutoresTobias Raisch, Dipankar Bhandari, Kevin Sabath, Sigrun Helms, Eugene Valkov, Oliver Weichenrieder, Elisa Izaurralde,
Tópico(s)CRISPR and Genetic Engineering
ResumoArticle11 March 2016Open Access Source DataTransparent process Distinct modes of recruitment of the CCR4–NOT complex by Drosophila and vertebrate Nanos Tobias Raisch Tobias Raisch Department of Biochemistry, Max Planck Institute for Developmental Biology, Tübingen, Germany Search for more papers by this author Dipankar Bhandari Dipankar Bhandari Department of Biochemistry, Max Planck Institute for Developmental Biology, Tübingen, Germany Search for more papers by this author Kevin Sabath Kevin Sabath Department of Biochemistry, Max Planck Institute for Developmental Biology, Tübingen, Germany Search for more papers by this author Sigrun Helms Sigrun Helms Department of Biochemistry, Max Planck Institute for Developmental Biology, Tübingen, Germany Search for more papers by this author Eugene Valkov Eugene Valkov Department of Biochemistry, Max Planck Institute for Developmental Biology, Tübingen, Germany Search for more papers by this author Oliver Weichenrieder Corresponding Author Oliver Weichenrieder Department of Biochemistry, Max Planck Institute for Developmental Biology, Tübingen, Germany Search for more papers by this author Elisa Izaurralde Corresponding Author Elisa Izaurralde Department of Biochemistry, Max Planck Institute for Developmental Biology, Tübingen, Germany Search for more papers by this author Tobias Raisch Tobias Raisch Department of Biochemistry, Max Planck Institute for Developmental Biology, Tübingen, Germany Search for more papers by this author Dipankar Bhandari Dipankar Bhandari Department of Biochemistry, Max Planck Institute for Developmental Biology, Tübingen, Germany Search for more papers by this author Kevin Sabath Kevin Sabath Department of Biochemistry, Max Planck Institute for Developmental Biology, Tübingen, Germany Search for more papers by this author Sigrun Helms Sigrun Helms Department of Biochemistry, Max Planck Institute for Developmental Biology, Tübingen, Germany Search for more papers by this author Eugene Valkov Eugene Valkov Department of Biochemistry, Max Planck Institute for Developmental Biology, Tübingen, Germany Search for more papers by this author Oliver Weichenrieder Corresponding Author Oliver Weichenrieder Department of Biochemistry, Max Planck Institute for Developmental Biology, Tübingen, Germany Search for more papers by this author Elisa Izaurralde Corresponding Author Elisa Izaurralde Department of Biochemistry, Max Planck Institute for Developmental Biology, Tübingen, Germany Search for more papers by this author Author Information Tobias Raisch1,‡, Dipankar Bhandari1,‡, Kevin Sabath1, Sigrun Helms1, Eugene Valkov1, Oliver Weichenrieder 1 and Elisa Izaurralde 1 1Department of Biochemistry, Max Planck Institute for Developmental Biology, Tübingen, Germany ‡These authors contributed equally to this work *Corresponding author. Tel: +49 7071 6011350; E-mail: [email protected] *Corresponding author. Tel: +49 7071 6011358; E-mail: [email protected] The EMBO Journal (2016)35:974-990https://doi.org/10.15252/embj.201593634 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Nanos proteins repress the expression of target mRNAs by recruiting effector complexes through non-conserved N-terminal regions. In vertebrates, Nanos proteins interact with the NOT1 subunit of the CCR4–NOT effector complex through a NOT1 interacting motif (NIM), which is absent in Nanos orthologs from several invertebrate species. Therefore, it has remained unclear whether the Nanos repressive mechanism is conserved and whether it also involves direct interactions with the CCR4–NOT deadenylase complex in invertebrates. Here, we identify an effector domain (NED) that is necessary for the Drosophila melanogaster (Dm) Nanos to repress mRNA targets. The NED recruits the CCR4–NOT complex through multiple and redundant binding sites, including a central region that interacts with the NOT module, which comprises the C-terminal domains of NOT1–3. The crystal structure of the NED central region bound to the NOT module reveals an