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

LETTER

doi:10.1038/nature13401

DENR–MCT-1 promotes translation re-initiation downstream of uORFs to control tissue growth Sibylle Schleich1,2, Katrin Strassburger1*, Philipp Christoph Janiesch2*, Tatyana Koledachkina2, Katharine K. Miller2, ¨ chler2, Georg Stoecklin1,3, Kent E. Duncan2* & Aurelio A. Teleman1* Katharina Haneke1,3, Yong-Sheng Cheng1, Katrin Ku

During cap-dependent eukaryotic translation initiation, ribosomes scan messenger RNA from the 59 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 process1,4–6. 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. Cellular protein abundance depends largely on mRNA translation8. Little is known about how translation of specific sets of mRNAs can be coordinately regulated9,10. mRNAs with uORFs require re-initiation11–14, whereby ribosomes translate the uORF, terminate and then restart translating the main ORF1,4,6,15. No metazoan trans-acting factors have yet been described that selectively affect re-initiation, enabling coordinate regulation of uORF-containing mRNAs. eIF2D (also called ligatin) and the related DENR–MCT-1 complex are candidate re-initiation regulators. They associate with 40S ribosomal subunits and have domains implicated in RNA binding and start codon recognition (Extended Data Fig. 1a). In vitro they can recycle post-termination complexes, recruit initiator methionyl-tRNA (Met-tRNAiMet) to mRNAs containing viral internal ribosome entry sites16,17, and affect movement of post-termination 80S complexes to nearby AUG codons6. DENR–MCT-1 has not previously been implicated in re-initiation, and MCT-1 is an oncogene affecting cellular mRNA translation by an unclear mechanism18–20. Collectively, these studies suggest that DENR–MCT-1 and eIF2D might regulate translation of cancer-relevant mRNAs through non-canonical mechanisms. To study DENR function, we generated Drosophila knockouts for the homologous gene (CG9099), lacking DENR transcript or protein (DENRKO, Extended Data Fig. 1b–d). DENRKO flies die as pharate adults with a larval-like epidermis (Fig. 1a), due to impaired proliferation of histoblast cells (Fig. 1b and Extended Data Fig. 1e). This is rescued by expressing DENR ubiquitously (Tubulin–GAL4) or specifically in histoblast cells (Escargot–GAL4) (x2 test P , 0.05, Extended Data Fig. 1f). Although DENR is expressed ubiquitously (Extended Data Fig. 1g), quickly proliferating histoblast cells appear more sensitive to DENR loss than non-proliferating tissues. DENRKO flies also have crooked legs and incorrectly rotated genitals (Fig. 1c and Extended Data Fig. 1h–h9). These phenotypes are not observed in mutants with generally impaired translation

(Minutes; ref. 21), but are found in flies with reduced cell-cycle regulators or Ecdysone Receptor signalling22,23, suggesting that DENR affects translation of a subset of mRNAs involved in cell proliferation and signalling. Similar phenotypes were observed in flies expressing RNA interference (RNAi) targeting MCT-1 (fly homologue CG5941; Extended Data Fig. 1i), which like human MCT-1 binds DENR (Extended Data Fig. 1j). Reducing ligatin (fly homologue of human eIF2D) gene dosage in DENRKO flies caused fewer animals to reach pupation (x2 test P , 0.05, Extended Data Fig. 1k), indicating that DENRKO phenotypes result from loss of DENR–MCT-1 complex with eIF2D-like activity. DENRKO larvae and DENR knockdown S2 cells grow slowly with reduced protein accumulation rates (Fig. 1d, e, g). Mutant polysome profiles show reduced polysome/monosome ratios (Fig. 1f, h and Extended Data Fig. 1l–n9), suggesting defective translation initiation. Despite more ribosomes and initiator tRNA per cell (Extended Data Fig. 1o, p), DENR knockdown cells have reduced protein synthesis rates when proliferating (Fig. 1i). When quiescent, DENR knockdown cells no longer display these phenotypes, and become enlarged compared to controls (Fig. 1h, i (right panel) and Extended Data Fig. 1q–r). Thus, DENR promotes translation of cellular mRNAs in proliferating but not quiescent cells. We identified ,100 mRNAs requiring DENR for efficient translation by profiling actively translated mRNAs from 80S and polysome fractions of control and DENR knockdown cells and normalizing to total mRNA (Supplementary Table 1). We further analysed myoblast city (mbc) because it was the second most under-translated mRNA and we could obtain antibody to detect it. Quantitative polymerase chain reaction with reverse transcription (RT–PCR) confirmed that mbc mRNA is under-represented in polysomes of DENR knockdown cells (Fig. 2a), leading to reduced Mbc protein but not mRNA (Fig. 2b), whereas other proteins were not reduced (Extended Data Fig. 2a). The mbc 59 UTR was sufficient to impart DENR-dependence to a Renilla luciferase (RLuc) reporter (Extended Data Fig. 2b). This DENR dependence requires the 59 cap and is not accompanied by a drop in general translation (Extended Data Fig. 2c, d9 and Fig. 2c). Combined knockdown of DENR and MCT-1 had no additive effect, as they are a functional complex (Extended Data Fig. 2e). In sum, the DENR–MCT-1 complex selectively promotes cap-dependent translation of mbc via its 59 UTR. Systematic 59 UTR truncations (Extended Data Fig. 2f–h) identified 175 nucleotides necessary and sufficient for DENR dependence (Fig. 2d and Extended Data Fig. 3a, b) containing 3 uORFs with strong Kozak sequences (stuORFs, red boxes in Fig. 2d). Mutating all three stuORF ATGs, or their Kozak sequences, abolished DENR dependence (Fig. 2e, f and Extended Data Fig. 3c), indicating that translation initiation on these stuORFs is necessary for DENR dependence. No additional cis-acting sequences were necessary; removing sequences upstream, downstream, or between the uORFs, or mutating the uORF coding sequences, did not affect DENR dependence (Fig. 2g and Extended Data Fig. 2f–h). Two possible explanations are: (1) DENR promotes bypass of stuORF initiation codons; and (2) DENR affects re-initiation after stuORF translation.

