Cap-Binding Protein 4EHP Effects Translation Silencing by Micrornas

Cap-binding protein 4EHP effects translation silencing by microRNAs

Clément Chapata,b,1, Seyed Mehdi Jafarnejada,b,1, Edna Matta-Camachoa,b, Geoffrey G. Heskethc, Idit A. Gelbartd, Jan Attige, Christos G. Gkogkasf, Tommy Alaing, Noam Stern-Ginossard, Marc R. Fabianh, Anne-Claude Gingrasc,i, Thomas F. Duchainea,b,2, and Nahum Sonenberga,b,2

aGoodman Cancer Research Center, McGill University, Montreal, QC H3A 1A3, Canada; bDepartment of Biochemistry, McGill University, Montreal, QC H3A 1A3, Canada; cCentre for Systems Biology, Lunenfeld-Tanenbaum Research Institute, Sinai Health System, Toronto, ON M5G 1X5, Canada; dDepartment of Molecular Genetics, Weizmann Institute of Science, Rehovot 7610001, Israel; eThe Francis Crick Institute, London NW1 1AT, United Kingdom; fPatrick Wild Centre, Centre for Integrative Physiology, University of Edinburgh, Edinburgh EH8 9XD, United Kingdom; gChildren’s Hospital of Eastern Ontario Research Institute, Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, ON K1H 8L1, Canada; hLady Davis Institute for Medical Research, McGill University, Montreal, QC H3A 1A3, Canada; and iDepartment of Molecular Genetics, University of Toronto, Toronto, ON M5S 1A1, Canada

