METTL13 Methylation of eEF1A Increases Translational Output to Promote Tumorigenesis.

Increased protein synthesis plays an etiologic role in diverse cancers. Here, we demonstrate that METTL13 (methyltransferase-like 13) dimethylation of eEF1A (eukaryotic elongation factor 1A) lysine 55 (eEF1AK55me2) is utilized by Ras-driven cancers to increase translational output and promote tumorigenesis in vivo. METTL13-catalyzed eEF1A methylation increases eEF1A's intrinsic GTPase activity in vitro and protein production in cells. METTL13 and eEF1AK55me2 levels are upregulated in cancer and negatively correlate with pancreatic and lung cancer patient survival. METTL13 deletion and eEF1AK55me2 loss dramatically reduce Ras-driven neoplastic growth in mouse models and in patient-derived xenografts (PDXs) from primary pancreatic and lung tumors. Finally, METTL13 depletion renders PDX tumors hypersensitive to drugs that target growth-signaling pathways. Together, our work uncovers a mechanism by which lethal cancers become dependent on the METTL13-eEF1AK55me2 axis to meet their elevated protein synthesis requirement and suggests that METTL13 inhibition may constitute a targetable vulnerability of tumors driven by aberrant Ras signaling.

SMYD5 methylation of rpL40 links ribosomal output to gastric cancer.

Dysregulated transcription due to disruption in histone lysine methylation dynamics is an established contributor to tumorigenesis1,2. However, whether analogous pathologic epigenetic mechanisms act directly on the ribosome to advance oncogenesis is unclear. Here we find that trimethylation of the core ribosomal protein L40 (rpL40) at lysine 22 (rpL40K22me3) by the lysine methyltransferase SMYD5 regulates mRNA translation output to promote malignant progression of gastric adenocarcinoma (GAC) with lethal peritoneal ascites. A biochemical-proteomics strategy identifies the monoubiquitin fusion protein partner rpL40 (ref. 3) as the principal physiological substrate of SMYD5 across diverse samples. Inhibiting the SMYD5-rpL40K22me3 axis in GAC cell lines reprogrammes protein synthesis to attenuate oncogenic gene expression signatures. SMYD5 and rpL40K22me3 are upregulated in samples from patients with GAC and negatively correlate with clinical outcomes. SMYD5 ablation in vivo in familial and sporadic mouse models of malignant GAC blocks metastatic disease, including peritoneal carcinomatosis. Suppressing SMYD5 methylation of rpL40 inhibits human cancer cell and patient-derived GAC xenograft growth and renders them hypersensitive to inhibitors of PI3K and mTOR. Finally, combining SMYD5 depletion with PI3K-mTOR inhibition and chimeric antigen receptor T cell administration cures an otherwise lethal in vivo mouse model of aggressive GAC-derived peritoneal carcinomatosis. Together, our work uncovers a ribosome-based epigenetic mechanism that facilitates the evolution of malignant GAC and proposes SMYD5 targeting as part of a potential combination therapy to treat this cancer.

Distinctive tRNA repertoires in proliferating versus differentiating cells.

Transfer RNAs (tRNAs) deliver amino acids to the ribosome during mRNA translation. Gingold et al. now provide evidence that alterations in the cellular tRNA repertoire are tightly coordinated with changes in mRNA expression. These changes in the tRNA repertoire dictate translational programs that distinguish differentiating from proliferating cells.

Adaptive translational pausing is a hallmark of the cellular response to severe environmental stress.

To survive, mammalian cells must adapt to environmental challenges. While the cellular response to mild stress has been widely studied, how cells respond to severe stress remains unclear. We show here that under severe hyperosmotic stress, cells enter a transient hibernation-like state in anticipation of recovery. We demonstrate this adaptive pausing response (APR) is a coordinated cellular response that limits ATP supply and consumption through mitochondrial fragmentation and widespread pausing of mRNA translation. This pausing is accomplished by ribosome stalling at translation initiation codons, which keeps mRNAs poised to resume translation upon recovery. We further show that recovery from severe stress involves ISR (integrated stress response) signaling that permits cell cycle progression, resumption of growth, and reversal of mitochondria fragmentation. Our findings indicate that cells can respond to severe stress via a hibernation-like mechanism that preserves vital elements of cellular function under harsh environmental conditions.