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German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany. 2Center for Molecular Neurobiology (ZMNH), University Medical Center Hamburg-Eppendorf (UKE), Falkenried 94, 20251 Hamburg, Germany. 3Zentrum fu¨r Molekulare Biologie der Universita¨t Heidelberg (ZMBH), DKFZ-ZMBH Alliance, 69120 Heidelberg, Germany. *These authors contributed equally to this work. 0 0 M O N T H 2 0 1 4 | VO L 0 0 0 | N AT U R E | 1

©2014 Macmillan Publishers Limited. All rights reserved

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Introducing synthetic stuORFs into a control reporter was sufficient to impart DENR dependence, with multiple stuORFs acting additively (Fig. 3a). Re-initiation efficiency is reportedly inversely related to uORF length, presumably because initiation factors dissociate from ribosomes as elongation proceeds7. Consistently, the ability of DENR to promote reinitiation dropped as uORFs became longer (Fig. 3a, right panel), reaching

In model 1, the stuORF stop codon is irrelevant because DENR would act at the stuORF start codon. In model 2 the stuORF stop codon is crucial as translation re-initiation on the main ORF only occurs after termination on the stuORF. Two point mutations removing the stuORF stop codons completely abolished DENR dependence (Fig. 2h and Extended Data Fig. 3d), indicating that DENR promotes translation re-initiation.

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Figure 1 | DENR promotes cell proliferation and boosts protein synthesis in proliferating but not quiescent cells. a, DENRKO flies die as pharate adults with larval-like abdominal epidermis. b, DENRKO anterior–dorsal histoblast nests have correct cell numbers at onset of pupation (0 h after puparium formation, APF), but impaired proliferation during the first 4 h of pupal development (4 h APF) (visualized by esgG4.GFP). c, DENRKO flies have crooked legs. d, e, DENRKO flies accrue mass (d) and protein (e) slowly, pupating with 1 day delay (day 6 data point, absent in controls). f, Polysome profiles from DENRKO larvae have reduced polysome/ monosome ratios. g, DENR knockdown cells proliferate slowly and enter quiescence at low cell density. h, Proliferating but not quiescent polysome profiles of DENR knockdown cells have reduced polysome/monosome ratios. i, Proliferating (left) but not quiescent (right) DENR knockdown cells have reduced de novo protein synthesis rates, quantified by metabolic labelling with methionine analogue. Error bars indicate standard deviation (s.d.) (d–f) or s.e.m. (g–i). t-test (d–f) or Mann–Whitney U-test (g–i9) *P , 0.05, **P , 0.01, ***P , 0.001.

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Figure 2 | DENR promotes re-initiation of translation downstream of uORFs in the mbc 59 UTR. a, qRT–PCR validation that the mbc mRNA is preferentially depleted from polysomes in DENR knockdown cells (DENR A and B) compared to controls (GFP A and B). b, Mbc protein (left) but not mRNA (right) levels are reduced in DENR and MCT-1 knockdown cells. c, Translation extracts from DENR knockdown cells are impaired in translating a reporter containing the mbc 59 UTR (left) but not in translating a control RLuc reporter mRNA without uORFs in the 59 UTR (right). d, Schematic overview of the mbc 59 UTR and the tested DNA reporter constructs, summarizing results from other panels as well as multiple ($3) additional replicates on all the luciferase assays, not shown. Details in Extended Data Fig. 3. e, f, Mutating the start codons of the three mbc uORFs with strong Kozak sequences (e) or their Kozak sequences to the less functional gtgtATG (f) blunts regulation by DENR. g, Mutating the coding sequence of mbc uORFs to poly-glutamine has no effect on DENR regulation. h, Mutation of the stop codons of mbc uORFs 218, 248 and 338, as diagrammatically shown in d, causing the uORFs to extend past the RLuc ATG, leads to loss of DENR-dependent regulation. i, DENR knockdown leads to impaired expression of the mbc 59 UTR RLuc reporter in proliferating but not quiescent S2 cells. Error bars: s.d. t-test *P , 0.05, ***P , 0.001.

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LETTER RESEARCH

Figure 3 | uORFs with strong Kozak sequences (stuORFs) are sufficient to impart DENR–MCT-1-dependent regulation. a, Introduction of synthetic uORFs bearing a ‘strong’ Kozak into a control 59 UTR imparts

DENR-dependent regulation (DNA reporters). b, c, 59 UTRs bearing stuORFs are all DENR-dependent (b) whereas 59 UTRs lacking uORFs (c) are not (DNA reporters). Error bars indicate s.d.

zero effect on a dicistronic transcript containing a long upstream ORF (not shown). In sum, mRNAs display a continuum of DENR dependence, depending on the number and length of the uORFs and the strength of their Kozak sequences. A computational search revealed thousands of 59 UTRs containing uORFs (Extended Data Fig. 4a). We generated a predicted ‘DENR-dependence score’ for all transcripts based on the number of uORFs they contain and the strength of their Kozak sequences (Extended Data Fig. 4b, b9 and Supplementary Table 2). Transcripts with high DENR-dependence scores were significantly enriched among the mRNAs with reduced translation on DENR knockdown (Extended Data Fig. 4c, d), suggesting a general mechanism. We tested ten 59 UTRs predicted to be DENR-dependent using luciferase assays. Six conferred DENR dependence (Fig. 3b), and four inhibited reporter translation too strongly to test experimentally. Conversely, 16 59 UTRs without uORFs were not DENR-dependent (Fig. 3c and data not shown). Therefore, 59 UTRs with stuORFs are DENR-dependent, identifying a new class of transcripts for which translation can be co-regulated. Gene Ontology analysis24 revealed that these genes are enriched for transcriptional regulators and kinases (Extended Data Fig. 4e, f). Immunoprecipitation of DENR showed that it binds mRNAs containing or lacking stuORFs (Extended Data Fig. 4g), suggesting that it interacts generally with initiating ribosomes, but is required on stuORFcontaining mRNAs. Because only 15% of genes contain stuORFs, we were surprised to see global effects on polysomes upon DENR knockdown (Extended Data Fig. 1l). A DENR knockdown time course revealed that stuORF-dependent translation drops before changes in polysome or ribosome levels (Extended Data Fig. 5), indicating that these are probably secondary consequences. Because Insulin and Ecdysone receptors (InR and EcR) contain DENRdependent 59 UTRs (Fig. 3b), we asked whether impaired InR and EcR translation contribute to DENRKO phenotypes. Loss of DENR–MCT-1 function in S2 cells or DENRKO animals leads to reduced InR and EcR proteins, but not mRNA levels, and reduced InR and EcR signalling (Fig. 4a–c and Extended Data Fig. 6a–c9). Reconstituting InR/EcR expression in DENRKO animals partially but significantly rescued developmental rate and histoblast proliferation (Fig. 4d, e and Extended Data Fig. 6d). Thus, loss of DENR–MCT-1 causes reduced InR/EcR translation and signalling, and consequently impaired cell proliferation and organismal development.

