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DENR–MCT-1 promotes translation re-initiation downstream of uORFs to control tissue growth

Nature volume 512, pages 208–212 (2014)Cite this article

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Abstract

During cap-dependent eukaryotic translation initiation, ribosomes scan messenger RNA from the 5′ end to the first AUG start codon with favourable sequence context1,2. For many mRNAs this AUG belongs to a short upstream open reading frame (uORF)3, and translation of the main downstream ORF requires re-initiation, an incompletely understood process1,4,5,6. Re-initiation is thought to involve the same factors as standard initiation1,5,7. It is unknown whether any factors specifically affect translation re-initiation without affecting standard cap-dependent translation. Here we uncover the non-canonical initiation factors density regulated protein (DENR) and multiple copies in T-cell lymphoma-1 (MCT-1; also called MCTS1 in humans) as the first selective regulators of eukaryotic re-initiation. mRNAs containing upstream ORFs with strong Kozak sequences selectively require DENR–MCT-1 for their proper translation, yielding a novel class of mRNAs that can be co-regulated and that is enriched for regulatory proteins such as oncogenic kinases. Collectively, our data reveal that cells have a previously unappreciated translational control system with a key role in supporting proliferation and tissue growth.

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Figure 1: DENR promotes cell proliferation and boosts protein synthesis in proliferating but not quiescent cells.
Figure 2: DENR promotes re-initiation of translation downstream of uORFs in the mbc 5′ UTR.
Figure 3: uORFs with strong Kozak sequences (stuORFs) are sufficient to impart DENR–MCT-1-dependent regulation.
Figure 4: Loss of DENR leads to reduced InR and EcR protein levels and signalling.

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Gene Expression Omnibus

Data deposits

Polysome microarray data are deposited at NCBI GEO under accession number GSE54625.

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Acknowledgements

We thank B. Bukau, R. Green and P. Soba for suggestions on the manuscript, V. Benes and T. Bähr-Ivacevic (EMBL Genomics Core Facility) for assistance with microarray experiments, S. Abmayr for anti-Mbc antibody, T. Hsu for anti-Awd antibody, C. Strein and M. Hentze for Drosophila DENR antibodies, P. Jakob for help with testing conditions for S2 in vitro translation extracts, L. Schibalski and J. Grawe for help with cloning, S. Hofmann for help with polysome analysis, and S. Lerch, A. Haffner and M. Schröder for technical assistance. K.K.M. is the recipient of an Alzheimer’s Research Scholarship from the Hans und Ilse Breuer Foundation. P.C.J. is supported in part by a grant from the Fritz Thyssen Foundation to K.E.D. This work was supported in part by a Deutsche Forschungsgemeinschaft (DFG) grant and ERC Starting Grant to A.A.T.

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Author notes

  1. Katrin Strassburger, Philipp Christoph Janiesch, Kent E. Duncan and Aurelio A. Teleman: These authors contributed equally to this work.

Authors and Affiliations

  1. German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany,

    Sibylle Schleich, Katrin Strassburger, Katharina Haneke, Yong-Sheng Cheng, Georg Stoecklin & Aurelio A. Teleman

  2. Center for Molecular Neurobiology (ZMNH), University Medical Center Hamburg-Eppendorf (UKE), Falkenried 94, 20251 Hamburg, Germany,

    Sibylle Schleich, Philipp Christoph Janiesch, Tatyana Koledachkina, Katharine K. Miller, Katrin Küchler & Kent E. Duncan

  3. Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ-ZMBH Alliance, 69120 Heidelberg, Germany,

    Katharina Haneke & Georg Stoecklin

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Contributions

S.S. performed most experiments, except as indicated below. K.S. analysed histoblast proliferation, EcR and InR protein levels and signalling in cells and animals, and performed EcR/InR rescue experiments in vivo. P.C.J. assessed proliferation, translation, rRNA and tRNA levels, as well as polysome profiles and genome-wide polysomal profiling of DENR knockdown S2 cells, and performed in vitro translation assays. T.K. established and performed inducible reporter assays in proliferating and quiescent cells (Extended Data Figs 7a–f and 10c). K.K.M. performed DENR–MCT-1 co-immunoprecipitation assays (Extended Data Fig. 10b). K.H. performed DENR RNA immunoprecipitation assays (Extended Data Fig. 4g). Y.-S.C. analysed MCT-1 levels and phosphorylation (Extended Data Fig. 10d′). K.K. established in vitro translation assays from S2 cells. A.A.T. performed the bioinformatic analyses. K.E.D. and A.A.T. jointly designed and coordinated the study and wrote the paper with input from G.S. All authors interpreted data, discussed results and contributed to writing the manuscript.