unanticipated bipartite binding interface that contacts NOT1 and NOT3 and is distinct from the NIM of vertebrate Nanos. Thus, despite the absence of sequence conservation, the N-terminal regions of Nanos proteins recruit CCR4–NOT to assemble analogous repressive complexes. Synopsis While Nanos represses target mRNAs by recruiting the CCR4–NOT complex in both flies and mammals, Drosophila Nanos uses a unique, bipartite peptide to contact another CCR4–NOT surface than vertebrate Nanos. Nanos proteins use short linear motifs to directly recruit the CCR4–NOT complex Crystal structure shows the NOT module in complex with a Drosophila Nanos peptide Drosophila and vertebrate Nanos peptides engage distinct NOT module surfaces Orthologous proteins can use distinct interaction modes to perform analogous functions Introduction Post-transcriptional mRNA regulation plays an essential role in embryonic development. This regulation is mediated by RNA-binding proteins that control the spatial and temporal expression of target mRNAs through the recruitment of effector complexes (Barckmann & Simonelig, 2013). The RNA-binding proteins of the Nanos family are conserved post-transcriptional mRNA regulators that play essential roles in embryonic germline specification, germline stem cell maintenance, and neuronal homeostasis in Drosophila melanogaster (Dm) and a wide range of other metazoans (Jaruzelska et al, 2003; Tsuda et al, 2003; Baines, 2005; Lai & King, 2013). The Dm Nanos protein is also required for posterior pattern formation in the embryo (Lehmann & Nüsslein-Volhard, 1991). Three Nanos paralogs (Nanos1–3) exist in vertebrates and various invertebrate species, whereas there is only one family member in Dm and other insects (Subramaniam & Seydoux, 1999; Mochizuki et al, 2000; Jaruzelska et al, 2003; Tsuda et al, 2003). This protein family is defined by a highly conserved CCHC-type zinc-finger (ZnF) domain (Curtis et al, 1997; Hashimoto et al, 2010) and divergent N- and C-terminal unstructured regions of variable lengths (Fig 1A). The ZnF domain mediates binding to RNA and to Pumilio, a conserved Nanos partner that confers mRNA target specificity (Murata & Wharton, 1995; Curtis et al, 1997; Wreden et al, 1997; Asaoka-Taguchi et al, 1999; Sonoda & Wharton, 1999; Jaruzelska et al, 2003). The unstructured regions are required for interaction with effector complexes (Verrotti & Wharton, 2000; Ginter-Matuszewska et al, 2011), which include the CCR4–NOT deadenylase complex (Kadyrova et al, 2007; Suzuki et al, 2010, 2012; Joly et al, 2013; Bhandari et al, 2014). Figure 1. Dm Nanos represses translation and promotes mRNA degradation Nanos comprises a highly conserved zinc-finger RNA binding domain (ZnF) and non-conserved N-terminal and C-terminal extensions (gray). NIM, NOT1-interacting motif; NED, Nanos effector domain; NBR, NOT module binding region; N1BM and N3BM, NOT1 and NOT3 binding motifs, respectively. Numbers above the bars indicate residues at domain/motif boundaries. Tethering assay using the F-Luc-5BoxB reporter and the indicated λN-HA-tagged proteins in S2 cells. A plasmid expressing R-Luc served as a transfection control. The F-Luc activity (black bars) and mRNA levels (green bars) were normalized to those of the R-Luc transfection control and set to 100 in the presence of the λN-HA-GST. The panel shows mean values ± standard deviations from three independent experiments. Northern blot of representative RNA samples corresponding to the experiment shown in (B). Western blot analysis of the expression of the λN-HA-tagged proteins used in the experiments shown in (B) and (C). GFP served as a transfection control. GFP-tagged Nanos fragments were tested for their ability to repress an F-Luc reporter containing the hb 3′ UTR. A plasmid expressing GFP served as a negative control. F-Luc activity (black bars) and mRNA levels (green bars) were normalized to those of an R-Luc transfection control and analyzed as described in (B). The panel shows mean values ± standard deviations from three independent experiments. Northern blot of representative RNA samples corresponding to the experiment shown in (E). Western blot showing the expression of the