A hydride transfer complex reprograms NAD metabolism and bypasses senescence.

Metabolic rewiring and redox balance play pivotal roles in cancer. Cellular senescence is a barrier for tumorigenesis circumvented in cancer cells by poorly understood mechanisms. We report a multi-enzymatic complex that reprograms NAD metabolism by transferring reducing equivalents from NADH to NADP+. This hydride transfer complex (HTC) is assembled by malate dehydrogenase 1, malic enzyme 1, and cytosolic pyruvate carboxylase. HTC is found in phase-separated bodies in the cytosol of cancer or hypoxic cells and can be assembled in vitro with recombinant proteins. HTC is repressed in senescent cells but induced by p53 inactivation. HTC enzymes are highly expressed in mouse and human prostate cancer models, and their inactivation triggers senescence. Exogenous expression of HTC is sufficient to bypass senescence, rescue cells from complex I inhibitors, and cooperate with oncogenic RAS to transform primary cells. Altogether, we provide evidence for a new multi-enzymatic complex that reprograms metabolism and overcomes cellular senescence.

Genome-Wide Screens Reveal that Resveratrol Induces Replicative Stress in Human Cells.

Resveratrol is a natural product associated with wide-ranging effects in animal and cellular models, including lifespan extension. To identify the genetic target of resveratrol in human cells, we conducted genome-wide CRISPR-Cas9 screens to pinpoint genes that confer sensitivity or resistance to resveratrol. An extensive network of DNA damage response and replicative stress genes exhibited genetic interactions with resveratrol and its analog pterostilbene. These genetic profiles showed similarity to the response to hydroxyurea, an inhibitor of ribonucleotide reductase that causes replicative stress. Resveratrol, pterostilbene, and hydroxyurea caused similar depletion of nucleotide pools, inhibition of replication fork progression, and induction of replicative stress. The ability of resveratrol to inhibit cell proliferation and S phase transit was independent of the histone deacetylase sirtuin 1, which has been implicated in lifespan extension by resveratrol. These results establish that a primary impact of resveratrol on human cell proliferation is the induction of low-level replicative stress.

A second-generation eIF4A RNA helicase inhibitor exploits translational reprogramming as a vulnerability in triple-negative breast cancer.

In this study, we aimed to address the current limitations of therapies for macro-metastatic triple-negative breast cancer (TNBC) and provide a therapeutic lead that overcomes the high degree of heterogeneity associated with this disease. Specifically, we focused on well-documented but clinically underexploited cancer-fueling perturbations in mRNA translation as a potential therapeutic vulnerability. We therefore developed an orally bioavailable rocaglate-based molecule, MG-002, which hinders ribosome recruitment and scanning via unscheduled and non-productive RNA clamping by the eukaryotic translation initiation factor (eIF) 4A RNA helicase. We demonstrate that MG-002 potently inhibits mRNA translation and primary TNBC tumor growth without causing overt toxicity in mice. Importantly, given that metastatic spread is a major cause of mortality in TNBC, we show that MG-002 attenuates metastasis in pre-clinical models. We report on MG-002, a rocaglate that shows superior properties relative to existing eIF4A inhibitors in pre-clinical models. Our study also paves the way for future clinical trials exploring the potential of MG-002 in TNBC and other oncological indications.

Translational control of immune responses: from transcripts to translatomes.

Selective translational control of gene expression is emerging as a principal mechanism for the regulation of protein abundance that determines a variety of functions in both the adaptive immune system and the innate immune system. The translation-initiation factor eIF4E acts as a node for such regulation, but non-eIF4E mechanisms are also prevalent. Studies of 'translatomes' (genome-wide pools of translated mRNA) have facilitated mechanistic discoveries by identifying key regulatory components, including transcription factors, that are under translational control. Here we review the current knowledge on mechanisms that regulate translation and thereby modulate immunological function. We further describe approaches for measuring and analyzing translatomes and how such powerful tools can facilitate future insights on the role of translational control in the immune system.