Contributed by Nahum Sonenberg, April 11, 2017 (sent for review January 30, 2017; reviewed by Reuven Agami and Nancy Standart) MicroRNAs (miRNAs) play critical roles in a broad variety of The 4EHP protein has primarily been documented as a trans- biological processes by inhibiting translation initiation and by lation repressor. In the Drosophila embryo, 4EHP associates with destabilizing target mRNAs. The CCR4–NOT complex effects the RNA binding protein Bicoid to repress caudal mRNA trans- miRNA-mediated silencing, at least in part through interactions lation (23). Similarly, 4EHP also represses the hunchback mRNA with 4E-T (eIF4E transporter) protein, but the precise mechanism by binding to the nanos repressive element complex, which con- is unknown. Here we show that the cap-binding eIF4E-homologous sists of nanos, pumilio, and brain tumor proteins (24). A similar protein 4EHP is an integral component of the miRNA-mediated si- mechanism functions in the mouse, where 4EHP binds the Prep1 lencing machinery. We demonstrate that the cap-binding activity of RNA-binding protein and inhibits Hoxb4 mRNA translation 4EHP contributes to the translational silencing by miRNAs through (25). Moreover, 4EHP forms a translational repressor complex – the CCR4 NOT complex. Our results show that 4EHP competes with with GIGYF2 (Grb10-interacting GYF protein 2), which acts eIF4E for binding to 4E-T, and this interaction increases the affinity as a cofactor in translational repression and mRNA decay of of 4EHP for the cap. We propose a model wherein the 4E-T/4EHP tristetraprolin-targeted mRNAs (26, 27). interaction engenders a closed-loop mRNA conformation that blocks In this work, we demonstrate that 4EHP interacts with the translational initiation of miRNA targets. mRNA-silencing machinery and engenders miRNA-mediated translational repression. Our data support a model wherein 4EHP microRNA | 4EHP | 4E-T | CCR4–NOT | mRNA translation interactions with miRISC/CCR4–NOT lead to the translational repression of miRNA targets. icroRNAs (miRNAs) are short, ∼22-nucleotide noncoding MRNAs that affect gene expression in most eukaryotes. Significance miRNAs mediate posttranscriptional silencing by guiding the miRNA-induced silencing complex (miRISC), an assembly of miRNAs are important components of gene regulatory net- Argonautes and GW182/TNRC6 proteins, to target mRNAs. works and affect all aspects of cell biology by controlling the Target recognition initiates a succession of events: mRNA trans- stability and translation efficiency of their target mRNAs. Here, lational repression, deadenylation, and mRNA decay (1). miRNAs we identified the mRNA cap-binding eIF4E-related protein impair the function of eIF4F, a three-subunit complex composed 7 4EHP as an effector of miRNA-mediated translation repression. BIOCHEMISTRY of eIF4E, the m GTP (cap)-interacting factor; eIF4G, a scaffolding Through screening for protein interactions in cells via the BioID – protein; and eIF4A, a DEAD-box RNA helicase (2 5). The si- method, we identified 4EHP as a component of the CCR4–NOT/ lencing activity of miRISC is mediated by the CCR4–NOT DDX6/4E-T axis. Direct interaction between 4E-T and 4EHP in- deadenylase complex through the scaffolding subunit, CNOT1 creases the latter’s cap-binding affinity, suggesting that this (6–8). CNOT1 recruits the DDX6 and 4E-T (eIF4E transporter, interaction potentiates its competition with the eIF4F complex also known as EIF4ENIF1) proteins, which are important for for binding to the mRNA 5′ cap. Our findings suggest that 4EHP miRNA-mediated silencing (9–16). The 4E-T protein is a con- facilitates the formation of a closed-loop structure between served eIF4E-binding protein, which directly binds to the dorsal the 3′ UTR of the mRNA and its 5′ cap, which causes repression surface of eIF4E through its canonical eIF4E-binding YX4LL of mRNA translation. (Y30TKEELL) motif and impairs the eIF4E/eIF4G interaction and translation initiation (17). The 4E-T protein also facilitates Author contributions: C.C. and S.M.J. designed research; C.C., S.M.J., E.M.-C., G.G.H., and – T.A. performed research; C.C., S.M.J., E.M.-C., G.G.H., J.A., C.G.G., M.R.F., and A.-C.G. the decay of CCR4 NOT-targeted mRNAs by linking the contributed new reagents/analytic tools; C.C., S.M.J., E.M.-C., G.G.H., I.A.G., J.A., N.S.-G., 3′-terminal mRNA decay machinery to the cap via its interaction A.-C.G., and N.S. analyzed data; and C.C., S.M.J., T.F.D., and N.S. wrote the paper. with eIF4E (13). Reviewers: R.A., The Netherlands Cancer Institute; and N.S., University of Cambridge. In mammals, eIF4E is the best-studied member of a family of The authors declare no conflict of interest. proteins composed of eIF4E (eIF4E1), 4EHP (4E-homologous Data deposition: The dataset has been deposited in the MassIVE database (ID protein; eIF4E2), and eIF4E3. The 4EHP and eIF4E proteins MSV000080503; available for FTP download at massive.ucsd.edu/ProteoSAFe/status.jsp? share 28% sequence identity (18, 19). The 4EHP protein is task=766a1eeee437427a95e3ca76806876b3). The dataset has also been deposited in the ubiquitously expressed, and it is 5–10 times less abundant than ProteomeXchange Consortium, proteomecentral.proteomexchange.org (identifier – PXD005789). eIF4E in a number of mammalian cell lines (18 20). Like eIF4E, 1 C.C. and S.M.J. contributed equally to this work. 4EHP binds to 4E-T, but in sharp contrast to eIF4E, it does not 2To whom correspondence may be addressed. Email: [email protected] or associate with eIF4G (18, 21). The 4EHP protein has a 30- to [email protected]. 100-fold weaker affinity for the cap than eIF4E due to a two- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. amino acid substitution in its cap-binding pocket (22). 1073/pnas.1701488114/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1701488114 PNAS | May 23, 2017 | vol. 114 | no. 21 | 5425–5430 Downloaded by guest on September 23, 2021 Results cofactors, including DDX6, CNOT1, PATL-1, and TNRC6A/B, The 4EHP Protein Is a Component of the miRISC Effector Machinery. the scaffolding proteins of miRISC (1). To confirm that 4EHP The specificity of eIF4E and 4EHP for their mRNA targets and physically interacts with these proteins, we carried out coim- their affinities for the cap are influenced by binding partners (2, munoprecipitation (co-IP) experiments. Because of the poor 26, 28). Thus, we used the BioID assay (29) to identify the eIF4E quality of the commercially available anti-4EHP antibodies for and 4EHP interacting proteins. We created stable cell lines IP assay, lysates prepared from human embryonic kidney (HEK) expressing 4EHP or eIF4E fused in-frame with an abortive biotin 293T cells transfected with a vector expressing 3xFlag–4EHP, or ligase BirA* (R118G) (Fig. S1 A and B). The 4EHP and eIF4E an empty vector, were immunoprecipitated with anti-Flag anti- proteins were used as baits to biotinylate and capture interactors body, followed by Western blot. Flag-tagged 4EHP coprecipitated and proteins in close proximity. Biotinylated proteins were iso- 4E-T and the CNOT1 subunit of the CCR4–NOT complex (Fig. lated by using streptavidin-affinity purification under denaturing 1C). Likewise, endogenous 4EHP coprecipitated with endoge- conditions and analyzed by mass spectrometry (MS). Each bait nous DDX6, as well as HA-tagged PATL-1, a physical partner of protein was fused at its N or C terminus to BirA*. Two in- CCR4–NOT, DDX6, and 4E-T (12, 32, 33) (Fig. 1 D and E). dependent replicates for each construct were analyzed (four Together, these data demonstrate that 4EHP associates with total for each bait protein) alongside negative controls. The MS several key proteins involved in miRISC/CCR4–NOT-mediated data were processed with the SAINT (Significance Analysis of gene silencing. Interactome) computational tool (30), using several controls for statistical analysis to assign confidence scores to interaction pairs The 4EHP Protein Effects miRNA-Dependent Translational Repression. (Dataset S1 and SI Materials and Methods). Data were highly con- The association of 4EHP with components of the miRISC and its sistent across replicates (mean correlation coefficient R2 = 0.89 and effector machinery (CCR4–NOT, PATL-1, 4E-T, and DDX6) 0.94 for 4EHP and eIF4E, respectively; Fig. S1 C and D). BioID raised the possibility that 4EHP plays a role in miRNA-mediated identified 30 high-confidence targets for 4EHP and 8 for eIF4E silencing. To investigate this possibility, we used a luciferase [false discovery rate (FDR) ≤ 1%; Fig. 1A and Dataset S1]. The list construct (Fig. 2A) fused to the wild-type (WT) 3′ UTR of the of proteins identified for 4EHP is consistent with previous reports Hmga2 mRNA (an endogenous target of the let-7 miRNA) or a showing that 4EHP interacts with GIGYF1, GIGYF2, ZNF598, modified version with point mutations disrupting all seven target and 4E-T proteins (26). Known interactions with eIF4E, such as sites (34). Reporter constructs were transfected into mouse em- 4E-BP1 (EIF4EBP1), eIF4G1, and eIF4G3, were also detected (31). bryonic fibroblasts (MEFs) from WT or 4EHP-knockout (KO) Gene ontology (GO) analysis showed that “translation” was mice (Fig. S2A) (26). The reporter containing the WT 3′ UTR of the biological process with the most significant representation Hmga2 was repressed 4.4-fold in WT MEFs compared with the among 4EHP interactions (Fig. 1B and Dataset S2). In contrast mutated, control reporter (Fig. 2A and Fig. S2B). This repression to eIF4E, BioID with 4EHP identified several known miRNA was significantly less (1.6-fold) in 4EHP-KO cells, demonstrating

