Mitosis-related phosphorylation of the eukaryotic translation suppressor 4E-BP1 and its interaction with eukaryotic translation initiation factor 4E (eIF4E)

Eukaryotic translation initiation factor 4E (eIF4E)–binding protein 1 (4E-BP1) inhibits cap-dependent translation in eukaryotes by competing with eIF4G for an interaction with eIF4E. Phosphorylation at Ser-83 of 4E-BP1 occurs during mitosis through the activity of cyclin-dependent kinase 1 (CDK1)/cyclin B rather than through canonical mTOR kinase activity. Here, we investigated the interaction of eIF4E with 4E-BP1 or eIF4G during interphase and mitosis. We observed that 4E-BP1 and eIF4G bind eIF4E at similar levels during interphase and mitosis. The most highly phosphorylated mitotic 4E-BP1 isoform (δ) did not interact with eIF4E, whereas a distinct 4E-BP1 phospho-isoform, EB-γ, phosphorylated at Thr-70, Ser-83, and Ser-101, bound to eIF4E during mitosis. Two-dimensional gel electrophoretic analysis corroborated the identity of the phosphorylation marks on the eIF4E-bound 4E-BP1 isoforms and uncovered a population of phosphorylated 4E-BP1 molecules lacking Thr-37/Thr-46–priming phosphorylation. Moreover, proximity ligation assays for phospho-4E-BP1 and eIF4E revealed different in situ interactions during interphase and mitosis. The eIF4E:eIF4G interaction was not inhibited but rather increased in mitotic cells, consistent with active translation initiation during mitosis. Phosphodefective substitution of 4E-BP1 at Ser-83 did not change global translation or individual mRNA translation profiles as measured by single-cell nascent protein synthesis and eIF4G RNA immunoprecipitation sequencing. Mitotic 5′-terminal oligopyrimidine RNA translation was active and, unlike interphase translation, resistant to mTOR inhibition. Our findings reveal the phosphorylation profiles of 4E-BP1 isoforms and their interactions with eIF4E throughout the cell cycle and indicate that 4E-BP1 does not specifically inhibit translation initiation during mitosis.