Data from DENRKO flies and DENR knockdown cells suggested that proliferating cells are phenotypically more sensitive to DENR loss-offunction than quiescent cells. One explanation could be that DENR activity is low in quiescent cells, hence its removal has little effect. Using mbc and stuORF reporters, and endogenous mbc translation, as readouts for DENR activity revealed that DENR loss had a larger impact in proliferating compared to quiescent cells (Fig. 2i and Extended Data Fig. 7). Hence DENR–MCT-1 present in quiescent cells is not very active. To study DENR function in vivo, we generated flies carrying fluorescent reporters with or without a stuORF (Extended Data Fig. 8a). These reporters have identical promoters, 59 UTRs and 39 UTRs, and are integrated in exactly the same genomic locus via phiC31-mediated recombination, ensuring their identical transcription. This revealed that DENR promotes stuORF reporter, but not control reporter, expression in animals (Extended Data Fig. 8). Because stuORF–GFP reporter expression is entirely DENR-dependent, it serves as an in vivo DENR activity readout. Interestingly, the larval anterior, which contains proliferating tissues like brain and imaginal discs, shows stronger DENR activity than other larval regions (Extended Data Fig. 8b). Inclusion of an RFP normalization control in trans, analogous to a dual-luciferase assay set-up, revealed high DENR activity (stuORF–GFP/normalization-RFP) in proliferating tissues (brain and imaginal discs), and low activity in tissues with growing, but non-proliferating, cells (salivary gland and fat body, Extended Data Fig. 9). We wondered how DENR–MCT-1 activity is regulated. Neither DENR protein levels nor DENR–MCT-1 binding dropped in quiescent S2 cells (Extended Data Fig. 10a–c). Phosphorylation of T82, T125 and a double phosphorylation on T118 and S119 in human and fly MCT-1 have been observed25. Using cells where endogenous MCT-1 is knocked down via its 39 UTR and then reconstituted with MCT-1 versions lacking the endogenous 39 UTR revealed that mutations blocking T118/S119 phosphorylation abolished MCT-1 activity (Extended Data Fig. 10d, d9). Notably, T118 and S119 are evolutionarily conserved in humans. Although MCT-1 was observed to be phosphorylatable in vitro by Erk and Cdc2 (ref. 26), we could not observe an effect of Erk, Cdc2, PI(3)K, Akt or TORC1 inhibition on stuORF reporter expression (Extended Data Fig. 10e–g). Further work will be required to identify upstream kinases regulating DENR– MCT-1. 0 0 M O N T H 2 0 1 4 | VO L 0 0 0 | N AT U R E | 3


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Figure 4 | Loss of DENR leads to reduced InR and EcR protein levels and signalling. a, DENR and MCT-1 knockdown cells have reduced InR and EcR protein levels. b, c, DENR knockdown cells are less sensitive to insulin stimulation (1 h) (b) and to ecdysone (20E, 1 mM, 4 h; c). d, e, Expression of EcR

(d) or InR (e) in histoblast cells and imaginal discs of DENRKO using escargot-GAL4 rescues their delayed pupation (d) and mildly but significantly their proliferation defect (e). Error bars indicate s.d. t-test ***P , 0.001.

We have identified a new translational control system regulating an abundant class of mRNAs, featuring: (1) stuORFs as the critical cis-element; (2) DENR–MCT-1 as the trans-acting factor; and (3) proliferation as an important cellular context. This system differs fundamentally from GCN4/ATF4 paradigms both mechanistically and functionally. Unlike GCN4-type mechanisms1,2,5,27–30, DENR–MCT-1 functions in non-stressed cells, when general translation is not compromised, and independently of uORF to main-ORF distance (Fig. 2h), to promote proliferation. Importantly, DENR–MCT-1 uncouples translation re-initiation from standard initiation, as it is not required for initiation (Fig. 2c (right)). In contrast, GCN4-type mechanisms rely on coupling of initiation and re-initiation to antagonistically regulate GCN4/ATF4 versus all other genes (Supplementary discussion). Our results suggest that re-initiation can be independently controlled via DENR–MCT-1 to modulate translation of a specific group of mRNAs.

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METHODS SUMMARY DENR knockout flies were generated by homologous recombination using pW25. Phospho-dAkt(T342) antibody was developed in collaboration with PhosphoSolutions. Luciferase assays were performed using a dual-luciferase set-up based on pGL3 vectors containing either firefly or Renilla luciferase, and the Drosophila hsp70 basal promoter. The number of replicates for each experiment are described in Supplementary Information. Online Content Methods, along with any additional Extended Data display items and Source Data, are available in the online version of the paper; references unique to these sections appear only in the online paper.

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13. 14. 15. 16. 17.

Received 2 July 2013; accepted 23 April 2014.

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Published online 6 July 2014. 1.

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LETTER RESEARCH 21. Kongsuwan, K. et al. A Drosophila Minute gene encodes a ribosomal protein. Nature 317, 555–558 (1985). 22. Hayashi, S., Hirose, S., Metcalfe, T. & Shirras, A. D. Control of imaginal cell development by the escargot gene of Drosophila. Development 118, 105–115 (1993). 23. Wilson, T. G., Yerushalmi, Y., Donnell, D. M. & Restifo, L. L. Interaction between hormonal signaling pathways in Drosophila melanogaster as revealed by genetic interaction between methoprene-tolerant and broad-complex. Genetics 172, 253–264 (2006). 24. Huang da, W., Sherman, B. T. & Lempicki, R. A. Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists. Nucleic Acids Res. 37, 1–13 (2009). 25. Bodenmiller, B. et al. PhosphoPep–a phosphoproteome resource for systems biology research in Drosophila Kc167 cells. Mol. Syst. Biol. 3, 139 (2007). 26. Nandi, S. et al. Phosphorylation of MCT-1 by p44/42 MAPK is required for its stabilization in response to DNA damage. Oncogene 26, 2283–2289 (2007). 27. Spriggs, K. A., Bushell, M. & Willis, A. E. Translational regulation of gene expression during conditions of cell stress. Mol. Cell 40, 228–237 (2010). 28. Harding, H. P. et al. Regulated translation initiation controls stress-induced gene expression in mammalian cells. Mol. Cell 6, 1099–1108 (2000). 29. Vattem, K. M. & Wek, R. C. Reinitiation involving upstream ORFs regulates ATF4 mRNA translation in mammalian cells. Proc. Natl Acad. Sci. USA 101, 11269–11274 (2004). 30. Hood, H. M., Neafsey, D. E., Galagan, J. & Sachs, M. S. Evolutionary roles of upstream open reading frames in mediating gene regulation in fungi. Annu. Rev. Microbiol. 63, 385–409 (2009). Supplementary Information is available in the online version of the paper. Acknowledgements We thank B. Bukau, R. Green and P. Soba for suggestions on the manuscript, V. Benes and T. Ba¨hr-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. Schro¨der 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. Author 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. 10d9). 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. Author Information Polysome microarray data are deposited at NCBI GEO under accession number GSE54625. Reprints and permissions information is available at www.nature.com/reprints. The authors declare no competing financial interests. Readers are welcome to comment on the online version of the paper. Correspondence and requests for materials should be addressed to K.E.D. ([email protected]) or A.A.T. ([email protected]).