Fig. 1. Proteomic identification of the 4EHP and eIF4E proximal proteins. (A) High-confidence protein interactions discovered by BioID for the indicated baits in HEK293 cells. CT (C-terminal) and NT (N-terminal) indicate the location of BirA* fusion protein in relation to the Bait protein. An average of two independent experiments for each tagged variant is presented. Interacting proteins were categorized according to their known functions. Avg-Spec shows the spectral counts for each indicated prey. BFDR, Bayesian FDR. A complete list of the proximal proteins for each bait is presented in Dataset S1.(B) GO analysis of the BirA*–4EHP proximal proteins. The 10 most significantly enriched biological processes identified by using prohits-viz.lunenfeld.ca/ running g:Profiler (51) software are presented. A complete list of the enriched biological processes and molecular pathways is presented in Dataset S2.(C) HEK293T cells were transiently transfected with control or 3xFlag-4EHP plasmids. Two days later, cytoplasmic cell lysate was immunoprecipitated by using anti-Flag antibody in the presence of RNase A. LE, long exposure; SE, short exposure. (D) Cytoplasmic cell lysate from HEK293T cells was immunoprecipitated by using anti- DDX6 or IgG antibodies. (E) HEK293T cells were transiently transfected with control or HA-PATL-1 plasmid. Two days later, cytoplasmic cell lysate was immunoprecipitated by using anti-HA antibody. Western blot was performed by using the specified antibodies.

