mRNA Translation Elongation Research Articles

The eEF2 kinase coordinates the DNA damage response to cisplatin by supporting p53 activation

Eukaryotic elongation factor 2 (eEF2) kinase (eEF2K) is a stress-responsive hub that inhibits the translation elongation factor eEF2, and consequently mRNA translation elongation, in response to hypoxia and nutrient deprivation. EEF2K is also involved in the response to DNA damage but its role in response to DNA crosslinks, as induced by cisplatin, is not known. Here we found that eEF2K is critical to mediate the cellular response to cisplatin. We uncovered that eEF2K deficient cells are more resistant to cisplatin treatment. Mechanistically, eEF2K deficiency blunts the activation of the DNA damage response associated ATM and ATR pathways, in turn preventing p53 activation and therefore compromising induction of cisplatin-induced apoptosis. We also report that loss of eEF2K delays the resolution of DNA damage triggered by cisplatin, suggesting that eEF2K contributes to DNA damage repair in response to cisplatin. In support of this, our data shows that eEF2K promotes the expression of the DNA repair protein ERCC1, critical for the repair of cisplatin-caused DNA damage. Finally, using Caenorhabditis elegans as an in vivo model, we find that deletion of efk-1, the worm eEF2K ortholog, mitigates the induction of germ cell death in response to cisplatin. Together, our data highlight that eEF2K represents an evolutionary conserved mediator of the DNA damage response to cisplatin which promotes p53 activation to induce cell death, or alternatively facilitates DNA repair, depending on the extent of DNA damage.

Abstract B044: Elongation control of mRNA translation drives Group 3 medulloblastoma adaptation to nutrient deprivation

Abstract Background: Group 3 affiliation and MYC genetic amplification are associated with poor life expectancy and substantial morbidity in children suffering from medulloblastoma (MB). The high metabolic demand induced by MYC-driven transformation sensitizes MYC-overexpressing MB to cell death under conditions of nutrient deprivation (ND). Additionally, MYC-driven transformation is known to promote mitochondrial oxidative phosphorylation (OXPHOS). We previously reported that eukaryotic Elongation Factor Kinase 2 (eEF2K), the master regulator of mRNA translation elongation, promotes survival of MYC-overexpressing tumors under ND. Interestingly, eEF2K is overexpressed in MYC-driven MB and our preliminary proteomics data highlight large-scale alterations in OXPHOS components affecting eEF2K deficient MB cells. We therefore hypothesized that eEF2K activity is required for the selective translation of mRNAs needed for efficient OXPHOS, and for the progression of MYC-driven MB. METHODS: Multiplexed enhanced Protein Dynamic Mass Spectrometry performed in eEF2K knockdown MYC-overexpressing D425 MB cells to identify mRNAs selectively translated upon eEF2K activation. Time course experiments under ND were conducted in eEF2K knockout (KO) D425 cells to assess the presence of electron transport chain (ETC) complexes I-IV in their native state (via BN-PAGE), as well as transcript expression of individual ETC complex components (by qPCR). The effects of eEF2K inactivation on oxygen consumption, metabolic fluxes and mitochondrial membrane potential were studied with Seahorse technology and JC1/TMRE staining. The viability of eEF2K KO D425 cells was assessed by Incucyte system. Finally, MB orthotopic xenograft mouse models were used to confirm in vitro observations. RESULTS: Multiple (9 out of 10 detected) components of the mitochondrial OXPHOS pathway are selectively translated upon eEF2K activation. Inactivation of eEF2K by genetic KO leads to the disassembly of ETC complexes I-IV without affecting mRNA levels of their respective components. Consistently, eEF2K KO MB cells display decreased mitochondrial membrane potential and ~20% increased proton leak through the mitochondrial membrane. In addition, eEF2K inactivation results in increased D425 cell death under ND and doubles survival of MB bearing mice fed with calorie restricted diets (p<0.05). CONCLUSION: Control of mRNA translation elongation by eEF2K is critical for mitochondrial ETC complex assembly and efficient OXPHOS in MYC-overexpressing MB, likely representing an adaptive response by which MYC-driven MB cells cope with acute metabolic stress. Future therapeutic studies will aim to combine eEF2K inhibition with caloric restriction mimetic drugs as eEF2K activity appears critical under metabolic stress conditions. Citation Format: Alberto Delaidelli, Fares Burwag, Jessica Oliveira de Santis, Betty Yao, Haifeng Zhang, Yue Zhou Huang, Gian Luca Negri, Christopher Hughes, Gabriel Leprivier, Poul Sorensen. Elongation control of mRNA translation drives Group 3 medulloblastoma adaptation to nutrient deprivation [abstract]. In: Proceedings of the AACR Special Conference on Brain Cancer; 2023 Oct 19-22; Minneapolis, Minnesota. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_1):Abstract nr B044.

