A novel uORF-based regulatory mechanism controls translation of the human MDM2 and eIF2D mRNAs during stress

Accepted Manuscript A novel uORF-based regulatory mechanism controls translation of the human MDM2 and eIF2D mRNAs during stress Kseniya A. Akulich, Pavel G. Sinitcyn, Desislava S. Makeeva, Dmitry E. Andreev, Ilya M. Terenin, Aleksandra S. Anisimova, Ivan N. Shatsky, Sergey E. Dmitriev PII:

S0300-9084(18)30320-1

DOI:

https://doi.org/10.1016/j.biochi.2018.11.005

Reference:

BIOCHI 5543

To appear in:

Biochimie

Received Date: 28 August 2018 Accepted Date: 6 November 2018

Please cite this article as: K.A. Akulich, P.G. Sinitcyn, D.S. Makeeva, D.E. Andreev, I.M. Terenin, A.S. Anisimova, I.N. Shatsky, S.E. Dmitriev, A novel uORF-based regulatory mechanism controls translation of the human MDM2 and eIF2D mRNAs during stress, Biochimie, https://doi.org/10.1016/ j.biochi.2018.11.005. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Short upstream open reading frames (uORFs) are the most prevalent cis-acting regulatory elements in the mammalian transcriptome which can orchestrate mRNA translation. Apart from being “passive roadblocks” that decrease expression of the main coding regions, particular uORFs can serve as specific sensors for changing conditions, thus regulating translation in response to cell stress. Here we

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report a novel uORF-based regulatory mechanism that is employed under conditions of hyperosmotic stress by at least two human mRNAs, coding for translation reinitiation/recycling factor eIF2D and E3 ubiquitin ligase MDM2. This novel mode of translational control selectively downregulates their expression and requires as few as one uORF. Using a set of reporter mRNAs and fleeting mRNA

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transfection (FLERT) technique, we provide evidence that the phenomenon does not rely on delayed reinitiation, altered AUG recognition, ribosome stalling, mRNA destabilization or other known

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mechanisms. Instead, it is based on events taking place at uORF stop codon or immediately downstream. Functional aspects and implications of the novel regulatory mechanism to cell

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physiology are discussed.

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A novel uORF-based regulatory mechanism controls translation of the human MDM2 and eIF2D mRNAs during stress

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Kseniya A. Akulich1,2, Pavel G. Sinitcyn1, Desislava S. Makeeva1,2, Dmitry E. Andreev2, Ilya M. Terenin2,3, Aleksandra S. Anisimova1,2, Ivan N. Shatsky2, and Sergey E. Dmitriev1,2,4,5* 1

School of Bioengineering and Bioinformatics, Lomonosov Moscow State University, Moscow, 119234

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Russia; 2Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, 119234 Russia; 3Sechenov First Moscow State Medical University, Institute of Molecular

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Medicine, 119991, Moscow, Russia 4Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, 119991, Russia; 5Department of Biochemistry, Biological Faculty, Lomonosov Moscow State University, Moscow, 119991 Russia

* To whom correspondence should be addressed: Belozersky Institute of Physico-Chemical Biology,

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Lomonosov Moscow State University, Bldg. A, Leninskie gory, 1-40, Moscow, 119234 Russia

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Tel: +7 903 2220066; Fax: +7 495 9393181; E-mail: [email protected]

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Running title: Regulation of MDM2 and eIF2D mRNA translation by uORF

Abbreviations used: FLERT, fleeting mRNA transfection; eIF, eukaryotic initiation factor; Fluc, firefly luciferase; Rluc, Renilla luciferase; 5’ UTR, 5’ untranslated region; ORF, open reading frame ; uORF, upstream open reading frame; uAUG, upstream AUG; CDS, coding sequence; ISR, integrated stress response; ICIER, initiation complexes interference with elongating ribosomes.

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Abstract

Short upstream open reading frames (uORFs) are the most prevalent cis-acting regulatory elements in the mammalian transcriptome which can orchestrate mRNA translation. Apart from being “passive roadblocks” that decrease expression of the main coding regions, particular uORFs can serve as

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specific sensors for changing conditions, thus regulating translation in response to cell stress. Here we report a novel uORF-based regulatory mechanism that is employed under conditions of hyperosmotic stress by at least two human mRNAs, coding for translation reinitiation/recycling factor eIF2D and E3

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ubiquitin ligase MDM2. This novel mode of translational control selectively downregulates their expression and requires as few as one uORF. Using a set of reporter mRNAs and fleeting mRNA

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transfection (FLERT) technique, we provide evidence that the phenomenon does not rely on delayed reinitiation, altered AUG recognition, ribosome stalling, mRNA destabilization or other known mechanisms. Instead, it is based on events taking place at uORF stop codon or immediately downstream. Functional aspects and implications of the novel regulatory mechanism to cell

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physiology are discussed.

Keywords Hyperosmotic stress, fleeting mRNA transfection assay (FLERT), p53 checkpoint, cell cycle arrest, translation termination, ribosome recycling

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1. Introduction

The ability of open reading frames, located upstream of the main coding sequence (uORFs), to regulate expression was first described in the 80s of the last century. In recent years, the biological significance of uORF-mediated translational control is getting more and more obvious and is becoming a very active

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area of research [1, 2]. Systems approaches identified short translated ORFs in the 5’ untranslated regions (5’ UTRs) of many human genes, including a substantial proportion of uORFs initiated from nonAUG codons [3, 4]. A few of these short ORFs encode stable functional peptides or even small proteins

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[5], while the overwhelming majority plays a regulatory role at the level of mRNA translation only. In most cases, uORFs inhibit translation of the corresponding main coding regions, sometimes leading to

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clinically relevant consequences [6-8]. However, there is a growing number of cases when uORFs can adjust the mRNA translation level in response to changing conditions [1, 2, 8]. Nevertheless, there are only a limited number of underlying regulatory mechanisms that have been well-studied to date. The most well-known is the so called delayed translation reinitiation, exemplified by the yeast GCN4

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mRNA, mammalian ATF4 mRNA and a number of other transcripts with more than one uORFs in their 5’ leaders (for review, see [2, 9]). Translation of these mRNAs is paradoxically activated in response to eIF2 phosphorylation (which inhibits translation in most cases). This mechanism strictly requires the presence

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of at least two separate uORFs, since it relies on bypassing of the second uORF by the 43S complex that reinitiates scanning after translation of the first one. Systematic studies of translational response to eIF2

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phosphorylation [10, 11] revealed, however, that a considerable number of mRNAs possess only one uORF, and still exhibit efficient translation under these conditions. The ICIER (Initiation Complexes Interference with Elongating Ribosomes) mechanism was proposed for some of these mRNAs to explain the phenomenon [12].

The limited number of other known uORF-mediated regulatory pathways includes the sequencedependent stalling of elongating ribosomes [13, 14], altered upstream AUG (uAUG) recognition [15-17], 43S sliding [18], ribosome shunting, mRNA destabilization and some others (for review, see [1, 2, 8]).

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Molecular mechanisms of start codon selection during a conventional translation initiation have been extensively studied in genetic, biochemical and structural terms [19, 20]. However, how the ribosome resumes scanning after reading an uORF and reinitiates translation of the downstream main coding sequence (CDS), is characterized much less thoroughly [21-23]. In recent years, there has been marked

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progress in analysis of both structural and functional aspects of this complicated process [24-28], but understanding of its regulation is still far from complete.

In this work we show that human mRNA encoding one of the translation reinitiation/ribosome recycling

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factors, eIF2D [29, 30], has a regulatory element within the 5’ UTR that drastically inhibits synthesis of the protein under certain conditions. This regulation requires an uORF and reinitiation after its

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translation, but differs from all known uORF-mediated pathways mentioned above. Based on the structural features of the eIF2D 5’ UTR, we found another human mRNA with a similar response to stress, encoding the oncoprotein MDM2 [31]. We believe that hypersensitivity of MDM2 mRNA translation to osmotic stress may play a role in activation of cell cycle checkpoints and control of cell

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death under certain conditions [32, 33].

