Role In Translational Control Research Articles

TRNA-derived RNAs: Biogenesis and roles in translational control.

Transfer RNA (tRNA)-derived RNAs (tDRs) are a class of small non-coding RNAs that play important roles in different aspects of gene expression. These ubiquitous and heterogenous RNAs, which vary across different species and cell types, are proposed to regulate various biological processes. In this review, we will discuss aspects of their biogenesis, and specifically, their contribution into translational control. We will summarize diverse roles of tDRs and the molecular mechanisms underlying their functions in the regulation of protein synthesis and their impact on related events such as stress-induced translational reprogramming. This article is categorized under: RNA Processing > Processing of Small RNAs Regulatory RNAs/RNAi/Riboswitches > Regulatory RNAs Regulatory RNAs/RNAi/Riboswitches > Biogenesis of Effector Small RNAs.

Abstract 3967: A novel translation program regulates tumor-initiating potential and chemoresistance in multiple myeloma

Abstract Background: Despite the introduction of novel therapies, the majority of the multiple myeloma (MM) patients eventually relapse and develop chemoresistance, and the factors responsible for disease progression are not fully understood. Relapse suggest that MM cells that are resistant to chemotherapy and have the clonogenic growth potential to produce tumor cells exist, and we have found that tumor cells lacking the plasma cell antigen CD138 are enriched in these properties. Understanding the mechanisms regulating these MM cancer stem cells (CSCs) may lead to novel targeting strategies. Aberrant translation is a widespread characteristic of tumor cells and plays a major role in drug resistance. Thus, therapeutic agents that target translation hold promise as novel anti-cancer drugs. MM cells are characterized by heightened protein production in the form of immunoglobulin (Ig) and are highly dependent on proteolytic pathways such as the proteasome to maintain protein homeostasis. As a consequence, proteasome inhibitors (PIs) such as bortezomib (BTZ) have been established as a frontline therapy for the disease. Immature plasma cells with diminished Ig production are resistant to BTZ, and patients with low Ig producing tumors are more likely to relapse. Therefore, low protein synthesis may confer growth advantages to cancer cells such as chemoresistance and enhanced tumor regrowth potential. Methods & Results: To examine alterations in the translational landscape in therapy resistant MM, we treated MM cells with BTZ, and found that eukaryotic translation initiation factor-5 (eIF5) was highly upregulated. Importantly, over-expression of eIF5 led to increased chemotherapy resistance and enhanced clonogenicity. O-propargyl-puromycin incorporation showed a global translational reduction in cells over-expressing eIF5. Matched transcriptional RNA sequencing (RNA-seq) and ribosome profiling revealed that a subset of genes, including MSI2 and EZH1, escaped translation suppression by maintaining efficient translation in eIF5 over-expressing cells. In addition, clonogenic (CD138neg) MM cells exhibited increased eIF5 expression and decreased protein synthesis. Discussion: Understanding the mechanisms of chemoresistance in the cell populations responsible for residual disease and re-growth may lead to novel targeting strategies. Recent studies have indicated that stem cell behavior is coupled to protein synthesis regulation. However, the role of translational control in the maintenance of tumor-initiating cells has been explored only recently. Studies have implicated deregulation of initiation factors in various types of cancer. However, the role of eIF5 in cancer remains elusive. Our data reveals, for the first time, a novel eIF5-directed translational program that enhances chemoresistance and tumor-initiating potential in MM cells, making it a promising therapeutic target. Citation Format: Kiera Rycaj, Eun-Hee Park, Tori Tonn, Qiuju Wang, Can Cenik, Christian Gocke, William Matsui. A novel translation program regulates tumor-initiating potential and chemoresistance in multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3967.

Cytosolic aspartate aminotransferase moonlights as a ribosome-binding modulator of Gcn2 activity during oxidative stress.

