Drosophila Bag-of-marbles directly interacts with the CAF40 subunit of the CCR4-NOT complex to elicit repression of mRNA targets

Abstract

Drosophila melanogaster Bag-of-marbles (Bam) promotes germline stem cell (GSC) differentiation by repressing the expression of mRNAs encoding stem cell maintenance factors. Bam interacts with Benign gonial cell neoplasm (Bgcn) and the CCR4 deadenylase, a catalytic subunit of the CCR4-NOT complex. Bam has been proposed to bind CCR4 and displace it from the CCR4-NOT complex. Here, we investigated the interaction of Bam with the CCR4-NOT complex by using purified recombinant proteins. Unexpectedly, we found that Bam does not interact with CCR4 directly but instead binds to the CAF40 subunit of the complex in a manner mediated by a conserved N-terminal CAF40-binding motif (CBM). The crystal structure of the Bam CBM bound to CAF40 reveals that the CBM peptide adopts an α-helical conformation after binding to the concave surface of the crescent-shaped CAF40 protein. We further show that Bam-mediated mRNA decay and translational repression depend entirely on Bam's interaction with CAF40. Thus, Bam regulates the expression of its mRNA targets by recruiting the CCR4-NOT complex through interaction with CAF40.

Keywords: deadenylation; germ cell differentiation; mRNA decay; translational repression.

FIGURE 1.

Bam induces degradation of bound mRNAs through its N-terminal region. (A) Bam consists of several predicted α-helices (shown in yellow) and a β-strand (shown in cyan). The position of the CAF40-binding motif (CBM, in red) as well as the boundaries of the Bam-N and Bam-C fragments are indicated. (B) Tethering assay using the F-Luc-5BoxB reporter and λN-HA-tagged Bam (full-length or the indicated fragments) in Dm S2 cells. A plasmid expressing R-Luc mRNA served as a transfection control. For each experiment, F-Luc activity and mRNA levels were normalized to those of the R-Luc transfection control and set to 100% in cells expressing the λN-HA peptide. (C) Northern blot of representative RNA samples shown in B. (D) Western blot analysis showing the equivalent expression of the λN-HA-tagged proteins used in the tethering assays shown in B and C. Protein size markers (kDa) are shown on the right of the panel. Full-length Bam and Bam-N display an aberrant mobility in SDS–PAGE, thus resulting in a higher apparent molecular weight. (E,F) Tethering assay using the F-Luc-5BoxB-A₉₅-C₇-HhR reporter and λN-HA-tagged Bam (full-length or the indicated fragments) in Dm S2 cells. The samples were analyzed as described in B and C. In B and E, bars represent mean values and error bars represent standard deviations from at least three independent experiments.

FIGURE 2.

Bam degrades mRNAs through the 5′-to-3′ mRNA decay pathway. (A) Tethering assay using the F-Luc-5BoxB reporter was performed in control S2 cells or cells depleted of the decapping enzyme DCP2 (DCP2 KD). The transfection mixture included plasmids expressing either GFP-V5 or a GFP-tagged catalytically inactive DCP2 mutant (DCP2*; E361Q). The F-Luc-5BoxB mRNA levels were normalized to those of the R-Luc transfection control and set to 100% in control and knockdown cells expressing the λN-HA peptide. The gray bars represent the normalized F-Luc-5BoxB mRNA values in control cells expressing GFP-V5. The black bars represent the normalized F-Luc-5BoxB mRNA values in DCP2-depleted cells expressing GFP-DCP2*-V5. (B) Northern blot of representative RNA samples shown in A. The positions of the polyadenylated (A_n) and deadenylated (A₀, dashed red line) mRNA reporter are indicated on the right of the panel. (C) Western blot analysis showing equivalent expression of λN-HA-tagged proteins in the experiments shown in A and B. (KD) Knockdown. (D,E) Tethering assay using the F-Luc-5BoxB reporter in control S2 cells or in NOT1-depleted cells. Samples were analyzed as described in Figure 1B–D. (F,G) Tethering assay using the F-Luc-5BoxB-A₉₅-C₇-HhR reporter in control cells and in NOT1-depleted cells. Samples were analyzed as described in Figure 1B–D. In A, D, and F, bars represent mean values and error bars represent standard deviations from at least three independent experiments. (H) Western blot analysis showing the efficiency of NOT1 depletion in the experiments shown in D–G. Dilutions of control cell lysates were loaded in lanes 1–4. PABP served as a loading control. Protein size markers (kDa) are shown on the right in each panel.