2. Materials and Methods

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2.1. Plasmid constructs and in vitro transcription

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eIF2D-Fluc (short variant, based on GenBank NM_006893.2 sequence), MDM2-Fluc (based on GenBank NM_002392.5 sequence), CDK4-Fluc, CFTR-Fluc, ATF4-Fluc, IFRD1-Fluc, Actin-Fluc and Actin-Rluc plasmids were described before [10, 18, 34, 35]. To prepare DHX9-Fluc, TUBA1B-Fluc, AQP9_1-Fluc and AQP9_2-Fluc, 5’ proximal regions of the human DHX9, TUBA1B and AQP9 (two variants) cDNAs were amplified from a total HEK293T cDNA by PCR with primer pairs CTAGGTAATACGACTCACTATAGATGCGTACGCTCGCTGGCCCCG and GCCAAGCTTACCCATGATTCAAGTGTCTTCTTC (for DHX9), CTAGGTAATACGACTCACTATAGGAGTGCGTTACTTACCTCGACTC and GCCAAGCTTGCCAACGTGGATGGAGATGC (for TUBA1B), GCCAAGCTTCATCTTGGGGCTTCTCTGAGG and 4

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CTAGGTAATACGACTCACTATAGGAACACAACTGGCACATCTCTTTTC or CTAGGTAATACGACTCACTATAGGAAGTCGCAGATTCAAACAAATAGC (for AQP9_1 or AQP9_2, respectively) and inserted into pGL3-CrPV construct replacing the CrPV sequence [36]. For eIF2DLong-Fluc and eIF2D_WT-45 (Fig. S6), PCR products similarly obtained with primers

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CGCGCCTAGGTAATACGACTCACTATAGGAGTTCCAGGTACCGCCACTG and CTTGATGGCCGTGTTGGACTT, or TCGAACTAGTCTTTTCGCGGCCGGGCCCCAGCATGGC and

CTAGCCATGGCTGCTGGGGTGGCCTGGGGAAGAG, respectively, were inserted into AvrII and PvuII sites of L1-Fluc vector [37], or into SpeI and NcoI sites of Actin-Fluc plasmid, accordingly. Other plasmids with

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eIF2D 5’ UTR derivatives were obtained on the basis of the eIF2D-Fluc construct by PCR mutagenesis with primers CTGCCCCCACGGCTGAGGG and CCTAGCTGGGGCCCGGCCGCGAA (No_uORF),

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CATTCCCTGGCTTCTGTGCTCT and TCCAGAAAGCGAGGGCGCAGCAGCT (uORF_77), CATTCCCTGGCTTCTGTGCTCT and CAAGAAAGCGAGGGCGCAGCAG (uORF_fusion), CTGCCCCCACGGCTGAGGG and CCCATGCTGGGGCCCGGCCGCGAA (uORF_6). Other eIF2D 5’ UTR derivatives were generated during transcription template preparation (see below).

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For synthesis of the polyadenylated reporter mRNAs, 50T-tailed PCR products were used as templates, as described previously [37]. The templates were obtained with T7 promoter-containing forward primers annealing to the beginning of the corresponding 5’ UTRs (e.g. T7eIF2D primer

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CGCCGTAATACGACTCACTATAGCTTTTCGCGGCCGGGCCCCA was used for eIF2D-Fluc). In the case of

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GL3_WT-45, CAA_WT-45, 1/2WT and uORF_2, the primers CGCCGTAATACGACTCACTATAGGGAGCTTATCGATACCGTCG, CGCCGTAATACGACTCACTATAGGGCAACAACAACAACAACAACAGCATGGCTGCCCCCACGGCTGAGGGCC, CTAGGTAATACGACTCACTATAGCCCTCGCTTTCTTGACATTCC and CTAGGTAATACGACTCACTATAGCTTTTCGCGGCCGGGCCCCAGCATGGCTTGACCCACGGCTGAGG, respectively, were used instead the above T7eIF2D primer (Fig. S5). Transcription was performed with RiboMAX kit (Promega). The resulting transcripts were precipitated with 2M LiCl and capped with Vaccinia Capping System (NEB), following by another LiCl precipitation. All mRNA transcripts were checked for integrity by denaturing urea polyacrylamide gel electrophoresis. 5

2.2. Cell culture and FLERT

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HEK293T or RKO cells were cultured and transferred into 24-well plates 12-16 h before transfection to obtain final confluency of ~70–80%, as described [37]. The transfection was performed using Unifectin56 (Unifect Group) according to [38]. 3M NaCl, 3M sorbitol or other stress inducers (100x stock solutions

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in PBS) were added to the medium right before (~5 min) the addition of the transfection complexes, and gently mixed. All manipulations were performed so as to minimize the time cells spend out of CO2 box and to avoid cooling the plate, as described previously [38]. Two hours after transfection, cells were

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harvested, and luciferase activities were analyzed using the Dual Luciferase Assay kit (Promega). All the transfections were repeated at least three times in different cell passages. The mean values ±SD were

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calculated.

2.3. Mammalian cell-free system and in vitro translation assays

Krebs-2 ascite cells S30 extract was prepared as described previously [34]. Translation experiments in the mammalian system were performed in a total volume of 10 μl, which contained 5 μl of the S30

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extract, translation buffer (20 mM Hepes-KOH pH 7.6, 1 mM DTT, 0.5 mM spermidine-HCl, 0.8 mM Mg(OAc)2, 8 mM creatine phosphate, 1 mM ATP, 0.2 mM GTP, 120 mM KOAc and 25 μM of each amino acid), 2U of RiboLock RNase inhibitor (Thermo Scientific), 0.5 mM D-luciferin (Promega), 100 ng mRNA

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and 1 μl of eiter water or 10x NaCl solution (as indicated). Translation mixtures were incubated in a white 384-well plate covered by a PCR plate seal, at 30°C, in the TECAN Infinite F200 Pro plate reader

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with continuous measurement of the luciferase activity, as described [38]. Light intensities at 60 min were taken as luciferase activity values.

3. Results 3.1. A prevailing shorter variant of the eIF2D mRNA leader possesses 5’ TOP and uORF During initial characterization of translation factor eIF2D [29], we noticed peculiar organization of its mRNA in mammals. Its 5’ UTR has a high GC content (>70%), implying a complex secondary structure,

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and one uORF with an AUG codon in a strong nucleotide context (Fig. S1A). In the human eIF2D mRNA, this uORF is 51 nt (17 codons) long and separated by a 46 nt spacer from the main coding region. While the uORF sequence is not conserved among mammals (neither at the nucleotide, nor amino acid level), its presence and the strong uAUG context is evolutionarily conserved (Fig. S1B). Interestingly, the main

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eIF2D CDS start codon, in contrast, lies within a sub-optimal nucleotide context [39], having U/C in +4 position in all mammalian eIF2D mRNAs. Analysis of publicly available ribosome profiling data in the GWIPS-viz browser [40] reveals high ribosome occupancy of the uORF from both human and mouse (Fig.

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S2).

To investigate translational properties of the human eIF2D mRNA, we decided to fuse its 5’ proximal

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part to the coding region of firefly luciferase (Fluc). The 5’ UTR of the mRNA annotated in GenBank (NM_006893) is 209 nt long. However, both EST analysis and Ensembl Gene Prediction strongly argue that the major (and probably the only authentic) transcription start site is located at position -122 nt relative to the eIF2D CDS (Fig. S3). Strikingly, the resulting transcript has a classical oligopyrimidine tract (CUUUUCG) at its 5’ end and thus belongs to the 5’ TOP class of mRNAs, which is characteristic of many

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other components of the translation machinery [41]. Both the longer (209 nt) and the shorter (122 nt) 5’ UTR variants share the same uORF, however. Thus, we created two luciferase reporter constructs having these two 5’ UTRs (Fig. 1A). We also included the first 45 nt of the eIF2D coding region in both

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constructs, as this mRNA fragment could potentially form a secondary structure contributing to start

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codon selection and translational control. This also preserved the original nucleotide context of the main AUG codon.