sequencing. Mitotic 5-terminal oligopyrimidine RNA translation was active and, unlike interphase translation, resistant to mTOR inhibition. Our findings reveal the phosphorylation profiles of 4E-BP1 isoforms and their interactions with eIF4E throughout the cell cycle and indicate that 4E-BP1 does not specifically inhibit translation initiation during mitosis.
4E-BP1, 5 also known as phosphorylated heat-and acid-stable protein regulated by insulin (PHAS-I), was first identified as a protein phosphorylated in response to insulin treatment (1). 4E-BP1 was subsequently isolated from a human cDNA library of eIF4E-binding proteins and shown to inhibit cap-dependent translation (2,3). Efficient cap-dependent translation requires assembly of the translation initiation complex eIF4F (composed of eIF4E, eIF4G, and eIF4A) on the mRNA 5Ј cap structure (4,5). 4E-BP1 inhibits translation by binding to eIF4E, which prevents eIF4G:eIF4E interaction, thus inhibiting assembly of the eIF4F complex (6 -14).
Here, we examined 4E-BP1 phosphorylation and 4E-BP1: eIF4E interaction throughout the cell cycle in HeLa cells. A distinct eIF4E-binding (EB)-␥ isoform of 4E-BP1, with a phosphorylated Ser-83 residue, was identified to bind eIF4E during mitosis, demonstrating that Ser-83 phosphorylation alone does not prevent 4E-BP1 from sequestering eIF4E. The combinatorial complexity of the various phosphorylation sites on 4E-BP1 have largely, and unsatisfactorily, been resolved using various phosphospecific antibodies via one-dimensional gel electrophoresis. At best, four closely migrating protein bands designated ␣, ␤, ␥, and ␦ are distinguishable. By differentiating 4E-BP1 isoforms on two-dimensional gel electrophoresis, multiple new phospho-isoforms of 4E-BP1 were identified, including phospho-isoforms lacking priming phosphorylations at Thr-37/Thr-46. Concurrently, we characterized the key 4E-BP1 phosphorylation events for the regulation of the 4E-BP1:eIF4E interaction, expanding the previously proposed two-step model. Proximity ligation assays (PLAs) provided visual localization of the in situ interaction between eIF4E and different phosphorylated 4E-BP1 isoforms during mitosis and interphase. Strong eIF4E:eIF4G PLA signals were present in mitotic cells, suggesting that assembly of the translation initiation eIF4F complex is not inhibited but rather increased in mitosis. In contrast to previously examined cell lines (35), 4E-BP1-independent global translation suppression was observed in HeLa cells by a flow cytometry-based Click-iT labeling assay, which indicates that mitotic translation inhibition occurs downstream of eIF4F complex loading to RNA. eIF4G RNA immunoprecipitation sequencing (RIP-Seq) validated active mitotic TOP gene translation initiation, consistent with 4E-BP1 not being responsible for mitotic translation suppression in HeLa cells. Alanine substitution mutation at 4E-BP1 S83 alone did not significantly alter eIF4G RIP-Seq pro-files. Taken together, these data reveal phosphorylation marks on eIF4E-associated 4E-BP1 isoforms throughout the cell cycle and update the understanding of various 4E-BP1 phosphorylation marks on 4E-BP1 function.
The lowest-order 4E-BP1 phospho-isoform in asynchronous cells (Fig. 3A, dot A1) showed PP242-sensitive Thr-37/ Thr-46 phosphorylation. This phosphorylation was present in most higher-order phospho-isoforms (B, C, D, and E), consistent with mTOR-dependent Thr-37/Thr-46 -priming phosphorylation during interphase as reported previously (20). For mitotic cells, the most highly phosphorylated isoforms (F), corresponding to the ␦ band seen on 1D gel, were abundant and resistant to PP242 treatment (Fig. 3B). Multiple mitotic lower-order phospho-isoforms lacked Thr-37/ Thr-46 -priming phosphorylations and were also resistant to PP242 treatment (e.g. dots A2, A3, B3, and C4). Based on its migration and phosphorylated residues, dot C4 most likely represents the EB-␥ band found in Fig. 1. This was confirmed by alanine substitution mutation at 4E-BP1 Ser- Transfected cells were split into two groups, 1) asynchronous and 2) synchronized at mitosis, by STLC treatment (5 M; 16 h). Cell lysates were immunoprecipitated with anti-FLAG antibodies followed by immunoblotting with corresponding antibodies. The intensities of immunoprecipitated bands were quantitated (underlined values). The ratio of each eIF4E-bound 4E-BP1 band in total was calculated (right panel). Results are presented as mean Ϯ S.D. Error bars represent S.D. The p value was calculated by t test with **, p Ͻ 0.01. At least three biological replicates were performed. Data shown here is a representative result. The immunoprecipitated 4E-BP1 and eIF4G levels are normalized to immunoprecipitated eIF4E band intensities. B, the membrane from A was stripped and reprobed with different phosphospecific 4E-BP1 antibodies. Total 4E-BP1 immunoblotting from A is shown for comparison. C, HeLa cells were split into asynchronous cells and STLC-treated (5 M; 16 h) mitosis-enriched cells. Cell lysates were incubated with m 7 GTP cap pulldown beads. Cap-bound proteins were detected by immunoblotting with the designated antibodies. The 4E-BP1 EB-␥ isoform is indicated by *, and the 4E-BP1 ␦ isoform is indicated by #. EB-␥ and ␥ are two different and distinct 4E-BP1 phospho-isoforms.
The dephosphorylation of 4E-BP1 has been proposed to be responsible for the shutdown of mitotic cap-dependent translation (38). This has been disputed in several recent studies showing high levels of 4E-BP1 phosphorylation (34,35,39,40) and active cap-dependent translation during mitosis using single-cell pulse-chase analysis (35). Even though a substantial fraction of eIF4E was found to be bound to 4E-BP1 during both mitosis and interphase (Fig. 5A), strong fluorescent eIF4E: eIF4G PLA signals were present in mitotic cells, suggesting that assembly of the translation initiation eIF4F complex is not inhibited, confirming the eIF4E coimmunoprecipitation (co-IP) results (Figs. 5B, 1A, and 2, A and B) as well as previously published studies (35,41).