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RESEARCH LETTER METHODS Fly stocks. Escargot-GAL4, UAS-nuclear-GFP, UAS-cytoplasmic-GFP (Kyoto DGRC); UAS-MCT-1-dsRNA (Vienna Drosophila RNAi Center). DENR knockout fly generation. DENR knockout flies were generated by homologous recombination as described31. To generate the knockout construct, upstream and downstream genomic flanks were amplified using oligonucleotides listed below and cloned into the NotI and AscI sites of pW25 (ref. 31), respectively. Antibodies and immunoblotting. Phospho-dAkt(T342) antibody was developed in collaboration with PhosphoSolutions. Anti-Mbc antibody was provided by S. Abmayr32. Antibodies anti-RpS6, anti-total Akt, anti-phosphoS6K (Cell Signaling); anti-tubulin and anti-EcR (Developmental Studies Hybridoma Bank); anti-dFOXO (ref. 33) and anti-InR (ref. 34) were used. Anti-dDENR and anti-MCT-1 antibodies used in this study were either generated at DKFZ by immunizing guinea-pigs with recombinant His-tagged full-length dDENR or MCT-1 or at Eurogentec by immunizing rabbits with two DENR-derived peptides selected to be specific and immunogenic (the latter are a gift from M. Hentze). Rabbit polyclonal antibodies to Drosophila eIF2D were raised to peptides from N- and C-terminal parts of the protein by Eurogentec (Seraing, Belgium), affinity purified and used 1:200 for western blotting. Mouse anti-tubulin antibody (Sigma; 1:20,000). Goat anti-rabbit-HRP and goat anti-mouseHRP secondary antibodies from Thermo Fisher Scientific and were used at 1:4,000– 1:20,000 depending on the primary antibody. Immunoblotting to nitrocellulose or PVDF was performed either with wet transfer under standard conditions35 or using an iBlot rapid transfer device (Life Technologies) according to the manufacturer’s guidelines. Blots were blocked in 5% milk/ TBS-T solution and probed with antibodies diluted as indicated in this solution or in TBS-T without milk. Signals were visualized using Super Signal Dura or Femto reagent (Thermo Fisher) and imaged on a Fujifilm LAS-4000 luminescent image analyser. Plasmids, cloning and in vitro transcription. Sequences of all oligonucleotides used for cloning are provided in Supplementary Information. All constructs were verified by sequencing. For most plasmid luciferase reporters, 59 UTRs were isolated by PCR using gene-specific primers as PstI-BstBI products (except the 59 UTRs of snoo and rbp6 which were isolated as PstI/ClaI products), and cloned into the respective sites of pAT1152, which contains the hsp70 basal promoter followed by a polylinker, the Renilla luciferase (RLuc) ORF and an SV40 polyadenylation signal. This cloning strategy retained the identical Kozak sequence for the RLuc ORF in all constructs. RLuc reporters were co-transfected with an equivalent firefly luciferase (FLuc) reporter containing the same hsp70 basal promoter, an FLuc ORF and the same SV40 polyadenylation signal (pAT1088). Mutations of the uORF ATGs in the mbc 59 UTR were generated by mutating a single nucleotide for each ATG by point mutagenesis (oligonucleotide sequences in Supplementary Information), in a manner predicted by mfold36 to not disrupt the secondary structure of the mbc 59 UTR. Likewise, mutagenesis of uORF Kozak sequences, uORF coding sequences and the intervening sequence between uORFs 218/248 and uORF338 were done by site-directed PCR mutagenesis using oligonucleotides described in Supplementary Information. Introduction of synthetic uORFs into an RLuc reporter containing a control 59 UTR was done as follows. The RLuc reporter containing the CG43674 59 UTR was mutagenized by site-directed PCR mutagenesis to introduce SpeI and AgeI sites into the middle of the 59 UTR. uORFs were then introduced by oligonucleotide cloning into the SpeI and AgeI sites. The cloned oligonucleotides (sequences in Supplementary Table 1) introduce a Kpn21 site at the 59 end of the insert, which is compatible with AgeI. This allows repeated rounds of oligonucleotide clonings using the SpeI and Kpn21 sites to introduce tandem copies of the uORFs. For stable cell line generation, the full ORF of dMCT-1 was cloned into pMT/ V5-HisA vector (Life Technologies) containing a copper-inducible metallothionein promoter, C-terminal V5 epitope tag and polyhistidine affinity tag and SV40 late polyadenylation signal using KpnI and XhoI sites. For inducible luciferase reporters, a complete ORF of firefly luciferase was cloned into pMT/V5-HisA (Life Technologies) using EcoRI and XhoI sites. Constructs for inducible synthetic RLuc reporters were generated by subcloning of the CG43674 59 UTR with or without 1aa uORF from corresponding original plasmids in pAT1152 into pTK122 vector (pMT-Renilla with a backbone BstBI site eliminated) using a polylinker EcoRI site and the BstBI site in RLuc. For luciferase assays with in vitro transcribed mRNA reporters, a synthetic poly(A)72 was introduced into the basal FLuc and RLuc reporter plasmids (pAT1088 and pAT1152, respectively) by oligo cloning, followed by the actin 59 UTR, yielding pSS72 and pSS73—the normalization control plasmid and the negative control plasmid, respectively. The actin 59 UTR was replaced with the mbc 59 UTR to generate pAT1337 (oligonucleotide sequences below). The mbc DATG version of this construct was generated by PCR mutagenesis. Coding sequences 1 59 UTR 1 39 UTR were PCR amplified from the pAT1152 backbone versions using the same T7 promoter forward primer and a RLuc reverse primer as for the wild-type mbc 59