To exclude possible effects related to transcription and other DNA-related events, we took advantage of the mRNA transfection technique [42]. We prepared m7G-capped and polyadenylated mRNAs and transfected them into HEK293T cells. For normalization, we used a similarly obtained Renilla luciferase mRNA with the human β-actin mRNA leader (Actin-Rluc), co-transfected with the Fluc mRNAs. The results of this in vivo translation (Fig. 1B) indicated that the mRNA with the longer 5’ UTR variant was translated very inefficiently. In contrast, the shorter leader provided only ~9-fold lower activity of Fluc than the classical β-globin 5’ UTR, a very strong mammalian leader [43]. The poor activity of the longer 7

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5’ UTR can be easily explained by its extremely GC-rich 5’ terminal sequence (Fig. S1), which suggests a developed secondary structure. It is well known that base pairing at the 5’ terminus dramatically inhibits binding of initiation factors and prevents ribosome loading [44]. In contrast, computer modeling of the shorter mRNA secondary structure suggested that its 5’ end is likely single stranded, while the AUG-

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proximal portion of the leader still has a developed secondary structure, which also involves the beginning of the coding region (Fig. 1C). We assumed that the shorter form of the 5’ UTR, which is more abundant in cells, mainly determines overall eIF2D production. Thus, in our subsequent study we mostly

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used this 5’ UTR variant (hereafter called eIF2D WT 5’ UTR) and the corresponding eIF2D-Fluc reporter.

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3.2. Translation of the eIF2D 5` UTR directed reporter is hypersensitive to moderate hyperosmotic stress in cultured cells

To look for a putative specific translational response of the eIF2D mRNA, we applied different types of stresses to the transfected cells. We monitored immediate effects of stress inducers on translation of

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our reporter mRNAs using the recently developed Fleeting mRNA Transfection (FLERT) technique, a method that allows narrowing the period of analysis down to 2 hours or less and thus helping unambiguously discriminate translational regulation from other types of gene expression control [38].

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An initial screen of stress inducers (Fig. 2A) showed that translation of the eIF2D-Fluc mRNA was specifically and highly sensitive to mild hyperosmotic stress (caused by addition of NaCl or sorbitol), as

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compared to translation directed by a non-specific leader from the control vector. We decided to investigate this phenomenon further. The FLERT technique allowed us to focus on the immediate osmotic stress response, which occurs within 2 hours [45, 46]. We repeated the experiment with different concentration of NaCl added right before mRNA transfection to achieve saline concentrations of 200 - 230 mM, as compared to the normal ~150 mM NaCl concentration in the medium (hereafter denoted as 50 – 80 mM NaCl excess). These concentrations are known to induce moderate hyperosmotic stress, which does not trigger irreversible arrest of proliferation, DNA damage, apoptosis induction or other dramatic consequences [45, 46]. To assess effects of this type of stress on 8

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general protein synthesis, we use an mRNA reporter with a representative mammalian 5’ UTR from the human β-actin mRNA. Indeed, it resulted in a moderate decrease (1/3 to 2/3 of the control level) of the Actin-Rluc mRNA translation (Fig. 2B), in accordance with expected values [47] and with a higher GC content in its leader [48], 77% vs. 51% GC in the artificial leader of the control GL3-Rluc mRNA we used

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previously for our initial screening (Fig. 2A). However, in the same cells, level of the eIF2D-Fluc mRNA translation fell to a much greater extent, down to nearly zero translation at the highest NaCl concentration tested (Fig. 2B). The difference was not caused by differences in the coding region used,

same inhibition pattern as Actin-Rluc (see below).

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since other Fluc-encoding mRNAs we tested (including that with the β-actin leader) demonstrated the

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Hypertonicity is known to affect stability of some specific mRNA species [49]. To check whether the observed phenomenon was caused by selective mRNA degradation, we performed a time-course experiment. HEK293T cells were co-transfected with the same two transcripts and harvested every hour for up to 4 h. Then the luciferase activities were analyzed. As shown in Fig. 2C, the Fluc/Rluc ratio did not change significantly over time under neither normal, nor stress conditions. This indicated that no

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selective degradation of transcripts occurred and thus most likely the phenomenon we observed was caused by translation regulation.

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Since HEK293T cells are derived from the kidney, they could have some peculiarities in osmotic response. Thus, we repeated our experiment in cell line of a different origin, human colon carcinoma

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cells RKO. We obtained essentially the same results (Fig. S4A), with a small difference that can be explained by specificity in translational response in these different cell types.

3.3. uORF determines translational response of the eIF2D 5’ UTR reporter mRNA to hyperosmotic stress The observed phenomenon could be explained by some structural features of the eIF2D 5’ UTR. Thus, we performed modifications of its sequence in the context of the Fluc reporter and used the resulting mRNA construct in a FLERT assay under normal and hyperosmotic stress conditions (Fig. 3). These experiments showed that neither the 45-nt fragment of the eIF2D coding region (providing the non9

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optimal context of the main AUG), nor the 5’ terminal region of the 5’ UTR affect the regulation significantly (Fig. 3, right panel, constructs WT-45, GL3_WT-45, CAA_WT-45; see Fig. S5 for the construct sequences). In contrast, deleting the first half of the 5’ UTR completely abrogated (and even partially reversed) the

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effect (Fig. 3, right panel, ½WT). This part of the eIF2D leader contained the start codon of the uORF. We therefore proposed that the uORF could be responsible for the observed phenomenon. Indeed, a mutation converting the uAUG codon to UAG abrogated mRNA hypersensitivity to osmotic stress (Fig. 3,

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No_uORF; Fig. 2D). The same result was obtained under different salt concentrations and with another cell line, RKO (Fig. S4B). Importantly, the presence of the uORF in the reporter mRNAs substantially

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inhibited their translation under normal conditions (Fig. 3, left panel). This indicates that the uAUG codon is efficiently recognized in the WT eIF2D 5’ UTR.

We then checked whether uAUG recognition per se is responsible for the hypersensititvity. For this, we extended the uORF far beyond the AUG of the main coding region by substituting its stop codon UGA

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with a sense GGA codon. The resulting uORF_77 construct had a much longer (231 nt long) uORF that overlapped with the main CDS for 43 codons (Fig. 3). In this case, the uAUG recognition should occur with the same efficiency, but no reinitiation is possible after uORF translation due to a limited ability of

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the eukaryotic ribosome to move backward during reinitiation [50]. Translation of the uORF_77 mRNA construct was ~30% as efficient as that of the WT, suggesting that ~2/3 of ribosomes reading the eIF2D

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coding region are involved in the original WT uORF translation (i.e. represented reinitiating ribosomes). Strikingly, the uORF_77 mRNA construct lost the translation hypersensitivity to osmotic stress (Fig. 3). It is therefore not the uAUG recognition but rather some uORF termination or reinitiation event that is responsible for the effect.

This conclusion was further supported by a result obtained with another mRNA construct, uORF_fusion. This construct had one nucleotide removed from UGA stop codon of the uORF, resulting in a single fused coding region starting with the former uAUG and then followed by the in-frame Fluc CDS. Its translation therefore followed uAUG recognition and reflected uORF translation in the original WT construct. The 10

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uORF_fusion mRNA translation was neither hypersensitive, no more resistant to the stress conditions (Fig. 3), while the latter could be expected if uAUG recognition was involved in the effects. Thus, we concluded that the eIF2D 5’ UTR uORF translation, followed by termination and renitiation at the next AUG codon, provides the specific translation regulation of the main ORF under these specific

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conditions. To address whether amino acid sequence of the uORF encoded peptide was important for the effect, we created uORF_6 mRNA construct with one nucleotide (G) inserted right after the uAUG codon. This resulted in a frameshift that simultaneously shortened the uORF (from 17 to 6 codons) and

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completely altered the encoded amino acid sequence (MAAPTAEGLAAAAPSLS to MGCPHG).

Importantly, the insertion preserved the uAUG nucleotide context (i.e. G at +4) and thus should not have

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affected its recognition. The frameshift also preserved UGA as the uORF stop codon. We found that this perturbation did not change the translation response significantly (Fig. 3). In accordance with this result, a similar observation was made with even a shorter uORF encoding only two amino acid peptide (MA)

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but still having both the same strong nucleotide contexts of uAUG and the same stop codon, UGA.