Global mitotic translation in HeLa cells
To determine whether Ser-83 phosphorylation of 4E-BP1 affects global translation, single-cell protein synthesis was measured in WT 4E-BP1 and 4E-BP1 S83A mutant HeLa cells by a flow cytometry-based Click-iT labeling assay (35). Newly synthesized proteins are labeled by the methionine analog L-homopropargylglycine (HPG) in a pulse-chase assay. To specifically label mitotic, newly synthesized proteins, cells were arrested at the G 2 /M boundary with the CDK1 inhibitor RO3306, and HPG was added to the methionine-depleted medium following RO3306 release. As shown in Fig. 6, repressed translation was Transfected cells were divided into two groups, 1) asynchronous and 2) synchronized at mitosis, by STLC treatment (5 M; 16 h). Cell lysates were immunoprecipitated for eIF4E with anti-HA antibodies. Cell lysates (Input) or immunoprecipitated elutes (IP) were subjected to 1D-and 2D-gel electrophoresis (isoelectric focusing at pH 3-6) followed by immunoblotting with total 4E-BP1 and p-4E-BP1 T37/T46 antibodies. B, FLAG-tagged eIF4E plasmids were transfected into HeLa cells. Transfected cells were synchronized at mitosis with STLC (5 M; 16 h). Cell lysates were immunoprecipitated with anti-FLAG antibodies. Cell lysates (Input) or immunoprecipitated elutes (IP) were subjected to 2D-gel electrophoresis (isoelectric focusing at pH 3-6) followed by immunoblotting with different phosphospecific and total 4E-BP1 antibodies. Blue circles indicate canonical phosphorylated 4E-BP1 isoforms (20,37), red circles indicate PP242-resistant isoforms of 4E-BP1 in mitosis, dashed-line circles indicate isoforms with weaker signals, filled circles indicate eIF4E-bound 4E-BP1 isoforms, and NP indicates nonphosphorylated 4E-BP1. The 4E-BP1 EB-␥ isoform is indicated by *.

Mitotic 4E-BP1:eIF4E interaction
observed in a large population of mitotic cells (p-H3 S10positive), consistent with previously reported translation assay results for HeLa cells (42, 43) but different from the observed results in BJ-T cells (35). This repression was not due to 4E-BP1 dephosphorylation as the same repression was also observed in 4E-BP1-knockout and native HeLa cells (Fig. S5). No significant differences were found between WT 4E-BP1 and 4E-BP1 S83A mutant HeLa cells, suggesting that Ser-83 phosphorylation of 4E-BP1 does not affect global translation in HeLa cells.