Regulation of translation is a fundamental facet of the cellular response to rapidly changing external conditions. Specific RNA-binding proteins (RBPs) co-ordinate the translational regulation of distinct mRNA cohorts during stress. To identify RBPs with previously under-appreciated roles in translational control, we used polysome profiling and mass spectrometry to identify and quantify proteins associated with translating ribosomes in unstressed yeast cells and during oxidative stress and amino acid starvation, which both induce the integrated stress response (ISR). Over 800 proteins were identified across polysome gradient fractions, including ribosomal proteins, translation factors, and many others without previously described translation-related roles, including numerous metabolic enzymes. We identified variations in patterns of PE in both unstressed and stressed cells and identified proteins enriched in heavy polysomes during stress. Genetic screening of polysome-enriched RBPs identified the cytosolic aspartate aminotransferase, Aat2, as a ribosome-associated protein whose deletion conferred growth sensitivity to oxidative stress. Loss of Aat2 caused aberrantly high activation of the ISR via enhanced eIF2α phosphorylation and GCN4 activation. Importantly, non-catalytic AAT2 mutants retained polysome association and did not show heightened stress sensitivity. Aat2 therefore has a separate ribosome-associated translational regulatory or 'moonlighting' function that modulates the ISR independent of its aspartate aminotransferase activity.

PUMILIO-mediated translational control of somatic cell cycle program promotes folliculogenesis and contributes to ovarian cancer progression.

Translational control is a fundamental mechanism regulating animal germ cell development. Gonadal somatic cells provide support and microenvironment for germ cell development to ensure fertility, yet the roles of translational control in gonadal somatic compartment remain largely undefined. We found that mouse homolog of conserved fly germline stem cell factor Pumilio, PUM1, is absent in oocytes of all growing follicles after the primordial follicle stage, instead, it is highly expressed in somatic compartments of ovaries. Global loss of Pum1, not oocyte-specific loss of Pum1, led to a significant reduction in follicular number and size as well as fertility. Whole-genome identification of PUM1 targets in ovarian somatic cells revealed an enrichment of cell proliferation pathway, including 48 key regulators of cell phase transition. Consistently granulosa cells proliferation is reduced and the protein expression of the PUM-bound Cell Cycle Regulators(PCCR) were altered accordingly in mutant ovaries, and specifically in granulosa cells. Increase in negative regulator expression and decrease in positive regulators in the mutant ovaries support a coordinated translational control of somatic cell cycle program via PUM proteins. Furthermore, postnatal knockdown, but not postnatal oocyte-specific loss, of Pum1 in Pum2 knockout mice reduced follicular growth and led to similar expression alteration of PCCR genes, supporting a critical role of PUM-mediated translational control in ovarian somatic cells for mammalian female fertility. Finally, expression of human PUM protein and its regulated cell cycle targets exhibited significant correlation with ovarian cancer and prognosis for cancer survival. Hence, PUMILIO-mediated cell cycle regulation represents an important mechanism in mammalian female reproduction and human cancer biology.

Reversal of G-Quadruplexes' Role in Translation Control When Present in the Context of an IRES.

G-quadruplexes (GQs) are secondary nucleic acid structures that play regulatory roles in various cellular processes. G-quadruplex-forming sequences present within the 5′ UTR of mRNAs can function not only as repressors of translation but also as elements required for optimum function. Based upon previous reports, the majority of the 5′ UTR GQ structures inhibit translation, presumably by blocking the ribosome scanning process that is essential for detection of the initiation codon. However, there are certain mRNAs containing GQs that have been identified as positive regulators of translation, as they are needed for translation initiation. While most cellular mRNAs utilize the 5′ cap structure to undergo cap-dependent translation initiation, many rely on cap-independent translation under certain conditions in which the cap-dependent initiation mechanism is not viable or slowed down, for example, during development, under stress and in many diseases. Cap-independent translation mainly occurs via Internal Ribosomal Entry Sites (IRESs) that are located in the 5′ UTR of mRNAs and are equipped with structural features that can recruit the ribosome or other factors to initiate translation without the need for a 5′ cap. In this review, we will focus only on the role of RNA GQs present in the 5′ UTR of mRNAs, where they play a critical role in translation initiation, and discuss the potential mechanism of this phenomenon, which is yet to be fully delineated.