3.4. The eIF2D 5’ UTR dependent translational control requires cell integrity We then tried to reproduce the effects of increasing NaCl on reporter mRNA translation in a cell-free

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system. We chose the Krebs-2 cells S30 extract, as this system closely recapitulates translation in living cells [34]. Even a slight excess of NaCl over conventional salt concentration in a reaction buffer led to a

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decrease in translation of both eIF2D WT and No_uORF mRNAs (Fig. 4). Notably, the addition of an extra 50 mM NaCl, which did not substantially affect No-uORF mRNA translation when added into cell culture medium, completely inhibits its translation in vitro. This difference between in vivo and in vitro systems can be explained by tight control of ion content by living cells even under conditions of osmotic stress [45]. Importantly, both eIF2D mRNA derivatives (with and without the uORF) demonstrated approximately the same degree of translation inhibition. Thus, we concluded that the phenomenon of differential inhibition, which we previously observed in cultured cells, was not caused by direct increase

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of the salt concentration (the ionic strength) in the cytoplasm, but by some signaling that requires cell integrity.

3.5. The presence of an uORF in mRNA is not sufficient for translational hypersensitivity to osmotic stress

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As the physiological significance of the eIF2D mRNA specific translational control under hyperosmotic conditions is not known, we decided to look for another case of the same regulation among genes with a clearer role in the stress response. For this, we took a list of human genes with documented decrease

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in their protein abundance under conditions of osmotic stress [45] and selected a number of examples having uORFs in their mRNAs. We prepared luciferase reporter constructs with these leaders (Fig. S6)

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and introduced the corresponding mRNAs into HEK293T cells, along with the control Actin-Rluc mRNA (Fig. 5).

We observed various translation efficiencies of the mRNAs under normal conditions (Fig. 5, left panel) and a differential response to hyperosmotic stress induced by NaCl (Fig. 5, right panel). Some mRNAs

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demonstrated translation that was more resistant to the stress conditions than the co-transfected ActinRluc mRNA. The latter could be explained by differences in mRNA secondary structure, which are known to affect stress sensitivity [48]. Notably, these results clearly showed that the presence of an uORF per

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se is not sufficient to provide a hypersensitive translation response to osmotic stress.

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Nevertheless, there were two mRNAs in our set that demonstrated effects similar to that of the eIF2DFluc mRNA. These were reporters with 5’ UTRs from human MDM2 and CDK4 mRNAs. The case of MDM2 seemed to be of special interest due to the well-known role of this oncoprotein in cell cycle control and stress response through regulating p53 degradation [31].

3.6. The first of two uORFs in the MDM2 mRNA 5’ UTR is responsible for specific translation regulation under hyperosmotic stress

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Hyperosmotic conditions are known to cause rapid elimination of MDM2 protein, which results in p53 accumulation and cell cycle arrest [32, 33]. Thus, we decided to further characterize translation regulation of its mRNA under these conditions. The human MDM2 5’ UTR contains two uORFs, encoding 14 amino acid peptides each (Figs. S6 and S7).

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Their inhibitory action on MDM2 translation was previously shown using plasmid DNA transfection [51, 52]. However, whether this inhibition is modulated in response to stress has not been investigated. We prepared four mRNA constructs with either none, one, two or both uORF deleted, and introduced

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them into HEK293T cells (Fig. 6A). In accordance with the published data [51, 52], the first uORF (uORF1) provided a severe (~3,5 fold) inhibitory effect on reporter translation under normal conditions (Fig. 6A,

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left panel; cf. no_ORFs and no_ORF1 constructs), while the second one (uORF2) had a minor impact. This correlated well with data available from ribosome profiling studies that indicated high ribosome occupancy at the uORF1 (Fig. S7C). Strikingly, under hyperosmotic conditions a single nucleotide change (AUG to AGG) that removed uORF1 eliminated the high stress sensitivity of MDM2-Fluc translation,

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while similar changes removing uORF2 had only a marginal effect (Fig. 6A). We further confirmed this observation in a more accurate analysis using gradually increasing NaCl concentration in the medium. The titration curve obtained for the MDM2 WT 5’ UTR reporter strikingly

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resembled that for eIF2D WT leader (Figs. 6B and 2B, respectively). As for the latter, relative expression level did not change during the 4 h of incubation, implying the MDM2-Fluc mRNA with 5` UTR of MDM2

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to be equally stable within the cell under both normal and hyperosmotic conditions (Fig. 6C). Elimination of uORF1 changed the curve dramatically, while uORF2 disruption still left the reporter translation highly sensitive to the stress (Fig. 6D). Interestingly, simultaneous removal of both uORF1 and uORF2 made it even less sensitive than the control Actin-Rluc mRNA translation.

3.7. Translational stress response provided by eIF2D and MDM2 5’ UTRs is of a novel type To date, the most studied mechanism of uORF-mediated translational control is the delayed reinitiation that operates on the yeast GCN4 and mammalian ATF4 mRNAs (for review, see [1, 9]). It provides a 13

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paradoxical effect of their translation activation under conditions of eIF2 inhibition, when the majority of cellular mRNAs are silenced. This mechanism requires at least two non-overlapping uORFs. Recently it was shown, however, that a similar result can be provided by a single uORF with certain features, leading to ICIER, as illustrated by the case of the human IFRD1 mRNA [10, 12]. Thus, we decided to

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compare translational response of the eIF2D and MDM2 5’ UTR containing mRNAs with that of ATF4 and IFRD1 directed reporters.

For this, we performed FLERT analysis of the Fluc-encoding mRNA constructs under conditions of

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arsenite-induced oxidative stress and NaCl-induced hyperosmosis (Fig. 7A and 7B, respectively). Sodium arsenite is a well-known inducer of integrated stress response (ISR) that is widely used for studying

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effects of eIF2 phosphorylation on mRNA translation [10, 53-58]. The compound is known to cause eIF2 inactivation in a few minutes after the treatment, leading to a robust general inhibition of cellular protein synthesis [55, 56]. However, translation of a few mRNA species, including the ATF4 mRNA encoding the major ISR transcription factor, either persists or is even activated [10, 11, 55, 59-61].

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In accordance with the above, FLERT performed under these conditions showed a drop of Actin-Fluc reporter activity, and resistance or activation of ATF4-Fluc and IFRD1-Fluc mRNA translation, depending on the arsenite concentration used (Fig. 7A). Under the same conditions, translation of eIF2D-Fluc and

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MDM2-Fluc reporters was inhibited similar to that of canonical Actin-Fluc mRNA. In contrast, under conditions of hyperosmotic stress, ATF4-Fluc and IFRD-Fluc translational behavior resembles that of

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Actin-Fluc, while eIF2D-Fluc and MDM2-Fluc demonstrated the hypersensitive response, as before (Fig. 7B). We concluded that the phenomenon under study is novel and differs mechanistically from that of the well-known translational control via delayed reinitiation.

4. Discussion Recent advances in genome sequencing, high-throughput transcriptome and translatome analysis have revealed that many mammalian mRNAs harbor translated uORFs in their 5’ UTRs [3, 4, 9]. Many of them probably play a direct inhibitory role only, nevertheless providing proper expression levels of 14

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clinically relevant genes [6, 8]. Some of these elements, however, have more complex regulatory functions (for review, see [1, 2, 9]). Here we describe a novel type of uORF-dependent translational control exemplified by 5’ UTRs of two human mRNAs, encoding translation factor eIF2D and E3 ubiquitin ligase MDM2. It operates under

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conditions of hyperosmotic stress and provides a dramatic drop of translation directed by these 5’ UTRs, as compared to that of mRNAs with conventional leaders. We show that this type of regulation requires at least one uORF non-overlapping with the main coding region. Using a set of reporter mRNA

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constructs, site-directed mutagenesis and FLERT assay, we prove that alterations of uAUG or main AUG recognition efficiency do not affect this regulation. Most likely, the control is achieved at the

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uORF stop codon or immediately downstream to it (Fig. 7C).