UTR. This fragment was digested with BglII and BstBI and cloned into the respective sites of pMbc wild type to generate pCJ1. Plasmids were linearized using a HindIII site directly downstream of the synthetic poly(A) tail for run-off transcription. In vitro transcription reactions to yield capped and A-capped mRNAs were performed as previously described37. Cell culture, RNAi, transfection and reporter assays. S2 cells were grown in flasks at 25 uC in either Express-Five serum-free medium (Life Technologies) or Schneider’s medium (Life Technologies or Bio&Sell) supplemented with 10% FBS (Life Technologies). For suspension culture RNAi experiments, S2 cells grown semi-adherently at 25 uC were re-suspended at 2 3 106 cells ml21 in Schneider’s Medium without FBS and dsRNA was added to 15 mg ml21. Cells were transferred to a T-25 flask and incubated semi-adherently for 1.5 h at 25 uC. Subsequently Complete Schneider’s medium was added (2 ml per 1 ml of dsRNA-treated culture), and cells were grown semi-adherently for 4 days. Cells were diluted to 0.7 3 106 cells ml21, the surfactant Pluronic F-68 (Life Technologies) was added to a final concentration of 0.1% (v/v) and cells were incubated with gentle agitation on a rocker at 25 uC. DENR knockdown was efficient throughout the duration of the experiment, as quantified by immunoblotting (Extended Data Fig. 7b). Cells were subsequently collected for quantification of cell density, polysome profiling, or preparation of extracts for in vitro translation assays. Cell numbers were assessed with a haemocytometer and cell viability was assessed in parallel by trypan blue staining. For standard luciferase reporter assays with DNA or RNA reporters, S2 cells were seeded in 6-well plates and treated with dsRNAs at 12 mg ml21. After 3 or 4 days of adherent culture, cells were diluted into a 96-well plate at a fixed low concentration of 90,000 cells per well. After 2 days of incubation, cells were transfected with reporters using Effectene (Qiagen) and 18 h later they were harvested for luciferase assays. RNAi, transfection and reporter assays: proliferative versus quiescent cells. To obtain proliferating versus quiescent cells in an adherent format for transfection followed by luciferase assays, 5 3 106 S2 cells were treated with 30 mg of dsRNA in 2 ml of medium in a T-25 flask. After 3 days, they were split into wells of a 24-well dish at a concentration of 0.5 3 106 or 2.0 3 106 cells per well in 1 ml of medium and allowed to grow for 3 more days to obtain a state of proliferation or quiescence. For luciferase assays, cells were transfected without replacing the medium 1 day before lysis. For inducible reporter assays, 1 ml of S2 cells at a concentration of 1.5 3 106 per ml was incubated in the presence of 15 mg of dsRNA in medium lacking FCS for 1 h. After that, 4 ml of complete medium was added and the culture was incubated in a T-25 flask for 3 days. Efficiency of the knockdown was assessed by western blotting. 1.5 3 106 S2 cells previously treated with dsRNA were plated onto a 6-well plate at a concentration of 1.5 3 106 cells per well in 2 ml of medium and grown overnight. On the next day, the cells were transfected with 10 ng of inducible FLuc reporter (transfection control), 20 ng of inducible RLuc reporter with either control or 1 amino acid (1aa) uORF-containing 59 UTR and 370 ng of a non-specific plasmid using Effectene transfection reagent (Qiagen). For proliferating cells, expression of luciferase reporters was induced by addition of CuSO4 (FC 5 0.1 mM) 3–6 h posttransfection. For quiescent conditions, the transfected cells were first grown for 6 days and then induced with CuSO4 (FC 5 0.1 mM). In both cases induction was for 18 h and the cells were then harvested and lysed with 1x Passive Lysis buffer (Promega). 10 ml of cell extracts were used for dual luciferase assays (Promega) in a multiwell plate luminometer (Perkin-Elmer). Generation of stable S2 cell lines. For stable line generation, S2 cells grown in complete Schneider’s medium (Bio&Sell) supplemented with 10% FBS (Life Technologies) were plated onto a 6-well plate at a concentration of 1 3 106 per well, grown for 18 h and transfected with 400 ng of pMT-MCT-1-V5His inducible construct together with 10 ng of pCoBlast plasmid (Life Technologies), which provides resistance to the antibiotic Blasticidin S. Transfection was performed using Effectene reagent (Qiagen) according to the manufacturer’s protocol and stable blasticidinresistant clones were selected by incubating the transfected cells in the presence of decreasing concentrations of blasticidin S (Life Technologies) from 25 mg ml21 to 10 mg ml21 over several weeks. The resulting polyclonal stable cell lines were confirmed to enable inducible transgene expression by western blotting. Co-immunoprecipitation from proliferating versus quiescent cells. Stable S2 cell lines with copper-inducible V5 epitope-tagged MCT-1 were grown under proliferative or quiescent conditions and MCT-1-v5 expression was induced by addition of CuSO4 (FC 5 0.5 mM) for 48 h before harvesting for immunoprecipitation. For the quiescent state, cells were plated at a density of 10 3 106 per well in a 6-well plate and grown for 4 days before induction. For proliferating conditions, the cells were plated at 1 3 106 per well in a 6-well plate and grown for 2 days before induction. On day 3 and 4 for quiescent and day 2 for proliferating cells, control cell counts were performed to verify the proliferation/quiescent status of the cells. The cells were harvested, washed with 1xPBS, flash-frozen in liquid nitrogen and kept at 280 uC.


