Distinct modes of recruitment of the CCR4-NOT complex by Drosophila and vertebrate Nanos

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.

Keywords: deadenylation; decapping; mRNA decay; translational repression.

Figure 1. Dm Nanos represses translation and promotes mRNA degradation

1. 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.

2. 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.

3. Northern blot of representative RNA samples corresponding to the experiment shown in (B).

4. 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.

5. 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.

6. Northern blot of representative RNA samples corresponding to the experiment shown in (E).

7. Western blot showing the expression of the GFP‐tagged proteins used in (E) and (F). R‐Luc‐V5 served as a transfection control.

8. 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.

9. Northern blot of representative RNA samples corresponding to the experiment shown in (H).

Source data are available online for this figure.

Figure EV1. Dm Nanos interacts with subunits of the deadenylase complexes

1. 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).

2. B

   Northern blot analysis showing the decay of the F‐Luc‐5BoxB mRNA in S2 cells expressing the indicated proteins. The mRNA half‐lives (t_1/2) ± standard deviations calculated from the decay curves of three independent experiments are indicated below the panels.

3. 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.

4. 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.

5. E

   Normalized luciferase activities corresponding to the experiment shown in Fig 3A and B.

6. 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.

7. 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.

Figure 2. Dm Nanos exhibits intrinsic repressive activity

1. Schematic representation of the Nanos response element in the 3′ UTR of hb mRNA.

2. 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.

3. Northern blot of representative RNA samples corresponding to the experiment shown in (B).

4. 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.

5. Northern blot of representative RNA samples corresponding to the experiment shown in (D).

6. Western blot analysis of S2 cells depleted of PUM and expressing HA‐PUM. Endogenous PABP served as a loading control.

7. 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.

8. Northern blot of representative RNA samples corresponding to the experiment shown in (G).

Source data are available online for this figure.

Figure 3. Dm Nanos induces deadenylation‐dependent decapping

1. 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.

2. Northern blot of representative RNA samples corresponding to the experiment shown in (A). The positions of the polyadenylated (A_n) and deadenylated (A₀) F‐Luc‐5BoxB mRNA are indicated on the right.

3. 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).

4. 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 (t_1/2) ± standard deviations calculated from the decay curves of three independent experiments are indicated below the panels.

Source data are available online for this figure.

Figure 4. The Nanos NED interacts with subunits of the deadenylase and decapping complexes

1. 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.

Figure EV2. Dm Nanos interacts with decapping factors and the NOT module

1. 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.

2. 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.

Figure 5. The central region of the Nanos NED interacts directly with the NOT module

1. GST pull‐down assay showing the interaction of the GST‐tagged Dm Nanos NED (wild type or lacking the NBR) and GST‐NBR with the recombinant Hs NOT module (NOT1–3; containing the NOT1 SHD and the NOT2 and NOT3 C‐terminal fragments).

2. GST pull‐down assay showing the interaction of the GST‐tagged Dm Nanos NBR with the assembled NOT module (NOT1–3), the isolated NOT1 SHD (NOT1), or the NOT2–NOT3 dimers (NOT2–3).

Source data are available online for this figure.

Figure EV3. Sequence alignment of the Nanos NED regions of the indicated Drosophila species

1. The residues conserved in all of the aligned sequences are shown with a red background, and the residues with > 70% similarity are highlighted with a salmon background. The residues interacting with NOT1 and NOT3 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 NOT module binding) and in orange (I123M mutation for the incorporation of selenomethionine as an anomalous scatterer).

2. Tethering assay using the F‐Luc‐5BoxB reporter in S2 cells expressing the indicated λN‐HA‐tagged NED fragments. A plasmid‐expressing R‐Luc mRNA was used as a transfection control. The F‐Luc activities 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.

3. Western blot analysis showing the expression of the λN‐HA‐tagged proteins used in the experiment described in (B). GFP served as a transfection control.

4. An experiment similar to that described in (B) was performed using an F‐Luc reporter that lacks the BoxB hairpins. The panel shows mean values ± standard deviations from three independent experiments.

5. GST pull‐down assay showing that the MBP‐tagged Nanos3 NIM peptide does not compete with the GST‐tagged Nanos NBR for binding to the purified NOT module.

Source data are available online for this figure.

Figure EV4. Comparison of NOT module conformations and details of Dm Nanos NBR binding

1. Superposition of the mutant NOT module (apo form) crystallized in space group P2₁ (blue; chains A, B, C), and the wild‐type NOT module crystallized in space group P2₁2₁2 (red; PDB code 4C0D; Boland et al, 2013).