FIGURE 3.

Bam binds directly to CAF40 by using an N-terminal CAF40-binding motif (CBM). (A) SBP pull-down assay in HEK293T cells expressing V5-SBP-tagged full-length Bam. V5-SBP-tagged MBP served as negative control. Input (1.5% for the V5-SBP tagged proteins and 1% for endogenous CCR4–NOT subunits) and bound fractions (10% for the V5-SBP tagged proteins and 30% for the CCR4–NOT subunits) were analyzed by western blotting using the indicated antibodies. (B) SBP pull-down assay in HEK293T cells expressing V5-SBP-tagged full-length Bam and HA-CCR4. V5-SBP-tagged MBP served as negative control. Samples were analyzed as described in A. (C) MBP pull-down assay testing the interaction of MBP-tagged full-length Bam, the CBM or Bam ΔCBM with the Dm CN9BD–CAF40 complex. MBP served as a negative control. The inputs (10%) and bound fractions (50%) were analyzed by SDS–PAGE and subsequent Coomassie staining. (D) MBP pull-down assay showing the interaction of MBP-tagged Bam CBM with the Dm and Hs CN9BD–CAF40 complex and Hs CAF40. Samples were analyzed as in C. (E) Tethering assay using the F-Luc-5BoxB reporter and λN-HA-tagged Bam (full-length or the indicated fragments) in S2 cells. The samples were analyzed as described in Figure 1B–D. The mean values ±SD from three independent experiments are shown. (F) Northern blot of representative RNA samples shown in E. (G) Western blot showing the equivalent expression of λN-HA-tagged proteins used in E and F. Protein size markers (kDa) are shown on the right in each panel.

FIGURE 4.

Structure of the Bam CBM bound to CAF40 and to the NOT1 CN9BD–CAF40 module. (A) The Bam CBM peptide (red, backbone shown in ribbon representation) bound to Hs CAF40 (gray). CAF40 helices are depicted as tubes and numbered in black. The orange semicircle marks the predominantly hydrophobic interface between the CBM peptide and CAF40. (B) Cartoon representation of the Bam CBM peptide bound to Hs CAF40. Selected CAF40 secondary structure elements are labeled in black. (C) Structure of the CBM peptide bound to the NOT1 CN9BD–CAF40 complex. (D) Superposition of the CAF40–Bam CBM structure with the structure of CAF40 bound to the Roq CBM (PDB 5LSW; Sgromo et al. 2017). The Roq CBM is shown in yellow and CAF40 from the Roq complex in blue. (E) In vitro competition assay. GST-tagged Hs CAF40 was incubated with equimolar amounts of MBP-tagged Bam or Roq CBMs and increasing amounts of His₆-NusA-tagged Bam CBM. His₆-NusA was used as a negative control. Proteins bound to GST-CAF40 were pulled down and analyzed by SDS-PAGE and subsequent Coomassie staining. Molar equivalents (eq) are relative to GST-CAF40.

FIGURE 5.