GFP-tagged proteins used in (E) and (F). R-Luc-V5 served as a transfection control. Tethering assay using the F-Luc-5BoxB-A95-C7-Hhr reporter and the indicated λN-HA-tagged proteins in S2 cells. F-Luc activity (black bars) and mRNA levels (green bars) were analyzed as described in (B). The panel shows mean values ± standard deviations from three independent experiments. Northern blot of representative RNA samples corresponding to the experiment shown in (H). Source data are available online for this figure. Source Data for Figure 1 [embj201593634-sup-0003-SDataFig1.pdf] Download figure Download PowerPoint The CCR4–NOT complex catalyzes the removal of mRNA poly(A) tails and consequently represses translation. In addition, dead-enylation by the CCR4–NOT complex is coupled to decapping and 5′-to-3′ exonucleolytic degradation by XRN1 and can therefore lead to full mRNA degradation in some cellular contexts (Temme et al, 2014). Furthermore, the CCR4–NOT complex can also repress translation independently of deadenylation (Cooke et al, 2010; Chekulaeva et al, 2011; Bawankar et al, 2013; Zekri et al, 2013). The CCR4–NOT complex consists of several independent modules that dock with NOT1, a central scaffold subunit (Temme et al, 2014). NOT1 consists of independently folded α-helical domains that provide binding sites for the individual modules (Fig EV1A). A central domain of NOT1, structurally related to the middle domain of eIF4G (the NOT1 MIF4G domain), provides a binding site for the catalytic module, which comprises two deadenylases, namely CAF1 (or its paralog POP2) and CCR4a (or its paralog CCR4b). Click here to expand this figure. Figure EV1. Dm Nanos interacts with subunits of the deadenylase complexes A. Domain organization of Hs and Dm NOT1. NOT1 consists of N-terminal (NOT1-N), middle (NOT1-M), and C-terminal regions (NOT1-C). NOT1-N contains two HEAT repeat domains (dark and light blue); NOT1-M contains a MIF4G domain that also consists of HEAT repeats and a three-helix bundle domain (CN9BD). NOT1-C contains another HEAT repeat domain, the NOT1 superfamily homology domain (SHD). B. Northern blot analysis showing the decay of the F-Luc-5BoxB mRNA in S2 cells expressing the indicated proteins. The mRNA half-lives (t1/2) ± standard deviations calculated from the decay curves of three independent experiments are indicated below the panels. C. Tethering assay corresponding to the experiment described in Fig 1B but using an F-Luc reporter that lacks the BoxB hairpins. F-Luc activity was normalized to R-Luc and set to 100 in cells expressing λN-HA. The panel shows mean values ± standard deviations from three independent experiments. D. GFP-tagged Nanos was coexpressed with an F-Luc reporter containing the oskar 3′ UTR. R-Luc served as a transfection control. F-Luc activity was normalized to that of the R-Luc transfection control and set to 100 in cells expressing GFP. The panel shows mean values ± standard deviations from three independent experiments. E. Normalized luciferase activities corresponding to the experiment shown in Fig 3A and B. F. Western blot analysis showing the expression of the DCP2 mutant (DCP2 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 GFP-tagged Dm Nanos (full length) with HA-tagged deadenylase subunits. GFP-tagged firefly luciferase (F-Luc) served as a negative control. Proteins were immunoprecipitated using a polyclonal anti-GFP antibody. Inputs and immunoprecipitates were analyzed by Western blotting using anti-GFP and anti-HA antibodies. For the GFP-tagged proteins, 3% of the inputs and 10% of the immunoprecipitates were loaded, whereas for the HA-tagged proteins, 1% of the input and 30% of the immunoprecipitates were analyzed. In each panel, cell lysates were treated with RNase A prior to immunoprecipitation. Source data are available online for this figure. Download figure Download PowerPoint The C-terminal region of NOT1 contains the NOT1 superfamily homology domain (SHD; Fig EV1A) and assembles with NOT2–NOT3 heterodimers to form the NOT module (Bhaskar et al, 2013; Boland et al, 2013). The NOT module provides binding sites for RNA-binding proteins, such as vertebrate Nanos and Dm