FAM86A methylation of eEF2 links mRNA translation elongation to tumorigenesis

eEF2 post-translational modifications (PTMs) can profoundly affect mRNA translation dynamics. However, the physiologic function of eEF2K525 trimethylation (eEF2K525me3), a PTM catalyzed by the enzyme FAM86A, is unknown. Here, we find that FAM86A methylation of eEF2 regulates nascent elongation to promote protein synthesis and lung adenocarcinoma (LUAD) pathogenesis. The principal physiologic substrate of FAM86A is eEF2, with K525me3 modeled to facilitate productive eEF2-ribosome engagement during translocation. FAM86A depletion in LUAD cells causes 80S monosome accumulation and mRNA translation inhibition. FAM86A is overexpressed in LUAD and eEF2K525me3 levels increase through advancing LUAD disease stages. FAM86A knockdown attenuates LUAD cell proliferation and suppression of the FAM86A-eEF2K525me3 axis inhibits cancer cell and patient-derived LUAD xenograft growth invivo. Finally, FAM86A ablation strongly attenuates tumor growth and extends survival in KRASG12C-driven LUAD mouse models. Thus, our work uncovers an eEF2 methylation-mediated mRNA translation elongation regulatory node and nominates FAM86A as an etiologic agent in LUAD.

TMET-08. ELONGATION CONTROL OF MRNA TRANSLATION DRIVES GROUP 3 MEDULLOBLASTOMA ADAPTATION TO NUTRIENT DEPRIVATION

Abstract Group 3 affiliation and MYC genetic amplification are associated with poor life expectancy and substantial morbidity in children suffering from medulloblastoma (MB). However, the high metabolic demand induced by MYC-driven transformation sensitizes MYC-overexpressing MB to cell death under conditions of nutrient deprivation (ND). Additionally, MYC-driven transformation is known to promote mitochondrial oxidative phosphorylation (OXPHOS). We previously reported that eukaryotic Elongation Factor Kinase 2 (eEF2K), the master regulator of mRNA translation elongation, promotes survival of MYC-overexpressing tumors under ND. Interestingly, eEF2K is overexpressed in MYC-driven MB and our preliminary proteomics data highlight large-scale alterations in OXPHOS components affecting eEF2K deficient MB cells. We therefore hypothesized that eEF2K activity is required for the selective translation of mRNAs needed for efficient OXPHOS, and for the progression of MYC-driven MB. We performed Multiplexed enhanced Protein Dynamic Mass Spectrometry in eEF2K knockdown MYC-overexpressing D425 MB cells to identify mRNAs selectively translated upon eEF2K activation. Messenger RNAs encoding multiple (9 out of 10 detected) components of the mitochondrial OXPHOS pathway are selectively translated upon eEF2K activation. Inactivation of eEF2K by genetic KO leads to the disassembly of electron transport chain (ETC) complexes I-IV without affecting mRNA levels of their respective components. Consistently, eEF2K KO MB cells display decreased mitochondrial membrane potential and 20% increased proton leak thorough the mitochondrial membrane. In addition, eEF2K inactivation results in increased Group 3 MB cell death under ND and doubles survival of MB bearing mice fed with calorie restricted diets (p< 0.05). Control of mRNA translation elongation by eEF2K is critical for mitochondrial ETC complex assembly and efficient OXPHOS in MYC-overexpressing MB, likely representing an adaptive response by which MYC-driven MB cells cope with acute metabolic stress. Future therapeutic studies will aim to combine eEF2K inhibition with caloric restriction mimetic drugs as eEF2K activity appears critical under metabolic stress conditions.

TMET-16. ELUCIDATING MECHANISMS OF ENHANCED OXIDATIVE PHOSPHORYLATION IN MYC-AMPLIFIED MEDULLOBLASTOMA

Abstract Medulloblastoma (MBL) is the most commonly diagnosed pediatric brain tumour and the leading cause of childhood cancer deaths. MYC overexpression often accompanies poor prognosis in MBL patients, as MYC-driven transformation is known to promote mitochondrial oxidative phosphorylation (OXPHOS). However, it also induces a high metabolic demand that impairs cell survival under acute nutrient deprivation. We previously reported that eukaryotic Elongation Factor Kinase 2 (eEF2K), the master regulator of mRNA translation elongation, is overexpressed in MYC-driven MBL. Preliminary data suggests a relationship between eEF2K-mediated selective translation of mRNAs needed for efficient OXPHOS, and for the progression of MYC-driven MBL. Multiplexed enhanced Protein Dynamic Mass Spectrometry was carried out in eEF2K knockdown MYC-overexpressing D425 MB cells to identify mRNAs selectively translated upon eEF2K activation. 9 of 10 detected components of the OXPHOS pathway were selectively translated in eEF2K-active MBL cells. Genetic eEF2K knockout led to the disassembly of electron transport chain (ETC) complexes I-V and reduced complex activity, as observed via Blue Native PAGE and colorimetric assays. Furthermore, siRNA knockdown of Cytochrome c oxidase assembly factor 7 (COA7), a mitochondrial OXPHOS assembly factor differentially expressed in eEF2K active or inactive D425 cells, produces the same pattern of supercomplex (SC) loss as the inactivation of eEF2K in wild-type D425 cells. The assembly of ETC SC across D425 eEF2K active and inactive cells was further studied using blue native gel-based mass spectrometry to further characterize patterns in SC loss. In summary, control of translation elongation by eEF2K is critical for mitochondrial ETC complex assembly and efficient OXPHOS in MYC-overexpressing MBL, likely representing an adaptive response against acute metabolic stress. Future therapeutic studies will aim to combine eEF2K inhibition with caloric restriction mimetics.