The Bam and Roq CBMs use a similar CAF40-binding mode. (A,B) Close-up views of the CAF40-Bam CBM-binding interface in two orientations. Selected residues of CAF40 and Bam are shown as orange and red sticks, respectively. Hydrogen bonds are indicated by red dashed lines. Residues mutated in this study are underlined. (C) Close-up view of the structural superposition of the CAF40-Bam CBM structure with the structure of the Roq CBM bound to CAF40. The Bam and Roq CBMs are shown in red and yellow, respectively. (D) (Upper panel) Superposition of the Bam and Roq CBM peptides bound to CAF40. The backbones are shown in ribbon representation, and side chains are shown as sticks. CAF40 is indicated as a thick gray line. (Lower panel) Sequence alignment of the Bam and Roq CBMs from the indicated Drosophila species. Hydrophobic residues interacting with CAF40 are highlighted by a light green background. Gray letters indicate residues that were not included in the crystallization setup. (E) MBP pull-down assay testing the interaction of MBP-tagged Bam (wild-type or mutants L17E and M24E) with the Dm NOT1-CN9BD–CAF40 complex. MBP served as a negative control. (F) MBP pull-down assay testing the interaction of MBP-tagged Bam with Dm NOT1-CN9BD–CAF40 complex (containing CAF40 wild-type or the indicated mutants). MBP served as a negative control.

FIGURE 6.

The CBM is necessary for Bam-mediated mRNA repression. (A) Tethering assay using the F-Luc-5BoxB reporter and λN-HA-tagged Bam (wild-type or the indicated mutants) in S2 cells. The samples were analyzed as described in Figure 1B. (B) Northern blot of representative RNA samples shown in A. (C) Western blot showing the equivalent expression of λN-HA-tagged proteins used in A and B. (D) SBP pull-down assay in control and CAF40-null HEK293T cells expressing V5-SBP-tagged full-length Bam. V5-SBP-tagged MBP served as a negative control. Input (1.5% for the V5-SBP tagged proteins and 1% for endogenous CCR4–NOT subunits) and bound fractions (10% for the V5-SBP tagged proteins and 30% for the CCR4–NOT subunits) were analyzed by western blotting using the indicated antibodies. (KO) Knockout. (E) SBP pull-down assay in control and CAF40-null HEK293T cells expressing V5-SBP-tagged full-length Bam and HA-CCR4. Samples were analyzed as in D. (F) SBP pull-down assay in HEK293T cells expressing V5-SBP-tagged full-length Bam or the 4xMut. V5-SBP-tagged MBP served as a negative control. Input (1.5% for the V5-SBP tagged proteins and 1% for CCR4–NOT subunits) and the bound fractions (10% for the V5-SBP tagged proteins and 30% for CCR4–NOT subunits) were analyzed by western blotting using the indicated antibodies. (G) SBP pull-down assay in HEK293T cells expressing V5-SBP-tagged full-length Bam or the 4xMut and HA-tagged CCR4.

FIGURE 7.

Bam depends on CCR4–NOT complex recruitment to induce mRNA decay. (A) Tethering assay using the F-Luc-5BoxB reporter and λN-HA-tagged Bam and Roq in S2 cells. The transfection mixtures also contained plasmids for expression of GFP (control) or GFP-CAF40 (wild-type or the V186E mutant) as indicated. The samples were analyzed as described in Figure 1B. (B) Northern blot of representative RNA samples shown in A. (C) Western blot showing the equivalent expression of the λN-HA-tagged proteins in cells expressing GFP or GFP-CAF40 (either wild-type or the V186E mutant) used in A and B. (D) Western blot showing the efficiency of the CAF40 depletion in Dm S2 cells. Dilutions of control cell lysates were loaded in lanes 1–4 to estimate the efficacy of the depletion. PABP served as a loading control. (KD) Knockdown. (E) Complementation assay using the F-Luc-5BoxB reporter and λN-HA-tagged Bam in S2 cells depleted of CAF40 (CAF40 KD) or in control cells (control). Samples were analyzed as described in Figure 1B. (F) Northern blot of representative RNA samples shown in E. (G) Western blot showing the equivalent expression of the λN-HA-tagged Bam constructs used in E and F.