Vectorial channeling as a mechanism for translational control by functional prions and condensates

Translation of messenger RNA (mRNA) is regulated through a diverse set of RNA-binding proteins. A significant fraction of RNA-binding proteins contains prion-like domains which form functional prions. This raises the question of how prions can play a role in translational control. Local control of translation in dendritic spines by prions has been invoked in the mechanism of synaptic plasticity and memory. We show how channeling through diffusion and processive translation cooperate in highly ordered mRNA/prion aggregates as well as in less ordered mRNA/protein condensates depending on their substructure. We show that the direction of translational control, whether it is repressive or activating, depends on the polarity of the mRNA distribution in mRNA/prion assemblies which determines whether vectorial channeling can enhance recycling of ribosomes. Our model also addresses the effect of changes of substrate concentration in assemblies that have been suggested previously to explain translational control by assemblies through the introduction of a potential of mean force biasing diffusion of ribosomes inside the assemblies. The results from the model are compared with the experimental data on translational control by two functional RNA-binding prions, CPEB involved in memory and Rim4 involved in gametogenesis.

Abstract 2441: Polysome profiling suggests VHL-dependent translational control in clear cell renal cell carcinoma

Abstract ccRCC is the most common type of renal carcinoma with 80% of incidence among all types of kidney neoplasms. Most cases are localized in the kidney and potentially curable after nephrectomy however about 30% of patients will relapse with distant metastasis. Metastatic patients comprehend one third of all cases and, despite the advances in therapies, they still have low response rates. The identification of molecular mechanisms associated with ccRCC is essential to understand disease progression and treatment resistance. Genes frequently mutated in ccRCC affect the activation of signaling pathways including the mTOR pathway which can cause an unbalance in translational control. Another frequent mutation is in the tumor suppressor gene VHL which regulates response under hypoxia. Hypoxia affects gene expression by both translational and transcriptional controls that contributes to tumor formation and disease progression. Here we aim to understand how translational control can contribute to ccRCC development. We evaluated the activity of mTOR pathway and translational control in cell lines and PDX models with VHL mutation through polysome profiling. We observed lower global translational rates in both VHL mutated models suggesting an important role in translational control. Differentially translated genes identified from polysome associated RNA show a specific translational signature in response to VHL deletion. For human tumors, a cohort of 118 cases was selected between metastatic and non-metastatic patients available at A.C. Camargo Cancer Center Tumor Tissue Biobank. Polysome profiling was performed for all cases and show that increased translational rates are associated with reduced overall and progression-free survival. Citation Format: Julia A. Vassalakis, Stenio C. Zequi, Stephania M. Bezerra, Walter H. da Costa, Ola Larsson, Ivan Topisirovic, Glaucia N. Hajj. Polysome profiling suggests VHL-dependent translational control in clear cell renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2441.

A p53-dependent translational program directs tissue-selective phenotypes in a model of ribosomopathies.

In ribosomopathies, perturbed expression of ribosome components leads to tissue-specific phenotypes. What accounts for such tissue-selective manifestations as a result of mutations in the ribosome, a ubiquitous cellular machine, has remained a mystery. Combining mouse genetics and invivo ribosome profiling, we observe limb-patterning phenotypes in ribosomal protein (RP) haploinsufficient embryos, and we uncover selective translational changes of transcripts that controlling limb development. Surprisingly, both loss of p53, which is activated by RP haploinsufficiency, and augmented protein synthesis rescue these phenotypes. These findings are explained by the finding that p53 functions as a master regulator of protein synthesis, at least in part, through transcriptional activation of 4E-BP1. 4E-BP1, a key translational regulator, in turn, facilitates selective changes in the translatome downstream of p53, and this thereby explains how RP haploinsufficiency may elicit specificity to gene expression. These results provide an integrative model to help understand how invivo tissue-specific phenotypes emerge in ribosomopathies.