Mitotic 5-TOP transcript translation in HeLa cells
To investigate mitotic translation of individual gene transcripts, RNA binding to the translation initiation complex eIF4F was examined by eIF4G RIP-Seq. As shown in Fig. 7A, HeLa cells were arrested at the G 2 /M boundary with RO3306, released, and synchronized for mitotic entry. Mitotic cells were collected by shake-off, whereas attached cells were allowed to progress into postmitosis. Harvested cell pellets were then subjected to RNA-Seq and eIF4G RIP-Seq. The results for 5Ј-TOP genes (44) are shown in Fig. 7. Total transcriptome RIP-Seq analyses are shown in Fig. S6.
Most 5Ј-TOP gene transcripts were abundantly expressed in cells and proportionally bound to eIF4G during mitosis and postmitosis (linear least-squares fit, R 2 ϭ 0.70 -0.81) (Fig. 7A). This expression-translation profile for 5Ј-TOP transcripts was not significantly changed by PP242 treatment in mitosis-enriched cells (Chow test, p ϭ 0.083; effect size, d ϭ 0.356), which is consistent with mTOR independence. Postmitotic eIF4G binding of 5Ј-TOP transcripts was significantly reduced compared with mitotic 5Ј-TOP eIF4G binding (Chow test, p ϭ 2.6EϪ12; effect size, d ϭ 1.261). Postmitotic cells treated with PP242 had a further decrease in eIF4G engagement compared HeLa cells were synchronized at the G 2 /M boundary with CDK1 inhibitor RO3306 treatment (10 M; 16 h) and then released into mitosis by removing RO3306. After 60 min, cells were fixed and permeabilized. A, PLAs were performed using mouse eIF4E and rabbit phosphospecific or total 4E-BP1 antibodies. Cell nuclei were stained with DAPI (blue). PLA signal was obtained from rolling circle amplification (red). Images were captured by fluorescence microscope (40ϫ). B, PLAs were performed using mouse eIF4E and rabbit eIF4G or eEF2 antibodies. Images were captured by fluorescence microscope (40ϫ). White arrows indicate mitotic cells; yellow arrows indicate interphase cells. PLA signals were quantitated using ImageJ (particle counting). Results are presented as mean Ϯ S.D. Error bars represent S.D. The p value was calculated by t test. NS indicates that the difference is not significant. ***, p Ͻ 0.001.

Discussion
Our study was performed to catalog mitotic and interphase 4E-BP1 phospho-isoforms and to assess their interactions with the translation initiation protein eIF4E. This was examined by eIF4E co-IP followed by 2D-gel electrophoresis and by 4E-BP1: eIF4E PLA. We found heterogeneous 4E-BP1 phosphorylations within both mitotic and interphase cells. The majority of mitotic 4E-BP1 isoforms are hyperphosphorylated at four or more sites (␦-4E-BP1) and do not bind eIF4E. A fraction of mitotic phosphorylated 4E-BP1 lacking Thr-37/Thr-46 phosphorylation retained their ability to interact with eIF4E, which has been overlooked in previous studies (34,35,39,40).
There are several important caveats that should be considered when interpreting our findings. 1) STLC-induced mitotic arrest was used in our study and is anticipated to inhibit protein synthesis as with nocodazole. This method achieves high rates (Ͼ60%) of mitotic arrest for HeLa cells but will still have substantial contamination of interphase cells (Fig. S2A), which complicates the analysis. For example, phospho-isoforms labeled B1, C1, and D1 disappear with mTOR inhibition in STLC-treated cells, but we cannot distinguish whether these isoforms represent true mitotic phospho-isoforms or contaminating interphase phospho-isoforms (Fig. 3). About 5% of untreated, asynchronous HeLa cells undergo mitosis at any given time, and so mitotic contamination of asynchronous cells Cycloheximide (CHX; 100 g/ml) was added at the same time to block new protein synthesis, functioning as the negative control. Cells were collected and fixed for subsequent staining. B, flow cytometry analysis of HPG incorporation (new protein synthesis). Cells were labeled with Alexa Fluor 488 azide using Click-iT HPG kits and stained with p-H3 S10 antibody to label the mitotic cell population.