SAT075 Beta Cells Adapt To Insulin Resistance Via Posttranslational Modification Of The Translation Factor eIF5A

Abstract Disclosure: C. Anderson: None. A. Kulkarni: None. B. Maier: None. S.A. Tersey: None. R. Anderson: None. R.G. Mirmira: None. Prior to type 2 diabetes onset, β cells adapt to insulin resistance through compensation—a process that maintains insulin secretion and glucose homeostasis. Our lab has previously shown that β cell compensation requires the activity of deoxyhypusine synthase (DHPS), which post-translationally catalyzes the formation of the amino acid hypusine at Lys50 of eukaryotic initiation factor eIF5A. Although hypusinated eIF5A is required for β cell compensation, it is unclear if unhypusinated eIF5A limits this compensatory response. To identify the role of unhypusinated eIF5A, we used the following animal and cell-based models: RNAi knockdown and transgenic zebrafish; inducible, β cell-specific knockout mice fed a high fat diet; and cultured HEK-293 cells. Zebrafish embryos injected with morpholinos to reduce global DHPS (and accumulate unhypusinated eIF5A) showed stunted exocrine pancreas growth at 3 days post-fertilization. Although, those injected with anti-eIF5A morpholinos (to deplete all eIF5A) showed normal pancreas growth. Transgenic overexpression of a mutant eIF5A incapable of hypusination resulted in reduced pancreas size in zebrafish. Although a unique function of unhypusinated eIF5A has not yet been documented, these findings suggest that the presence of unhypusinated eIF5A may be the major driver of altered pancreas phenotypes. Similarly, following 4 weeks of high fat diet feeding and obesity, mice lacking total eIF5A in β cells had improved glucose tolerance compared to mice lacking DHPS in β cells, despite similar weight gain and insulin sensitivity. To clarify the molecular mechanisms behind the damaging role of unhypusinated eIF5A, we performed polyribosome profiling to measure translation in lysates from HEK-293 cells in which either DHPS or total eIF5A were depleted by RNAi. Whereas loss of total eIF5A slightly reduced mRNA translation elongation, loss of DHPS (and accumulation of unhypusinated eIF5A) reduced mRNA translation initiation. Further experiments in HEK-293 cells suggest that unhypusinated eIF5A increases levels of phosphorylated eIF2α (an inhibitor of global mRNA translation) by specifically interacting with the kinase GCN2. Taken together, our data provide evidence that DHPS deficiency and obesity conditions impair β cell function, in part, from the accumulation of the unhypusinated form of eIF5A. Our studies reveal a mechanism in which β cells respond to obesity by regulating mRNA translation through the balance between hypusinated and unhypusinated forms of eIF5A. Presentation: Saturday, June 17, 2023

Antibody toolkit to investigate eEF1A methylation dynamics in mRNA translation elongation

Protein synthesis is a fundamental step in gene expression, with modulation of mRNA translation at the elongation step emerging as an important regulatory node in shaping cellular proteomes. In this context, five distinct lysine methylation events on eukaryotic elongation factor 1A (eEF1A), a fundamental nonribosomal elongation factor, are proposed to influence mRNA translation elongation dynamics. However, a lack of affinity tools has hindered progress in fully understanding how eEF1A lysine methylation impacts protein synthesis. Here we develop and characterize a suite of selective antibodies to investigate eEF1A methylation and provide evidence that methylation levels decline in aged tissue. Determination of the methyl state and stoichiometry on eEF1A in various cell lines by mass spectrometry shows modest cell-to-cell variability. We also find by Western blot analysis that knockdown of individual eEF1A-specific lysine methyltransferases leads to depletion of the cognate lysine methylation event and indicates active crosstalk between different sites. Further, we find that the antibodies are specific in immunohistochemistry applications. Finally, application of the antibody toolkit suggests that several eEF1A methylation events decrease in aged muscle tissue. Together, our study provides a roadmap for leveraging methyl state and sequence-selective antibody reagents to accelerate discovery of eEF1A methylation-related functions and suggests a role for eEF1A methylation, via protein synthesis regulation, in aging biology.