Indeed, at least in the case of the eIF2D mRNA leader, we show that: (i) the regulation element tolerates wide changes in nucleotide sequence of the mRNA 5’ terminus (Fig. 3, constructs GL3_WT-45 and CAA_WT-45); (ii) it still persists if uORF length and sequence are altered (up to complete sequence

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replacement in the case of uORF_6 or length degeneration to only 6 nt in uORF_2 constructs), but (iii) completely disappears when the uORF start codon is removed (1/2WT and No_uORF constructs); (iv) extension of the uORF far beyond the main AUG codon removes the effect completely (uORF_77

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construct); (v) the phenomenon survives an alteration of the main AUG nucleotide context (WT-45 construct). From these data we concluded that this novel regulation mechanism is not based on

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changes in efficiency of the 43S complex assembly, initial ribosome binding to the mRNA 5’ end, 43S migration toward uAUG codon, subunit joining step or uORF translation elongation (see the model in Fig. 7C). Importantly, we also excluded changes in efficiency of uAUG recognition (otherwise we should have been able to observe an effect for the uORF_77 construct, and also observe its reversion for the uORF_fusion construct, which was clearly not the case for both situations, see Fig. 3). As the same mechanism operates on two mRNAs with very different spacers between uORF and main coding region (46 in the eIF2D 5’ UTR and 186 nt in the MDM2 one), it is very unlikely that the regulation occurs at a step when the reinitiating 43S complexes move along and scan the spacer.

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When the ribosome translating uORF encounters the stop codon, it should be recognized by eRF1/eRF3 complex that mediates translation termination and peptide release [21, 23, 62, 63]. Then the 60S ribosome subunit is removed with the help of ABCE1/RLI1 protein, followed by deacylated tRNA removal and 40S subunit recycling (Fig. 7C). The latter two steps are mediated by special

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translation factors, eIF2D or MCTS1•DENR heterodimer (TMA64, TMA20•TMA22 in yeast) [26, 30] and is probably the point where a decision is made, whether the 40S is recycled or resumes scanning. It could be that competition between the 40S recycling factors and the canonical translation initiation factor (eIF1, eIF3, eIF5 and eIF2 with Met-tRNAi and GTP bound, together called multifactor complex,

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MFC) determines the outcome [26, 27]. We believe that the above few steps occurring at the stop codon and immediately downstream (Fig. 7, steps 7-10) may be involved in the described novel type

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of mRNA-specific regulation, exemplified by eIF2D and MDM2 translational control under osmotic stress. Intriguingly, both eIF2D uORF and MDM2 uORF1 have a “weak” termination codon UGA-C [64, 65] which could be relevant to this mechanism.

Such regulation is clearly distinct from the previously described cases. In particular, the most

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acknowledged mechanism of uORF dependent translational control, the delayed reinitiation exemplified by the yeast GCN4 and mammalian ATF4 mRNAs, is based on a decreased (delayed) MFC binding during resumed scanning of the uORF-CDS spacers (Fig. 7C, step 11). We clearly show that the

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case we describe here is distinct from the ATF4-like regulation, as well as from ICIER mechanism

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exemplified by IFRD1 (Fig. 7A and 7B). It also differs from that of C/EBPα and C/EBPβ translation regulation [66], as in the latter case a modulation of eIF4E-mediated 43S binding to the 5’ end (Fig. 7, step 2) strongly affects alternative start site selection, while we did not observe any significant changes in eIF2D-Fluc translation response when we altered its 5’ terminal sequence (Fig. 3, GL3_WT45 and CAA_WT-45 constructs), and for the human MDM2 mRNA translation a relatively high resistance to eIF4E inhibitors was reported [67]. The cases of DDIT3 [13] and AdoMetDC [14] mRNAs deal with ribosome stalling during translation elongation while reading uORF (Fig. 7, step 6), the phenomenon distinct from described here, as changing amino acid sequence of eIF2D uORF did not abrogate the regulation (Fig. 3, uORF_6 and uORF_2 constructs). Other known examples of uORF16

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dependent translational tuning under changing conditions are related to alteration of uAUG recognition rate: due to eIF1 modification [15], elevation of intracellular Mg2+ concentration [16] or eIF5 activity [17]. Also, the deficiency in eIF5 or eIF5B activities was reported to provoke 43S sliding from the already recognized AUG codon, thus substantially altering start codon selection on many

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uORF containing mRNAs [18]. Again, all these cases (attributed to steps 4-5 on Fig. 7C) have nothing in common with the described novel regulation pathway, since the latter is not related to modulation of initiation frequency at the uAUG codon (see above). Some uORFs are known to destabilize mRNAs under certain conditions [8], but we excluded explanations related to mRNA stability and decay (Fig.

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2C and 6C), as well.

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Unfortunately, we do not yet understand which modification(s) of translation apparatus during hyperosmotic stress response cause the alterations in termination, ribosome recycling or reinitiation rate, although we showed that some signaling is involved, since the response requires intact cells (Fig. 4). We hypothesize that it could be a modification of some translation initiation, termination or ribosome release factor(s), but we also cannot exclude regulation by a specific mRNA binding protein,

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similar to the recently described msl-2/SXL module [68]. At this stage, we can only speculate about the physiological relevance of the regulated eIF2D and MDM2

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expression under conditions of hyperosmosis. In the latter case, it could be a part of stress response program leading to proliferation pause. Since MDM2 has a very short half-life [69, 70], the protein is

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rapidly eliminated under hyperosmotic conditions, resulting in p53 accumulation and cell cycle arrest [32, 33]. Intriguingly, another important cell cycle regulator, CDK4, is also controlled posttranscriptionally during osmotic stress [71] and could be targeted by the same translation regulation (Fig. 5). A role for eIF2D in the hyperosmotic cell response is obscure, although it could be involved in modulation of protein synthesis that occurs under these conditions [47, 48]. We should also draw the attention to the involvement of this factor in ribosome recycling (see above), one of the points where the new mechanism may operate. Thus, a self-regulating circuit cannot be ruled out.

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In summary, we described a novel type of regulation controlling the human MDM2 and eIF2D mRNAs translation under conditions of hyperosmotic stress. The mechanism requires uORF and is most likely based on delaying translation termination or modulating efficiency of ribosome recycling at its stop

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codon, or changing the proportion of ribosomes that resume scanning under conditions of stress.

Conflicts of interest

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The authors declare no conflict of interest.

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Acknowledgments

We are grateful to K.Vassilenko (Institute for Protein Research RAS) and A.Alexandrov (MSU) for reading the manuscript and valuable suggestions. This work was supported by the Russian Science Foundation (grant RSF 18-14-00291 to S.E.D.) The in vitro translation experiments were supported by the Russian

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Foundation for Basic Research (grant RFBR 16-04-01271 to S.E.D.)

Appendix A. Supplementary data

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Supplementary data to this article can be found online at https://xxxx

References [1] [2] [3]

[4] [5]

K. Wethmar, The regulatory potential of upstream open reading frames in eukaryotic gene expression, Wiley Interdiscip Rev RNA 5 (2014) 765-778. S.K. Young,R.C. Wek, Upstream Open Reading Frames Differentially Regulate Gene-specific Translation in the Integrated Stress Response, J Biol Chem 291 (2016) 16927-16935. D.E. Andreev, P.B. O'Connor, G. Loughran, S.E. Dmitriev, P.V. Baranov,I.N. Shatsky, Insights into the mechanisms of eukaryotic translation gained with ribosome profiling, Nucleic Acids Res 45 (2017) 513-526. G.A. Brar,J.S. Weissman, Ribosome profiling reveals the what, when, where and how of protein synthesis, Nat Rev Mol Cell Biol 16 (2015) 651-664. A. Chugunova, T. Navalayeu, O. Dontsova,P. Sergiev, Mining for Small Translated ORFs, J Proteome Res 17 (2018) 1-11. 18

ACCEPTED MANUSCRIPT

[11] [12]

[13] [14]

[15] [16]

[17] [18]

[19] [20]

AC C

[21]

RI PT

[10]

SC

[9]