Corresponding authors

Correspondence to Kent E. Duncan or Aurelio A. Teleman.

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Extended data figures and tables

Extended Data Figure 1 DENR knockout strategy and phenotypes.

a, Domain structures of eIF2D, DENR and MCT-1. b, DENR (CG9099) genomic locus, indicating the knockout region that was replaced with the mini-white cassette via homology-mediated recombination. c, d, DENRKO animals have no detectable DENR transcript by quantitative RT–PCR (c) or protein (d). e, DENRKO anterior–dorsal histoblast nests have correct cell numbers at onset of pupation (0 h APF), but impaired proliferation the first 4 h of pupal development (4 h APF). Quantification shown here; representative images shown in Fig. 1b. Cells visualized by esgG4> cytoplasmic GFP+nuclear GFP. f, DENR expression in histoblast cells of DENRKO animals rescues their viability and abdominal phenotypes (DENRKO, escargot-GAL4, UAS-DENR). g, DENR transcript is expressed in all tissues and developmental stages tested, detected by quantitative RT–PCR, normalized to rp49. h, h′, DENRKO animals have genitals that are not properly rotated. i, MCT-1 (CG5941) knockdown animals (Tubulin-GAL4>UAS-CG5941 RNAi) phenocopy DENRKO animals. They are pupal lethal, with abdominal epidermis that is soft and transparent, whereas head and thorax structures are comparatively better-formed. j, HA-tagged Drosophila MCT-1 can co-immunoprecipitate endogenous Drosophila DENR from S2 cells. k, Crossing scheme to test for genetic interaction between ligatin and DENR. DENRKO heterozygous females were crossed to males heterozygously carrying a deficiency for the ligatin locus. Resulting non-FM7 hemizygous DENR knockout male pupae were scored for presence of the TM6B balancer (using the Tubby marker) or the ligatin deficiency. If the ligatin deficiency had no effect on survival of DENR mutants, it would be present in the expected 50% Mendelian ratio, in equal proportion to the FM7 balancer. Instead, only 18 out of the scored 53 animals were carrying the ligatin deficiency. l, Polysome profiles from DENRKO larvae have high 80S and low polysomal peaks. Quantification of polysome/monosome (p/m) ratio for 5 independent replicates is shown in Fig. 1f. m, n, Polysome profiles of DENR knockdown cells have increased 80S peaks, reduced p/m ratios, and increased total areas under the curve at both 4 (m, m′) and 7 (n, n′) days post dilution into suspension culture. o, DENR knockdown cells have elevated levels of 18S rRNA, 28S rRNA and initiator tRNA by qRT–PCR. Experiment was performed 8 days after treatment with DENR dsRNA. p, DENR knockdown S2 cells have elevated levels of ribosomal protein S6, quantified by western blot relative to tubulin. q, r, Quiescent (r) but not proliferating (q) DENR knockdown cells have high protein per cell. Error bars indicate s.d. (b–g) or s.e.m. (o–r). Mann–Whitney U-test *P < 0.05; t-test ***P < 0.001.

Extended Data Figure 2 DENR promotes re-initiation of translation downstream of uORFs in the mbc 5′ UTR.