TMET-33. ELONGATION CONTROL OF MRNA TRANSLATION SUPPORTS GROUP 3 MEDULLOBLASTOMA ADAPTATION TO NUTRIENT DEPRIVATION

Abstract Group 3 affiliation and MYC genetic amplification are associated with poor life expectancy and substantial morbidity in children suffering from medulloblastoma (MB). However, the high metabolic demand induced by MYC-driven transformation sensitizes MYC-overexpressing MB to cell death under conditions of nutrient deprivation (ND). Additionally, MYC-driven transformation is known to promote mitochondrial oxidative phosphorylation (OXPHOS). We previously reported that eukaryotic Elongation Factor Kinase 2 (eEF2K), the master regulator of mRNA translation elongation, promotes survival of MYC-overexpressing tumors under ND. Interestingly, eEF2K is overexpressed in MYC-driven MB and our preliminary proteomics data highlight large-scale alterations in OXPHOS components affecting eEF2K deficient MB cells. We therefore hypothesized that eEF2K activity is required for the selective translation of mRNAs needed for efficient OXPHOS, and for the progression of MYC-driven MB. We performed Multiplexed enhanced Protein Dynamic Mass Spectrometry in eEF2K knockdown MYC-overexpressing D425 MB cells to identify mRNAs selectively translated upon eEF2K activation. Messenger RNAs encoding multiple (9 out of 10 detected) components of the mitochondrial OXPHOS pathway are selectively translated upon eEF2K activation. Inactivation of eEF2K by genetic KO leads to the disassembly of electron transport chain (ETC) complexes I-IV without affecting mRNA levels of their respective components. Consistently, eEF2K KO MB cells display decreased mitochondrial membrane potential and 20% increased proton leak thorough the mitochondrial membrane. In addition, eEF2K inactivation results in increased Group 3 MB cell death under ND and doubles survival of MB bearing mice fed with calorie restricted diets (p< 0.05). Control of mRNA translation elongation by eEF2K is critical for mitochondrial ETC complex assembly and efficient OXPHOS in MYC-overexpressing MB, likely representing an adaptive response by which MYC-driven MB cells cope with acute metabolic stress. Future therapeutic studies will aim to combine eEF2K inhibition with caloric restriction mimetic drugs as eEF2K activity appears critical under metabolic stress conditions.

Manipulation of mRNA translation elongation influences the fragmentation of a biotherapeutic Fc-fusion protein produced in CHO cells.

Mammalian cells, particularly Chinese hamster ovary cells, are the dominant system for the production of protein-based biotherapeutics, however, product degradation, particularly of Fc-fusion proteins, is sometimes observed that impacts the quality of the protein generated. Here, we identify the site of fragmentation of a model immunoglobulin G1 Fc-fusion protein, show that the observed clipping and aggregation are decreased by reduced temperature culturing, that the fragmentation/clipping is intracellular, and that reduced clipping at a lower temperature (<37°C) relates to mesenger RNA (mRNA) translation elongation. We subsequently show that reduced fragmentation can be achieved at 37°C by addition of chemical reagents that slow translation elongation. We then modified mRNA translation elongation speeds by designing different transcript sequences for the Fc-fusion protein based on alternative codon usage and improved the product yield at 37°C, and the ratio of intact to a fragmented product. Our data suggest that rapid elongation results in misfolding that decreases product fidelity, generating a region susceptible to degradation/proteolysis, whilst the slowing of mRNA translation improves the folding, reducing susceptibility to fragmentation. Manipulation of mRNA translation and/or the target Fc-fusion transcript is, therefore, an approach that can be applied to potentially reduce fragmentation of clipping-prone Fc-fusion proteins.

Recognition of 3′ nucleotide context and stop codon readthrough are determined during mRNA translation elongation

The nucleotide context surrounding stop codons significantly affects the efficiency of translation termination. In eukaryotes, various 3′ contexts that are unfavorable for translation termination have been described; however, the exact molecular mechanism that mediates their effects remains unknown. In this study, we used a reconstituted mammalian translation system to examine the efficiency of stop codons in different contexts, including several previously described weak 3′ stop codon contexts. We developed an approach to estimate the level of stop codon readthrough in the absence of eukaryotic release factors (eRFs). In this system, the stop codon is recognized by the suppressor or near-cognate tRNAs. We observed that in the absence of eRFs, readthrough occurs in a 3′ nucleotide context-dependent manner, and the main factors determining readthrough efficiency were the type of stop codon and the sequence of the 3′ nucleotides. Moreover, the efficiency of translation termination in weak 3′ contexts was almost equal to that in the tested standard context. Therefore, the ability of eRFs to recognize stop codons and induce peptide release is not affected by mRNA context. We propose that ribosomes or other participants of the elongation cycle can independently recognize certain contexts and increase the readthrough of stop codons. Thus, the efficiency of translation termination is regulated by the 3′ nucleotide context following the stop codon and depends on the concentrations of eRFs and suppressor/near-cognate tRNAs.