M AN U

[8]

TE D

[7]

S.E. Calvo, D.J. Pagliarini,V.K. Mootha, Upstream open reading frames cause widespread reduction of protein expression and are polymorphic among humans, Proc Natl Acad Sci U S A 106 (2009) 7507-7512. A.S. Anisimova, P.M. Rubtsov, K.A. Akulich, S.E. Dmitriev, E. Frolova,A. Tiulpakov, Late Diagnosis of POMC Deficiency and In Vitro Evidence of Residual Translation From Allele With c.-11C>A Mutation, J Clin Endocrinol Metab 102 (2017) 359-362. C. Barbosa, I. Peixeiro,L. Romao, Gene expression regulation by upstream open reading frames and human disease, PLoS Genet 9 (2013) e1003529. A.G. Hinnebusch, I.P. Ivanov,N. Sonenberg, Translational control by 5'-untranslated regions of eukaryotic mRNAs, Science 352 (2016) 1413-1416. D.E. Andreev, P.B. O'Connor, C. Fahey, E.M. Kenny, I.M. Terenin, S.E. Dmitriev, P. Cormican, D.W. Morris, I.N. Shatsky,P.V. Baranov, Translation of 5' leaders is pervasive in genes resistant to eIF2 repression, Elife 4 (2015) e03971. C. Sidrauski, A.M. McGeachy, N.T. Ingolia,P. Walter, The small molecule ISRIB reverses the effects of eIF2alpha phosphorylation on translation and stress granule assembly, Elife 4 (2015). D.E. Andreev, M. Arnold, S.J. Kiniry, G. Loughran, A.M. Michel, D. Rachinskii,P.V. Baranov, TASEP modelling provides a parsimonious explanation for the ability of a single uORF to derepress translation during the integrated stress response, Elife 7 (2018). S.K. Young, L.R. Palam, C. Wu, M.S. Sachs,R.C. Wek, Ribosome Elongation Stall Directs Genespecific Translation in the Integrated Stress Response, J Biol Chem 291 (2016) 6546-6558. J.R. Hill,D.R. Morris, Cell-specific translational regulation of S-adenosylmethionine decarboxylase mRNA. Dependence on translation and coding capacity of the cis-acting upstream open reading frame, J Biol Chem 268 (1993) 726-731. L. Zach, I. Braunstein,A. Stanhill, Stress-induced start codon fidelity regulates arsenite-inducible regulatory particle-associated protein (AIRAP) translation, J Biol Chem 289 (2014) 20706-20716. I.A. Nikonorova, N.V. Kornakov, S.E. Dmitriev, K.S. Vassilenko,A.G. Ryazanov, Identification of a Mg2+-sensitive ORF in the 5'-leader of TRPM7 magnesium channel mRNA, Nucleic Acids Res 42 (2014) 12779-12788. G. Loughran, M.S. Sachs, J.F. Atkins,I.P. Ivanov, Stringency of start codon selection modulates autoregulation of translation initiation factor eIF5, Nucleic Acids Res 40 (2012) 2898-2906. I.M. Terenin, K.A. Akulich, D.E. Andreev, S.A. Polyanskaya, I.N. Shatsky,S.E. Dmitriev, Sliding of a 43S ribosomal complex from the recognized AUG codon triggered by a delay in eIF2-bound GTP hydrolysis, Nucleic Acids Res 44 (2016) 1882-1893. A.G. Hinnebusch, Structural Insights into the Mechanism of Scanning and Start Codon Recognition in Eukaryotic Translation Initiation, Trends Biochem Sci 2017). A.G. Hinnebusch, The scanning mechanism of eukaryotic translation initiation, Annu Rev Biochem 83 (2014) 779-812. C.U.T. Hellen, Translation Termination and Ribosome Recycling in Eukaryotes, Cold Spring Harb Perspect Biol 2018). S. Gunisova, V. Hronova, M.P. Mohammad, A.G. Hinnebusch,L.S. Valasek, Please do not recycle! Translation reinitiation in microbes and higher eukaryotes, FEMS Microbiol Rev 42 (2018) 165192. T.E. Dever,R. Green, The elongation, termination, and recycling phases of translation in eukaryotes, Cold Spring Harb Perspect Biol 4 (2012) a013706. M.A. Skabkin, O.V. Skabkina, C.U. Hellen,T.V. Pestova, Reinitiation and other unconventional posttermination events during eukaryotic translation, Mol Cell 51 (2013) 249-264. S. Schleich, K. Strassburger, P.C. Janiesch, T. Koledachkina, K.K. Miller, K. Haneke, Y.S. Cheng, K. Kuechler, G. Stoecklin, K.E. Duncan,A.A. Teleman, DENR-MCT-1 promotes translation reinitiation downstream of uORFs to control tissue growth, Nature 512 (2014) 208-212. D.J. Young, D.S. Makeeva, F. Zhang, A.S. Anisimova, E.A. Stolboushkina, F. Ghobakhlou, I.N. Shatsky, S.E. Dmitriev, A.G. Hinnebusch,N.R. Guydosh, Tma64/eIF2D, Tma20/MCT-1, and Tma22/DENR Recycle Post-termination 40S Subunits In Vivo, Mol Cell 71 (2018) 761-774 e765.

EP

[6]

[22]

[23]

[24] [25]

[26]

19

[32] [33]

[34]

[35]

[36]

[37]

[38]

[39] [40]

AC C

[41]

RI PT

[31]

SC

[30]

M AN U

[29]

TE D

[28]