LETTER RESEARCH Frozen cells were thawed and lysed for 30 min on ice with ES2 lysis buffer (100 mM KCl, 20 mM HEPES pH 7.5, 2.5 mM EDTA, 5 mM DTT, 0.05% Triton X-100 with protease and phosphatase inhibitors) and spun at 10,000g for 10 min at 4 uC. Lysate input was normalized by Bradford assay. 50 ml of Protein G Dynabeads were used for each sample. Beads were blocked by washing 53 with 3% BSA in PBS, and were incubated with 20 ml of either V5 or Flag antibodies in 1 ml PBS-T for 1 h at room temperature, rotating. Beads were then washed 23 with PBS-T, and 23 with 0.1 M sodium borate, pH 9. Antibodies were coupled to the beads during 23 30 min incubations rotating at room temperature in a 1 ml 20 mM DMP/0.1 M sodium borate solution. The reaction was stopped by 23 washes of 50 mM glycine, and beads were washed 33 with PBS-T. Lysate was added to the antibody-coupled beads, and samples were rotated at room temperature for 10 min followed by washing 33 with lysis buffer. 13 Laemmli loading buffer was added directly to the beads, and beads were boiled for 5 min at 95 uC. Samples were run on a 15% SDS–PAGE gel, and probed using either V5 or DENR antibody. Crosslinking and RNA immunoprecipitation. S2 cells were grown to subconfluency in 10-cm dishes, two dishes were used per immunoprecipitation (IP) sample. Cells were washed once with PBS and cellular proteins were crosslinked with 0.5% formaldehyde in PBS for 10 min at room temperature. Further crosslinking was stopped by the addition of 0.25 M glycine (pH 7.0) for 5 min at room temperature. Cells were then scraped on ice in 1 ml RNA-IP lysis buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1.5 mM MgCl2, 1 mM DTT, 1% NP-40, Mini Complete protease inhibitors EDTA-free (Roche) and 0.2 U ml21 RNasin (Promega)). Lysates were cleared by centrifugation (5 min at 4 uC and 2,000 r.p.m. in a table-top centrifuge) and split into input (1:5) and IP samples (4:5). The IP samples were pre-cleared with Protein A/G UltraLink Resin (Thermo Pierce) for 1 h at 4 uC and incubated with 2 ml anti-DENR or 3 ml anti-GFP serum for 1 h at 4 uC. Immuno-complexes were precipitated by incubation with protein A/G beads for 1 h at 4 uC. Beads were washed three times with low salt RNA IP wash buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1.5 mM MgCl2), followed by 3 washes with high salt RNA-IP wash buffer (50 mM Tris-HCl (pH 7.5), 300 mM NaCl, 1.5 mM MgCl2). RNA was eluted from the beads using 1 ml ml21 TriFast reagent (PeqLab) according to the manufacturer’s instructions. 15 mg GlycoBlue (Ambion) was added to the RNA before precipitation. 4 ml RNA were subjected to reverse transcription using random hexamer primers and Superscript III Reverse Transcriptase (Invitrogen) according to the manufacturer’s instructions, followed by qPCR analysis. Quantitative RT–PCR for relative mRNA levels. RNA purification was performed with Trizol reagent according to the manufacturer’s recommendations. qRT–PCRs were done as previously described41. rp49 was used as a normalization control for experiments analysing mRNAs. Primer sequences appear in the oligonucleotide table in Supplementary Table 1. Quantitative RT–PCR for rRNA and tRNA levels. To quantify 18S, 28S rRNA and initiator tRNA levels in control or DENR knockdown cells, total RNA was extracted from an equal number of cells in the presence of a defined amount of spiked in RLuc RNA that had been in vitro transcribed. This RLuc spike-in was then used for normalization in qRT–PCR assays with primers and conditions described previously35,37. Primer sequences appear in the oligonucleotide table in Supplementary Information. Quantification of de novo protein synthesis by metabolic labelling. Nascent protein synthesis rates of DENR-depleted and control S2 cultures in either the proliferative ($4 independent samples) or quiescent ($3 independent samples) growth state were assayed using the Click-IT L-Azidohomoalanine kit (Life Technologies) according to the manufacturer’s instructions, but adapted for use with S2 cells. 2–6 3 106 cells (with or without cycloheximide present as a control) were washed in 25 uC PBS, re-suspended in 1 ml pre-warmed Schneider’s Complete Drosophila medium lacking methionine (Bio&Sell) transferred to 6-well plates and incubated at 25 uC for methionine depletion. After 1 h, the methionine analogue Click-IT LAzidohomoalanine (final concentration 5 50 mM) was added for nascent protein labelling and cells were subsequently incubated at 25 uC for an additional 2 h. Cells were harvested and lysed in lysis buffer (1% SDS in 50 mM Tris-HCl pH 8.0). Lysates were sonicated, centrifuged briefly to remove debris, and protein concentration in the resulting supernatant was measured by Bradford protein assay (BioRad). Between 0.8 and 7.8 mg of total protein were used for the chemoselective ‘click’ reaction (20 min at room temperature) between the azide (l-azidohomoalanine) and alkyne (tetramethylrhodamine, TAMRA). Proteins were pelleted by methanol precipitation, solubilized in NuPAGE LDS sample buffer (Life Technologies) and incubated for 10 min at 70 uC. Equal amounts of total protein were run on 4–12% Bis-Tris NuPAGE gels (Life Technologies) and TAMRA signals for nascent proteins were detected using the Fujifilm Fluorescent Image Analyzer FLA-9000 system. For quantification of total protein for normalization, gels were subsequently stained with SYPRORuby (Life Technologies) and re-scanned on the FLA-9000. TAMRA and SYPRORuby signals for each sample were quantified using FLA-9000 software and processed in Microsoft Excel.