MEDB-18. Elongation control of mRNA translation supports Group 3 medulloblastoma adaptation to nutrient deprivation

Group 3 affiliation and MYC genetic amplification are associated with poor life expectancy and substantial morbidity in children suffering from medulloblastoma (MB). However, the high metabolic demand induced by MYC-driven transformation sensitizes MYC-overexpressing MB to cell death under conditions of nutrient deprivation (ND). Additionally, MYC-driven transformation is known to promote mitochondrial oxidative phosphorylation (OXPHOS). We previously reported that eukaryotic Elongation Factor Kinase 2 (eEF2K), the master regulator of mRNA translation elongation, promotes survival of MYC-overexpressing tumors under ND. Interestingly, eEF2K is overexpressed in MYC-driven MB and our preliminary proteomics data highlight large-scale alterations in OXPHOS components affecting eEF2K deficient MB cells. We therefore hypothesized that eEF2K activity is required for the selective translation of mRNAs needed for efficient OXPHOS, and for the progression of MYC-driven MB. We pefrormed Multiplexed enhanced Protein Dynamic Mass Spectrometry in eEF2K knockdown MYC-overexpressing D425 MB cells to identify mRNAs selectively translated upon eEF2K activation. Messenger RNAs encoding multiple (9 out of 10 detected) components of the mitochondrial OXPHOS pathway are selectively translated upon eEF2K activation. Inactivation of eEF2K by genetic KO leads to the disassembly of electron transport chain (ETC) complexes I-IV without affecting mRNA levels of their respective components. Consistently, eEF2K KO MB cells display decreased mitochondrial membrane potential and 20% increased proton leak thorough the mitochondrial membrane. In addition, eEF2K inactivation results in increased Group 3 MB cell death under ND and doubles survival of MB bearing mice fed with calorie restricted diets (p< 0.05).Control of mRNA translation elongation by eEF2K is critical for mitochondrial ETC complex assembly and efficient OXPHOS in MYC-overexpressing MB, likely representing an adaptive response by which MYC-driven MB cells cope with acute metabolic stress. Future therapeutic studies will aim to combine eEF2K inhibition with caloric restriction mimetic drugs as eEF2K activity appears critical under metabolic stress conditions.

CSIG-23. ELONGATION CONTROL OF mRNA TRANSLATION DRIVES GROUP 3 MEDULLOBLASTOMA ADAPTATION TO NUTRIENT DEPRIVATION

Abstract Group 3 affiliation and MYC genetic amplification are associated with poor life expectancy and substantial morbidity in children suffering from medulloblastoma (MB). However, the high metabolic demand induced by MYC-driven transformation sensitizes MYC-overexpressing MB to cell death under conditions of nutrient deprivation (ND). Additionally, MYC-driven transformation is known to promote mitochondrial oxidative phosphorylation (OXPHOS). We previously reported that eukaryotic Elongation Factor Kinase 2 (eEF2K), the master regulator of mRNA translation elongation, promotes survival of MYC-overexpressing tumors under ND. Interestingly, eEF2K is overexpressed in MYC-driven MB and our preliminary proteomics data highlight large-scale alterations in OXPHOS components affecting eEF2K deficient MB cells. We therefore hypothesized that eEF2K activity is required for the selective translation of mRNAs needed for efficient OXPHOS, and for the progression of MYC-driven MB. We pefrormed Multiplexed enhanced Protein Dynamic Mass Spectrometry in eEF2K knockdown MYC-overexpressing D425 MB cells to identify mRNAs selectively translated upon eEF2K activation. Messenger RNAs encoding multiple (9 out of 10 detected) components of the mitochondrial OXPHOS pathway are selectively translated upon eEF2K activation. Inactivation of eEF2K by genetic KO leads to the disassembly of electron transport chain (ETC) complexes I-IV without affecting mRNA levels of their respective components. Consistently, eEF2K KO MB cells display decreased mitochondrial membrane potential and 20% increased proton leak thorough the mitochondrial membrane. In addition, eEF2K inactivation results in increased Group 3 MB cell death under ND and doubles survival of MB bearing mice fed with calorie restricted diets (p&amp;lt; 0.05). Control of mRNA translation elongation by eEF2K is critical for mitochondrial ETC complex assembly and efficient OXPHOS in MYC-overexpressing MB, likely representing an adaptive response by which MYC-driven MB cells cope with acute metabolic stress. Future therapeutic studies will aim to combine eEF2K inhibition with caloric restriction mimetic drugs as eEF2K activity appears critical under metabolic stress conditions.