2. Superposition of the mutant NOT module structure obtained in the absence (blue, chains A, B, C) and presence of the Dm Nanos NBR (orange; chains A, B, C). The NBR peptide is shown in red.

3. Superposition of the two NOT module complexes from the asymmetric unit of the crystals obtained in the presence of the Dm Nanos NBR peptide. Complex 1 (chains A, B, C) is colored in orange and complex 2 (chains E, F, G) in green. The NBR peptide is shown in red and dark green.

4. Crystal packing of the NOT module mutant bound to the Dm Nanos NBR peptide.

5. Orientation of NOT1 helix α23 in the wild type (PDB code 4C0D; Boland et al, 2013) and mutant NOT module apo complexes. Colors are as in (A). The black lines indicate the change in the relative orientation of the helix axes.

6. Orientation of the NOT1 helix α23 in the mutant NOT module complex as compared to the orientation in the complex of the NOT1 SHD with the human Nanos1 NIM (PDB code 4CQO; Bhandari et al, 2014).

7. Alternative view of the N1BM binding pocket centered on Dm Nanos F130. Selected residues of NOT1 and of the NBR peptide are shown as gray and red sticks, respectively. Residues mutated in this study are underlined.

8. Alternative view of the N3BM binding pocket centered on Dm Nanos F152. Selected residues of NOT3 and of the NBR peptide are shown as cyan and red sticks, respectively. Residues mutated in this study are underlined.

9. Additional close‐up of the N3BM binding site emphasizing the role of NOT3 K737 with hydrogen bonds as dashed green lines. Residues mutated in this study are underlined.

Figure 6. Structure of the Dm Nanos NBR bound to the NOT module

1. A, B

   Cartoon representation of the Dm NBR peptide (red) bound to the NOT module in two orientations. The NOT1 SHD is colored in gray. NOT2 and NOT3 are shown in green and cyan, respectively.

2. C

   Model including the Nanos1 NIM peptide (from PDB entry 4CQO, Bhandari et al, 2014) obtained by superimposing the NOT1 SHD domains.

3. D

   Surface representation of the N1BM binding pocket of NOT1 with residues colored in a gradient from white to yellow with increasing hydrophobicity according to Kyte and Doolittle (1982).

4. E

   Cartoon representation of the N1BM binding pocket. Selected residues of NOT1 and of the NBR peptide are shown as gray and red sticks, respectively. Residues mutated in this study are underlined.

5. F

   Alternative view of the N1BM binding pocket. NOT1 residues 1883–1894 of loop L2 have been omitted for clarity. Residues mutated in this study are underlined.

6. G

   Surface representation of the N3BM binding pocket of NOT3 colored as described in (D).

7. H

   Cartoon representation of the N3BM binding pocket. Selected residues of NOT3 and of the NBR peptide are shown as cyan and red sticks, respectively. Residues mutated in this study are underlined.

8. I

   Alternative view of the N3BM binding pocket including selected hydrogen bonds as dashed green lines. Residues mutated in this study are underlined.

9. J–L

   Conservation of the NBR binding sites on the NOT module surface. The NOT module is shown in surface representation. Surface residues that are identical between Hs and Dm are shown in orange, all other residues are shown in white. The views shown in (K) and (L) are in the same orientation as those shown in (D) and (G), respectively.

Figure EV5. Validation of the sequence assignment of the NBR peptide bound to the NOT module and activity of NBR mutants

1. A–D

   Anomalous difference Fourier map (black mesh) calculated at a resolution of 7.5 Å and contoured at the 4.0 σ level. Data (available as source data) were collected at the Selenium K‐edge peak wavelength from a crystal containing selenomethionine‐substituted Dm Nanos NBR peptide (I123M mutant). The panels show close‐up views of the binding sites of the N1BM (A, B) and the N3BM (C, D) in the same orientations as in Fig 6. Residues mutated in this study are underlined.

2. E–G

   Western blot analysis showing the interaction of GFP‐tagged Dm Nanos (wild type or 3xMut) with HA‐tagged NOT1, NOT2, and NOT3. 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 as described in Fig EV1G–M.

3. H

   A tethering assay using the F‐Luc‐5BoxB reporter and the indicated λN‐HA‐tagged proteins was performed in S2 as described in Fig 1B. The panel shows mean values ± standard deviations from three independent experiments.