a, western blotting of extracts from control, DENR knockdown and MCT-1 knockdown S2 cells using a panel of antibodies does not show dramatic differences in the level of most proteins. Quantification of luminescence on a LICOR Oddesey FC relative to control knockdown cells is indicated. b, Levels of Renilla luciferase (RLuc) reporters containing the mbc 5′ UTR and either the mbc or hsp70 promoters are reduced in DENR and MCT-1 knockdown cells. DNA reporters were transfected into S2 cells together with a firefly luciferase (FLuc) normalization control containing an hsp70 promoter. c, In vitro transcribed RLuc reporter mRNAs containing mbc or actin 5′ UTR, co-transfected with a control FLuc reporter into DENR, MCT-1 or control knockdown cells. mRNAs were capped with the normal m7G cap (cap) or with an adenosine cap structure (A-cap) which does not bind eIF4E. Below, representative raw counts (from the control) for the three reporters shows that the signal obtained with A-capped reporters is roughly one-half to one-quarter the signal observed with a normal m7G cap, and well above background. d, Reduced translation in DENR knockdown cells of an mRNA luciferase reporter containing the mbc 5′ UTR is not accompanied by a reduction in the intracellular levels of the reporter mRNA. In vitro transcribed mRNA consisting of the mbc 5′ UTR, the Renilla luciferase ORF and a synthetic poly(A) was co-transfected with a normalization control mRNA consisting of a short 5′ UTR, firefly luciferase ORF and a synthetic poly(A) into control or DENR knockdown cells. Relative luminescence counts (d) and relative mRNA levels determined by quantitative RT–PCR (d′) are shown. e, Combined knockdown of DENR and MCT-1 does not have additive effects compared to the single knockdowns on translation of a luciferase reporter containing the mbc 5′ UTR, consistent with them working together in one functional complex. f–h, High-resolution deletion series of the mbc Renilla luciferase reporter, summarized in Fig. 2d, identifies nucleotides 200–375 of the mbc 5′ UTR as the minimum region required to impart DENR-dependent regulation. Error bars indicate s.d.

Extended Data Figure 3 Detailed sequence information for the mbc 5′ UTR.

a, Full sequence of the mbc 5′ UTR. ATGs are indicated in bold. Open reading frames are shown as red or grey boxes depending on whether they have a good or bad Kozak sequences, respectively. Stop codons are indicated with asterisks. b, Schematic overview of the mbc 5′ UTR and the tested reporter constructs, summarizing results from other panels. Light-peach-coloured shading indicates the minimal region required to impart DENR-dependent regulation to a luciferase reporter. Red and grey boxes indicate upstream open reading frames (uORFs) with strong and weak Kozak sequences, respectively. ΔATG construct: single point mutations were introduced to abolish the three indicated uORFs. ΔKozak construct: Kozak sequences of the indicated uORFs were mutated to gtgtATG, thereby disfavouring translation initiation at these sites. Δstop construct: stop codons of all three in-frame uORFs were mutated, resulting in a construct where the uORF stop codon is just downstream of the RLuc ATG. c, Translation extracts from DENR knockdown cells are impaired in translating a reporter containing the mbc 5′ UTR. mRNA reporters containing the full-length mbc 5′ UTR, or a mutated ΔATG version where the ATGs at positions 218, 248, 338 and 415 were mutated, were prepared by in vitro transcription and introduced into translation extracts prepared from S2 cells treated with either control (GFP) or DENR dsRNA. Mutating the ATGs both increases the overall translation of the reporter, and renders the reporter insensitive to DENR knockdown. Statistics performed using the two-tailed, two-sided Student’s t-test. d, Full sequence of the 5′ UTR in the mbc Δstop construct. Mutated stop codons are shown with underlining. The same sequence (nucleotides 200–375) without the two point mutations is regulated by DENR (Fig. 2h).

Extended Data Figure 4 Genome-wide analysis of stuORF transcripts.