Mitotic 4E-BP1:eIF4E interaction
is less of a concern. Furthermore, STLC treatment, like nocodazole treatment, may nonspecifically inhibit translation. This effect, if present, is downstream of 4E-BP1 phosphorylation, and we see similar 4E-BP1 phosphorylation patterns for STLCarrested cells compared with mitotic cells isolated by shake-off without pharmacologic mitotic arrestors (Fig. S2B) 2) Commercial p-4E-BP1 S65 antibody has specific reactivity to the p-4E-BP1 S65 epitope but cross-reacts with human p-4E-BP1 S101 , depending on the dilution of the antibody and the amount of p-4E-BP1 S101 epitope (15). The positive p-4E-BP1 S65 signal for EB isoforms of 4E-BP1 might represent Ser-101 phosphorylation of 4E-BP1 because Ser-65 phosphorylation has been previously described only in hyperphosphorylated iso-forms of 4E-BP1 that have no interaction with eIF4E (19,20), consistent with the weak or undetectable PLA signals between eIF4E and p-4E-BP1 S65/S101 (Fig. 5A) 3) The two priming threonine sites, Thr-37 and Thr-46, have identical epitope sequences, and the available commercial p-4E-BP1 T37/T46 antibody cannot distinguish between single Thr-37 or Thr-46 phosphorylation or between combined Thr-37/Thr-46 phosphorylations. Also, priming-site phosphorylation does not change the electrophoretic mobility of 4E-BP1 on one-dimensional SDS-PAGE (19, 45) 4) Isoelectric focusing resolves protein by charge; some of the species ("dots") observed on 2D gel may well be composed of a mixture of species with similar charge but are actually phosphorylated at different sites.

Mitotic 4E-BP1:eIF4E interaction
Cap-dependent translation during mitosis is technically difficult to measure because mitosis is short (Ͻ1.5 h) as well as rare in cultured cells (ϳ5%), and spindle assembly inhibitors nonspecifically inhibit protein synthesis, possibly through activated downstream phosphorylation of eIF2␣ (35,40). There is, however, substantial evidence from multiple studies that cap-dependent translation is active during mitosis (35,39,43), suggesting that the accepted dogma for a shift from cap-dependent to cap-independent translation during mitosis should be revisited. We found that eIF4G:eIF4E interaction was not inhibited during mitosis but was slightly increased (Figs. 5B, 1A, and 2, A  and B). Intriguingly, previous studies on eIF4G also demonstrated enhanced assembly of eIF4F complex (eIF4G:eIF4A interaction) during nocodazole-induced mitosis in which protein synthesis was inhibited (41). Consistent with these findings, eIF4G RIP-Seq in HeLa cells demonstrated that 5Ј-TOP gene translation initiation is still active and mTOR-independent during mitosis (Fig. 7). However, we did not find that translation of these transcripts was related to the status of 4E-BP1 S83 phosphorylation in HeLa cells. We cannot exclude the possibility that this effect is cell line-dependent; for example, HeLa cells have reduced mitotic translation compared with BJ-T cells (35). Alternatively, 4E-BP1 S83 phosphorylation may be related to a nontranslational signaling pathway or may be coincidental to CDK1.
It is widely accepted that the interaction between eIF4E and 4E-BP1 is regulated by the multisite phosphorylation of 4E-BP1. However, some studies have shown that Thr-37/ Thr-46 phosphorylation is sufficient to dissociate 4E-BP1 from eIF4E and that Ser-65 phosphorylation is dispensable for the regulation of 4E-BP1:eIF4E interaction (16,25,45,46). A recent study using far-Western blotting supported Thr-46 phosphorylation as key in controlling 4E-BP1:eIF4E interaction (37). In this study, our co-IP data as well as PLA data indicate that no single 4E-BP1 phosphorylation is sufficient to block 4E-BP1 sequestration of eIF4E in vivo; rather it is a combination of phosphorylations that results in the loss of eIF4E interaction with 4E-BP1. The 2D-gel data (Fig. 4) suggest that phosphorylation at both Thr-37 and Thr-46 on 4E-BP1 is the critical event in the dissociation of 4E-BP1 from eIF4E and support the notion that further Thr-70 or Ser-65 phosphorylation is dispensable in controlling 4E-BP1:eIF4E interaction (46). This differs from the canonical two-step model (19). Usually, the eIF4E-bound 4E-BP1 migrated into two or more bands after one-dimensional SDS-PAGE. These two bands both can be visualized using p-4E-BP1 T37/T46 and p-4E-BP1 T70 antibodies, leading to the misinterpretation that only hyperphosphorylated 4E-BP1 (p-4E-BP1 T37/T46, S65, T70 ) can dissociate from eIF4E. However, due to the limitation of the resolution of SDS-PAGE, the two bands actually correspond to multiple, alternative overlapping isoforms of 4E-BP1 as demonstrated by 2D-gel electrophoresis (Fig. 4). Unlike the previously proposed canonical model for the dissociation of eIF4E from 4E-BP1, wherein higher-order phosphorylations are entirely predicated upon priming phosphorylations at Thr-37 and Thr-46, these priming phosphorylations are not required for 4E-BP1 hyperphosphorylation during mitosis because CDK1/cyclin B can substitute for mTOR to phosphorylate 4E-BP1 at multiple sites (35). Pre-vious studies have shown that Ser-2448 phosphorylation of mTORC1 is reduced during mitosis (47); however, assessment of mitotic mTOR activity is complicated. 4E-BP1 or ribosomal S6 kinase (S6K1) phosphorylations are frequently used as a surrogate readouts for mTORC1 activity, but both of these proteins are also phosphorylated by kinases other than mTORC1 during mitosis (35,48,49). The bulk of mitotic 4E-BP1 phosphorylation remains resistant to PP242, suggesting that kinases other than mTOR are primarily responsible for mitotic 4E-BP1 phosphorylation. We cannot conclude that mTORC1 plays no role in mitotic 4E-BP1 phosphorylation and it may act in concert with CDK1/cyclin B to generate fully phosphorylated 4E-BP1 isoforms during mitosis. It is desirable to directly determine the status of mTORC1 in mitosis, for example whether mTORC1 is still in a dimer active form (50,51). Our study also confirms that p-4E-BP1 S83 is a unique marker for mitotic cells. Ser-83 phosphorylation alone is insufficient to block 4E-BP1 sequestration of eIF4E. Interestingly, a recent study reported another CDK could phosphorylate 4E-BP1, relying on mTORpriming phosphorylation (52), whereas CDK1 can phosphorylate 4E-BP1 at various residues independently of mTOR kinase. Taken together, our investigation of 4E-BP1:eIF4E interaction during the cell cycle reveals a complex accounting of the phosphorylation profile of 4E-BP1 isoforms bound to eIF4E.

