Translational Control Of Gene Expression Research Articles

Navigating translational control of gene expression in satellite cells.

Satellite cells, named for their satellite position around the sarcolemma of the myofibre, are responsible for skeletal muscle regeneration. Satellite cells normally reside in a quiescent state, but rapidly activate the myogenic program and the cell cycle in response to injury. Translational control of gene expression has emerged as an important regulator of satellite cell activity. Quiescent satellite cells maintain low levels of protein synthesis and selectively translate specific mRNAs to conserve limited energy. Activated satellite cells rapidly restore global protein synthesis to meet the demands of proliferating myogenic progenitors that participate in muscle repair. We propose a model by which translational control enables rapid protein level changes in response to injury-induced environmental shifts, serving as both a brake mechanism during quiescence and an accelerator for injury response. In this Chapter, we navigate the processing, translation and metabolism of newly transcribed mRNAs. We review the modifications of mRNA that occur during mRNA processing in the nucleus of satellite cells, and illustrate how these modifications impact the translation and stability of mRNAs. In the cytoplasm, we review how pathways work in concert to regulate protein synthesis globally, while trans acting microRNAs and RNA binding proteins modify specific mRNA translation within a context of tightly regulated protein synthesis. While navigating translational control of gene expression in satellite cells, this chapter reveals that despite significant progress, the field remains nascent in the broader scope of translational control in cell biology. We propose that future investigations will benefit from incorporating emerging global analyses to study translational control of gene expression in rare satellite cells, and we pose unanswered questions that warrant future exploration.

Enhancement of protein translation by CRISPR/dCasRx coupled with SINEB2 repeat of noncoding RNAs.

The use of new long noncoding RNAs (lncRNAs) as biotechnological or therapeutic tools is still in its infancy, despite recent efforts to uncover their involvement in various biological processes including mRNA translation. An important question is whether lncRNA functional elements can be used to target translation of mRNAs of interest by incorporating the RNA-targeting CRISPR tools. The CRISPR/dCasRx-SINEB2 technology was developed in this research by coupling the sgRNA of a catalytically inactive Type VI-D Cas13 enzyme (CasRx) to an integrated SINEB2 domain of uchl1 lncRNA that promotes the translation of targeted mRNA. It has been demonstrated to be effective and adaptable in selectively increasing the expression of a variety of exogenous and endogenous proteins with a variety of functions with minimal off-target effects. dCasRx-SINEB2 is currently the sole CRISPR-related technique for translational control of gene expression, and works just as well or even better than the traditional RNAe tool under comparable conditions. Additionally, human cancer cells can be prevented from proliferating and migrating both in vitro and in vivo by dCasRx-SINEB2-targeted mRNA translation of transcripts encoding for antitumor proteins, including PTEN and P53. The present study provides an innovative protein enhancement method that will have several applications in biopharmaceuticals production and cancer research.

Inhibition of the Translation Initiation Factor eIF4A Enhances Tumor Cell Radiosensitivity.

A fundamental component of cellular radioresponse is the translational control of gene expression. Because a critical regulator of translational control is the eukaryotic translation initiation factor 4F (eIF4F) cap binding complex, we investigated whether eIF4A, the RNA helicase component of eIF4F, can serve as a target for radiosensitization. Knockdown of eIF4A using siRNA reduced translational efficiency, as determined from polysome profiles, and enhanced tumor cell radiosensitivity as determined by clonogenic survival. The increased radiosensitivity was accompanied by a delayed dispersion of radiation-induced γH2AX foci, suggestive of an inhibition of DNA double-strand break repair. Studies were then extended to (-)-SDS-1-021, a pharmacologic inhibitor of eIF4A. Treatment of cells with the rocaglate (-)-SDS-1-021 resulted in a decrease in translational efficiency as well as protein synthesis. (-)-SDS-1-021 treatment also enhanced the radiosensitivity of tumor cell lines. This (-)-SDS-1-021-induced radiosensitization was accompanied by a delay in radiation-induced γH2AX foci dispersal, consistent with a causative role for the inhibition of double-strand break repair. In contrast, although (-)-SDS-1-021 inhibited translation and protein synthesis in a normal fibroblast cell line, it had no effect on radiosensitivity of normal cells. Subcutaneous xenografts were then used to evaluate the in vivo response to (-)-SDS-1-021 and radiation. Treatment of mice bearing subcutaneous xenografts with (-)-SDS-1-021 decreased tumor translational efficiency as determined by polysome profiles. Although (-)-SDS-1-021 treatment alone had no effect on tumor growth, it significantly enhanced the radiation-induced growth delay. These results suggest that eIF4A is a tumor-selective target for radiosensitization.