Bicaudal-C, which recruit the CCR4–NOT complex to their mRNA targets (Chicoine et al, 2007; Suzuki et al, 2012; Bhandari et al, 2014). The three vertebrate Nanos paralogs contain a 17-amino acid NOT1-interacting motif (NIM) that binds directly to the NOT1 SHD domain (Suzuki et al, 2012; Bhandari et al, 2014). Although the NIM is conserved in vertebrate Nanos, Nanos proteins of insects and worms do not have a detectable NIM (Lai et al, 2011; Suzuki et al, 2012; Bhandari et al, 2014). Nevertheless, Dm Nanos has been reported to interact with NOT4 through its unstructured N-terminus (Kadyrova et al, 2007). However, because NOT4 is not stably associated with the CCR4–NOT complex in metazoans (Lau et al, 2009; Temme et al, 2010), it has remained unclear whether Dm Nanos recruits the CCR4–NOT complex to mRNA targets directly or rather relies on its interaction with additional partners, such as Pumilio (PUM) and Brain tumor (BRAT), to exert its repressive function (Wreden et al, 1997; Sonoda & Wharton, 1999, 2001). In this study, we show that although Dm Nanos does not contain a NIM, it interacts directly with the CCR4–NOT complex using an extended region that we termed the Nanos effector domain (NED). The NED overlaps with a region previously shown to contribute to Nanos function in Dm embryos (Curtis et al, 1997; Arrizabalaga & Lehmann, 1999). The crystal structure of a central region of the NED (termed the NOT module binding region, NBR) bound to the NOT module revealed a bipartite interface that contacts both the NOT1 SHD and the NOT3 NOT-box domains. The binding site for the Dm NBR on NOT1 does not overlap with the vertebrate NIM-binding site. These results indicate that Nanos proteins have maintained the ability to interact with the CCR4–NOT complex using divergent motifs in disordered protein regions. Results Identification of the Dm Nanos effector domain (NED) To investigate whether Dm Nanos possesses intrinsic mRNA repressive activity, we used a λN-based tethering assay in Dm S2 cells (Behm-Ansmant et al, 2006). This assay enabled the study of Nanos function independently of RNA-binding activity. Tethered Dm Nanos caused fourfold repression of a firefly luciferase (F-Luc) reporter containing five binding sites (Box B hairpins) for the λN-tag that were inserted in the 3′ UTR (Fig 1B and C). The reduction in F-Luc activity was predominantly explained by a corresponding decrease in the mRNA abundance (Fig 1B and C) and a shortening of the mRNA half-life (Fig EV1B), indicating that Nanos induces mRNA degradation in S2 cells. Nanos did not affect the expression of an F-Luc reporter lacking the BoxB hairpins (Fig EV1C); thus, Nanos must bind to the mRNA to induce degradation. To define the sequences in Dm Nanos required for the repressive activity, we designed a series of deletion mutants. A Nanos protein lacking the ZnF domain retained the activity of the full-length protein in the tethering assay, whereas the isolated ZnF domain was inactive (Fig 1A–C), as shown previously for vertebrate Nanos (Lai et al, 2011; Bhandari et al, 2014). Further analyses indicated that a region comprising residues 50–236 retained full repressive activity (Fig 1B and C) and was therefore termed the Nanos effector domain (NED). Conversely, deletion of residues 50–236 (ΔNED) abolished the ability of Nanos to repress the expression of the F-Luc-5BoxB reporter (Fig 1B and C). All protein fragments tested were expressed at comparable levels (Fig 1D), and none of them affected the expression of an F-Luc reporter lacking the BoxB hairpins (Fig EV1C). The results of the tethering assay were confirmed using a reporter containing the F-Luc ORF fused to the 3′ UTR of the hunchback (hb) mRNA, a known Nanos target (Wang & Lehmann, 1991; Wreden et al, 1997; Sonoda & Wharton, 1999). Nanos degraded this mRNA (Fig 1E–G) but did not significantly affect the expression of an F-Luc reporter containing the oskar 3′ UTR (Fig EV1D). Thus, Nanos causes mRNA degradation in S2 cells irrespective of whether it is artificially tethered or binds directly to an mRNA. Importantly, deletion of the NED strongly impaired the ability of Nanos to repress the F-Luc-hb reporter (Fig 1E