Translational control by the eukaryotic ribosome.

The ribosome plays a universally conserved role in catalyzing protein synthesis. Kondrashov et al. (2011) now provide evidence that the loss of function of ribosomal protein L38 in mice leads to a selective reduction in the translation of Hox mRNAs, thus suggesting that ribosomal proteins play a critical role during embryonic development.

mTOR Controls Mitochondrial Dynamics and Cell Survival via MTFP1.

The mechanisms that link environmental and intracellular stimuli to mitochondrial functions, including fission/fusion, ATP production, metabolite biogenesis, and apoptosis, are not well understood. Here, we demonstrate that the nutrient-sensing mechanistic/mammalian target of rapamycin complex 1 (mTORC1) stimulates translation of mitochondrial fission process 1 (MTFP1) to control mitochondrial fission and apoptosis. Expression of MTFP1 is coupled to pro-fission phosphorylation and mitochondrial recruitment of the fission GTPase dynamin-related protein 1 (DRP1). Potent active-site mTOR inhibitors engender mitochondrial hyperfusion due to the diminished translation of MTFP1, which is mediated by translation initiation factor 4E (eIF4E)-binding proteins (4E-BPs). Uncoupling MTFP1 levels from the mTORC1/4E-BP pathway upon mTOR inhibition blocks the hyperfusion response and leads to apoptosis by converting mTOR inhibitor action from cytostatic to cytotoxic. These data provide direct evidence for cell survival upon mTOR inhibition through mitochondrial hyperfusion employing MTFP1 as a critical effector of mTORC1 to govern cell fate decisions.

A Unique ISR Program Determines Cellular Responses to Chronic Stress.

The integrated stress response (ISR) is a homeostatic mechanism induced by endoplasmic reticulum (ER) stress. In acute/transient ER stress, decreased global protein synthesis and increased uORF mRNA translation are followed by normalization of protein synthesis. Here, we report a dramatically different response during chronic ER stress. This chronic ISR program is characterized by persistently elevated uORF mRNA translation and concurrent gene expression reprogramming, which permits simultaneous stress sensing and proteostasis. The program includes PERK-dependent switching to an eIF3-dependent translation initiation mechanism, resulting in partial, but not complete, translational recovery, which, together with transcriptional reprogramming, selectively bolsters expression of proteins with ER functions. Coordination of transcriptional and translational reprogramming prevents ER dysfunction and inhibits "foamy cell" development, thus establishing a molecular basis for understanding human diseases associated with ER dysfunction.

Translational control of the activation of transcription factor NF-κB and production of type I interferon by phosphorylation of the translation factor eIF4E.

Type I interferon is an integral component of the antiviral response, and its production is tightly controlled at the levels of transcription and translation. The eukaryotic translation-initiation factor eIF4E is a rate-limiting factor whose activity is regulated by phosphorylation of Ser209. Here we found that mice and fibroblasts in which eIF4E cannot be phosphorylated were less susceptible to virus infection. More production of type I interferon, resulting from less translation of Nfkbia mRNA (which encodes the inhibitor IκBα), largely explained this phenotype. The lower abundance of IκBα resulted in enhanced activity of the transcription factor NF-κB, which promoted the production of interferon-ß (IFN-ß). Thus, regulated phosphorylation of eIF4E has a key role in antiviral host defense by selectively controlling the translation of an mRNA that encodes a critical suppressor of the innate antiviral response.

Distinct, opposing functions for CFIm59 and CFIm68 in mRNA alternative polyadenylation of Pten and in the PI3K/Akt signalling cascade.