a, Genome-wide histogram of transcripts containing uORFs in their 5′ UTRs. b, b′, Frequency distribution of nucleotides found surrounding the ATG of all main ORFs annotated in the fly genome. Frequency matrix in b′ was used to generate a multiplicative score for the strength of any Kozak sequence of interest. c, Transcripts that were translationally downregulated in S2 polysome profiles upon DENR knockdown are enriched for stuORF-containing transcripts. A translation score was measured for each transcript in the genome, consisting of the ratio of transcript levels in 80S + polysome fractions (indicating active translation) divided by transcript levels in total mRNA, yielding the percentage of mRNA being actively translated. This was calculated for both control and DENR knockdown S2 cells, and compared. Transcripts were then sorted into quintiles according to this comparison, with the first quintile containing transcripts that are most downregulated in DENR knockdown cells compared to controls, and quintile 5 containing transcripts that were upregulated in DENR knockdown cells compared to controls. For each quintile, the average stuORF score was calculated, as described in the main text. A clear correlation can be observed, with downregulated quintiles being enriched for transcripts containing stronger stuORF score. Error bars indicate s.e.m. d, Analysis the other way around: stuORF-containing transcripts are preferentially downregulated in the polysome analysis from DENR knockdown cells compared to transcripts containing no uORFs. Two categories of transcripts were selected genome-wide: the top 210 with high stuORF scores, and the bottom 6,000 transcripts containing no uORFs. For each category of transcripts, histograms of the translation score were plotted, with negative numbers representing transcripts that were translationally downregulated in DENR knockdown cells compared to controls. Transcripts containing stuORFs are preferentially downregulated genome-wide upon DENR knockdown in a highly statistically significant manner (Mann–Whitney U-test P = 10−14) compared to transcripts lacking uORFs. e, Gene ontology enrichment using DAVID on stuORF-bearing transcripts. f, Examples of genes predicted via the DENR-dependence score to be DENR-dependent in Drosophila. g, DENR binds both mRNAs containing and lacking stuORFs. Cellular proteins were crosslinked with 0.5% formaldehyde and then DENR-containing complexes were immunoprecipitated. RNA was purified from the immunoprecipitation, reverse-transcribed with random hexamers, and subjected to quantitative PCR. Both transcripts containing stuORFs (mbc, InR) and transcripts containing no or weak uORFs (RpS13, cdc2, RpL32), as well as initiator tRNA, were found to be significantly enriched compared to RNA from a control immunoprecipitation using anti-GFP antibody. Error bars indicate s.d.

Extended Data Figure 5 Time course of DENR knockdown reveals that changes in stuORF translation precede global changes in polysome profiles or ribosome levels.

a, Translation of a stuORF reporter (bearing 1× ATGTAA, as in Fig. 3) progressively drops upon DENR knockdown from 1–4 days after treatment with dsRNA. A significant drop in translation can be observed already 2 and 3 days after DENR knockdown. In contrast, a control reporter is unperturbed. b, b′, DENR protein levels 2 (b) and 3 (b′) days after knockdown in S2 cells, assessed by immunoblotting. c, c′, Polysome profile of S2 cells treated for 2 (c) or 3 (c′) days with DENR dsRNA (red traces) shows no significant drop in polysome fractions compared to cells treated with control (GFP) dsRNA (grey traces), suggesting that the drop in polysome levels observed at later time points (for example, Extended Data Fig. 1) is in vivo secondary consequences (for example, due to reduced translation of stuORF-containing transcripts such as Insulin Receptor, leading to a global reduction in translation rates). d, d′, Quantification of 18S RNA, 28S RNA and initiator tRNA levels in S2 cells treated with dsRNA for 2 (d) or 4 (d′) days shows little to no increase upon DENR knockdown compared to control.

Extended Data Figure 6 Loss of DENR leads to reduced InR and EcR protein levels and signalling in vivo—support data.

a, a′, DENR and MCT-1 knockdown in S2 cells does not lead to reduced EcR-B1 and InR mRNA levels. b, DENR knockdown cells are less sensitive to ecdysone (1 μM, 4 h), assayed by qRT–PCR of two EcR target genes BR-C (Fig. 4c) and E75 (shown here), normalized to rp49. c, c′, DENRKO pupae 4 h APF have reduced levels of InR and EcR-A protein (c) but not mRNA (c′). d, Expression of InR in histoblast cells and imaginal discs of DENRKO animals using escargot-GAL4 rescues their delayed pupation.

Extended Data Figure 7 DENR promotes expression of an inducible 1aa stuORF reporter in proliferating cells more efficiently than in quiescent cells.