5426 | www.pnas.org/cgi/doi/10.1073/pnas.1701488114 Chapat et al. Downloaded by guest on September 23, 2021 Fig. 2. miRISC/CCR4–NOT-mediated translational silencing is impaired by 4EHP depletion. (A, Upper) Schematic representation of the RL-Hmga2 3′ UTR reporter. (A, Lower) WT and 4EHP-KO MEFs were cotransfected with RL-Hmga2 3′ UTR (WT) or mutant (Mut), along with Firefly luciferase (FL). Luciferase activity was measured 24 h after transfection. Renilla luciferase (RL) values were normalized against FL levels, and repression fold was calculated for the RL-Hmga2 3′ UTR (WT) relative to RL-Hmga2 3′ UTR (Mut) level for each population. The same data are shown as relative RL/FL levels in Fig. S2B.(B, Upper) Schematic representation of the FL-E2f 3′ UTR reporter. (B, Lower) shCTR and sh4EHP U251 cells were cotransfected with pGL3-FL-E2f 3′ UTR (WT) or a variant with mutations disrupting the two miR-17/20a–binding sites (Mut), along with RL. FL values were normalized against RL levels, and repression fold was calculated for the FL-E2f 3′ UTR (WT) relative to FL-E2f 3′ UTR (Mut) level for each population. (C) Diagram of full-length CNOT1 and the N-terminal, middle, and C-terminal fragments used in D. The binding partners in CNOT1 are also depicted and the main domains are shown in green. Amino acid positions at domain boundaries are indicated below the protein outlines. (D, Upper) Vectors expressing V5-CNOT1 and 3xFlag–4EHP (or control plasmid) were transfected into HEK293T cells. IP of V5-CNOT1 from RNase A-treated extracts was performed by using anti-V5 antibody. Purified proteins were analyzed by Western blot. (D, Lower) HEK293T cells were transfected with vectors expressing V5-CNOT1 fragments (as described in C): N, N-terminal region (amino acids 1–690); M, middle repressive module (amino acids 1,030–1,600); and C, C-terminal region (amino acids 1,830–2,376) and 3xFlag-4EHP. Extracts were subjected to anti-V5 IP, and the eluted fractions were analyzed by Western blot. (E) The 4EHP protein mediates translational repression by tethered CNOT1 and GW182(SD). (E, Upper)

Schematic representation of the RL-5boxB-A114-N40-HhR reporter. Control HEK293T cells (shCTR) or cells depleted of 4EHP (sh4EHP) were cotransfected with vectors expressing either λN-CNOT1, λN-GW182(SD) or λN control, along with RL-5boxB-A114-N40-HhR or RL-A114-N40-HhR, and FL. RL luminescence was normalized against the FL level. (E, Lower) Fold repression of RL-5boxB-A114-N40-HhR relative to RL-A114-N40-HhR expression is shown. Repression of RL-5boxB-A114-N40-HhR by λN alone in shCTR cells was set as 1. (F)RescueassayforλN-CNOT1–dependent silencing was performed, as described in E,in cells depleted of 4EHP. shCTR and sh4EHP HEK293T cells were transfected with the indicated plasmids in combination with constructs expressing shRNA- resistantversionsof3xFlag–4EHP (WT), 3xFlag–4EHPW124A cap-binding mutant (Mut), or empty vector (EV). The experiments illustrated in A, B, E,andF are represented as mean values (±SD) of three independent experiments. The P value was determined by two-tailed Student’s t test. ns, nonsignificant. *P < 0.05; **P < 0.01; ***P < 0.001.

that 4EHP is required for the efficient silencing of the Hmga2 3xFlag–4EHP and V5-tagged full-length or truncated variants of 3′ UTR reporter by the let-7 miRNA. We also examined the CNOT1 (Fig. 2C) and examined their interaction by co-IP. The impact of 4EHP depletion on silencing of two additional miRNA 4EHP protein coprecipitated with either full-length CNOT1 or the BIOCHEMISTRY reporters, in the U251 human glioblastoma cell line and in the middle domain (M) of CNOT1 (Fig. 2D). Importantly, the middle human cervical adenocarcinoma HeLa cell line. A luciferase con- domain also interacts with 4E-T via DDX6 (9, 10, 14–16). To in- struct containing a portion of the E2f 3′ UTR, which is repressed by vestigate whether 4EHP plays a role in translation repression by the the miR-17/20a paralogs in control U251 cells, was significantly less CCR4–NOT complex, we used the λN-BoxB tethering approach repressed upon knockdown of 4EHP (1.5-fold in shCTR compared (35). We used a Renilla luciferase (RL) reporter containing five with 1.1-fold repression in sh4EHP; Fig. 2B and Fig. S2C). Similarly, BoxB hairpins in its 3′ UTR. The 3′ end of the reporter contains a depletion of 4EHP (si4EHP) in HeLa cells significantly derepressed self-cleaving hammerhead ribozyme (HhR) to generate an in- a luciferase reporter harboring three miR-19a binding sites, in ternalized poly(A) stretch of 114 nucleotides, followed by 40 nu- comparison with cells treated with a control siRNA (siCTR) cleotides, to prevent deadenylation and subsequent degradation (1.6-fold in siCTR compared with 1.2-fold repression in si4EHP; (36). The reporter was cotransfected into HEK293T cells along with Fig. S2 D and E). Derepression was not due to the differential a plasmid encoding a fusion of CNOT1 to a λN peptide, which expression of the miRNAs in 4EHP-depleted cells (Fig. S2F). binds to BoxB elements. Silencing through CNOT1 was examined Repression of both miR-17/20a– and miR-19a–targeted re- by comparing the luciferase activity in cells wherein 4EHP was porters in the control cells was less pronounced than that of the depleted via shRNA (sh4EHP) with nondepleted control cells Hmga2 3′ UTR construct, likely due to fewer miRNA-binding (shCTR). CNOT1 tethering to the BoxB reporter in shCTR cells sites in the respective UTRs (seven miRNA-binding sites for resulted in a 3.7-fold repression of RL activity, whereas depletion of Hmga2 compared with three for miR-19a and two for miR-17/20a 4EHP resulted in significant derepression of the reporter (1.8-fold; reporters). Together, these data show that 4EHP promotes Fig. 2E and Fig. S2G). Similarly, siRNA knockdown of 4E-T par- miRNA-mediated silencing by several miRNAs and in various tially relieved the repression exerted by tethered CNOT1 (1.8-fold mammalian cell lines. vs.2.9-foldrepressioninsiCTR)(Fig. S2 H and I). We also ex- CNOT1, the scaffolding subunit of the CCR4–NOT complex, amined the role of 4EHP in the repressive activity of the C-terminal is an established player in miRNA-mediated silencing (6–8). We silencing domain of human GW182/TNRC6C [GW182(SD); amino therefore sought to examine the interaction of 4EHP with CNOT1. acids 1,382–1,690], which functions through CNOT1 recruitment To this end, we transfected HEK293T cells with plasmids encoding (6–8). Consistent with the effects on the miRNA reporters and with