Polysome profiling from S2 cells. Cells growing in suspension with gentle rocking were first treated with cycloheximide (CHX) to freeze polysomes before lysis. CHX (final concentration 5 100 mg ml21) was added to cultures followed by further incubation for 30 min at 25 uC on a rocker. Cells were counted and equal cell numbers were subsequently centrifuged at 800g for 3 min. The supernatant was discarded and the cell pellet was washed 2 times with 13 PBS, pH 5 7.4. Cells were resuspended in 175 ml polysome lysis buffer (PLB: 20 mM Tris, 20 mM NaCl, 3 mM MgCl2, 100 U ml21 Recombinant RNasin Ribonuclease Inhibitor (Promega, Mannheim, Germany) and Roche Complete Protease Inhibitor Cocktail (Roche, GrenzachWyhlen, Germany)). 175 ml of Polysome Extraction Buffer (PEB: PLB 1 1% Triton X-100, 2% Tween-20 and 1% Na-deoxycholate; all final concentration) was added. Lysates were mixed, incubated on ice for 10 min and then spun at maximum speed for 10 min at 4 uC in an Eppendorf microcentrifuge. Supernatants were loaded onto 14 3 95 mm Polyclear centrifuge tubes (Seton, Petaluma, CA) containing 17.5–50% sucrose gradients generated using the Gradient Master 108 programmable gradient pourer (Biocomp, New Brunswick, Canada) and centrifuged for 2.5 h at 35,000 r.p.m. in an SW40Ti rotor in a Beckman L7 ultracentrifuge (Beckman Coulter, Krefeld, Germany). After centrifugation gradients were simultaneously fractionated and measured for RNA content using a Piston Gradient Fractionator (Biocomp) attached to a UV monitor (BioRad, Hercules, CA). For RNA and/or protein isolation after fractionation, Trizol reagent (Life Technologies, Carlsbad, CA) was added to fractions and either protein (followed by TCA precipitation) or RNA (by Pure Link RNA Mini Kit purification (Life Technologies, Carlsbad, CA)) was isolated for downstream applications according to the manufacturer’s recommendations. For quantitative analysis of individual elements (that is, 80S monosomes, polysomes, etc) from the polysome profiles, all traces were first exported to Microsoft Excel and adjusted as necessary to ensure matching x/y-axis scales. Traces were then exported to Adobe Photoshop and ImageJ for processing, baseline setting, cropping and pixel counting. These pixel counts were then used for determining polysome/ monosome values, plot generation, and statistical analysis in Microsoft Excel. For immunoblotting of polysome gradient fractions, proteins in fractions were precipitated with TCA, washed 23 with acetone, and resuspended in protein gel sample buffer before electrophoresis and immunoblotting under standard conditions. Polysome profiling from Drosophila larvae. Polysome profiles from Drosophila larvae were performed by crushing male larvae in lysis buffer (50 mM Tris pH 7.4, 15 mM MgCl2, 300 mM NaCl, 1% Triton X-100, 100 mg ml21 cycloheximide, 0.1% b-mercaptoethanol, 1x protease inhibitor cocktail from Roche, 200 U ml21 RiboLock RNase inhibitor from Fermentas) on ice. Lysates were cleared by centrifugation, equalized in protein concentration and loaded onto a 17.5–50% sucrose gradient and centrifuged for 2.5 h at 35,000 r.p.m. In vitro translation assays with extracts from S2 cells. S2 cell extracts for in vitro translation assays were generated according to a modified version of a protocol previously used for embryo translation extracts38, adapted for smaller cell volumes. Cells were grown in suspension culture as described above to a density of 5–6 3 106 cells ml21, harvested by low-speed centrifugation (5 min, 800g), and cell pellets were re-suspended in ice-cold PBS to wash and then respun. This PBS wash step was repeated a further 23. After the final wash, pellet volume was carefully estimated and cells were re-suspended in precisely 2 pellet volumes of DEIKM Buffer (10 mM HEPES pH 7.4, 80 mM KOAc, 0.5 mM Mg(OAc)2, 5 mM dithiothreitol (DTT), plus Complete Mini, EDTA-free Protease Inhibitor Cocktail (Roche,1 tablet per 10 ml)) and then incubated for 30 to 45 min on ice. Lysis was by ,20 strokes with either a 1 ml or 2 ml syringe. Extracts were spun for 20 min at 20,000g at 4 uC. Supernatants were carefully removed and protein concentrations were checked by Bradford assay (BioRad). Extracts were adjusted to a final concentration of 10 mg ml21 with DEIKM buffer as necessary and adjusted to a final concentration of 10% glycerol. Extracts with concentrations below 10 mg ml21 were discarded, as our experience was that they displayed higher variability. Translation extracts were aliquotted, flashfrozen in liquid nitrogen, and stored at 280 uC. Translation extracts were used at a final concentration of 40% for in vitro translation reactions, which were performed essentially as described (Gebauer et al.38). The extracts were verified to be cap and poly(A) tail responsive. We also performed a titration of mRNA concentration for each reporter mRNA and used mRNA concentrations within the linear range of translation for all of the experiments shown. Bioinformatics. All bioinformatics are based on Flybase release 5.46 (ref. 39). A ‘DENR-dependence’ score was calculated based on the number of ATG start codons in the mRNA 59 UTR and the strength of their Kozaks. To predict Kozak strength, the frequency of each nucleotide found at positions 24 to 21 relative to the ATG for ‘main ORFs’ in the genome was quantified, yielding a frequency table (Extended Data Fig. 4d9) and the corresponding consensus of cAaaAUG (Extended Data Fig. 4d), similar to that of other species and the ‘pre-genomic’ Drosophila consensus defined with a much smaller set of genes40. A score for the strength of any Kozak in Drosophila was then generated using a multiplicative model whereby the frequencies indicated


RESEARCH LETTER in Extended Data Fig. 4d9 for each nucleotide position from 24 to 21 relative to the ATG were multiplied together. In this manner, 4-mer sequences frequently observed upstream of main ORF ATGs in the fly genome obtain high scores (maximum of 0.0496) whereas infrequent ones obtain low scores (minimum of 0.00012). The score from all individual ATGs in a 59 UTR were summed to reach the combined score for the 59 UTR. Oligonucleotide sequences and number of replicates for figure data. Full oligonucleotide sequences for all oligonucleotides used, as well as information about replicate numbers for each figure panel, are in Supplementary Information. 31. Huang, J., Zhou, W., Watson, A. M., Jan, Y. N. & Hong, Y. Efficient ends-out gene targeting in Drosophila. Genetics 180, 703–707 (2008). 32. Erickson, M. R., Galletta, B. J. & Abmayr, S. M. Drosophila myoblast city encodes a conserved protein that is essential for myoblast fusion, dorsal closure, and cytoskeletal organization. J. Cell Biol. 138, 589–603 (1997). 33. Puig, O., Marr, M. T., Ruhf, M. L. & Tjian, R. Control of cell number by Drosophila FOXO: downstream and feedback regulation of the insulin receptor pathway. Genes Dev. 17, 2006–2020 (2003).

