Genome-wide Translational Profiling Research Articles

The eukaryotic translation initiation factor eIF4E regulates flowering and circadian rhythm in Arabidopsis.

Translation initiation is a critical, rate-limiting step in protein synthesis. The eukaryotic translation initiation factor 4E (eIF4E) plays an essential role in this process. However, the mechanisms by which eIF4E-dependent translation initiation regulates plant growth and development remain not fully understood. In this study, we found that Arabidopsis eIF4E proteins are distributed in both the nucleus and cytoplasm, with only the cytoplasmic eIF4E being involved in the control of photoperiodic flowering. Genome-wide translation profiling using Ribo-tag sequencing reveals that eIF4E may regulate plant flowering by maintaining the homeostatic translation of components in the photoperiodic flowering pathway. eIF4E not only regulates the translation of flowering genes such as FLOWERING LOCUS T (FT) and FLOWERING LOCUS D (FLD) but also influences the translation of circadian genes like CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) and PSEUDO-RESPONSE REGULATOR 9 (PRR9). Consistently, our results show that the eIF4E modulates the rhythmic oscillation of the circadian clock. Together, our study provides mechanistic insights into how the protein translation regulates multiple developmental processes in Arabidopsis, including the circadian clock and photoperiodic flowering.

The transcription factor DUX4 orchestrates translational reprogramming by broadly suppressing translation efficiency and promoting expression of DUX4-induced mRNAs.

Translational control is critical for cell fate transitions during development, lineage specification, and tumorigenesis. Here, we show that the transcription factor double homeobox protein 4 (DUX4), and its previously characterized transcriptional program, broadly regulates translation to change the cellular proteome. DUX4 is a key regulator of zygotic genome activation in human embryos, whereas misexpression of DUX4 causes facioscapulohumeral muscular dystrophy (FSHD) and is associated with MHC-I suppression and immune evasion in cancer. We report that translation initiation and elongation factors are disrupted downstream of DUX4 expression in human myoblasts. Genome-wide translation profiling identified mRNAs susceptible to DUX4-induced translation inhibition, including those encoding antigen presentation factors and muscle lineage proteins, while DUX4-induced mRNAs were robustly translated. Endogenous expression of DUX4 in human FSHD myotubes and cancer cell lines also correlated with reduced protein synthesis and MHC-I presentation. Our findings reveal that DUX4 orchestrates cell state conversion by suppressing the cellular proteome while maintaining translation of DUX4-induced mRNAs to promote an early developmental program.

Neuronal cell types, projections, and spatial organization of the central amygdala.

The central amygdala (CEA) has been richly studied for interpreting function and behavior according to specific cell types and circuits. Such work has typically defined molecular cell types by classical inhibitory marker genes; consequently, whether marker-gene-defined cell types exhaustively cover the CEA and co-vary with connectivity remains unresolved. Here, we combined single-cell RNA sequencing, multiplexed fluorescent in situ hybridization, immunohistochemistry, and long-range projection mapping to derive a "bottom-up" understanding of CEA cell types. In doing so, we identify two major cell types, encompassing one-third of all CEA neurons, that have gone unresolved in previous studies. In spatially mapping these novel types, we identify a non-canonical CEA subdomain associated with Nr2f2 expression and uncover an Isl1-expressing medial cell type that accounts for many long-range CEA projections. Our results reveal new CEA organizational principles across cell types and spatial scales and provide a framework for future work examining cell-type-specific behavior and function.

Origin of translational control by eIF2α phosphorylation: insights from genome-wide translational profiling studies in fission yeast.

During amino acid limitation, the protein kinase Gcn2 phosphorylates the α subunit of eIF2, thereby regulating mRNA translation. In yeast Saccharomyces cerevisiae and mammals, eIF2α phosphorylation regulates translation of related transcription factors Gcn4 and Atf4 through upstream open reading frames (uORFs) to activate transcription genome wide. However, mammals encode three more eIF2α kinases activated by distinct stimuli. Did the translational control system involving eIF2α phosphorylation evolve from so simple (as found in yeast S. cerevisiae) to complex (as found in humans)? Recent genome-wide translational profiling studies of amino acid starvation response in the fission yeast Schizosaccharomyces pombe provide an unexpected answer to this question.

Genome-wide translational profiling of amygdala Crh-expressing neurons reveals role for CREB in fear extinction learning

Fear and extinction learning are adaptive processes caused by molecular changes in specific neural circuits. Neurons expressing the corticotropin-releasing hormone gene (Crh) in central amygdala (CeA) are implicated in threat regulation, yet little is known of cell type-specific gene pathways mediating adaptive learning. We translationally profiled the transcriptome of CeA Crh-expressing cells (Crh neurons) after fear conditioning or extinction in mice using translating ribosome affinity purification (TRAP) and RNAseq. Differential gene expression and co-expression network analyses identified diverse networks activated or inhibited by fear vs extinction. Upstream regulator analysis demonstrated that extinction associates with reduced CREB expression, and viral vector-induced increased CREB expression in Crh neurons increased fear expression and inhibited extinction. These findings suggest that CREB, within CeA Crh neurons, may function as a molecular switch that regulates expression of fear and its extinction. Cell-type specific translational analyses may suggest targets useful for understanding and treating stress-related psychiatric illness.