RNA and neuronal function: the importance of post-transcriptional regulation.

The brain represents an organ with a particularly high diversity of genes that undergo post-transcriptional gene regulation through multiple mechanisms that affect RNA metabolism and, consequently, brain function. This vast regulatory process in the brain allows for a tight spatiotemporal control over protein expression, a necessary factor due to the unique morphologies of neurons. The numerous mechanisms of post-transcriptional regulation or translational control of gene expression in the brain include alternative splicing, RNA editing, mRNA stability and transport. A large number of trans-elements such as RNA-binding proteins and micro RNAs bind to specific cis-elements on transcripts to dictate the fate of mRNAs including its stability, localization, activation and degradation. Several trans-elements are exemplary regulators of translation, employing multiple cofactors and regulatory machinery so as to influence mRNA fate. Networks of regulatory trans-elements exert control over key neuronal processes such as neurogenesis, synaptic transmission and plasticity. Perturbations in these networks may directly or indirectly cause neuropsychiatric and neurodegenerative disorders. We will be reviewing multiple mechanisms of gene regulation by trans-elements occurring specifically in neurons.

Combined analyses of translatome and transcriptome in Arabidopsis reveal new players responding to magnesium deficiency.

Translational control of gene expression, including recruitment of ribosomes to messenger RNA (mRNA), is particularly important during the response to stress. Purification of ribosome-associated mRNAs using translating ribosome affinity purification (TRAP) followed by RNA-sequencing facilitates the study of mRNAs undergoing active transcription and better proxies the translatome, or protein response, to stimuli. To identify plant responses to Magnesium (Mg) deficiency at the translational level, we combined transcriptome and translatome analyses. Excitingly, we found 26 previously unreported Mg-responsive genes that were only regulated at the translational level and not the transcriptional level, during the early response to Mg deficiency. In addition, mutants of the transcription factor ELONGATED HYPOCOTYL 5 (HY5), the H+ /CATION EXCHANGER 1 and 3 (CAX1 and CAX3), and UBIQUITIN 11 (UBQ11) exhibited early chlorosis phenotype under Mg deficiency, supporting their functional involvement in ion homeostasis. Overall, our study strongly supports that TRAP-seq combined with RNA-seq followed by phenotype screening could facilitate the identification of novel players during stress responses.

Translation Initiation Machinery as a Tumor Selective Target for Radiosensitization.

Towards improving the efficacy of radiotherapy, one approach is to target the molecules and processes mediating cellular radioresponse. Along these lines, translational control of gene expression has been established as a fundamental component of cellular radioresponse, which suggests that the molecules participating in this process (i.e., the translational machinery) can serve as determinants of radiosensitivity. Moreover, the proteins comprising the translational machinery are often overexpressed in tumor cells suggesting the potential for tumor specific radiosensitization. Studies to date have shown that inhibiting proteins involved in translation initiation, the rate-limiting step in translation, specifically the three members of the eIF4F cap binding complex eIF4E, eIF4G, and eIF4A as well as the cap binding regulatory kinases mTOR and Mnk1/2, results in the radiosensitization of tumor cells. Because ribosomes are required for translation initiation, inhibiting ribosome biogenesis also appears to be a strategy for radiosensitization. In general, the radiosensitization induced by targeting the translation initiation machinery involves inhibition of DNA repair, which appears to be the consequence of a reduced expression of proteins critical to radioresponse. The availability of clinically relevant inhibitors of this component of the translational machinery suggests opportunities to extend this approach to radiosensitization to patient care.