Single‐Molecule Dynamics of Eukaryotic Translation Initiation

Translation initiation is a critical control point for eukaryotic gene expression. The molecular mechanism of initiation requires a highly dynamic and exquisitely coordinated interplay of mRNA with the translation machinery. Recognition of the mRNA 5ʹ cap structure by initiation factor eIF4F is a key early initiation step, and plays important roles in translational control. We have developed single-molecule fluorescence approaches to directly observe eIF4F–mRNA interaction in real time, both on individual mRNAs and at transcriptome scale with single-molecule resolution. Our data reveal order-of-magnitude variability across mRNAs for interaction of the cap-binding eIF4F subunit, eIF4E, and provide insights into how the eIF4F subunits eIF4G and eIF4A dynamically modulate cap recognition. The data implicate features throughout the mRNA as controlling cap recognition at the 5ʹ end, including a relationship between coding-sequence length and the accessibility of the cap for recognition. We have also developed a single-molecule assay to directly observe the initiation steps that follow eIF4F–mRNA recognition, i.e. 43S ribosomal pre-initiation complex recruitment and pre-initiation complex scanning to locate the start codon. Taken together, our results evoke a multi-layered, mRNA-centric mechanism to kinetically control translation initiation and confer differential sensitivity to distinct regulatory mechanisms.

Ribosomal protein S7 ubiquitination during ER stress in yeast is associated with selective mRNA translation and stress outcome

eIF2α phosphorylation-mediated translational regulation is crucial for global translation repression by various stresses, including the unfolded protein response (UPR). However, translational control during UPR has not been demonstrated in yeast. This study investigated ribosome ubiquitination-mediated translational controls during UPR. Tunicamycin-induced ER stress enhanced the levels of ubiquitination of the ribosomal proteins uS10, uS3 and eS7. Not4-mediated monoubiquitination of eS7A was required for resistance to tunicamycin, whereas E3 ligase Hel2-mediated ubiquitination of uS10 was not. Ribosome profiling showed that the monoubiquitination of eS7A was crucial for translational regulation, including the upregulation of the spliced form of HAC1 (HAC1i) mRNA and the downregulation of Histidine triad NucleoTide-binding 1 (HNT1) mRNA. Downregulation of the deubiquitinating enzyme complex Upb3-Bre5 increased the levels of ubiquitinated eS7A during UPR in an Ire1-independent manner. These findings suggest that the monoubiquitination of ribosomal protein eS7A plays a crucial role in translational controls during the ER stress response in yeast.

New Pancreatic Cancer Biomarkers eIF1, eIF2D, eIF3C and eIF6 Play a Major Role in Translational Control in Ductal Adenocarcinoma.

Pancreatic cancer is one of the deadliest forms of cancer and ranks among the leading causes of cancer-related death worldwide. The most common histological type is ductal adenocarcinoma (PDAC), accounting for approximately 95% of cases. Deregulation of protein synthesis has been found to be closely related to cancer. The rate-limiting step of translation is initiation, which is regulated by a broad range of eukaryotic translation initiation factors (eIFs). Human PDAC samples were biochemically analyzed for the expression of various eIF subunits on the protein level (immunohistochemistry, immunoblot analyses) in 174 cases of PDAC in comparison with non-neoplastic pancreatic tissue (n=10). Our investigation revealed a significant down-regulation of four specific eIF subunits, namely eIF1, eIF2D, eIF3C and eIF6. Concomitantly, the protein (immunoblot) levels of eIF1, eIF2D, eIF3C and eIF6 were reduced in PDAC samples as compared with non-neoplastic pancreatic tissue. Members of the eIF family are of relevance in pancreatic tumor biology and may play a major role in translational control in PDAC. Consequently, they might be useful as potential new biomarkers and therapeutic targets in PDAC.

The Role of Translation Control in Tumorigenesis and Its Therapeutic Implications

As a convergent mechanism downstream of most oncogenic signals, control of mRNA translation has emerged as a key driver in establishing and tuning gene expression at specific steps in cancer development. Translation control is the most energetically expensive molecular process in the cell that needs to be modulated upon adaption to limited cellular resources, such as cellular stress. It thereby serves as the Achilles’ heel for cancer cells, particularly in response to changes in the microenvironment as well as to nutrient and metabolic shifts characteristic of cancer cell growth and metastasis. In this review, we discuss emerging discoveries that reveal how cancer cells modulate the translation machinery to adapt to oncogenic stress, the mechanisms that guide mRNA translation specificity in cancer, and how this selective mode of gene regulation provides advantages for cancer progression. We also provide an overview of promising preclinical and clinical efforts aimed at targeting the unique vulnerabilities of cancer cells that rely on the remodeling of mRNA translation for their infinite growth and survival.

