Search

Here, we present a protocol using MATRIX (mass spectrometry analysis of active translation factors using ribosome density fractionation and isotopic labeling experiments) platform to investigate changes of the protein synthesis machinery in U87MG glioblastoma cells in response to the rocaglate silvestrol. This protocol describes steps to perform SILAC (stable isotope labeling by amino acids in cell culture), ribosome density fractionation, protein isolation, and mass spectrometry analysis. This approach can be applied to study any adaptive remodeling of protein synthesis machineries. For complete details on the use and execution of this protocol, please refer to Ho et al. (2021). 1 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Competing Interests: Declaration of interests No interests are declared.

Kaposi's sarcoma herpesvirus (KSHV) is an angiogenesis-inducing oncovirus whose ability to usurp the oxygen-sensing machinery is central to its oncogenicity. By upregulating the hypoxia-inducible factors (HIFs), KSHV reprograms infected cells to a hypoxia-like state, triggering angiogenesis. Here we identify a link between KSHV replicative biology and oncogenicity by showing that KSHV's ability to regulate HIF2α levels and localization to the endoplasmic reticulum (ER) in normoxia enables translation of viral lytic mRNAs through the HIF2α-regulated eIF4E2 translation-initiation complex. This mechanism of translation in infected cells is critical for lytic protein synthesis and contributes to KSHV-induced PDGFRA activation and VEGF secretion. Thus, KSHV regulation of the oxygen-sensing machinery allows virally infected cells to initiate translation via the mTOR-dependent eIF4E1 or the HIF2α-dependent, mTOR-independent, eIF4E2. This "translation initiation plasticity" (TRIP) is an oncoviral strategy used to optimize viral protein expression that links molecular strategies of viral replication to angiogenicity and oncogenesis., Competing Interests: Declaration of interests The authors declare no competing interests, (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Tactical disruption of protein synthesis is an attractive therapeutic strategy, with the first-in-class eIF4A-targeting compound zotatifin in clinical evaluation for cancer and COVID-19. The full cellular impact and mechanisms of these potent molecules are undefined at a proteomic level. Here, we report mass spectrometry analysis of translational reprogramming by rocaglates, cap-dependent initiation disruptors that include zotatifin. We find effects to be far more complex than simple "translational inhibition" as currently defined. Translatome analysis by TMT-pSILAC (tandem mass tag-pulse stable isotope labeling with amino acids in cell culture mass spectrometry) reveals myriad upregulated proteins that drive hitherto unrecognized cytotoxic mechanisms, including GEF-H1-mediated anti-survival RHOA/JNK activation. Surprisingly, these responses are not replicated by eIF4A silencing, indicating a broader translational adaptation than currently understood. Translation machinery analysis by MATRIX (mass spectrometry analysis of active translation factors using ribosome density fractionation and isotopic labeling experiments) identifies rocaglate-specific dependence on specific translation factors including eEF1ε1 that drive translatome remodeling. Our proteome-level interrogation reveals that the complete cellular response to these historical "translation inhibitors" is mediated by comprehensive translational landscape remodeling., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Responsible for generating the proteome that controls phenotype, translation is the ultimate convergence point for myriad upstream signals that influence gene expression. System-wide adaptive translational reprogramming has recently emerged as a pillar of cellular adaptation. As classic regulators of mRNA stability and translation efficiency, foundational studies established the concept of collaboration and competition between RNA-binding proteins (RBPs) and noncoding RNAs (ncRNAs) on individual mRNAs. Fresh conceptual innovations now highlight stress-activated, evolutionarily conserved RBP networks and ncRNAs that increase the translation efficiency of populations of transcripts encoding proteins that participate in a common cellular process. The discovery of post-transcriptional functions for long noncoding RNAs (lncRNAs) was particularly intriguing given their cell-type-specificity and historical definition as nuclear-functioning epigenetic regulators. The convergence of RBPs, lncRNAs, and microRNAs on functionally related mRNAs to enable adaptive protein synthesis is a newer biological paradigm that highlights their role as "translatome (protein output) remodelers" and reinvigorates the paradigm of "RNA operons." Together, these concepts modernize our understanding of cellular stress adaptation and strategies for therapeutic development. This article is categorized under: RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications Translation > Translation Regulation Regulatory RNAs/RNAi/Riboswitches > Regulatory RNAs., (© 2021 Wiley Periodicals LLC.)

Global translational remodeling has emerged as a principal mechanism of biological adaptation. Oxygen deficiency (hypoxia) disables the basal protein synthesis machinery ('Jekyll') and activates a hypoxic translational architecture ('Hyde') to drive translatome remodeling. Independent from mRNA-level fluctuations, this newer paradigm modernizes a field traditionally dominated by the hypoxia-inducible factor (HIF) transcriptional program., (Crown Copyright © 2020. Published by Elsevier Ltd. All rights reserved.)

