CUP promotes deadenylation and inhibits decapping of mRNA targets

Abstract

CUP is an eIF4E-binding protein (4E-BP) that represses the expression of specific maternal mRNAs prior to their posterior localization. Here, we show that CUP employs multiple mechanisms to repress the expression of target mRNAs. In addition to inducing translational repression, CUP maintains mRNA targets in a repressed state by promoting their deadenylation and protects deadenylated mRNAs from further degradation. Translational repression and deadenylation are independent of eIF4E binding and require both the middle and C-terminal regions of CUP, which collectively we termed the effector domain. This domain associates with the deadenylase complex CAF1-CCR4-NOT and decapping activators. Accordingly, in isolation, the effector domain is a potent trigger of mRNA degradation and promotes deadenylation, decapping and decay. However, in the context of the full-length CUP protein, the decapping and decay mediated by the effector domain are inhibited, and target mRNAs are maintained in a deadenylated, repressed form. Remarkably, an N-terminal regulatory domain containing a noncanonical eIF4E-binding motif is required to protect CUP-associated mRNAs from decapping and further degradation, suggesting that this domain counteracts the activity of the effector domain. Our findings indicate that the mode of action of CUP is more complex than previously thought and provide mechanistic insight into the regulation of mRNA expression by 4E-BPs.

Figure 1.

CUP represses translation and promotes the deadenylation of bound mRNAs. (A–D) S2 cells were transfected with a mixture of three plasmids: one expressing the F-Luc-5BoxB reporter, another expressing the Renilla luciferase (R-Luc) as a transfection control, and a third expressing λN-HA or λN-HA-CUP, as indicated. Additionally, all transfection mixtures contained a plasmid expressing a short uncapped and unadenylated RNA derived from the 7SL RNA. (A) Firefly luciferase activity was normalized to Renilla luciferase and set to 100 in cells expressing λN-HA. (B) F-Luc-5BoxB mRNA levels were normalized to those of the Renilla mRNA or 7SL RNA and set to 100 in cells expressing λN-HA. Similar values were obtained independently of the normalization control. Mean values ± standard deviations from three independent experiments are shown. (C) The normalized F-luc activity values were divided by the corresponding normalized mRNA levels for each condition. These ratios were set to 100 in cells expressing λN-HA. (D) Northern blot analysis of RNA samples corresponding to those shown in B. F-Luc-5Box mRNA lacking a poly(A) tail (A₀) was loaded as reference. The dashed line indicates the position of the deadenylated F-Luc-5BoxB mRNA. (E) RNA samples isolated from tethering assays (e.g., D) were treated with RNase H in the absence or presence of oligo(dT) and analyzed by Northern blot; rp49 mRNA served as a positive control for the RNase H treatment. (F) S2 cells were transfected as described in A. Three days after transfection, cells were treated with actinomycin D (5 μg/mL) and harvested at the indicated time points. F-Luc-5BoxB mRNA lacking a poly(A) tail was loaded as reference, and rp49 mRNA served as a loading control.

Figure 2.

CUP represses mRNA expression independently of eIF4E binding. (A) Domain organization of CUP protein. CUP contains an N-terminal regulatory domain (N-term) containing two eIF4E-binding motifs (4E-BM1 and 4E-BM2), a middle region (Mid), and a glutamine-rich C-terminal region (Q-rich). The Mid and Q-rich regions define the effector domain. The bottom panel shows the CUP point mutants used in this study and a summary of their activities. (stabil.) mRNA stabilization; (repres.) repression of protein production; (dead.) deadenylation; (decay) degradation of the mRNA body. (B) Interactions between HA-CUP wild type or mutants and endogenous eIF4E. Proteins were immunoprecipitated from cell lysates using a monoclonal anti-HA antibody. An HA-tagged version of maltose-binding protein (MBP) served as negative control. Inputs (1%) and immunoprecipitates (10%) were analyzed by Western blotting using anti-HA and anti-eIF4E antibodies. (C–F) The activity of CUP mutants relative to wild type was tested in tethering assays using the F-Luc-5BoxB reporter and analyzed as described in Figure 1. The mean values ± standard deviations from three independent experiments are shown. (G) Wild-type CUP and mutants were expressed at comparable levels. R-Luc-V5 served as a transfection control.

Figure 3.