4. I

   Tethering assays in human HEK293T cells, using a β‐globin reporter containing 6 binding sites (6xbs) for the MS2 protein and MS2‐HA‐tagged Hs Nanos2 (wild type or the indicated variants, Nanos2 ΔNIM and Nanos2 NIM‐to‐NBR). In the Nanos2 NIM‐to‐NBR protein, the NIM was replaced by the Dm Nanos NBR. A plasmid expressing an mRNA lacking MS2 binding sites (Control) served as a transfection control. The β‐globin‐6xbs mRNA levels were normalized to those of the control mRNA and set to 100 in the presence of MS2‐HA. The panel shows mean values ± standard deviations from three independent experiments.

5. J

   Northern blot of representative RNA samples corresponding to the experiment shown in (I).

6. 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.

7. L

   Co‐immunoprecipitation assay in human HEK293T cells showing the interaction of V5‐SBP‐tagged Nanos2 (wild type or the indicated variants) with endogenous NOT1 and NOT3. V5‐SBP‐tagged GFP‐MBP served as a negative control.

Source data are available online for this figure.

Figure 7. Validation of the interaction interfaces

1. GST pull‐down assay showing the interaction of the GST‐Dm Nanos NBR [wild type or 2xMut (L127D F130D)] with the recombinant NOT module (NOT1–3) containing the NOT1 SHD (either wild type or V1880E and H1949D). GST served as a negative control. Note that the GST‐Dm Nanos NBR 2xMut exhibits abnormal electrophoretic migration most likely due to the introduction of two negatively charged residues.

2. GST pull‐down assay showing the interaction of GST‐Dm Nanos NBR [wild type, F152E and 3xMut (E151A, F152A, N155A)] with the recombinant NOT module containing wild‐type NOT3 or the Y702A mutant. GST served as a negative control.

3. GST pull‐down assay showing the interaction of GST‐tagged Dm Nanos NBR (wild type, F152E, 2xMut, and 3xMut) with the Dm CCR4–NOT complex in S2 cell lysates. GST served as a negative control.

4. Tethering assay using the Nanos NBR mutants and the F‐Luc‐5BoxB reporter in S2 cells. A plasmid expressing R‐Luc mRNA served as a transfection control. The 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.

5. Northern blot of representative RNA samples corresponding to the experiment shown in (D).

6. Western blot showing the expression of the λN‐HA‐tagged proteins used in the experiments shown in (D) and (E). GFP served as a transfection and loading control.

Source data are available online for this figure.

Figure 8. The NED and NIM are functionally equivalent

1. Tethering assay using a chimeric Nanos protein and the F‐Luc‐5BoxB reporter in S2 cells. The chimeric Nanos protein contains the NIM of human Nanos2 (either wild type (NIM‐ZnF) or mutated (NIM*‐ZnF)), fused to the Dm ZnF domain. A plasmid expressing GST served as a negative control. F‐Luc activity and mRNA levels were analyzed as described in Fig 1B. The panel shows mean values ± standard deviations from three independent experiments.

2. Northern blot of representative RNA samples corresponding to the experiment shown in (A).

3. The activity of GST‐HA‐tagged Nanos chimeric protein was tested in S2 cells expressing an F‐Luc‐hb reporter. A plasmid expressing GST served as a negative control. F‐Luc activity and mRNA levels were analyzed as described in Fig 1B. The panel shows mean values ± standard deviations from three independent experiments.

4. Northern blot of representative RNA samples corresponding to the experiment shown in (C).

5. Western blot analysis showing the interaction of the HA‐tagged Nanos chimeric protein (NIM‐ZnF or NIM*‐ZnF) with endogenous Dm NOT1 and NOT3. HA‐MBP served as a negative control.

6. Tethering assays in human HEK293T cells, using a β‐globin reporter containing 6 binding sites (6xbs) for the MS2 protein and MS2‐HA‐tagged Dm Nanos or NED fragment. A plasmid expressing an mRNA lacking MS2 binding sites (Control) served as a transfection control. The β‐globin‐6xbs mRNA levels were normalized to those of the control mRNA and set to 100 in the presence of MS2‐HA. The panel shows mean values ± standard deviations from three independent experiments.

7. Northern blot of representative RNA samples corresponding to the experiment shown in (F).

8. Co‐immunoprecipitation assay in human HEK293T cells showing the interaction of the HA‐tagged Dm Nanos and the NED fragment with GFP‐tagged NOT3 in human cells. HA‐MBP served as a negative control. The inputs (1%) and bound fractions (10% of HA‐tagged proteins and 30% of GFP‐NOT3) were analyzed by Western blotting.

Source data are available online for this figure.