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.

Identification and characterization of hippuristanol-resistant mutants reveals eIF4A1 dependencies within mRNA 5' leader regions.

Hippuristanol (Hipp) is a natural product that selectively inhibits protein synthesis by targeting eukaryotic initiation factor (eIF) 4A, a DEAD-box RNA helicase required for ribosome recruitment to mRNA templates. Hipp binds to the carboxyl-terminal domain of eIF4A, locks it in a closed conformation, and inhibits its RNA binding. The dependencies of mRNAs for eIF4A during initiation is contingent on the degree of secondary structure within their 5′ leader region. Interest in targeting eIF4A therapeutically in cancer and viral-infected settings stems from the dependencies that certain cellular (e.g. pro-oncogenic, pro-survival) and viral mRNAs show towards eIF4A. Using a CRISPR/Cas9-based variomics screen, we identify functional EIF4A1 Hipp-resistant alleles, which in turn allowed us to link the translation-inhibitory and cytotoxic properties of Hipp to eIF4A1 target engagement. Genome-wide translational profiling in the absence or presence of Hipp were undertaken and our validation studies provided insight into the structure-activity relationships of eIF4A-dependent mRNAs. We find that mRNA 5′ leader length, overall secondary structure and cytosine content are defining features of Hipp-dependent mRNAs.

NcRNA-Encoded Peptides or Proteins and Cancer.

Non-coding RNAs (ncRNAs) are unique RNA transcripts that have been widely identified in the eukaryotic genome and have been shown to play key roles in the development of many cancers. However, the rapid development of genome-wide translation profiling and ribosome profiling has revealed that a small number of small open reading frames (sORFs) within ncRNAs actually have peptide- or protein-coding potential. The peptides or proteins encoded by ncRNA (HOXB-AS3, encoded by long ncRNA [lncRNA]; FBXW7-185aa, PINT-87aa, and SHPRH-146aa, encoded by circular RNA [circRNA]; and miPEP-200a and miPEP-200b, encoded by primary miRNAs) have been shown to be critical players in cancer development and progression, through effects upon the regulation of glucose metabolism, the epithelial-to-mesenchymal transition, and the ubiquitination pathway. In this review, we summarize the reported peptides or proteins encoded by ncRNAs in cancer and explore the application of these peptides or proteins in the development of anti-tumor drugs and the identification of relevant therapeutic targets and tumor biomarkers.

Nutrient Deprivation Elicits a Transcriptional and Translational Inflammatory Response Coupled to Decreased Protein Synthesis

SUMMARYNutrient deprivation inhibits mRNA translation through mTOR and eIF2α signaling, but it is unclear how the translational program is controlled to reflect the degree of a metabolic stress. In a model of breast cellular transformation, various forms of nutrient deprivation differentially affect the rate of protein synthesis and its recovery over time. Genome-wide translational profiling of glutamine-deprived cells reveals a rapid upregulation of mRNAs containing uORFs and downregulation of ribosomal protein mRNAs, which are followed by selective translation of cytokine and inflammatory mRNAs. Transcription and translation of inflammatory and cytokine genes are stimulated in response to diverse metabolic stresses and depend on eIF2α phosphorylation, with the extent of stimulation correlating with the decrease in global protein synthesis. In accord with the inflammatory stimulus, glutamine deprivation stimulates the migration of transformed cells. Thus, pro-inflammatory gene expression is coupled to metabolic stress, and this can affect cancer cell behavior upon nutrient limitation.

Translational profiling of dorsal root ganglia and spinal cord in a mouse model of neuropathic pain

Acute pain serves as a protective mechanism, guiding the organism away from actual or potential tissue injury. In contrast, chronic pain is a debilitating condition without any obvious physiological function. The transition to, and the maintenance of chronic pain require new gene expression to support biochemical and structural changes within the pain pathway. The regulation of gene expression at the level of mRNA translation has emerged as an important step in the control of protein expression in the cell. Recent studies show that signaling pathways upstream of mRNA translation, such as mTORC1 and ERK, are upregulated in chronic pain conditions, and their inhibition effectively alleviates pain in several animal models. Despite this progress, mRNAs whose translation is altered in chronic pain conditions remain largely unknown. Here, we performed genome-wide translational profiling of dorsal root ganglion (DRG) and spinal cord dorsal horn tissues in a mouse model of neuropathic pain, spared nerve injury (SNI), using the ribosome profiling technique. We identified distinct subsets of mRNAs that are differentially translated in response to nerve injury in both tissues. We discovered key converging upstream regulators and pathways linked to mRNA translational control and neuropathic pain. Our data are crucial for the understanding of mechanisms by which mRNA translation promotes persistent hypersensitivity after nerve injury.