and F). However, the isolated NED did not repress the expression of this reporter (Fig 1E and F), most likely because it lacks RNA binding capacity. These results indicate that the Nanos NED confers repressive activity but requires the ZnF domain to bind to natural mRNA targets, as reported previously (Curtis et al, 1997; Hashimoto et al, 2010). Nanos mediates translational repression in the absence of mRNA deadenylation In addition to promoting mRNA target degradation, vertebrate Nanos proteins can repress translation in a deadenylation-independent manner (Lai et al, 2011; Bhandari et al, 2014). Similarly, Dm Nanos promotes deadenylation and represses translation in the absence of mRNA degradation during oogenesis and early embryogenesis (Wharton & Struhl, 1991; Wreden et al, 1997; Chagnovich & Lehmann, 2001; Kadyrova et al, 2007). Therefore, we investigated whether Dm Nanos can repress translation in the absence of mRNA deadenylation in S2 cells. We used an mRNA with a 3′-end generated by a self-cleaving hammerhead ribozyme (HhR). This reporter is neither polyadenylated nor deadenylated (Zekri et al, 2013). Additionally, the reporter contains a DNA-encoded poly(A) stretch of 95 nucleotides and a 3′ poly(C) stretch of seven nucleotides upstream of the ribozyme cleavage site (F-Luc-5BoxB-A95C7-HhR). This reporter is efficiently translated in S2 cells (Zekri et al, 2013). Tethered Nanos caused a threefold reduction in F-Luc activity but only a 1.2-fold reduction in mRNA levels, indicating that Nanos represses the expression of this reporter primarily at the translational level (Fig 1H and I). Furthermore, a Nanos protein lacking the NED had no repressive activity. Conversely, the NED was sufficient to repress translation of this reporter in the absence of mRNA degradation (Fig 1H and I). Thus, the NED is a major determinant for the repression mediated by Dm Nanos irrespective of whether the repression is caused by mRNA deadenylation and decay or by translational repression in the absence of deadenylation. Nanos has intrinsic repressive activity independent of PUM and BRAT Nanos functions together with PUM and BRAT to repress hb mRNA in Dm embryos (Sonoda & Wharton, 1999, 2001). The sequence in the hb 3′ UTR that binds PUM, BRAT, and Nanos consists of a duplicated Nanos response element (NRE). Each NRE is composed of one BoxA and one BoxB motif (Fig 2A; Wharton & Struhl, 1991; Murata & Wharton, 1995; Zamore et al, 1997; Wreden et al, 1997; Wharton et al, 1998; Sonoda & Wharton, 1999, 2001; Gupta et al, 2009; Loedige et al, 2014). BRAT binds to the BoxA motif of the NRE. PUM binds with high affinity to the BoxB motif but also exhibits low affinity for the BoxA motif (Zamore et al, 1997; Wang et al, 2002; Gupta et al, 2009; Loedige et al, 2014, 2015). Figure 2. Dm Nanos exhibits intrinsic repressive activity Schematic representation of the Nanos response element in the 3′ UTR of hb mRNA. The activity of GFP-tagged Dm Nanos was tested in S2 cells expressing an F-Luc reporter containing the hb 3′ UTR (either wild type or mutants lacking the BoxA or BoxB sequences). A plasmid expressing GFP served as a negative control. F-Luc activity (black bars) and mRNA levels (green bars) were analyzed as described in Fig 1B. The panel shows mean values ± standard deviations from three independent experiments. Northern blot of representative RNA samples corresponding to the experiment shown in (B). Tethering assay using Dm Nanos and the F-Luc-5BoxB reporter in S2 cells depleted of PUM or control cells treated with a dsRNA targeting bacterial GST. A plasmid expressing R-Luc mRNA served as a transfection control. The F-Luc activity (black bars) and mRNA levels (green bars) were normalized to those of the R-Luc transfection control and analyzed as described in Fig 1B. The panel shows mean values ± standard deviations from three independent experiments. Northern blot of representative RNA samples corresponding to the experiment shown in (D). Western blot analysis of S2 cells depleted of PUM and expressing HA-PUM. Endogenous PABP served as a loading control. The ability of Nanos to repress the F-Luc-hb reporter was tested in S2 cells depleted