a, Schematic overview of a cell-based inducible dual luciferase reporter assay to enable systematic evaluation of the effect of DENR knockdown on expression of stuORF or control reporters under different growth states. b, DENR knockdown in S2 cells leads to efficient depletion of DENR protein for up to 18 days post-dsRNA treatment. 4 days after treatment with dsRNA, cells were diluted into either suspension culture at a density of 106 per ml (for Fig. 1g–i) in adherent culture (for the remaining panels in this figure). Graded loading of the GFP dsRNA control sample on the blot shows that DENR knockdown yields persistent depletion of DENR protein by ≥90%. c, Growth curve post-dilution of S2 cells treated with or without dsRNAs, indicating optimal days for reporter induction that correspond to proliferating or quiescent populations (2 d or 7 d, respectively). Under proliferative conditions both DENR knockdown and control cells double within ∼24 h. Conversely, under quiescent conditions no significant increase in cell number is observed, implying that the population has essentially stopped proliferating. d, Presence of a 1aa stuORF (ATGTAA) reduces translation of a Renilla reporter by 50% in control (GFP knockdown) cells. Error bars indicate s.e.m. Data from 2 independent knockdowns and 4 transfections. e, Effect of DENR knockdown on the inducible 1aa stuORF reporter expression is significantly stronger in proliferating compared to quiescent cells. Error bars indicate s.e.m.; t-test P < 0.0001. f, f′, Raw luciferase counts for the experiment shown in e demonstrating that upon DENR knockdown, control reporters increase in expression (consistent with what is also observed in translation extracts, Fig. 2c, right) whereas the stuORF reporter decreases in expression (f). g, Representative raw luciferase counts for Fig. 2i. h, h′, DENR knockdown affects Mbc protein levels more strongly in proliferating than in quiescent cells. DENR knockdown leads to reduced endogenous Mbc protein (h) but not mRNA (h′) in proliferating but not quiescent S2 cells. Cells were treated with dsRNA for 4 d, then seeded in parallel at two different densities to achieve proliferation or quiescence at time of assay. Error bars indicate s.d.

Extended Data Figure 8 DENR is required to express stuORF-containing transcripts in vivo in Drosophila.

a, Schematic of the fluorescent reporters introduced into Drosophila. Identical reporters were generated containing a Tubulin promoter, the 5′ UTR of CG43674, which does not contain any uORFs, the GFP open reading frame, and the SV40 poly-A. A 6-nucleotide stuORF (ATGTAA) was introduced by mutagenesis into the 5′ UTR of one construct, generating the stuORF reporter. Both constructs were inserted via phiC31-mediated recombination into the VK33 landing site at 65B2 on chromosome 3L, ensuring identical transcriptional regulation. b, The control reporter is well expressed in both larvae containing (DENR+/+) or lacking DENR (DENRKO). Immunoblotting of larval extracts (c) shows that GFP levels of the control reporter do not drop in DENR knockouts compared to controls, indicating that DENR is not required for translation of transcripts lacking uORFs. Introduction of the 6-nucleotide stuORF causes the reporter to be less expressed in a control DENR+/+ background, consistent with uORFs having a repressive role in translation of the main downstream ORF. In contrast to the control reporter, expression of the stuORF reporter is entirely dependent on DENR in vivo, as no GFP expression could be observed when the stuORF reporter is introduced into a DENRKO genetic background. This was confirmed by immunoblotting (c). c, Immunoblot to detect GFP and a loading control (awd) in larval extracts of animals bearing a control or a stuORF reporter, either in a control DENR+/+ or in a DENR knockout genetic background.

Extended Data Figure 9 DENR activity in the Drosophila larva is higher in proliferating tissues compared to non-proliferating tissues.

Expression of the stuORF GFP reporter is entirely DENR-dependent (Extended Data Fig. 8), hence it serves as a read-out for DENR activity. Flies were generated bearing either a control or a stuORF–GFP reporter, combined in trans with a normalization control RFP reporter, generated by replacing the GFP of the control reporter with RFP, and inserting it into the same VK33 landing site as the GFP reporters. This set-up is analogous to the dual FLuc/RLuc set-up used for luciferase assays and completely controls for transcriptional effects. Various tissues were dissected and imaged by confocal microscopy, the GFP/RFP ratio was calculated, and is displayed in pseudocolour. Flies bearing a control GFP reporter and the control RFP reporter have the same ratio of GFP to RFP in all tissues (a). For instance, the strong spot in the middle of the wing disc which is due to transcriptional effects is present in both the GFP reporter and the RFP normalization control, and is normalized out when the GFP/RFP ratio is calculated. In contrast, the stuORF reporter (b) is more strongly expressed in proliferating tissues such as the brain and associated imaginal discs, or the wing disc, compared to non-proliferating tissues such as fat body or salivary glands. This can be observed both on the overlay, which is yellow for brain and wing discs, but red for fat body and salivary gland, as well as in the pseudo-coloured GFP/RFP ratio panel which is high in brain and wing disc, but low in fat body or salivary gland.