Cell culture and transfection
HEK 293 and HeLa cells were maintained in Dulbecco's modified Eagle's medium (Corning Cellgro) supplemented with 10% fetal bovine serum. HEK 293 and HeLa cells were transfected with eIF4E expression plasmids using polyethylenimine (Sigma-Aldrich) and reseeded 12-16 h post-transfection to avoid confluence. Transfected cells were harvested 48 h post-transfection.
The following primary antibodies were used in this study:

Cell cycle synchronization
Mitotic cells were enriched by STLC treatment (5 M; 16 h) (56) or by mitotic shake-off. For the latter, cells were treated with 10 M CDK1 inhibitor RO3306 for 16 h to arrest cells at the G 2 /M boundary, and then the cells were released into mitosis by removing RO3306. After 30 min, mitotic cells were collected by mechanical shake-off.

Immunoprecipitation and immunoblotting
Cells were lysed in nondenaturing RIPA buffer (50 mM Tris⅐HCl (pH 7.4), 150 mM NaCl, 0.5% Triton X-100, 0.5% sodium deoxycholate, 2 mM Na 3 VO 4 , 2 mM NaF) supplemented with protease inhibitors (Roche Applied Science). Lysates were incubated with protein A/G-Sepharose beads (Santa Cruz Biotechnology) and anti-FLAG or anti-HA tag antibodies overnight at 4°C. Beads were collected, washed four times with RIPA buffer, and boiled in SDS loading buffer. Samples were subjected to 12% SDS-PAGE and transferred to nitrocellulose membranes. Membranes were blocked with 5% milk and incubated with primary antibodies overnight at 4°C. After washing, blots were subsequently incubated with IRDye-labeled anti-rabbit or anti-mouse secondary antibodies (LI-COR Biosciences) and analyzed by Odyssey IR scanning (LI-COR Biosciences).

m 7 GTP cap-binding assay
Cells were lysed in nondenaturing RIPA buffer supplemented with protease inhibitors (Roche Applied Science). Lysates were incubated with 30 l of m 7 GTP-Sepharose beads (Jena Bioscience) overnight at 4°C. Beads were collected, washed four times with RIPA buffer, and boiled in 1ϫ SDS loading buffer. Samples were subjected to 12% SDS-PAGE and immunoblotting.