EMBR-20. ELONGATION CONTROL OF MRNA TRANSLATION DRIVES GROUP 3 MEDULLOBLASTOMA

Medulloblastoma (MB) is the most common pediatric intracranial tumor and leading cause of childhood related cancer deaths. Group 3 affiliation and genetic amplifications of the MYC oncogene are predictors of adverse outcome in MB, underscoring a dire need for novel and more effective therapeutic approaches. The let-7 family of small non-coding RNAs (miRNAs) is known to inhibit tumor progression and regulate metabolism by targeting and degrading several cellular mRNAs, including MYC. Indeed, let-7 miRNAs are frequently repressed in several cancer types, including in MYC-driven MB. We previously reported that the mRNA translation elongation regulator eukaryotic Elongation Factor-2 Kinase (eEF2K) is a pivotal mediator of cancer cell adaptation to nutrient deprivation. In the current work, we identified a potential binding site for let-7 miRNAs on the eEF2K 3’ untranslated region (UTR). In addition, eEF2K mRNA and let-7 miRNA expressions negatively correlate in MB, suggesting a potential regulation of the former by the latter. Let-7 miRNAs transfection decreases eEF2K mRNA and protein levels (by ~40–50%). Down-regulation of luciferase activity by let-7 miRNAs is impaired upon mutation of the let-7 binding site on the eEF2K 3’UTR. Inhibition of eEF2K significantly reduces survival of MYC-amplified MB cell lines under nutrient deprivation, altering their mRNA translation rates. Knockout of eEF2K increases survival of MYC-amplified MB xenografts when mice are kept under calorie restricted diets. We conclude that let-7 miRNAs degrade the eEF2K mRNA by binding to its 3’UTR, indicating that let-7 repression in MYC-driven MB is partially responsible for increased eEF2K levels. Moreover, the let-7-eEF2K axis constitutes a critical mechanism for MYC-driven MB adaptation to acute metabolic stress, representing a promising therapeutic target. Future therapeutic studies will aim to combine eEF2K inhibition with caloric restriction mimetic drugs, as eEF2K activity appears critical under metabolic stress conditions.

Wobble tRNA modification and hydrophilic amino acid patterns dictate protein fate

Regulation of mRNA translation elongation impacts nascent protein synthesis and integrity and plays a critical role in disease establishment. Here, we investigate features linking regulation of codon-dependent translation elongation to protein expression and homeostasis. Using knockdown models of enzymes that catalyze the mcm5s2 wobble uridine tRNA modification (U34-enzymes), we show that gene codon content is necessary but not sufficient to predict protein fate. While translation defects upon perturbation of U34-enzymes are strictly dependent on codon content, the consequences on protein output are determined by other features. Specific hydrophilic motifs cause protein aggregation and degradation upon codon-dependent translation elongation defects. Accordingly, the combination of codon content and the presence of hydrophilic motifs define the proteome whose maintenance relies on U34-tRNA modification. Together, these results uncover the mechanism linking wobble tRNA modification to mRNA translation and aggregation to maintain proteome homeostasis.

Abstract B27: A link between small noncoding RNAs and mRNA translation elongation: The let7-eEF2K axis in pediatric tumor adaptation to nutrient deprivation

Abstract Background: Medulloblastoma (MB) and neuroblastoma (NB) are the most common pediatric solid cancers and the leading cause of childhood cancer-related deaths. Genetic amplifications of the MYC and MYCN oncogenes are the strongest independent predictor of adverse outcomes in MB and NB, underscoring a dire need to improve therapeutic approaches. Tumors continually weather stress from acute microenvironmental changes, including limited nutrient availability. The let-7 family of small noncoding RNAs (miRNAs) inhibits tumor progression and regulates metabolism by selectively targeting and degrading mRNAs, including MYC and MYCN. Indeed, let-7 miRNAs are frequently repressed in several cancers, including in MYC-driven MB and NB. We previously reported that the mRNA translation elongation regulator eukaryotic eEF2K is a pivotal mediator of cancer cell adaptation to nutrient deprivation. Our preliminary data indicate that the eEF2K 3´ untranslated region (UTR) harbors a potential binding site for let-7 miRNAs. In addition, eEF2K mRNA and let-7 miRNA expressions negatively correlate in MB and NB. We therefore hypothesized that let-7 downregulation induces eEF2K expression, thereby supporting MYC-driven MB and NB adaptation to nutrient deprivation and tumor progression. Methods: Effects of eEF2K pharmacologic and genetic inhibition on MB and NB cell death were evaluated in vitro by flow cytometry, while cellular mRNA translation rates were studied by polysome fractionation and Click chemistry. The ability of let-7 to degrade eEF2K mRNA was assessed by let-7 miRNAs transfection into MB cells, followed by RT-PCR and Western blotting for eEF2K. Binding of let-7 to the eEF2K 3´UTR was validated by luciferase reporter assay. Finally, NB xenograft mouse models confirmed in vitro observations. Results: Inhibition of eEF2K significantly reduces survival of MYC/MYCN-amplified NB and MB cell lines under nutrient deprivation, altering their mRNA translation rates. Let-7 miRNAs transfection decreases eEF2K mRNA and protein levels by 40-50%. Downregulation of luciferase activity by let-7 miRNAs is impaired upon mutation of the let-7 binding site on the eEF2K 3´UTR. eEF2K knockdown decreases growth twofold in MYCN-amplified NB xenografts in mice under calorie-restriction diets. Conclusions: Let-7 miRNAs degrade the eEF2K mRNA by binding to its 3´UTR, indicating that let-7 repression in MYC-driven NB and MB is partially responsible for increased eEF2K levels. Moreover, the let-7-eEF2K axis constitutes a critical mechanism for MYC-driven MB and NB adaptation to acute metabolic stress, representing a promising therapeutic target. Future therapeutic studies will aim to combine eEF2K inhibition with caloric restriction mimetic drugs, as eEF2K activity appears critical under metabolic stress conditions. Citation Format: Alberto Delaidelli, Gian Luca Negri, Xue Qi Wang, Simran Sidhu, Brian Cho, Gabriel Leprivier, Volker Hovestadt, Stefan Pfister, Michael Taylor, Marcel Kool, Poul Sorensen. A link between small noncoding RNAs and mRNA translation elongation: The let7-eEF2K axis in pediatric tumor adaptation to nutrient deprivation [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr B27.