Chapat et al. PNAS | May 23, 2017 | vol. 114 | no. 21 | 5427 Downloaded by guest on September 23, 2021 the CNOT1 tethering experiment, 4EHP depletion relieved the amounts of Flag-4EHP (Fig. 3C). Although the interaction between repression exerted by tethered GW182(SD) (1.8-fold derepression; HA–4E-T and endogenous eIF4E could be detected in the absence Fig. 2E and Fig. S2G). of exogenous 4EHP, overexpression of 4EHP dramatically reduced We next examined the importance of the cap-binding activity of 4E-T binding to eIF4E (Fig. 3C). To further examine these inter- 4EHP for miRNA-mediated silencing. We performed a comple- actions, we used recombinant 4EHP, eIF4E, and 4E-T proteins and mentation assay using tethered λN-CNOT1 in the presence of WT performed in vitro binding assays. We compared the 4E-T/4EHP 4EHP or the 4EHPW124A mutant, which is incapable of binding to interaction with the 4E-T/eIF4E interaction in vitro by using con- the cap (19). Transient transfection of shRNA-resistant 3xFlag– stant amounts of HA–4E-T and increasing concentrations of His- 4EHP in 4EHP-knockdown cells restored λN-CNOT1–mediated 4EHP or -eIF4E (Fig. S3 A and B). In such conditions, 4EHP was translation repression (Fig. 2F and Fig. S2J), whereas transfection 20–30 times more potent than eIF4E in binding to 4E-T (Fig. 3D with 4EHPW124A did not. These results demonstrate that cap and Fig. S3C). To corroborate the preference of 4E-T for 4EHP binding by 4EHP is required for translation repression effected over eIF4E, we performed an in vitro displacement assay using through CCR4–NOT. preassociated HA–4E-T/eIF4E immobilized on anti-HA agarose beads. An excess amount of eIF4E was incubated with the 4E-T– The 4EHP and eIF4E Proteins Compete for Binding to 4E-T. The 4E-T bound beads to saturate the binding sites on 4E-T. Preassembled protein is enriched among 4EHP BioID targets (Fig. 1A), but eIF4E/4E-T complexes were next incubated with increasing 4E-T also interacts with eIF4E, and both eIF4E and 4EHP bind amounts of recombinant 4EHP. In such conditions, addition of 30 the same eIF4E-binding motif of 4E-T (Y TKEELL) (21). We 1.8 μM 4EHP displaced >60% of eIF4E bound to 4E-T (Fig. 3 E compared the effects of mutations in this motif on the interactions and F). Together these results suggest that 4EHP has a com- of 4E-T with 4EHP and eIF4E. We cotransfected HEK293T cells petitive advantage over eIF4E for binding to 4E-T. with constructs encoding 3xFlag–4EHP and either WT HA-tagged 4E-T or 4E-T bearing point mutations in the Y30TKEELL motif Interaction with 4E-T Increases the Affinity of 4EHP to m7GTP Cap. In (Y30→Aand4A;Y30TKEELL→A30AKEEAA)andtestedtheirin- addition to the canonical YTKEELL motif, both 4EHP and teractions by IP. WT 4E-T coimmunoprecipitated with 4EHP eIF4E interact with a secondary noncanonical sequence of 4E-T and eIF4E, but these interactions were lost with either mutant of (21). However, the contribution of the noncanonical motif ap- the YTKEELL motif (Fig. 3A). To analyze the distribution of the pears more important for the interaction of 4E-T with 4EHP endogenous 4E-T with eIF4E and 4EHP proteins among native (21). We thus hypothesized that the extended interaction surface protein complexes, we performed size-exclusion chromatography of 4E-T and its greater affinity for 4EHP (Fig. 3) may influence of HEK293T lysates. The 4E-T protein associated with 4EHP at a the ability of 4EHP to bind the m7GTP cap. To address this molecular mass of ∼1 MDa, whereas eIF4E was detected in lower- hypothesis, we performed an isothermal titration calorimetry molecular-mass fractions (Fig. 3B). We thus hypothesized that (ITC) assay to examine whether the interaction of 4EHP with 4E-T may bind better to 4EHP than to eIF4E. To address this 4E-T peptides impacted the 4EHP affinity for the cap. We used hypothesis, we performed an IP assay in transfected HEK293T cells, three different 4E-T peptides: one encoding the canonical – which coexpressed constant amounts of HA–4E-T and increasing YTKEELL motif alone (4E-T28 37; Fig. S4A), the same sequence