Genome-wide Translation Profiling by Ribosome-Bound tRNA Capture

In the ribosome complex, tRNA is a critical element of mRNA translation. A rich repertoire of cell regulation is hypothesized to occur during the recruitment of specific tRNAs in polypeptide formation. However, this basic question in nascent chain biology remains unaddressed due to the lack of technologies to report the complete tRNA complement inside ribosomes during active translation. Here, we characterize a technique for profiling ribosome-embedded tRNA and their modifications. With this method, wegenerated a comprehensive survey of the quantity and quality of intra-ribosomal tRNAs. In cells underenvironmental stress, we show that methionine tRNA inside ribosomes is a robust biomarker for the impairment of translation initiation or elongation steps. Concurrent tRNA/mRNA ribosome profiling revealed a stress-dependent incorporation of damaged and uncharged tRNAs into ribosomes causing translation arrest. Thus, tRNA ribosome profiling can provide insights on translation control mechanisms in diverse biological contexts.

In Search of Lost Small Peptides.

A large body of evidence indicates that genome annotation pipelines have biased our view of coding sequences because they generally undersample small proteins and peptides. The recent development of genome-wide translation profiling reveals the prevalence of small/short open reading frames (smORFs or sORFs), which are scattered over all classes of transcripts, including both mRNAs and presumptive long noncoding RNAs. Proteomic approaches further confirm an unexpected variety of smORF-encoded peptides (SEPs), representing an overlooked reservoir of bioactive molecules. Indeed, functional studies in a broad range of species from yeast to humans demonstrate that SEPs can harbor key activities for the control of development, differentiation, and physiology. Here we summarize recent advances in the discovery and functional characterization of smORF/SEPs and discuss why these small players can no longer be ignored with regard to genome function.

MTOR inhibition favors the differentiation of human in vitro-induced regulatory T cell through selective protein synthesis

The immunological composition of the primary cancer, metastatic sites, and stromal tissue determine cancer progression and treatment response. Breast cancers produce cytokines and chemokines that attract and polarize immune cells in a manner that promotes disease progression and metastasis. For example, macrophages are polarized to anti-inflammatory type II tumor activating macrophages (TAMs), and CD4+ T cells into tumor promoting, immune suppressing T regulatory cells (Tregs). Studies in mice and humans show that Tregs also are developed when inhibiting the kinase mTOR (mammalian target of rapamycin) via a poorly understood mechanism. mTOR forms two complexes in the cell, mTORC1 and mTORC2, which regulate multiple metabolic processes. In particular, mTORC1 is inhibited by the immunosuppressant rapamycin and stimulates the translation of mRNAs involved in cell growth and proliferation. Protein synthesis is a highly regulated process involving general mRNA and selective mRNA translation. Our hypothesis is that mTOR downregulation alters lymphocyte gene expression by favoring the translation of specific mRNAs required for Treg differentiation. We generated human Tregs induced in culture (iTregs) by mTORC1 inhibition through RAD001 (everolimus), a rapalog used as an anti-cancer drug. By contrast, mTORC1/2 dual inhibitor PP242 blocked the growth of all the lymphocytes, pointing to a specific role of mTORC1 in Treg differentiation. In fact, we found that the proliferation of highly suppressive iTregs requires co-treatment with RAD001 and the cytokine TGFβ. Murine models do not require TGFβ, suggesting that caution should be used in extending findings from mouse to human Treg studies. Protein synthesis analysis in double-treated (RAD001+TGFβ), control-treated, RAD001- and TGFβ-treated cells shows that translation is greatly inhibited in the double-treated cells. Genome-wide translation profiling of mRNAs associated with actively translating ribosomes confirmed that only a selective pool of specific mRNAs is translated in the iTreg population. These specific mRNAs may be recruited to ribosome via an mTOR-independent mechanism involving PAIP2, and eIF4G adapter protein, and the ribosomal protein S25. Our work indicates the importance of selective translational regulation as an additional determinant of gene expression regulating T cell fate. We suggest that iTreg development is facilitated by selective translation of specific mRNAs whose recruitment to the ribosome is augmented when mTORC1 is inhibited. These findings also suggest that cancer treatments causing an improper balance of mTORC1 inhibition might attenuate the anti-tumor immune response through development of Tregs in the tumor microenvironment

Differential Requirements for eIF4E Dose in Normal Development and Cancer

eIF4E, the major cap-binding protein, has long been considered limiting for translating the mammalian genome. However, the eIF4E dose requirement at an organismal level remains unexplored. By generating an Eif4e haploinsufficient mouse, we found that a 50% reduction in eIF4E expression, while compatible with normal development and global protein synthesis, significantly impeded cellular transformation. Genome-wide translational profiling uncovered a translational program induced by oncogenic transformation and revealed a critical role for the dose of eIF4E, specifically in translating a network of mRNAs enriched for a unique 5' UTR signature. In particular, we demonstrate that the dose of eIF4E is essential for translating mRNAs that regulate reactive oxygen species, fueling transformation and cancer cell survival in vivo. Our findings indicate eIF4E is maintained at levels in excess for normal development that are hijacked by cancer cells to drive a translational program supporting tumorigenesis.