Component of splicing factor SF3b plays a key role in translational control of polyribosomes on the endoplasmic reticulum

One of the morphological hallmarks of terminally differentiated secretory cells is highly proliferated membrane of the rough endoplasmic reticulum (ER), but the molecular basis for the high rate of protein biosynthesis in these cells remains poorly documented. An important aspect of ER translational control is the molecular mechanism that supports efficient use of targeted mRNAs in polyribosomes. Here, we identify an enhancement system for ER translation promoted by p180, an integral ER membrane protein we previously reported as an essential factor for the assembly of ER polyribosomes. We provide evidence that association of target mRNAs with p180 is critical for efficient translation, and that SF3b4, an RNA-binding protein in the splicing factor SF3b, functions as a cofactor for p180 at the ER and plays a key role in enhanced translation of secretory proteins. A cis-element in the 5' untranslated region of collagen and fibronectin genes is important to increase translational efficiency in the presence of p180 and SF3b4. These data demonstrate that a unique system comprising a p180-SF3b4-mRNA complex facilitates the selective assembly of polyribosomes on the ER.

EIF4E-Binding Proteins 1 and 2 Limit Macrophage Anti-Inflammatory Responses through Translational Repression of IL-10 and Cyclooxygenase-2.

Macrophages represent one of the first lines of defense during infections and are essential for resolution of inflammation following pathogen clearance. Rapid activation or suppression of protein synthesis via changes in translational efficiency allows cells of the immune system, including macrophages, to quickly respond to external triggers or cues without de novo mRNA synthesis. The translational repressors eIF4E-binding proteins 4E-BP1 and 4E-BP2 (4E-BP1/2) are central regulators of proinflammatory cytokine synthesis during viral and parasitic infections. However, it remains to be established whether 4E-BP1/2 play a role in translational control of anti-inflammatory responses. By comparing translational efficiencies of immune-related transcripts in macrophages from wild-type and 4E-BP1/2 double-knockout mice, we found that translation of mRNAs encoding two major regulators of inflammation, IL-10 and PG-endoperoxide synthase 2/cyclooxygenase-2, is controlled by 4E-BP1/2. Genetic deletion of 4E-BP1/2 in macrophages increased endogenous IL-10 and PGE2 protein synthesis in response to TLR4 stimulation and reduced their bactericidal capacity. The molecular mechanism involves enhanced anti-inflammatory gene expression (sIl1ra, Nfil3, Arg1, Serpinb2) owing to upregulation of IL-10-STAT3 and PGE2-C/EBPβ signaling. These data provide evidence that 4E-BP1/2 limit anti-inflammatory responses in macrophages and suggest that dysregulated activity of 4E-BP1/2 might be involved in reprogramming of the translational and downstream transcriptional landscape of macrophages during pathological conditions, such as infections and cancer.

The Long Non-coding RNA lnc-31 Interacts with Rock1 mRNA and Mediates Its YB-1-Dependent Translation

SummaryCytoplasmic long non-coding RNAs have been shown to act at many different levels to control post-transcriptional gene expression, although their role in translational control is poorly understood. Here, we show that lnc-31, a non-coding RNA required for myoblast proliferation, promotes ROCK1 protein synthesis by stabilizing its translational activator, YB-1. We find that lnc-31 binds to the Rock1 mRNA as well as to the YB-1 protein and that translational activation requires physical interaction between the two RNA species. These results suggest a localized effect of YB-1 stabilization on the Rock1 mRNA. ROCK1 upregulation by lnc-31, in proliferative conditions, correlates well with the differentiation-repressing activity of ROCK1. We also show that, upon induction of differentiation, the downregulation of lnc-31, in conjunction with miR-152 targeting of Rock1, establishes a regulatory loop that reinforces ROCK1 repression and promotes myogenesis.

The 5' Untranslated Region of the Major Immediate Early mRNA Is Necessary for Efficient Human Cytomegalovirus Replication.