Fig. 3. The 4EHP protein competes with eIF4E for binding to 4E-T. (A) HEK293T cells were transfected with vectors expressing HA-tagged WT 4E-T or the eIF4E/4EHP-binding mutant variants and 3xFlag–4EHP (or control vector). Lysates were immunoprecipitated by using the anti-HA antibody. Western blot was performed by using the specified antibodies. (B) Fractionation of endogenous 4E-T, 4EHP, and eIF4E by size-exclusion chromatography. A total of 5 mg of proteins from HEK293T cells was loaded onto a Superose 6 column and run at a flow rate of 0.5 mL/min. Fractions of 0.5 mL were collected, and 50 μL of each fraction was analyzed by Western blot. The elution position of the molecular size markers is shown. A total of 50 μg (1%) of input was used for Western blot. (C) HEK293T cells were transfected with vectors expressing HA–4E-T and increasing amounts of 3xFlag–4EHP. Anti-HA IP and Western blot were performed as described in A.(D) Binding assays with 4E-T/4EHP and 4E-T/eIF4E complexes. Constant amounts of the HA–4E-T–bound beads were incubated with increasing concentrations of recombinant His-4EHP or His-eIF4E (0, 0.03, 0.06, 0.12, 0.25, 0.5, and 1 μM). After washing the beads, the retained complexes were eluted and analyzed by Western blot with anti-His and -HA antibodies. (E) In vitro displacement assay. Preassembled eIF4E/4E-T complexes bound on anti-HA agarose beads were incubated with increasing amounts of 4EHP (0, 0.11, 0.22, 0.44, 0.89, and 1.78 μM). Proteins were eluted and analyzed by Western blot. (F) Bar graph, obtained by densitometry analysis of Western blot data from E, shows quantified intensities of eIF4E and 4EHP signals normalized against 4E-T. The data are expressed as mean values (±SD) of two independent experiments.