Extended Data Figure 10 DENR activity is higher in proliferating cells compared to quiescent cells.

a, b, DENR–MCT-1 binding is not different in proliferating versus quiescent S2 cells. a, Immunoprecipitation of endogenous DENR from proliferating or quiescent S2 cells shows similar amounts of co-immunoprecipitating endogenous MCT-1 in the two conditions. b, A stably transfected S2 cell line bearing a copper-inducible pMT-V5-MCT-1 construct was grown in proliferative or quiescent conditions, as in Extended Data Fig. 7, and then induced to express MCT-1. V5-tagged MCT-1 was immunoprecipitated and endogenous DENR was detected by immunoblotting. An anti-Flag immunoprecipitation was performed as a negative control. Near-equal levels of DENR protein co-immunoprecipitated with MCT1 in both proliferative and quiescent conditions. c, Expression levels of DENR and eIF2D do not depend on each other and do not change in proliferating versus quiescent cells. Immunoblot of S2 cells treated with DENR, eIF2D or control (GFP) dsRNA, in proliferative or quiescent conditions. Two different amounts of the control samples were loaded to quantitatively assess knockdown efficiencies. A non-specific band on eIF2D blot is marked with an asterisk. d, d′, Abolishing phosphorylation on T118 and S119 blunts MCT-1 activity. d, Luciferase assay using a stuORF reporter in S2 cells depleted of endogenous MCT-1 via dsRNA targeting its 3′ UTR, and re-constituted to express wild-type or mutated forms of MCT-1 (using constructs with an exogenous 3′ UTR), identifies T118 and S119 as important phosphorylation sites. Four sites were tested—T118, S119, T82 and T125—for all of which phosphorylations on endogenous MCT-1 were observed by publicly available proteome-wide mass spectrometry analyses (PhosphoPep and PhosphoSite.org). Whereas wild-type MCT-1 is able to restore expression of the stuORF reporter, a T118A/S119A mutant form of MCT-1 cannot. Testing each site individually identifies S119 as the more important of the two. In contrast, mutating T82 and T125 to alanine does not impair their ability to promote stuORF translation. t-test **P < 0.01. Error bars indicate s.d. d′, MCT-1(T118A/S119A) is expressed and stable. Immunoblot of S2 cells transfected with control plasmid or plasmid expressing wild-type Flag–MCT-1 or Flag–MCT-1(T118A/S119A) shows that the mutant MCT-1 is expressed at roughly equivalent levels as the wild-type protein. e, f, Inhibition of Erk, PI(3)K, Akt or TORC1 does not have an effect on stuORF translation. Luciferase assay with a stuORF reporter in S2 cells treated with inhibitors for Erk (U0126, f), TOR complex 1 (rapamycin, e), Akt (Akt Inhibitor VIII, e) or PI(3)K (Wortmannin, e) shows that inhibition of any of these kinases has little to no effect on stuORF translation. g, Knockdown of Drosophila cdc2 (CG10498) does not reduce expression of a stuORF reporter. S2 cells were treated with either GFP dsRNA or CG10498 dsRNA, and then transfected with a stuORF reporter. GFP dsRNA was applied to 90 wells in a 96-well plate and the values displayed represent the average and s.d. (error bars). CG10498 knockdown results for two separate wells are shown.

Supplementary information

Supplementary Information

This file contains a Supplementary Discussion, a complete list of oligos and sequences used, and the number of replicates for each figure panel. (PDF 255 kb)

Supplementary Table 1

Microarray profiling of polysome gradients from control and DENR knockdown cells. (XLSX 4062 kb)

Supplementary Table 2

stuORF scores for all transcripts. (XLSX 1341 kb)

Supplementary Table 3

Top transcripts with high stuORF scores. (XLSX 111 kb)

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Schleich, S., Strassburger, K., Janiesch, P. et al. DENR–MCT-1 promotes translation re-initiation downstream of uORFs to control tissue growth. Nature 512, 208–212 (2014). https://doi.org/10.1038/nature13401

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