Abstract 3660: A link between miRNAs and mRNA translation elongation: The let7-eEF2K axis in MYC-driven pediatric tumors adaptation to nutrient deprivation

Abstract BACKGROUND/OBJECTIVES: MYC family proteins are implicated in many human cancers, but their therapeutic targeting has proven challenging. MYCN and MYC amplification in childhood neuroblastoma (NB) and medulloblastoma (MB) are associated with aggressive disease and high mortality, underscoring a dire need for novel therapies. Let-7 microRNAs (miRNAs) inhibit tumor progression and regulate metabolism by degrading several mRNAs, including MYC. Let-7 miRNAs are therefore frequently repressed in cancer, including MYC-driven NB and MB. We previously reported that the mRNA translation elongation regulator eukaryotic Elongation Factor-2 Kinase (eEF2K) is a pivotal mediator of cancer cells adaptation to nutrient deprivation (ND). Publicly available transcriptomic database analyses indicate that eEF2K expression significantly correlate with MYCN and MYC expression in multiple tumor cohorts. Our preliminary data also indicate that the eEF2K 3’ untranslated region (UTR) harbors a potential binding site for let-7 miRNAs. In addition, eEF2K mRNA and let-7 miRNA expression negatively correlates in NB and MB, suggesting a potential regulation of the former by the latter. We therefore hypothesized that let-7 down-regulation induces eEF2K expression, thereby supporting MYC-driven NB and MB adaptation to ND and tumor progression. METHODS: Immunohistochemistry for eEF2K substrate (p-eEF2) was performed on NB and MB tissue microarrays to link results with MYC expression and outcome. Effects of eEF2K pharmacological and genetic inhibition on NB and MB cell survival were evaluated in vitro by MTT assay and PI staining. The ability of let-7 to degrade eEF2K mRNA was assessed by let-7 miRNAs transfection into MB cells, followed by RT-PCR and Western Blotting for eEF2K. Binding of let-7 to the eEF2K 3’UTR was validated by luciferase reporter assay. Finally, NB xenograft mouse models were used to confirm in vitro observations. RESULTS: High eEF2K activity is linked to MYC over-expression and reduced survival in NB and MB (p&amp;lt;0.05). Pharmacological and genetic inhibition of eEF2K significantly reduces survival of MYC/MYCN-amplified NB and MB cell lines under ND. Let-7 miRNAs transfection decreases eEF2K mRNA and protein levels (by ~40-50%), and down-regulation of luciferase activity by let-7 miRNAs is impaired upon mutation of the let-7 binding site on the eEF2K 3’UTR. Knockdown of eEF2K determines a twofold growth decrease of MYCN-amplified NB xenografts when mice are kept under caloric restriction diet. CONCLUSIONS: Let-7 miRNAs degrade eEF2K mRNA by binding to its 3'UTR, indicating that let-7 repression in MYC-driven NB and MB is partially responsible for eEF2K increased levels and activity. Moreover, the let-7-eEF2K axis represents a critical mechanism for MYC-driven NB and MB adaptation to ND, constituting a promising therapeutic target. Citation Format: Alberto Delaidelli, Gian Luca Negri, Brian Cho, Simran Sidhu, Stefan Pfister, Michael Taylor, Gabriel Leprivier, Marcel Kool, Poul Soresnsen. A link between miRNAs and mRNA translation elongation: The let7-eEF2K axis in MYC-driven pediatric tumors adaptation to nutrient deprivation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3660.