Glucose treatment of human pancreatic β-cells enhances translation of mRNAs involved in energetics and insulin secretion

Glucose-mediated signaling regulates the expression of a limited number of genes in human pancreatic β-cells at the transcriptional level. However, it is unclear whether glucose plays a role in posttranscriptional RNA processing or translational control of gene expression. Here, we asked whether glucose affects posttranscriptional steps and regulates protein synthesis in human β-cell lines. We first showed the involvement of the mTOR pathway in glucose-related signaling. We also used the surface sensing of translation technique, based on puromycin incorporation into newly translated proteins, to demonstrate that glucose treatment increased protein translation. Among the list of glucose-induced proteins, we identified the proconvertase PCSK1, an enzyme involved in the proteolytic conversion of proinsulin to insulin, whose translation was induced within minutes following glucose treatment. We finally performed global proteomic analysis by mass spectrometry to characterize newly translated proteins upon glucose treatment. We found enrichment in proteins involved in translation, glycolysis, TCA metabolism, and insulin secretion. Taken together, our study demonstrates that, although glucose minorly affects gene transcription in human β-cells, it plays a major role at the translational level.

Translational control of gene expression by eIF2 modulates proteostasis and extends lifespan.

Although the stress response in eukaryotes depends on early events triggered in cells by environmental insults, long-term processes such as aging are also affected. The loss of cellular proteostasis greatly impacts aging, which is regulated by the balancing of protein synthesis and degradation systems. As translation is the input event in proteostasis, we decided to study the role of translational activity on cell lifespan. Our hypothesis was that a reduction on translational activity or specific changes in translation may increase cellular longevity.Using mutant strains of Schizosaccharomyces pombe and various stress conditions, we showed that translational reduction caused by phosphorylation of eukaryotic translation initiation factor 2 (eIF2) during the exponential growth phase enhances chronological lifespan (CLS). Furthermore, through next-generation sequence analysis, we found eIF2α phosphorylation-dependent translational activation of some specific genes, especially those involved in autophagy. This fact, together with the observed regulation of autophagy, points to a conserved mechanism involving general and specific control of translation and autophagy as mediators of the role of eIF2α phosphorylation in aging.

Global phosphoproteomics pinpoints uncharted Gcn2-mediated mechanisms of translational control

The conserved Gcn2 protein kinase mediates cellular adaptations to amino acid limitation through translational control of gene expression that is exclusively executed by phosphorylation of the α-subunit of the eukaryotic translation initiation factor 2 (eIF2α). Using quantitative phosphoproteomics, however, we discovered that Gcn2 targets auxiliary effectors to modulate translation. Accordingly, Gcn2 also phosphorylates the β-subunit of the trimeric eIF2G protein complex to promote its association with eIF5, which prevents spontaneous nucleotide exchange on eIF2 and thereby restricts the recycling of the initiator methionyl-tRNA-bound eIF2-GDP ternary complex in amino-acid-starved cells. This mechanism contributes to the inhibition of translation initiation in parallel to the sequestration of the nucleotide exchange factor eIF2B by phosphorylated eIF2α. Gcn2 further phosphorylates Gcn20 to antagonize, in an inhibitory feedback loop, the formation of the Gcn2-stimulatory Gcn1-Gcn20 complex. Thus, Gcn2 plays a substantially more intricate role in controlling translation initiation than hitherto appreciated.

A translation enhancer element from black beetle virus engages yeast eIF4G1 to drive cap-independent translation initiation

Cap-independent translation initiation plays crucial roles in fine-tuning gene expression under global translation shutdown conditions. Translation of uncapped or de-capped transcripts can be stimulated by Cap-independent translation enhancer (CITE) elements, but the mechanisms of CITE-mediated translation initiation remain understudied. Here, we characterized a short 5ʹ-UTR RNA sequence from black beetle virus, BBV-seq. Mutational analysis indicates that the entire BBV-seq is required for efficient translation initiation, but this sequence does not operate as an IRES-type module. In yeast cell-free translation extracts, BBV-seq promoted efficient initiation on cap-free mRNA using a scanning mechanism. Moreover, BBV-seq can increase translation efficiency resulting from conventional cap-dependent translation initiation. Using genetic approaches, we found that BBV-seq exploits RNA-binding properties of eIF4G1 to promote initiation. Thus, BBV-seq constitutes a previously uncharacterized short, linear CITE that influences eIF4G1 to initiate 5′ end-dependent, cap-independent translation. These findings bring new insights into CITE-mediated translational control of gene expression.