Precise maintenance of PTEN dosage is crucial for tumor suppression across a wide variety of cancers. Post-transcriptional regulation of Pten heavily relies on regulatory elements encoded by its 3'UTR. We previously reported the important diversity of 3'UTR isoforms of Pten mRNAs produced through alternative polyadenylation (APA). Here, we reveal the direct regulation of Pten APA by the mammalian cleavage factor I (CFIm) complex, which in turn contributes to PTEN protein dosage. CFIm consists of the UGUA-binding CFIm25 and APA regulatory subunits CFIm59 or CFIm68. Deep sequencing analyses of perturbed (KO and KD) cell lines uncovered the differential regulation of Pten APA by CFIm59 and CFIm68 and further revealed that their divergent functions have widespread impact for APA in transcriptomes. Differentially regulated genes include numerous factors within the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) signalling pathway that PTEN counter-regulates. We further reveal a stratification of APA dysregulation among a subset of PTEN-driven cancers, with recurrent alterations among PI3K/Akt pathway genes regulated by CFIm. Our results refine the transcriptome selectivity of the CFIm complex in APA regulation, and the breadth of its impact in PTEN-driven cancers.

Translational offsetting as a mode of estrogen receptor α-dependent regulation of gene expression.

Estrogen receptor alpha (ERα) activity is associated with increased cancer cell proliferation. Studies aiming to understand the impact of ERα on cancer-associated phenotypes have largely been limited to its transcriptional activity. Herein, we demonstrate that ERα coordinates its transcriptional output with selective modulation of mRNA translation. Importantly, translational perturbations caused by depletion of ERα largely manifest as "translational offsetting" of the transcriptome, whereby amounts of translated mRNAs and corresponding protein levels are maintained constant despite changes in mRNA abundance. Transcripts whose levels, but not polysome association, are reduced following ERα depletion lack features which limit translation efficiency including structured 5'UTRs and miRNA target sites. In contrast, mRNAs induced upon ERα depletion whose polysome association remains unaltered are enriched in codons requiring U34-modified tRNAs for efficient decoding. Consistently, ERα regulates levels of U34-modifying enzymes and thereby controls levels of U34-modified tRNAs. These findings unravel a hitherto unprecedented mechanism of ERα-dependent orchestration of transcriptional and translational programs that may be a pervasive mechanism of proteome maintenance in hormone-dependent cancers.

Selective inhibitors of mTORC1 activate 4EBP1 and suppress tumor growth.

The clinical benefits of pan-mTOR active-site inhibitors are limited by toxicity and relief of feedback inhibition of receptor expression. To address these limitations, we designed a series of compounds that selectively inhibit mTORC1 and not mTORC2. These 'bi-steric inhibitors' comprise a rapamycin-like core moiety covalently linked to an mTOR active-site inhibitor. Structural modification of these components modulated their affinities for their binding sites on mTOR and the selectivity of the bi-steric compound. mTORC1-selective compounds potently inhibited 4EBP1 phosphorylation and caused regressions of breast cancer xenografts. Inhibition of 4EBP1 phosphorylation was sufficient to block cancer cell growth and was necessary for maximal antitumor activity. At mTORC1-selective doses, these compounds do not alter glucose tolerance, nor do they relieve AKT-dependent feedback inhibition of HER3. Thus, in preclinical models, selective inhibitors of mTORC1 potently inhibit tumor growth while causing less toxicity and receptor reactivation as compared to pan-mTOR inhibitors.

Translation Links Nutrient Availability with Inflammation.

Translation plays a crucial role in shaping the proteome during adaptation to various types of stress. A recent study by Gameiro and Struhl identified an inflammatory response which comprises coordination of transcriptional and translational programs, and which appears to be required for recovery from nutrient deprivation.

Enhanced translation expands the endo-lysosome size and promotes antigen presentation during phagocyte activation.