ACCEPTED MANUSCRIPT

I.B. Lomakin, E.A. Stolboushkina, A.T. Vaidya, C. Zhao, M.B. Garber, S.E. Dmitriev,T.A. Steitz, Crystal Structure of the Human Ribosome in Complex with DENR-MCT-1, Cell Rep 20 (2017) 521528. M. Weisser, T. Schafer, M. Leibundgut, D. Bohringer, C.H.S. Aylett,N. Ban, Structural and Functional Insights into Human Re-initiation Complexes, Mol Cell 2017). S.E. Dmitriev, I.M. Terenin, D.E. Andreev, P.A. Ivanov, J.E. Dunaevsky, W.C. Merrick,I.N. Shatsky, GTP-independent tRNA delivery to the ribosomal P-site by a novel eukaryotic translation factor, J Biol Chem 285 (2010) 26779-26787. M.A. Skabkin, O.V. Skabkina, V. Dhote, A.A. Komar, C.U. Hellen,T.V. Pestova, Activities of Ligatin and MCT-1/DENR in eukaryotic translation initiation and ribosomal recycling, Genes Dev 24 (2010) 1787-1801. W. Hu, Z. Feng,A.J. Levine, The Regulation of Multiple p53 Stress Responses is Mediated through MDM2, Genes Cancer 3 (2012) 199-208. M. Iizuka, O.F. Sarmento, T. Sekiya, H. Scrable, C.D. Allis,M.M. Smith, Hbo1 Links p53-dependent stress signaling to DNA replication licensing, Mol Cell Biol 28 (2008) 140-153. H. Kishi, K. Nakagawa, M. Matsumoto, M. Suga, M. Ando, Y. Taya,M. Yamaizumi, Osmotic shock induces G1 arrest through p53 phosphorylation at Ser33 by activated p38MAPK without phosphorylation at Ser15 and Ser20, J Biol Chem 276 (2001) 39115-39122. S.E. Dmitriev, D.E. Andreev, Z.V. Adyanova, I.M. Terenin,I.N. Shatsky, Efficient cap-dependent translation of mammalian mRNAs with long and highly structured 5'-untranslated regions in vitro and in vivo, Mol Biol (Mosk) 43 (2009) 108-113. L.I. Shagam, I.M. Terenin, D.E. Andreev, J.E. Dunaevsky,S.E. Dmitriev, In vitro activity of human translation initiation factor eIF4B is not affected by phosphomimetic amino acid substitutions S422D and S422E, Biochimie 94 (2012) 2484-2490. I.V. Prokhorova, K.A. Akulich, D.S. Makeeva, I.A. Osterman, D.A. Skvortsov, P.V. Sergiev, O.A. Dontsova, G. Yusupova, M.M. Yusupov,S.E. Dmitriev, Amicoumacin A induces cancer cell death by targeting the eukaryotic ribosome, Sci Rep 6 (2016) 27720. S.E. Dmitriev, D.E. Andreev, I.M. Terenin, I.A. Olovnikov, V.S. Prassolov, W.C. Merrick,I.N. Shatsky, Efficient translation initiation directed by the 900-nucleotide-long and GC-rich 5' untranslated region of the human retrotransposon LINE-1 mRNA is strictly cap dependent rather than internal ribosome entry site mediated, Mol Cell Biol 27 (2007) 4685-4697. K.A. Akulich, D.E. Andreev, I.M. Terenin, V.V. Smirnova, A.S. Anisimova, D.S. Makeeva, V.I. Arkhipova, E.A. Stolboushkina, M.B. Garber, M.M. Prokofjeva, P.V. Spirin, V.S. Prassolov, I.N. Shatsky,S.E. Dmitriev, Four translation initiation pathways employed by the leaderless mRNA in eukaryotes, Sci Rep 6 (2016) 37905. M. Kozak, The scanning model for translation: an update, J Cell Biol 108 (1989) 229-241. A.M. Michel, S.J. Kiniry, P.B.F. O'Connor, J.P. Mullan,P.V. Baranov, GWIPS-viz: 2018 update, Nucleic Acids Res 46 (2018) D823-D830. O. Meyuhas,T. Kahan, The race to decipher the top secrets of TOP mRNAs, Biochim Biophys Acta 1849 (2015) 801-811. D.E. Andreev, I.M. Terenin, S.E. Dmitriev,I.N. Shatsky, Pros and cons of pDNA and mRNA transfection to study mRNA translation in mammalian cells, Gene 578 (2016) 1-6. D.E. Andreev, S.E. Dmitriev, I.M. Terenin, V.S. Prassolov, W.C. Merrick,I.N. Shatsky, Differential contribution of the m7G-cap to the 5' end-dependent translation initiation of mammalian mRNAs, Nucleic Acids Res 37 (2009) 6135-6147. M. Kozak, Circumstances and mechanisms of inhibition of translation by secondary structure in eucaryotic mRNAs, Mol Cell Biol 9 (1989) 5134-5142. M.B. Burg, J.D. Ferraris,N.I. Dmitrieva, Cellular response to hyperosmotic stresses, Physiol Rev 87 (2007) 1441-1474. R.R. Alfieri,P.G. Petronini, Hyperosmotic stress response: comparison with other cellular stresses, Pflugers Arch 454 (2007) 173-185. E. Robbins, T. Pederson,P. Klein, Comparison of mitotic phenomena and effects induced by hypertonic solutions in HeLa cells, J Cell Biol 44 (1970) 400-416.

EP

[27]

[42] [43]

[44] [45] [46] [47]

20

[53] [54]

[55]

[56]

[57] [58]

[59]

[60] [61] [62]

AC C

[63]

RI PT

[52]

SC

[51]

M AN U

[50]

TE D

[49]

ACCEPTED MANUSCRIPT

M. Kozak, Leader length and secondary structure modulate mRNA function under conditions of stress, Mol Cell Biol 8 (1988) 2737-2744. Q. Cai, J.D. Ferraris,M.B. Burg, High NaCl increases TonEBP/OREBP mRNA and protein by stabilizing its mRNA, Am J Physiol Renal Physiol 289 (2005) F803-807. M. Kozak, Constraints on reinitiation of translation in mammals, Nucleic Acids Res 29 (2001) 5226-5232. C.Y. Brown, G.J. Mize, M. Pineda, D.L. George,D.R. Morris, Role of two upstream open reading frames in the translational control of oncogene mdm2, Oncogene 18 (1999) 5631-5637. X. Jin, E. Turcott, S. Englehardt, G.J. Mize,D.R. Morris, The two upstream open reading frames of oncogene mdm2 have different translational regulatory properties, J Biol Chem 278 (2003) 25716-25721. L. Bernstam,J. Nriagu, Molecular aspects of arsenic stress, J Toxicol Environ Health B Crit Rev 3 (2000) 293-322. C.O. Brostrom, C.R. Prostko, R.J. Kaufman,M.A. Brostrom, Inhibition of translational initiation by activators of the glucose-regulated stress protein and heat shock protein stress response systems. Role of the interferon-inducible double-stranded RNA-activated eukaryotic initiation factor 2alpha kinase, J Biol Chem 271 (1996) 24995-25002. H.Y. Jiang, L. Jiang,R.C. Wek, The eukaryotic initiation factor-2 kinase pathway facilitates differential GADD45a expression in response to environmental stress, J Biol Chem 282 (2007) 3755-3765. L. Lu, A.P. Han,J.J. Chen, Translation initiation control by heme-regulated eukaryotic initiation factor 2alpha kinase in erythroid cells under cytoplasmic stresses, Mol Cell Biol 21 (2001) 79717980. I.M. Terenin, S.E. Dmitriev, D.E. Andreev,I.N. Shatsky, Eukaryotic translation initiation machinery can operate in a bacterial-like mode without eIF2, Nat Struct Mol Biol 15 (2008) 836-841. E. McEwen, N. Kedersha, B. Song, D. Scheuner, N. Gilks, A. Han, J.J. Chen, P. Anderson,R.J. Kaufman, Heme-regulated inhibitor kinase-mediated phosphorylation of eukaryotic translation initiation factor 2 inhibits translation, induces stress granule formation, and mediates survival upon arsenite exposure, J Biol Chem 280 (2005) 16925-16933. H.P. Harding, I. Novoa, Y. Zhang, H. Zeng, R. Wek, M. Schapira,D. Ron, Regulated translation initiation controls stress-induced gene expression in mammalian cells, Mol Cell 6 (2000) 10991108. P.D. Lu, H.P. Harding,D. Ron, Translation reinitiation at alternative open reading frames regulates gene expression in an integrated stress response, J Cell Biol 167 (2004) 27-33. K.M. Vattem,R.C. Wek, Reinitiation involving upstream ORFs regulates ATF4 mRNA translation in mammalian cells, Proc Natl Acad Sci U S A 101 (2004) 11269-11274. R.J. Jackson, C.U. Hellen,T.V. Pestova, Termination and post-termination events in eukaryotic translation, Adv Protein Chem Struct Biol 86 (2012) 45-93. S. Franckenberg, T. Becker,R. Beckmann, Structural view on recycling of archaeal and eukaryotic ribosomes after canonical termination and ribosome rescue, Curr Opin Struct Biol 22 (2012) 786796. K.K. McCaughan, C.M. Brown, M.E. Dalphin, M.J. Berry,W.P. Tate, Translational termination efficiency in mammals is influenced by the base following the stop codon, Proc Natl Acad Sci U S A 92 (1995) 5431-5435. A.G. Cridge, C. Crowe-McAuliffe, S.F. Mathew,W.P. Tate, Eukaryotic translational termination efficiency is influenced by the 3' nucleotides within the ribosomal mRNA channel, Nucleic Acids Res 46 (2018) 1927-1944. C.F. Calkhoven, C. Muller,A. Leutz, Translational control of C/EBPalpha and C/EBPbeta isoform expression, Genes Dev 14 (2000) 1920-1932. R. Genolet, G. Rahim, P. Gubler-Jaquier,J. Curran, The translational response of the human mdm2 gene in HEK293T cells exposed to rapamycin: a role for the 5'-UTRs, Nucleic Acids Res 39 (2011) 989-1003. J. Medenbach, M. Seiler,M.W. Hentze, Translational control via protein-regulated upstream open reading frames, Cell 145 (2011) 902-913.