2D-gel electrophoresis
Cells were lysed in RIPA buffer supplemented with protease inhibitors (Roche Applied Science) with a final lysate protein concentration above 10 g/l. Cleared lysates (400 -500 g) were diluted with rehydration buffer (Bio-Rad) to 220 l and then loaded to immobilized pH 3-6 gradient strips (Bio-Rad) for rehydration overnight. The rehydrated strips were focused with linear voltage ramping for 2 h at 200 V, 2 h at 500 V, and 16 h at 800 V. After focusing, the balanced strips were subjected to SDS-PAGE for second-dimensional separation and immunoblotting.
For immunoprecipitated samples, the final collected beads were boiled with 20 l of 2% SDS (57) and centrifuged to collect the supernatants (cooled to room temperature). The samples were then diluted with rehydration buffer to 220 l prior to two-dimensional gel electrophoresis as described above.

Click-iT labeling assay
Cells were cultured in 6-well plates with or without drug treatment. For labeling newly synthesized proteins, cells were washed with methionine-depleted medium once and cultured with methionine-depleted medium. After incubating for 15 min, cells were treated with HPG (50 M) for 30 min. Cycloheximide (100 g/ml) was added concurrently to block new protein synthesis. Cells were collected and fixed with 4% paraformaldehyde for 30 min followed by permeabilization with 0.2% Triton X-100 for 10 min. Incorporated HPG was labeled with Alexa Fluor 488 azide using Click-iT HPG kits (Life Technologies). Cells were stained with p-H3 S10 antibody (3458, Cell Signaling Technology) to label the mitotic cell population. HPG incorporation in cells was analyzed by flow cytometry.

RIP-Seq
eIF4G RIP were performed using the RIP-Assay kit (RN1001, MBL International). Collected cell pellets were lysed in 800 l of kit-provided lysis buffer supplemented with protease inhibitors (Roche Applied Science), RiboLock RNase inhibitor (Thermo Fisher), and dithiothreitol (DTT) on ice for 10 min. Lysed samples were centrifuged at 12,000 ϫ g for 5 min at 4°C to collect the supernatant (cell lysate). 80 l of supernatant was set aside as input, and the remaining supernatant was divided into two groups. Lysates were incubated with 30 l of protein A/G-Sepharose beads (Santa Cruz Biotechnology) and 5 l of kit-provided rabbit IgG or eIF4G antibody (RN002P, MBL International) overnight at 4°C. Beads were collected and washed three times with kit-provided wash buffer supplemented with DTT. The immunoprecipitated and input RNA was extracted using TRIzol (Thermo Fisher). Double-strand cDNA libraries were prepared with a SMART-seq Ultra Low Input kit (Takara Clontech). Double-strand cDNA libraries were fragmented and indexed using a Nextera XT DNA library preparation kit (Illumina). The quality of extracted RNA, double-strand cDNA libraries, and Nextera XT DNA libraries was Mitotic 4E-BP1:eIF4E interaction determined on a Bioanalyzer2100 (Agilent). Illumina NextSeq 500 sequencing was performed in paired-end read mode with 75 cycles.
Reads were trimmed and filtered to remove adaptor sequences with Trim Galore and Cutadapt programs. Trimmed sequences were aligned to human genome (hg19) with CLC Genomics Workbench (Qiagen). Data were analyzed using CLC Genomics Workbench and R. The 5Ј-TOP gene list was adapted from a previous study (44). The sequencing data reported in this paper have been deposited in the Gene Expression Omnibus database under accession number GSE131668.