of PUM as described in (D). The panel shows mean values ± standard deviations from three independent experiments. Northern blot of representative RNA samples corresponding to the experiment shown in (G). Source data are available online for this figure. Source Data for Figure 2 [embj201593634-sup-0004-SDataFig2.pdf] Download figure Download PowerPoint To investigate whether Nanos requires BRAT or PUM to repress hb mRNA, we tested the effect of deleting the BoxA or BoxB motifs individually from the F-Luc-hb reporter. As shown in Fig 1E, transfected Nanos repressed and degraded the F-Luc-hb reporter in S2 cells (Fig 2B and C). These cells express both BRAT and PUM but not endogenous Nanos (Miles et al, 2012; Weidmann & Goldstrohm, 2012; Loedige et al, 2014). The deletion of the BoxA motif did not change the ability of Nanos to repress the reporter. By contrast, deletion of the BoxB sequences abrogated the ability of Nanos to degrade the reporter (Fig 2B), indicating that Nanos cooperates with endogenous PUM to repress the F-Luc-hb reporter, as expected (Murata & Wharton, 1995; Wreden et al, 1997; Sonoda & Wharton, 1999). To determine whether Nanos activity in tethering assays was also dependent on its interaction with PUM, we performed the tethering assay in PUM-depleted cells. PUM depletion did not affect Nanos activity in tethering assays (Fig 2D–F) but partially suppressed its ability to repress the F-Luc-hb reporter (Fig 2G and H). In summary, Nanos requires the ZnF domain, PUM, and the BoxB motif to bind to the hb 3′ UTR and the NED to repress this mRNA. The requirement for the ZnF domain and PUM is bypassed when Nanos is directly tethered to the mRNA, indicating that Dm Nanos possesses intrinsic repressive activity, which is provided by the NED. Nanos triggers deadenylation-dependent decapping The vertebrate Nanos proteins trigger deadenylation of mRNA targets by interacting with the CCR4–NOT complex (Suzuki et al, 2010, 2012; Bhandari et al, 2014). Deadenylation by CCR4–NOT is typically coupled to decapping and 5′-to-3′ exonucleolytic degradation by XRN1 (Temme et al, 2014). We therefore investigated whether Dm Nanos degrades bound mRNAs by promoting deadenylation-dependent decapping. If deadenylation precedes decapping and 5′-to-3′ mRNA degradation, then deadenylated mRNA decay intermediates are expected to accumulate in cells in which decapping is inhibited. To inhibit decapping, we overexpressed a catalytically inactive mutant of the decapping enzyme (DCP2*, E361Q) in cells depleted of endogenous DCP2 (Fig 3A and B). In these cells, degradation of the F-Luc-5BoxB reporter by Dm Nanos was inhibited and the F-Luc-5BoxB mRNA accumulated as a fast-migrating form, corresponding to the deadenylated decay intermediate (A0; Fig 3B, lane 5). The luciferase activity is not restored despite restoration of mRNA levels (Fig EV1E), most likely because deadenylated transcripts are translated less efficiently. A similar fast-migrating form accumulated when the GW182 protein was tethered (Fig 3B, lane 6). GW182 is known to cause deadenylation-dependent decapping and thus served as a positive control (Behm-Ansmant et al, 2006). The DCP2 mutant was expressed at comparable levels in each condition (Fig EV1F). Figure 3. Dm Nanos induces deadenylation-dependent decapping Nanos tethering assay using the F-Luc-5BoxB reporter in control cells (treated with GST dsRNA) or in cells depleted of the decapping enzyme DCP2 (DCP2 KD) and expressing a catalytically inactive DCP2 mutant (E361Q). F-Luc activity and mRNA levels were normalized to those of an R-Luc transfection control and analyzed as described in Fig 1B. Normalized F-Luc activities are shown in Fig EV1E. The panel shows mean values ± standard deviations from three independent experiments. Northern blot of representative RNA samples corresponding to the experiment shown in (A). The positions of the polyadenylated (An) and deadenylated (A0) F-Luc-5BoxB mRNA are indicated on the right. Western blot analysis of S2 cells depleted of NOT3. Dilutions of control cell lysates were loaded in lanes 1–4 to estimate the efficacy of the depletion. α-Tubulin served as a loading control. KD: knockdown. Endogenous NOT1 and NOT2 are co-depleted with NOT3 as described previously (Boland et al, 2013). Northern blot analysis showing the decay of the F-Luc-5BoxB mRNA in control cells (treated with GST dsRNA) or in cells depleted of NOT3 expressing the indicated proteins. The mRNA half-lives (t1/2) ± standard deviations calculated from the decay curves of three independent experiments are indicated below the panels. Source data are available online for this figure. Source Data for Figure 3 [embj201593634-sup-0005-SDataFig3.pdf] Download figure Download PowerPoint To further investigate the dependence on the CCR4–NOT complex for Nanos-mediated mRNA degradation, we depleted NOT3 in S2 cells. NOT3 depletion results in co-depletion of NOT1 and NOT2 (Fig 3C; Temme et al, 2010; Boland et al, 2013). The ability of Nanos to elicit the degradation of F-Luc-5BoxB mRNA was partially suppressed in NOT3-depleted cells (Fig 3D). Indeed, the half-life of the F-Luc-5BoxB mRNA was increased fourfold in NOT3-depleted cells expressing Nanos relative to that of control cells (Fig 3D). The NED mediates binding to decay factors Given that Nanos promotes deadenylation-dependent decapping, we sought to determine whether it interacts with factors involved in the 5′-to-3′ decay pathway. We expressed Nanos with a GFP tag in S2 cells and tested for interactions with HA-tagged subunits of the CCR4–NOT and PAN2–PAN3 deadenylase complexes as well as with decapping factors. Nanos interacted with NOT1, NOT2, NOT3, PAN2, and PAN3 (Figs 4A–D and EV1G–M). These interactions were observed in the presence of RNase A, suggesting that they are not mediated by RNA. In agreement with the tethering assays, the Nanos NED exhibited the same binding properties as full-length Nanos, whereas a Nanos protein lacking the NED did not interact with the deadenylase subunits (Fig 4A–D). Figure 4. The Nanos NED interacts with subunits of the deadenylase and decapping complexes A–F. Western blots showing the interaction of GFP-tagged Dm Nanos (either full length, NED, or ΔNED) and the indicated HA-tagged proteins. The co-immunoprecipitations were performed using a polyclonal anti-GFP antibody. GFP-tagged firefly luciferase (F-Luc) served as a negative control. Inputs and immunoprecipitates were analyzed using anti-GFP and anti-HA antibodies. For the GFP-tagged proteins, 3% of the inputs and 10% of the immunoprecipitates were loaded, whereas for the HA-tagged proteins, 1% of the input and 30% of the immunoprecipitates were analyzed. In all panels, cell lysates were treated with RNase A prior to immunoprecipitation. Source data are available online for this figure. Source Data for Figure 4 [embj201593634-sup-0006-SDataFig4.pdf] Download figure Download PowerPoint Nanos also interacted with the decapping enzyme DCP2 and the decapping factor HPat in an RNA independent manner but not with additional decapping factors (Figs 4E and F, and EV2A–F). Importantly, the NED was also necessary and sufficient for the interactions with DCP2 and HPat (Fig 4E and F). Click here to expand this figure. Figure EV2. Dm Nanos interacts with decapping factors and the NOT module A–F. Co-immunoprecipitation assays using GFP-tagged Dm Nanos (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 GFP-tagged Dm Nanos and HA-tagged NOT1, NOT2, and NOT3 (either full length or the indicated fragments). Proteins were immunoprecipitated from RNase A-treated cell lysates using anti-GFP antibodies. GFP-F-Luc served as a negative control. For the detection of GFP-tagged proteins, 3% of the input and 10% of the bound fractions were analyzed by Western blotting. For the detection of HA-tagged NOT1, 1.5% of the input and 35% of the bound fractions were analyzed, whereas for HA–NOT2 and HA–NOT3 proteins, 1% of the input and 30% of the immunoprecipitates were analyzed. Source data are available online for this figure. Download figure Download PowerPoint The NED interacts directly with the NOT module of the CCR4–NOT complex To define more precisely how the CCR4–NOT complex interacts with Dm Nanos, we used truncated constructs of NOT1, NOT2, and NOT3 in co-immunopreci
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