The human cytomegalovirus (HCMV) immediate early 1 (IE1) and IE2 proteins are critical regulators of virus replication. Both proteins are needed to efficiently establish lytic infection, and nascent expression of IE1 and IE2 is critical for reactivation from latency. The regulation of IE1 and IE2 protein expression is thus a central event in the outcome of HCMV infection. Transcription of the primary transcript encoding both IE1 and IE2 is well studied, but relatively little is known about the posttranscriptional mechanisms that control IE1 and IE2 protein synthesis. The mRNA 5' untranslated region (5' UTR) plays an important role in regulating mRNA translation. Therefore, to better understand the control of IE1 and IE2 mRNA translation, we examined the role of the shared 5' UTR of the IE1 and IE2 mRNAs (MIE 5' UTR) in regulating translation. In a cell-free system, the MIE 5' UTR repressed translation, as predicted based on its length and sequence composition. However, in transfected cells we found that the MIE 5' UTR increased the expression of a reporter gene and enhanced its association with polysomes, demonstrating that the MIE 5' UTR has a positive role in translation control. We also found that the MIE 5' UTR was necessary for efficient IE1 and IE2 translation during infection. Replacing the MIE 5' UTR with an unstructured sequence of the same length decreased IE1 and IE2 protein expression despite similar levels of IE1 and IE2 mRNA and reduced the association of the IE1 and IE2 mRNAs with polysomes. The wild-type MIE 5'-UTR sequence was also necessary for efficient HCMV replication. Together these data identify the shared 5' UTR of the IE1 and IE2 mRNAs as an important regulator of HCMV lytic replication.IMPORTANCE The HCMV IE1 and IE2 proteins are critical regulators of HCMV replication, both during primary infection and during reactivation from viral latency. Thus, defining factors that regulate IE1 and IE2 expression is important for understanding the molecular events controlling the HCMV replicative cycle. Here we identify a positive role for the MIE 5' UTR in mediating the efficient translation of the IE1 and IE2 mRNAs. This result is an important advance for several reasons. To date, most studies of IE1 and IE2 regulation have focused on defining events that regulate IE1 and IE2 transcription. Our work reveals that in addition to the regulation of transcription, IE1 and IE2 are also regulated at the level of translation. Therefore, this study is important in that it identifies an additional layer of regulation controlling IE1 and IE2 expression and thus HCMV pathogenesis. These translational regulatory events could potentially be targeted by novel antiviral therapeutics that limit IE1 and IE2 mRNA translation and thus inhibit lytic replication or prevent HCMV reactivation.

Separation of low and high grade colon and rectum carcinoma by eukaryotic translation initiation factors 1, 5 and 6.

Colorectal cancer (CRC) is the third most common cause of cancer related death worldwide. Furthermore, with more than 1.2 million cases registered per year, it constitutes the third most frequent diagnosed cancer entity worldwide. Deregulation of protein synthesis has received considerable attention as a major step in cancer development and progression. Eukaryotic translation initiation factors (eIFs) are involved in the regulation of protein synthesis and are functionally linked to the phosphatidylinositol-3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling pathway.The identification of factors accounting for colorectal carcinoma (CRC) development is a major gap in the field. Besides the importance of eIF3 subunits and the eIF4 complex, eIF1, eIF5 and eIF6 were found to be altered in primary and metastatic CRC. We observed significant difference in the expression profile between low and high grade CRC. eIF1, eIF5 and eIF6 are involved in translational control in CRC. Our findings also indicate a probable clinical impact when separating them into low and high grade colon and rectum carcinoma.eIF and mTOR expression were analysed on protein and mRNA level in primary low and high grade colon carcinoma (CC) and rectum carcinoma (RC) samples in comparison to non-neoplastic tissue without any disease-related pathology. To assess the therapeutic potential of targeting eIF1, eIF5 and eIF6 siRNA knockdown in HCT116 and HT29 cells was performed. We evaluated the eIF knockdown efficacy on protein and mRNA level and investigated proliferation, apoptosis, invasion, as well as colony forming and polysome associated fractions.These results indicate that eIFs, in particular eIF1, eIF5 and eIF6 play a major role in translational control in colon and rectum cancer.