5428 | www.pnas.org/cgi/doi/10.1073/pnas.1701488114 Chapat et al. Downloaded by guest on September 23, 2021 – in combination with the noncanonical motif (4E-T28 71), and an- – other, which covers the entire N-terminal extremity (4E-T1 265; Fig. S4A). The 4E-T peptides were preincubated with recombinant 4EHP protein and titrated with m7GTP. The affinity of − 4EHP for m7GTP was 8.0 ± 0.56 × 10 6 M in the absence of 4E-T – peptides (Table 1 and Fig. S4B). Adding the 4E-T28 37 peptide did not significantly change this affinity. However, the affinity of 4EHP for the m7GTP cap was fourfold greater in the presence of ± × −6 ± × the N terminus of 4E-T (KD of 5.9 0.7 10 M and 1.8 0.4 Fig. 4. Model of 4EHP-mediated translation repression by miRNAs. The re- −6 28–71 1–265 10 Mfor4E-T and 4E-T , respectively; Table 1 and Fig. cruitment of 4EHP to the miRNA target mRNA through the CCR4–NOT/DDX6/4E- 7 S4 B–E). In comparison, the affinity of eIF4E for the m GTP cap T axis promotes its binding to the cap. The assembly of this complex is −8 (KD of 8.8 ± 1.1 × 10 M) remained virtually unchanged by the likely to initiate the formation of a closed-loop structure (Right)resembling 4E-T peptides (Table 1 and Fig. S4 F–I). Together, these results the cap-to-tail closed-loop mRNA conformation involving eIF4G/PABP in- suggest that 4E-T interactions with 4EHP enhance the binding of teraction (Left). the latter to the m7GTP cap structure. Discussion affinity for the cap by approximately fourfold, it remains much weaker than eIF4E (Fig. S4 and Table 1). The solution to this Here, we describe a role for 4EHP in miRNA-mediated trans- discrepancy may be explained by additional interactions that lation repression. Our results demonstrate that miRISC recruits prevail within the native miRISC effector complex and are absent 4EHP to target mRNAs through the CCR4–NOT complex. We in the in vitro assays. Other interactions may further improve the propose that these interactions engender a closed-loop mRNP affinity of 4EHP for the cap. Further studies of the protein–protein structure (Fig. 4), which resembles the cap-to-tail closed-loop interactions identified in our BioID survey may help resolve this mRNA conformation (37). A similar mechanism of trans- conundrum. Alternatively, because the affinity of 4E-T for 4EHP lational inhibition had been described in Drosophila embryos is greater than that for eIF4E, it is possible that the interaction of wherein an interaction between 4EHP and Bicoid bridges the 4E-T with 4EHP increases the concentration of 4EHP near the 5′ and 3′ ends of caudal mRNA (23). Thus, 4EHP-mediated mRNA cap to compete with eIF4E for binding to the cap. looping appears as a general repressive mechanism implicated in It was recently reported that 4E-T is recruited to mRNAs various posttranscriptional regulatory pathways. This mechanism targeted by CCR4–NOT and that 4E-T functions as an impor- can also be used by other RNA-binding proteins, such as pumilio tant cofactor of the mRNA decay machinery (13). This finding family members, tristetraprolin, and nanos, which recruit CCR4– raises the intriguing possibility that 4E-T may play a dual role in NOT to mRNAs (38). miRNA-mediated silencing: interaction of 4E-T with 4EHP An additional layer of complexity in understanding the exact potentiates translation repression, whereas its direct binding to mechanism of 4EHP action stems from the interaction of 4EHP eIF4E enables mRNA decay. Therefore, it would be important to with other protein partners. For instance, the GIGYF1/2 and 4E-T determine the relative importance of eIF4E/4E-T vs. 4EHP/4E-T proteins bind to the same motif on the 4EHP protein and thus contributions under different circumstances. GIGYF1/2 must compete with 4E-T for this binding site. The Although significant progress had been made in understanding GIGYF1/2–4EHP complex had been characterized as a trans- how miRNAs instigate mRNA deadenylation and decay, the lational repressor, notably as a cofactor of tristetraprolin (26, 27). mechanisms by which they repress translation remained unclear Interestingly, human GIGYF1/2 and the related yeast protein (1). Several recent studies demonstrated translational repression Smy2 also associate with CCR4–NOT (39, 40). Furthermore, it as an early step of miRNA-mediated silencing, which is followed was reported that GIGYF2 coimmunoprecipitates with AGO2, by mRNA deadenylation and decay (4, 42, 43). Multiple studies and tethering of GIGYF2 to an mRNA leads to its silencing now have shown that miRNAs interfere with translation initiation,

– BIOCHEMISTRY (41). Therefore, it is conceivable that the miRISC/CCR4 NOT axis specifically with cap recognition by eIF4E (3–5), and may induce the may engage 4EHP via several parallel or exclusive interactions, such dissociation of eIF4E and eIF4G from target mRNAs (44). The as GIGYF1/2 and 4E-T. Additional details of the recruitment of – model outlined here wherein 4E-T/4EHP interactions potentiate an 4EHP by CCR4 NOT remain to be determined. interaction with the cap shines a new light on prior findings. Our model provides a tenable mechanism of miRNA-mediated translation repression, but further investigation is necessary to Materials and Methods fully resolve the interactions that occur at the cap of miRNA Cell Lines and Culture Conditions. MEFs, HeLa, U251, HEK293FT, and H and targets. An important conundrum is that the affinity of recombi- T cells were routinely maintained in DMEM supplemented with 10% FBS and

nant eIF4E for the cap is 30- to 100-fold greater than that of penicillin/streptomycin in a humidified atmosphere of 5% CO2 at 37 °C. Flp-In recombinant 4EHP. Although binding to 4E-T increases 4EHP’s T-REx HEK293 cells (Thermo Scientific, catalog no. R78007) were grown in