Stress-Induced Translation Inhibition through Rapid Displacement of Scanning Initiation Factors

SummaryCellular responses to environmental stress are frequently mediated by RNA-binding proteins (RBPs). Here, we examined global RBP dynamics in Saccharomyces cerevisiae in response to glucose starvation and heat shock. Each stress induced rapid remodeling of the RNA-protein interactome without corresponding changes in RBP abundance. Consistent with general translation shutdown, ribosomal proteins contacting the mRNA showed decreased RNA association. Among translation components, RNA association was most reduced for initiation factors involved in 40S scanning (eukaryotic initiation factor 4A [eIF4A], eIF4B, and Ded1), indicating a common mechanism of translational repression. In unstressed cells, eIF4A, eIF4B, and Ded1 primarily targeted the 5′ ends of mRNAs. Following glucose withdrawal, 5′ binding was abolished within 30 s, explaining the rapid translation shutdown, but mRNAs remained stable. Heat shock induced progressive loss of 5′ RNA binding by initiation factors over ∼16 min and provoked mRNA degradation, particularly for translation-related factors, mediated by Xrn1. Taken together, these results reveal mechanisms underlying translational control of gene expression during stress.

Abstract IA11: Translating the cancer genome one codon at a time and its therapeutic implications

Abstract Our research is centered on understanding translational control of gene expression in both normal health and disease in a cell- and tissue-specific manner, with a particular focus on cancer biology. Our research combines mouse genetics with genome-wide translational profiling, in-depth molecular biology, and pharmacology to systematically define the points of regulation, in cis and trans, by which the genome is selectively decoded into proteins. We have uncovered that a common denominator of multiple oncogenic pathways is their ability to directly control the core translation machinery of a cell, resulting in the rapid rewiring of mRNA translation programs that promote distinct hallmarks of cancer development such as cell growth, metabolism, and increased motility. For example, our most recent findings delineate the in vivo requirements for a distinct threshold of the major cap-binding protein, eIF4E, a key translation factor downstream of mTOR signaling, in normal organismal development compared to that required for translating the cancer genome. We show that cancer cells require increased eIF4E activity for their survival as distinct subsets of mRNAs that regulate the cancer cell oxidative response are marked by the presence of a novel eIF4E-dependent cis-acting translational control motif present in their 5'UTRs. I will also discuss the generation of the first comprehensive systems-level analysis of the “cancer translatome” during each phase of cancer development in vivo that highlights a dichotomy in transcriptional vs. translational control of gene expression guiding key, select steps in cancer development and evolution. These studies have uncovered a previously unappreciated translation program guiding tumor cell evasion of immune checkpoints. The immediate impact of our research has been the design of a new generation of compounds to target the aberrant translation machinery in cancer cells, which are currently in clinical trials and may reflect a new frontier in cancer therapy. Citation Format: Davide Ruggero. Translating the cancer genome one codon at a time and its therapeutic implications [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr IA11.

Modulation of Escherichia coli Translation by the Specific Inactivation of tRNAGly Under Oxidative Stress.

Bacterial oxidative stress responses are generally controlled by transcription factors that modulate the synthesis of RNAs with the aid of some sRNAs that control the stability, and in some cases the translation, of specific mRNAs. Here, we report that oxidative stress additionally leads to inactivation of tRNAGly in Escherichia coli, inducing a series of physiological changes. The observed inactivation of tRNAGly correlated with altered efficiency of translation of Gly codons, suggesting a possible mechanism of translational control of gene expression under oxidative stress. Changes in translation also depended on the availability of glycine, revealing a mechanism whereby bacteria modulate the response to oxidative stress according to the prevailing metabolic state of the cells.

1H, 13C, and 15N backbone chemical shift assignments of m7GTP cap-bound Leishmania initiation factor 4E-1

Most of the translational control of gene expression in higher eukaryotes occurs during the initiation step of protein synthesis. While this process is well characterized in mammalian cells, it is less defined in parasites, including the ones that cause human Leishmaniasis. The Leishmania cap-binding isoform 1 (LeishIF4E-1) is the only isoform that binds the specific trypanosomatids-specific hypermethylated 5′ cap, called cap-4, in the human stage of the parasite life cycle. We report here the extensive NMR resonance assignment of LeishIF4E-1 bound to a cap analog, m7GTP. The chemical shift data constitute a prerequisite to understanding specific translation initiation mechanisms used in Leishmania parasites and to developing antiparasitic drugs targeting their translation initiation factors.