The mechanisms that govern organelle adaptation and remodelling remain poorly defined. The endo-lysosomal system degrades cargo from various routes, including endocytosis, phagocytosis, and autophagy. For phagocytes, endosomes and lysosomes (endo-lysosomes) are kingpin organelles because they are essential to kill pathogens and process and present antigens. During phagocyte activation, endo-lysosomes undergo a morphological transformation, going from a collection of dozens of globular structures to a tubular network in a process that requires the phosphatidylinositol-3-kinase-AKT-mechanistic target of rapamycin (mTOR) signalling pathway. Here, we show that the endo-lysosomal system undergoes an expansion in volume and holding capacity during phagocyte activation within 2 h of lipopolysaccharides (LPS) stimulation. Endo-lysosomal expansion was paralleled by an increase in lysosomal protein levels, but this was unexpectedly largely independent of the transcription factor EB (TFEB) and transcription factor E3 (TFE3), which are known to scale up lysosome biogenesis. Instead, we demonstrate a hitherto unappreciated mechanism of acute organelle expansion via mTOR Complex 1 (mTORC1)-dependent increase in translation, which appears to be mediated by both S6Ks and 4E-BPs. Moreover, we show that stimulation of RAW 264.7 macrophage cell line with LPS alters translation of a subset but not all of mRNAs encoding endo-lysosomal proteins, thereby suggesting that endo-lysosome expansion is accompanied by functional remodelling. Importantly, mTORC1-dependent increase in translation activity was necessary for efficient and rapid antigen presentation by dendritic cells. Collectively, we identified a previously unknown and functionally relevant mechanism for endo-lysosome expansion that relies on mTORC1-dependent translation to stimulate endo-lysosome biogenesis in response to an infection signal.

Largen: a molecular regulator of mammalian cell size control.

Little is known about how mammalian cells maintain cell size homeostasis. We conducted a novel genetic screen to identify cell-size-controlling genes and isolated Largen, the product of a gene (PRR16) that increased cell size upon overexpression in human cells. In vitro evidence indicated that Largen preferentially stimulates the translation of specific subsets of mRNAs, including those encoding proteins affecting mitochondrial functions. The involvement of Largen in mitochondrial respiration was consistent with the increased mitochondrial mass and greater ATP production in Largen-overexpressing cells. Furthermore, Largen overexpression led to increased cell size in vivo, as revealed by analyses of conditional Largen transgenic mice. Our results establish Largen as an important link between mRNA translation, mitochondrial functions, and the control of mammalian cell size.

Hepatic posttranscriptional network comprised of CCR4-NOT deadenylase and FGF21 maintains systemic metabolic homeostasis.

Whole-body metabolic homeostasis is tightly controlled by hormone-like factors with systemic or paracrine effects that are derived from nonendocrine organs, including adipose tissue (adipokines) and liver (hepatokines). Fibroblast growth factor 21 (FGF21) is a hormone-like protein, which is emerging as a major regulator of whole-body metabolism and has therapeutic potential for treating metabolic syndrome. However, the mechanisms that control FGF21 levels are not fully understood. Herein, we demonstrate that FGF21 production in the liver is regulated via a posttranscriptional network consisting of the CCR4-NOT deadenylase complex and RNA-binding protein tristetraprolin (TTP). In response to nutrient uptake, CCR4-NOT cooperates with TTP to degrade AU-rich mRNAs that encode pivotal metabolic regulators, including FGF21. Disruption of CCR4-NOT activity in the liver, by deletion of the catalytic subunit CNOT6L, increases serum FGF21 levels, which ameliorates diet-induced metabolic disorders and enhances energy expenditure without disrupting bone homeostasis. Taken together, our study describes a hepatic CCR4-NOT/FGF21 axis as a hitherto unrecognized systemic regulator of metabolism and suggests that hepatic CCR4-NOT may serve as a target for devising therapeutic strategies in metabolic syndrome and related morbidities.

Generally applicable transcriptome-wide analysis of translation using anota2seq.

mRNA translation plays an evolutionarily conserved role in homeostasis and when dysregulated contributes to various disorders including metabolic and neurological diseases and cancer. Notwithstanding that optimal and universally applicable methods are critical for understanding the complex role of translational control under physiological and pathological conditions, approaches to analyze translatomes are largely underdeveloped. To address this, we developed the anota2seq algorithm which outperforms current methods for statistical identification of changes in translation. Notably, in contrast to available analytical methods, anota2seq also allows specific identification of an underappreciated mode of gene expression regulation whereby translation acts as a buffering mechanism which maintains protein levels despite fluctuations in corresponding mRNA abundance ('translational buffering'). Thus, the universal anota2seq algorithm allows efficient and hitherto unprecedented interrogation of translatomes which is anticipated to advance knowledge regarding the role of translation in homeostasis and disease.