Abstract 4481: Translation initiation factor DAP5 plays an essential role in translational control of breast cancer metastasis

Abstract Metastasis is the cause of 90% of cancer-related deaths. Selective mRNA translation has been found to be crucial for breast cancer development, progression and metastasis; however, there is a poor biological and mechanistic understanding of the role of translation in these processes. Our work and others have pioneered an understanding of translational regulation in breast cancer. We have previously shown that overexpression of initiation factor eIF4GI in advanced breast cancer cells selectively increases the translation of mRNAs encoding survival, cell cycle inhibition, and DNA damage and repair proteins, among other DNA damage-protective mRNAs. eIF4G consists of three family members: eIF4GI, eIF4GII, and the poorly studied eIF4G homolog, DAP5, the subject of this study. DAP5 lacks the N-terminal domain for eIF4E and PABP binding. DAP5 is therefore suspected to promote eIF4E-independent or IRES-driven translation of mRNAs involved in cell stress and oncogenesis, but its role in metastasis hasn’t been investigated. We analyzed the NCI human tissue genome cancer atlas (TCGA) and found that higher DAP5 mRNA expression is strongly associated with estrogen receptor negative breast cancer metastasis with lower overall survival. We then engineered a highly metastatic breast cancer cell line MDA-MB-231TR to express dox-inducible shRNA to DAP5 or a non-silencing (NS) control. Tumors were developed in clinically relevant orthotopic mouse models, with and without DAP5 silencing by addition of dox to the drinking water. Remarkably, reduction of DAP5 expression by ~70% had no impact on primary tumor growth but fully eliminated metastasis to the lung. Genome-wide transcriptome and translatome analysis of DAP5 and in vivo ultraviolet-crosslinking and high-throughput sequencing (HITS-CLIP) identified specific mRNAs that interact directly with DAP5. A significant fraction of DAP5 mRNA targets are involved in cell death and survival, cell proliferation, cell mobility, DNA repair and translation initiation and do not have IRESs, suggesting a novel role for DAP5 in a non-classical translation initiation process upon cellular stress and metastasis. Our results suggest that the translation initiation factor DAP5 likely plays a critical role in breast cancer metastasis by a unique mechanism for metastasis-specific translation initiation and provides new concepts for therapeutic strategies involving translational regulation. Citation Format: Columba de la Parra, Amandine Alard, Amanda Ernlund, Robert J. Schneider. Translation initiation factor DAP5 plays an essential role in translational control of breast cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4481. doi:10.1158/1538-7445.AM2017-4481

Human Keratinocyte Differentiation Requires Translational Control by the eIF2α Kinase GCN2

Appropriate and sequential differentiation of keratinocytes is essential for all functions of the human epidermis. Although transcriptional regulation has proven to be important for keratinocyte differentiation, little is known about the role of translational control. A key mechanism for modulating translation is through phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2). A family of different eIF2α kinases function in the integrative stress response to inhibit general protein synthesis coincident with preferential translation of select mRNAs that participate in stress alleviation. Here we demonstrate that translational control through eIF2α phosphorylation is required for normal keratinocyte differentiation. Analyses of polysome profiles revealed that key differentiation genes, including involucrin, are bound to heavy polysomes during differentiation, despite decreased general protein synthesis. Induced eIF2α phosphorylation by the general control nonderepressible 2 (GCN2) protein kinase facilitated translational control and differentiation-specific protein expression during keratinocyte differentiation. Furthermore, loss of GCN2 thwarted translational control, normal epidermal differentiation, and differentiation gene expression in organotypic skin culture. These findings underscore a previously unknown function for GCN2 phosphorylation of eIF2α and translational control in the formation of an intact human epidermis.

Erratum: New frontiers in translational control of the cancer genome

The past several years have seen dramatic leaps in our understanding of how gene expression is rewired at the translation level during tumorigenesis to support the transformed phenotype. This work has been driven by an explosion in technological advances and is revealing previously unimagined regulatory mechanisms that dictate functional expression of the cancer genome. In this Review we discuss emerging trends and exciting new discoveries that reveal how this translational circuitry contributes to specific aspects of tumorigenesis and cancer cell function, with a particular focus on recent insights into the role of translational control in the adaptive response to oncogenic stress conditions.