Ribosome profiling in plants: what is not lost in translation?

Translation is a highly dynamic cellular process whereby genetic information residing in an mRNA molecule is converted into a protein that in turn executes specific functions. However, pre-synthesized mRNA levels do not always correlate with corresponding protein levels, suggesting that translational control plays an essential role in gene regulation. A better understanding of how gene expression is regulated during translation will enable the discovery of new genes and mechanisms that control important traits in plants. Therefore, in recent years, several methods have been developed to analyse the translatome; that is, all mRNAs being actively translated at a given time, tissue, and/or developmental stage. Ribosome profiling or ribo-seq is one such technology revolutionizing our ability to analyse the translatome and in turn understand translational control of gene expression. Ribo-seq involves isolating mRNA-ribosome complexes, treating them with a RNase, and then identifying ribosome-protected mRNA regions by deep sequencing. Here, we briefly review recent ribosome profiling studies that revealed new insights into plant biology. Manipulation of novel genes identified using ribosome profiling could prove useful for increasing yield through improved biotic and abiotic stress tolerance.

Satellite cell expansion is mediated by P-eIF2α-dependent Tacc3 translation.

Translational control of gene expression is an important regulator of adult stem cell quiescence, activation and self-renewal. In skeletal muscle, quiescent satellite cells maintain low levels of protein synthesis, mediated in part through the phosphorylation of eIF2α (P-eIF2α). Pharmacological inhibition of the eIF2α phosphatase with the small molecule sal003 maintains P-eIF2α and permits the expansion of satellite cells ex vivo Paradoxically, P-eIF2α also increases the translation of specific mRNAs, which is mediated by P-eIF2α-dependent read-through of inhibitory upstream open reading frames (uORFs). Here, we ask whether P-eIF2α-dependent mRNA translation enables expansion of satellite cells. Using transcriptomic and proteomic analyses, we show a number of genes associated with the assembly of the spindle pole to be upregulated at the level of protein, without corresponding change in mRNA levels, in satellite cells expanded in the presence of sal003. We show that uORFs in the 5' UTR of mRNA for the mitotic spindle stability gene Tacc3 direct P-eIF2α-dependent translation. Satellite cells deficient for TACC3 exhibit defects in expansion, self-renewal and regeneration of skeletal muscle.

A short translational ramp determines the efficiency of protein synthesis

Translation initiation is a major rate-limiting step for protein synthesis. However, recent studies strongly suggest that the efficiency of protein synthesis is additionally regulated by multiple factors that impact the elongation phase. To assess the influence of early elongation on protein synthesis, we employed a library of more than 250,000 reporters combined with in vitro and in vivo protein expression assays. Here we report that the identity of the amino acids encoded by codons 3 to 5 impact protein yield. This effect is independent of tRNA abundance, translation initiation efficiency, or overall mRNA structure. Single-molecule measurements of translation kinetics revealed pausing of the ribosome and aborted protein synthesis on codons 4 and 5 of distinct amino acid and nucleotide compositions. Finally, introduction of preferred sequence motifs only at specific codon positions improves protein synthesis efficiency for recombinant proteins. Collectively, our data underscore the critical role of early elongation events in translational control of gene expression.

Translational control of gene expression via interacting feedback loops.

Translation is a key step in the synthesis of proteins. Accordingly, cells have evolved an intricate array of control mechanisms to regulate this process. By constructing a multicomponent mathematical framework we uncover how translation may be controlled via interacting feedback loops. Our results reveal that this interplay gives rise to a remarkable range of protein synthesis dynamics, including oscillations, step change, and bistability. This suggests that cells may have recourse to a much richer set of control mechanisms than was previously understood.