34. Puig, O. & Tjian, R. Transcriptional feedback control of insulin receptor by dFOXO/ FOXO1. Genes Dev. 19, 2435–2446 (2005). 35. Duncan, K. et al. Sex-lethal imparts a sex-specific function to UNR by recruiting it to the msl-2 mRNA 39 UTR: translational repression for dosage compensation. Genes Dev. 20, 368–379 (2006). 36. Zuker, M. Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res. 31, 3406–3415 (2003). 37. Duncan, K. E., Strein, C. & Hentze, M. W. The SXL-UNR corepressor complex uses a PABP-mediated mechanism to inhibit ribosome recruitment to msl-2 mRNA. Mol. Cell 36, 571–582 (2009). 38. Gebauer, F., Corona, D. F., Preiss, T., Becker, P. B. & Hentze, M. W. Translational control of dosage compensation in Drosophila by Sex-lethal: cooperative silencing via the 59 and 39 UTRs of msl-2 mRNA is independent of the poly(A) tail. EMBO J. 18, 6146–6154 (1999). 39. Marygold, S. J. et al. FlyBase: improvements to the bibliography. Nucleic Acids Res. 41, D751–D757 (2013). 40. Cavener, D. R. Comparison of the consensus sequence flanking translational start sites in Drosophila and vertebrates. Nucleic Acids Res. 15, 1353–1361 (1987). 41. Teleman, A. A., Hietakangas, V., Sayadian, A. C. & Cohen, S. M. Nutritional control of protein biosynthetic capacity by insulin via Myc in Drosophila. Cell metab. 7, 21–32 (2008).


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RESEARCH LETTER 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 GFP1nuclear 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, h9, 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, m9) and 7 (n, n9) 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.


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RESEARCH LETTER Extended Data Figure 2 | DENR promotes re-initiation of translation downstream of uORFs in the mbc 59 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 59 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 59 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 59 UTR is not accompanied by a reduction in the intracellular levels of the reporter mRNA. In vitro transcribed mRNA consisting of the mbc 59 UTR, the Renilla luciferase ORF and a synthetic poly(A) was co-transfected with a normalization control mRNA consisting of a short 59 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 (d9) 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 59 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 59 UTR as the minimum region required to impart DENR-dependent regulation. Error bars indicate s.d.


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Extended Data Figure 3 | Detailed sequence information for the mbc 59 UTR. a, Full sequence of the mbc 59 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 59 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. DATG construct: single point mutations were introduced to abolish the three indicated uORFs. DKozak construct: Kozak sequences of the indicated uORFs were mutated to gtgtATG, thereby disfavouring translation initiation at these sites. Dstop 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 59 UTR. mRNA reporters containing the full-length mbc 59 UTR, or a mutated DATG 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 59 UTR in the mbc Dstop 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).


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LETTER RESEARCH Extended Data Figure 4 | Genome-wide analysis of stuORF transcripts. a, Genome-wide histogram of transcripts containing uORFs in their 59 UTRs. b, b9, Frequency distribution of nucleotides found surrounding the ATG of all main ORFs annotated in the fly genome. Frequency matrix in b9 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 1 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 5 10214) 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.


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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 13 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, b9, DENR protein levels 2 (b) and 3 (b9) days after knockdown in S2 cells, assessed by immunoblotting. c, c9, Polysome profile of S2 cells treated for 2 (c) or 3 (c9) 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, d9, Quantification of 18S RNA, 28S RNA and initiator tRNA levels in S2 cells treated with dsRNA for 2 (d) or 4 (d9) days shows little to no increase upon DENR knockdown compared to control.


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Extended Data Figure 6 | Loss of DENR leads to reduced InR and EcR protein levels and signalling in vivo—support data. a, a9, 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 mM, 4 h), assayed by qRT–PCR of two EcR target genes BR-C (Fig. 4c) and E75 (shown here),

normalized to rp49. c, c9, DENRKO pupae 4 h APF have reduced levels of InR and EcR-A protein (c) but not mRNA (c9). d, Expression of InR in histoblast cells and imaginal discs of DENRKO animals using escargot-GAL4 rescues their delayed pupation.


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LETTER RESEARCH 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, f9, 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, h9, 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 (h9) 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.


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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 59 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 59 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 (DENR1/1) 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 DENR1/1 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 DENR1/1 or in a DENR knockout genetic background.


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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.


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LETTER RESEARCH 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, d9, 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 39 UTR, and re-constituted to express wild-type or mutated forms of MCT-1 (using constructs with an exogenous 39 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. d9, 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.


hnRNPs and ELAVL1 cooperate with uORFs to inhibit protein translation.

Auxin Signaling in Regulation of Plant Translation Reinitiation.

Ribosome reinitiation at leader peptides increases translation of bacterial proteins.

eIF1 modulates the recognition of suboptimal translation initiation sites and steers gene expression via uORFs.

Phosphodiesterase 4D acts downstream of Neuropilin to control Hedgehog signal transduction and the growth of medulloblastoma.

On the reinitiation of cell growth in culture.

Translation of the rat LINE bicistronic RNAs in vitro involves ribosomal reinitiation instead of frameshifting.

Repeat Associated Non-AUG Translation (RAN Translation) Dependent on Sequence Downstream of the ATXN2 CAG Repeat.

ABCE1 Recycles Terminating Ribosomes and Controls Translation Reinitiation in 3'UTRs In Vivo.

Starting too soon: upstream reading frames repress downstream translation.

Fail-safe mechanism of GCN4 translational control--uORF2 promotes reinitiation by analogous mechanism to uORF1 and thus secures its key role in GCN4 expression.

Ribosomal protein L13a deficiency in macrophages promotes atherosclerosis by limiting translation control-dependent retardation of inflammation.

Activation of HuR downstream of p38 MAPK promotes cardiomyocyte hypertrophy.

Translation control of gene expression.

Control of growth of Hydra cultures by tissue potassium.

Nemo promotes Notch-mediated lateral inhibition downstream of proneural factors.

Cellular and geometric control of tissue growth and mitotic instability.

NSun2 Promotes Cell Growth via Elevating Cyclin-Dependent Kinase 1 Translation.

Drosophila casein kinase 2 (CK2) promotes warts protein to suppress Yorkie protein activity for growth control.

Overview: phosphorylation and translation control.

Why should cancer biologists care about tRNAs? tRNA synthesis, mRNA translation and the control of growth.

Growth hormone, cyclic nucleotides and the rapid control of translation in heart muscle.

Hormonal control of reinitiation of meiosis in starfish. The requirement of 1-methyladenine during nuclear maturation.

Reinitiation of mRNA translation in a patient with X-linked infantile spasms with a protein-truncating variant in ARX.