The Mid and Q-rich regions define the CUP effector domain. (A) Schematic representation of CUP deletion mutants used in this study and a summary of their activities, as described in Figure 2A. (B–D) The activities of CUP deletion mutants relative to wild type were tested using the F-Luc-5BoxB reporter as described in Figure 1. (E) The interaction between HA-tagged wild-type or mutant CUP protein and endogenous eIF4E was analyzed by coimmunoprecipitation as described in Figure 2B. (F) Wild-type and mutant CUP proteins were expressed at comparable levels. R-Luc-V5 served as a transfection control.

Figure 4.

CUP represses an mRNA reporter containing the oskar 3′ UTR. (A–D) S2 cells were transfected with a mixture of three plasmids: one expressing the F-Luc-oskar reporter, another expressing the Renilla luciferase (R-Luc) as a transfection control, and a third expressing GFP or CUP. Additionally, transfection mixtures contained a plasmid expressing GST or Bruno as indicated. (A) Firefly luciferase activity was normalized to Renilla luciferase and set to 100 in control cells (i.e., expressing GFP and GST). (B) F-Luc-oskar mRNA levels were normalized to those of the Renilla mRNA and set to 100 in control cells. Mean values ± standard deviations from three independent experiments are shown. (C) F-Luc activity was normalized to the mRNA levels. (D) Northern blot analysis of RNA samples corresponding to those shown in B. (E–H) S2 cells were transfected with a mixture of three plasmids: one expressing the F-Luc-oskar reporter, another expressing the Renilla luciferase (R-Luc) as a transfection control, and a third expressing MBP or CUP. Additionally, all transfection mixtures contained a plasmid expressing Bruno. Luciferase activity and mRNA levels were analyzed as described in A–D.

Figure 5.

The effector domain of CUP confers binding to deadenylase and decapping complexes. (A,B) Interaction between HA-CUP and GFP-tagged subunits of the CAF1–CCR4–NOT complex. Cell lysates were immunoprecipitated using a polyclonal anti-GFP antibody (A) or a monoclonal anti-HA antibody (B). When indicated, cell lysates were treated with RNase A before immunoprecipitation. F-Luc-GFP and HA-MBP served as negative controls. (C–E) Interactions between wild-type CUP or the indicated CUP fragments with binding partners. (C) Interaction of CUP and GFP-NOT1. (D,E) Interactions of HA-CUP with endogenous Me31B or Tral.

Figure 6.

CUP Mut2 promotes deadenylation-dependent decapping. (A–C) A tethering assay with CUP Mut2 or GW182 was performed in control cells (treated with GST dsRNA and expressing GFP) and in cells depleted of DCP2 (DCP2 knockdown; KD) and expressing a catalytically inactive GFP-DCP2 mutant (E361Q). Samples were analyzed as described in Figure 1. The dashed line in A indicates the position of the deadenylated F-Luc-5BoxB mRNA.

Figure 7.

The CAF1–CCR4–NOT complex deadenylates mRNAs associated with CUP. (A,B) S2 cells were treated with the indicated dsRNAs on days 0 and 4. Control cells were treated with an unrelated dsRNA targeting Neomycin. On day 6, cells were cotransfected with a mixture of three plasmids: one expressing the F-Luc-5BoxB mRNA, another expressing Renilla luciferase (R-Luc), and a third expressing the indicated λN-HA-tagged proteins. (A) Northern blot analysis of representative RNA samples. (B) Western blot analysis of control and NOT1-depleted cells. α-Tubulin served as a loading control. Dilutions of control cell lysates were loaded in lanes 1–4 to estimate the efficacy of the depletion. (C) Firefly luciferase activity was normalized to that of Renilla luciferase. For each condition, the normalized values of F-Luc activity were set to 100 in cells expressing the λN-HA tag. Mean values ± standard deviations from three independent experiments are shown. (D) The normalized F-luc activity values were divided by the corresponding normalized mRNA levels for each condition. These ratios were set to 100 in cells expressing λN-HA.

Figure 8.

Deadenylation is not required for CUP-mediated mRNA repression. (A–F) S2 cells were transfected with F-Luc5BoxB reporters in which the cleavage and polyadenylation signal had been substituted with either a histone H4 3′-terminal stem–loop (HSL; A,C,E) or a self-cleaving hammerhead ribozyme (HhR; B,D,F). Plasmids expressing Renilla luciferase (R-Luc) or 7SL RNA served as transfection controls. Firefly luciferase activity and mRNA levels were analyzed as described in Figure 1. A Northern blot analysis of representative RNA samples is shown below the corresponding graphs.