Table 1. Thermodynamic parameters for the interaction of eIF4E and 4EHP with m7GTP in the presence of 4E-T peptides, as determined by ITC

Protein/peptide KD,M ΔH, kcal/mol −TΔS, kcal/mol Molar ratio

− 4EHP/m7GTP 8.0 ± 0.56 × 10 6 −10.8 ± 1.5 1.26 1.10 ± 0.07 – − 4EHP + 4E-T28 37/m7GTP 7.8 ± 1.0 × 10 6 −10.2 ± 0.4 3.32 0.91 ± 0.02 4EHP + 4E-T28–71/m7GTP 5.9 ± 0.7 × 10−6 −10.8 ± 0.3 3.81 0.95 ± 0.01 – − 4EHP + 4E-T1 265/m7GTP 1.8 ± 0.4 × 10 6 −17.1 ± 0.2 9.42 1.02 ± 0.006 − eIF4E/m7GTP 8.8 ± 1.1 × 10 8 −11.12 ± 0.1 1.66 1.02 ± 0.12 – − eIF4E + 4E-T28 37/m7GTP 8.2 ± 0.4 × 10 8 −9.0 ± 0.1 0.43 0.98 ± 0.007 eIF4E + 4E-T28–71/m7GTP 7.3 ± 2.5 × 10−8 −18.4 ± 0.5 8.60 1.03 ± 0.01 – − eIF4E + 4E-T1 265/m7GTP 6.2 ± 3.0 × 10 8 −22.3 ± 0.3 12.70 1.04 ± 0.08

See Fig. S4A for a schematic description of the peptides.

Chapat et al. PNAS | May 23, 2017 | vol. 114 | no. 21 | 5429 Downloaded by guest on September 23, 2021 high-glucose DMEM (Thermo Scientific, catalog no. 11965118) supplemented at 4 °C. Beads were washed four times with lysis buffer and directly resuspended with 5% FBS, 5% Cosmic calf serum, 100 U/mL penicillin, 100 μg/mL strepto- in protein sample buffer. mycin, 2 mM L-glutamine, 100 μg/mL zeocin, and 15 μg/mL blasticidin. Cell lines expressing inducible BirA-4EHP or -eIF4E were generated as described (45) and ACKNOWLEDGMENTS. We thank Devon Merkley, Pudchalaluck Panichnantakul, selected and maintained in medium supplemented with 100 μg/mL hygrom- and Eliana Sacher for technical assistance; Maayan Shapiro, Mark A. ycin. Expression of tagged proteins was induced for 24 h by addition of tet- Hancock, and Nadeem Siddiqui for discussions; and Chris Rouya, Masahiro racycline to 1 μg/mL final concentration. Morita, T. Yamamoto, N. Gehring, and Y. Tomari for reagents. This work was supported by Canadian Institute of Health Research (CIHR) Grants MOP-7214 Extract Preparation and Immunoprecipitation. Cells were resuspended in a lysis (to N.S.), MOP 123352 (to T.F.D.), and FDN 143301 (to A.-C.G.); Terry Fox buffer containing 25 mM HEPES-KOH, pH 7.4, 150 mM KCl, 75 mM KOAc, Research Institute Grant TFF-122868 (to N.S.); and National Science and 2 mM MgCl2, 0.5% NP40, supplemented with complete EDTA-free protease Engineering Research Council Grant RGPIN-2014-06434 (to A.-C.G.). T.F.D. is inhibitor cocktail and 1 mM NaF, 1 mM Na3VO4,and1mMβ-glycerophosphate supported by the Fonds de la Recherche en Santé du Québec (FRQS) phosphatase inhibitors, and incubated for 20 min on ice. The lysate was clar- Chercheur-Boursier Senior Salary Award. S.M.J. is the recipient of a CIHR ified by centrifugation at 15,000 × g for 10 min at 4 °C. One milligram of postdoctoral fellowship. C.C. is supported by FRQS and Fondation pour la extract was used for immunoprecipitation with the indicated antibodies. Recherche Médicale postdoctoral fellowships. E.M.-C. is supported by Thirty microliters of preequilibrated protein G-agarose (Roche) and RNase A Groupe de Recherche Axé sur la Structure des Protéines. G.G.H. is supported (Thermo Scientific) were added, and the mixtures were rotated overnight by a Parkinson Canada Basic Research Fellowship.

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