Translational control maintains cartilage homeostasis and regulates osteoarthritis progression

Purpose: The unique properties of articular cartilage are defined by quiescent, differentiated articular chondrocytes whose homeostasis is maintained through a strict balance between anabolic and catabolic processes. This balance is shifted toward catabolic activities in osteoarthritis (OA), a disease that involves all joint tissues and disrupts the structural integrity of articular cartilage. In OA, articular chondrocytes become more hypertrophic-ike, and abnormally increase not only extracellular matrix (ECM) production but also the expression of ECM-degrading enzymes. While chondrocyte hypertrophy is indispensable for endochondral bone formation, in OA it mediates detrimental changes that lead to cartilage destruction. Our previous work established that translational control of gene expression is critical for dedifferentiation of articular chondrocytes in OA. We showed that fine-tuned control of protein synthesis by translational repressor 4E-BP (eIF4E-Binding Protein) is required for proper chondrocyte homeostasis. By binding to the m7-GTP cap-binding protein eIF4E, 4E-BP inhibits cap-dependent mRNA translation by restricting the incorporation of eIF4E into a multi-subunit initiation factor called eIF4F, which is needed to recruit 40S ribosomes to mRNA. We hypothesized that proper translational control is crucial for maintaining healthy cartilage phenotype. We therefore investigated expression of which genes is controlled (completely or partially) at the translational level. Methods: To identify the pool of mRNAs that are translationally control in OA cartilage we first compared translation efficiencies of mRNAs by analyzing polysome profiles of rat articular chondrocytes (RAC) untreated and treated with Interleukin 1β (IL-1β). Expression of several translationally regulated mRNAs was then compared to their expression in human and rodent OA cartilage. Additionally, the eIF4F inhibitor was used in vivo to determine if restriction of eIF4F activity affects OA progression in ACLT rat model. Results: We identified 617 mRNAs that are translationally regulated by IL-1β. One of them is the orphan receptor Nr4a1, whose protein but not mRNA level is significantly increased in OA cartilage in vivo, highlighting the exceptional potential of our approach for discovering new possible targets in OA cartilage. Modulating Nr4a1 expression in IL-1β-treated articular chondrocytes effects expression of MMP13, the metalloproteinase that has a predominant role in OA. Intra-articular injection of 4E1RCat, an inhibitor of cap-dependent translation not only precludes Fn and Nr4a upregulation in a rodent OA model, but also significantly delays cartilage degeneration in the ACLT rat model. Conclusions: We identified 617 mRNAs that are translationally regulated by IL-1β. One of them is the orphan receptor Nr4a1, whose protein but not mRNA level is significantly increased in OA cartilage in vivo, highlighting the exceptional potential of our approach for discovering new possible targets in OA cartilage. Modulating Nr4a1 expression in IL-1β-treated articular chondrocytes effects expression of MMP13, the metalloproteinase that has a predominant role in OA. Intra-articular injection of 4E1RCat, an inhibitor of cap-dependent translation not only precludes Fn and Nr4a upregulation in a rodent OA model, but also significantly delays cartilage degeneration in the ACLT rat model.

EIF4E-Dependent Translational Control: A Central Mechanism for Regulation of Pain Plasticity.

Translational control of gene expression has emerged as a key mechanism in regulating different forms of long-lasting neuronal plasticity. Maladaptive plastic reorganization of peripheral and spinal nociceptive circuits underlies many chronic pain states and relies on new gene expression. Accordingly, downregulation of mRNA translation in primary afferents and spinal dorsal horn neurons inhibits tissue injury-induced sensitization of nociceptive pathways, supporting a central role for translation dysregulation in the development of persistent pain. Translation is primarily regulated at the initiation stage via the coordinated activity of translation initiation factors. The mRNA cap-binding protein, eukaryotic translation initiation factor 4E (eIF4E), is involved in the recruitment of the ribosome to the mRNA cap structure, playing a central role in the regulation of translation initiation. eIF4E integrates inputs from the mTOR and ERK signaling pathways, both of which are activated in numerous painful conditions to regulate the translation of a subset of mRNAs. Many of these mRNAs are involved in the control of cell growth, proliferation, and neuroplasticity. However, the full repertoire of eIF4E-dependent mRNAs in the nervous system and their translation regulatory mechanisms remain largely unknown. In this review, we summarize the current evidence for the role of eIF4E-dependent translational control in the sensitization of pain circuits and present pharmacological approaches to target these mechanisms. Understanding eIF4E-dependent translational control mechanisms and their roles in aberrant plasticity of nociceptive circuits might reveal novel therapeutic targets to treat persistent pain states.