Sustained protein synthesis and reduced eEF2K levels in TAp73-\\- mice brain: a possible compensatory mechanism

ABSTRACTThe transcription factor p73 is a member of the p53 family, of which the transactivation domain containing isoform (TAp73) plays key roles in brain development and neuronal stem cells. TAp73 also facilitates homoeostasis and prevents oxidative damage in vivo by inducing the expression of its target genes. Recently, we found that in addition to its role in regulation of transcription, TAp73 also affects mRNA translation. In cultured cells, acute TAp73 depletion activates eEF2K, which phosphorylates eEF2 reducing mRNA translation elongation. As a consequence, there is a reduction in global proteins synthesis rates and reprogramming of the translatome, leading to a selective decrease in the translation of rRNA processing factors. Given the dramatic effects of Tap73 depletion in vitro it was important to determine whether similar effects were observed in vivo. Here, we report the surprising finding that in brains of TAp73 KO mice there is a reduced level of eEF2K, which allows protein synthesis rates to be maintained suggesting a compensation model. These data provide new insights to the role of TAp73 in translation regulation and the eEF2K pathway in the brain.

Issue Cover (July 2018)

Front cover: Background: Long-lasting forms of memory and synaptic plasticity such as late long-term potentiation (L-LTP) are dependent on protein synthesis. The aim of our study is to investigate potential interactions between signaling pathways controlling protein synthesis in hippocampal L-LTP. Main findings: We found in mice that L-LTP failure caused by inhibition of the mammalian target of rapamycin complex 1 (mTORC1) is prevented by genetic deletion of PERK, a kinase for eukaryotic initiation factor 2α (eIF2α). A key mechanism responsible for the L-LTP rescue may involve down-regulation of translational factor eEF2 phosphorylation, which is associated with increased protein synthesis capacity via its upregulation on mRNA translation elongation. Implication: These data, for the first time, connect reduction of PERK activity with the regulation of translation elongation in enabling L-LTP independent of mTORC1. Image content: This image (63x) taken by confocal laser scanning microscope shows area CA1 of an acute hippocampal slice from wild-type mouse that was labeled for phospho-eEF2 (green) and DAPI (blue). Phospho-eEF2 is distributed in both soma and dendrites. Read the full article ‘Genetic removal of eIF2α kinase PERK in mice enables hippocampal L-LTP independent of mTORC1 activity’ by H. R. Zimmermann, W. Yang, B. C. Beckelman, N. P. Kasica, X. Zhou, L. D. Galli, A. G. Ryazanov, T. Ma (J. Neurochem. 2018, vol. 146 (2), pp. 133–144) on doi: 10.1111/jnc.14306

Codon usage regulates protein structure and function by affecting translation elongation speed in Drosophila cells.

Codon usage biases are found in all eukaryotic and prokaryotic genomes and have been proposed to regulate different aspects of translation process. Codon optimality has been shown to regulate translation elongation speed in fungal systems, but its effect on translation elongation speed in animal systems is not clear. In this study, we used a Drosophila cell-free translation system to directly compare the velocity of mRNA translation elongation. Our results demonstrate that optimal synonymous codons speed up translation elongation while non-optimal codons slow down translation. In addition, codon usage regulates ribosome movement and stalling on mRNA during translation. Finally, we show that codon usage affects protein structure and function in vitro and in Drosophila cells. Together, these results suggest that the effect of codon usage on translation elongation speed is a conserved mechanism from fungi to animals that can affect protein folding in eukaryotic organisms.

Alcohol intoxication following muscle contraction in mice decreases muscle protein synthesis but not mTOR signal transduction.

Alcohol (ethanol [EtOH]) intoxication antagonizes stimulation of muscle protein synthesis and mammalian target of rapamycin (mTOR) signaling. However, whether the anabolic response can be reversed when alcohol is consumed after the stimulus is unknown. A single bout of electrically stimulated muscle contractions (10 sets of 6 contractions) was induced in fasted male C57BL/6 mice 2hours prior to alcohol intoxication. EtOH was injected intraperitoneally (3g/kg), and the gastrocnemius/plantaris muscle complex was collected 2hours later from the stimulated and contralateral unstimulated control leg. Muscle contraction increased protein synthesis 28% in control mice, while EtOH abolished this stimulation-induced increase. Further, EtOH suppressed the rate of synthesis ~75% compared to control muscle irrespective of stimulation. This decrease was associated with impaired protein elongation as EtOH increased the phosphorylation of eEF2 Thr(56) . In contrast, stimulation-induced increases in mTOR protein complex-1 (mTORC1) (S6K1 Thr(421) /Ser(424) , S6K1 Thr(389) , rpS6 Ser(240/244) , and 4E-BP1 Thr(37/46) ) and mitogen-activated protein kinase (MAPK) (JNK Thr(183) /Tyr(185) , p38 Thr(180) /Tyr(182) , and rpS6S(235/236) ) signaling were not reversed by acute EtOH. These data suggest that EtOH-induced decreases in protein synthesis in fasted mice may be independent of mTORC1 and MAPK signaling following muscle contraction and instead due to the antagonistic actions of EtOH on mRNA translation elongation. Therefore, EtOH suppresses the contraction-induced increase in protein synthesis, and over time has the potential to prevent skeletal muscle hypertrophy induced by repeated muscle contraction.