EP

[48]

[64]

[65]

[66] [67]

[68]

21

[70]

[71]

[72]

ACCEPTED MANUSCRIPT

Y. Pan,D.S. Haines, The pathway regulating MDM2 protein degradation can be altered in human leukemic cells, Cancer Res 59 (1999) 2064-2067. D.C. Olson, V. Marechal, J. Momand, J. Chen, C. Romocki,A.J. Levine, Identification and characterization of multiple mdm-2 proteins and mdm-2-p53 protein complexes, Oncogene 8 (1993) 2353-2360. G.Z. Tao, L.S. Rott, A.W. Lowe,M.B. Omary, Hyposmotic stress induces cell growth arrest via proteasome activation and cyclin/cyclin-dependent kinase degradation, J Biol Chem 277 (2002) 19295-19303. M. Zuker, Mfold web server for nucleic acid folding and hybridization prediction, Nucleic Acids Res 31 (2003) 3406-3415.

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[69]

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Figure Legends

Fig. 1. Structure and translational properties of reporter mRNA constructs with the human eIF2D mRNA

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leader. (A) Schematic representation of luciferase mRNAs with long and short variants of the eIF2D 5’ UTR. (B) Translation efficiency of the mRNA constructs as compared to that of luciferase mRNA with rabbit β-globin 5’ UTR. HEK293T cells were transfected with capped polyadenylated transcripts, together with the similarly prepared Actin-Rluc mRNA. Fluc activity was measured after 2 h incubation,

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normalized to Rluc activity units and represented as a ratio to the value for the eIF2D_short construct. (C) Predicted secondary structure of the short eIF2D 5’ UTR variant. The prediction was made using the MFold server (http://unafold.rna.albany.edu/?q=mfold/rna-folding-form, ref.[72]). uAUG and main AUG

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codons are shown by circles with arrows inside (white and black, respectively), uORF stop codon is

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depicted as STOP traffic sign.

Fig. 2. Differential inhibition of reporter mRNA translation in HEK293T cells under conditions of hyperosmotic stress. (A) The primary screening of stress inducers revealed hyperosmosis as a condition specifically affecting eIF2D 5’ UTR directed translation in mammalian cells. HEK293T cells were transfected by a mixture of eIF2D-Fluc and GL3-Rluc mRNAs. Various stresses were applied immediately before the transfection (except for Etoposide, Thymidine and Hydroxyurea that were added 6 h earlier). 2 h after transfection, the cells were harvested; Fluc and Rluc activity were measured and normalized to the values in Control. Final concentrations of inducers in the medium are shown. (B) Capped 22

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polyadenylated eIF2D-Fluc and Actin-Rluc mRNAs were co-transfected to HEK293T cells exposed to the increasing concentrations of NaCl (additional to that already contained in the medium). NaCl solution was added 5 min before the transfection. Fluc and Rluc activities measured 2 h after the transfection are represented as a ratio to the values in unstressed cells. (C) Time-course of Fluc to Rluc ratio in the same

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experiment under normal (“Control”) and stress (NaCl concentration increased by 50 mM) conditions. Cells were taken for the analysis every hour during a 4 h incubation period following the transfection. The value for the unstressed cells at 1 h time point was taken as 1. (D) The same experiment as in (B) was performed with the mixture of eIF2D No_uORF-Fluc and Actin-Rluc reporters. For all panels, 3

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replicates each containing 2 or more mRNA preparations, were used for each experiment. The mean

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values ± SD are shown.

Fig. 3. Translation efficiency and stress sensitivity of mRNA reporters with eIF2D 5’ UTR derivatives. The reporter mRNAs (schematically shown in the middle) were introduced into HEK293T cells for 2 h under

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normal or both normal and hyperosmotic (75 mM NaCl excess, right panel) conditions. Fluc activities were measured and normalized to Rluc values. In the left panel, the Fluc/Rluc values for different constructs under normal conditions are shown (with that for eIF2D WT taken as 100 units). On the right,

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Fluc/Rluc ratios for stressed and control cells are presented for each constructs separately, with the latter taken as 100 units. On the schematics, black boxes indicate sequence of eIF2D coding region,

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uORFs are shown by grey boxes, arrows point to nucleotide changes, wavy lines denote additional exogenous sequences.

Fig. 4. Effects of NaCl addition on mRNA translation in a mammalian cell-free system. Capped polyadenylated eIF2D WT and eIF2D No_uORF mRNAs were translated in S30 extract of mouse Krebs-2 ascite cells for 1 h. The luciferase activities were continuously measured, the final values were determined as described in Materials and Methods section, and normalized to the values obtained in the reaction mixture supplemented with water. 23

ACCEPTED MANUSCRIPT Fig. 5. Translation efficiency and sensitivity of mRNA reporters with a number of human uORFcontaining 5’ UTRs (shown in the middle). The transcripts were introduced into HEK293T cells for 2 h under normal or both normal and hyperosmotic (75 mM NaCl excess, right panel) conditions. Fluc/Rluc

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values were calculated and presented as in Fig. 3.

Fig. 6. Translation efficiency and sensitivity of mRNA reporters with MDM2 5’ UTR derivatives. (A) The

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transcripts (shown in the middle) were introduced into HEK293T cells for 2 h under normal or both normal and hyperosmotic (75 mM NaCl excess, right panel) conditions. Fluc/Rluc values were calculated

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and presented as in Figs. 3 and 4. (B) A comprehensive analysis of MDM2-Fluc and Actin-Rluc translation sensitivity to increasing NaCl concentration. Fluc and Rluc activities are depicted as in Fig. 2B. (C) Timecourse (1 to 4 h) of Fluc to Rluc ratio in FLERT experiment under normal and stress conditions. The value for the unstressed cells at 1 h time point was taken as 1. (D) The same experiment as in (A) was

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performed with the indicated MDM2-Fluc derivatives.

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Fig. 7. Novel translation regulation mechanism operates on mRNAs with the human eIF2D and MDM2 5’ UTRs. (A) Differential translational response of five Fluc encoding mRNAs to the arsenic (III)-induced

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oxidative stress. FLERT analysis was performed with the indicated mRNA constructs in HEK293T cells. Sodium dihydroarsenite (NaH2AsO3) was added ~5 min before the transfection. After 2h incubation, Fluc activities were measured and normalized to the values produced by unstressed cells. Translational resistance/activation of ATF4-Fluc and IFRD1-Fluc contrasts to a luciferase activity decrease in the case of eIF2D-Fluc, MDM2-Fluc and Actin-Fluc. (B) Translation of the same mRNA constructs was analyzed under conditions of hyperosmotic stress. A different translational response of eIF2D-Fluc and MDM2Fluc is revealed. (C) A model of translation reinitiation after uORF translation. Steps 1 and 2, 40S acquires the multifactor complex (eIF1, eIF3, eIF5 and eIF2-GTP-Met-tRNAi) and binds to the capped 5’ end of the mRNA. Steps 3 and 4, scanning and uAUG recognition occurs, followed by productive 80S 24

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complex formation (step 5). Alternatively, uAUG can be skipped by leaky scanning (if no AUG recognition occurs) or 43S sliding (if eIF2-bound GTP hydrolysis is delayed after successful AUG recognition). Step 6, the 80S ribosome elongates the uORF encoded peptide until the stop codon is encountered. Here, translation termination and peptide release take place (step 7), followed by dissociation of the 60S

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subunit (step 8). After that, the remaining deacylated tRNA should be evicted from the P-site of the 40S subunit, and then two outcomes are normally allowed: 40S should either dissociate and be recycled (step 9) or resume movement along the mRNA (step 10). During this futile movement, it acquires another MFC (step 11), which makes it initiation-competent. The newly formed 43S complex scans the

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spacer (step 12). This process can culminate in recognition of the next start codon (represented here by AUG codon of the main CDS, step 13), 60S joining and another round of elongation (steps 14-15). The

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process can be regulated at different steps, as indicated by colors and illustrated by specific examples described in the text. The steps which are regulated in the case of eIF2D and MDM2 mRNAs, as

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ACCEPTED MANUSCRIPT uORFs regulate translation of more than half of human mRNAs A novel uORF-based regulatory mechanism is employed by human MDM2 and eIF2D mRNAs Translation of MDM2 and eIF2D mRNAs is specifically inhibited during osmotic stress The inhibition is based on events occurring at uORF stop codon or downstream

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Translation repression of MDM2 mRNA contributes to stress-induced cell cycle arrest