Translatome reprogramming is a primary determinant of protein levels during stimuli adaptation. This raises the question: what are the translatome remodelers that reprogram protein output to activate biochemical adaptations. Here, we identify a translational pathway that represses metabolism to safeguard genome integrity. A system-wide MATRIX survey identified the ancient eIF5A as a pH-regulated translation factor that responds to fermentation-induced acidosis. TMT-pulse-SILAC analysis identified several pH-dependent proteins, including the mTORC1 suppressor Tsc2 and the longevity regulator Sirt1. Sirt1 operates as a pH-sensor that deacetylates nuclear eIF5A during anaerobiosis, enabling the cytoplasmic export of eIF5A/Tsc2 mRNA complexes for translational engagement. Tsc2 induction inhibits mTORC1 to suppress cellular metabolism and prevent acidosis-induced DNA damage. Depletion of eIF5A or Tsc2 leads to metabolic re-initiation and proliferation, but at the expense of incurring substantial DNA damage. We suggest that eIF5A operates as a translatome remodeler that suppresses metabolism to shield the genome.

Protein expression evolves under greater evolutionary constraint than mRNA levels, and translation efficiency represents a primary determinant of protein levels during stimuli adaptation. This raises the question as to the translatome remodelers that titrate protein output from mRNA populations. Here, we uncover a network of RNA-binding proteins (RBPs) that enhances the translation efficiency of glycolytic proteins in cells responding to oxygen deprivation. A system-wide proteomic survey of translational engagement identifies a family of oxygen-regulated RBPs that functions as a switch of glycolytic intensity. Tandem mass tag-pulse SILAC (TMT-pSILAC) and RNA sequencing reveals that each RBP controls a unique but overlapping portfolio of hypoxic responsive proteins. These RBPs collaborate with the hypoxic protein synthesis apparatus, operating as a translation efficiency checkpoint that integrates upstream mRNA signals to activate anaerobic metabolism. This system allows anoxia-resistant animals and mammalian cells to initiate anaerobic glycolysis and survive hypoxia. We suggest that an oxygen-sensitive RBP cluster controls anaerobic metabolism to confer hypoxia tolerance.

Homogeneous populations of mature differentiated primary cell types can display variable responsiveness to extracellular stimuli, although little is known about the underlying mechanisms that govern such heterogeneity at the level of gene expression. In this article, we show that morphologically homogenous human endothelial cells exhibit heterogeneous expression of VCAM1 after TNF-α stimulation. Variability in VCAM1 expression was not due to stochasticity of intracellular signal transduction but rather to preexisting established heterogeneous states of promoter DNA methylation that were generationally conserved through mitosis. Variability in DNA methylation of the VCAM1 promoter resulted in graded RelA/p65 and RNA polymerase II binding that gave rise to a distribution of VCAM1 transcription in the population after TNF-α stimulation. Microarray analysis and single-cell RNA sequencing revealed that a number of cytokine-inducible genes shared this heterogeneous response pattern. These results show that heritable epigenetic heterogeneity is fundamental in inflammatory signaling and highlight VCAM1 as a metastable epiallele., (Copyright © 2020 by The American Association of Immunologists, Inc.)

Amyloid bodies (A-bodies) are inducible membrane-less nuclear compartments composed of heterogeneous proteins that adopt an amyloid-like state. A-bodies are seeded by noncoding RNA derived from stimuli-specific loci of the rDNA intergenic spacer (rIGSRNA). This raises the question of how rIGSRNA recruits a large population of diverse proteins to confer A-body identity. Here, we show that long low-complexity dinucleotide repeats operate as the architectural determinants of rIGSRNA. On stimulus, clusters of rIGSRNA with simple cytosine/uracil (CU) or adenosine/guanine (AG) repeats spanning hundreds of nucleotides accumulate in the nucleolar area. The low-complexity sequences facilitate charge-based interactions with short cationic peptides to produce multiple nucleolar liquid-like foci. Local concentration of proteins with fibrillation propensity in these nucleolar foci induces the formation of an amyloidogenic liquid phase that seeds A-bodies. These results demonstrate the physiological importance of low-complexity RNA and repetitive regions of the genome often dismissed as "junk" DNA., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Although the functional role of chromatin marks at promoters in mediating cell-restricted gene expression has been well characterized, the role of intragenic chromatin marks is not well understood, especially in endothelial cell (EC) gene expression. Here, we characterized the histone H3 and H4 acetylation profiles of 19 genes with EC-enriched expression via locus-wide chromatin immunoprecipitation followed by ultra-high-resolution (5 bp) tiling array analysis in ECs versus non-ECs throughout their genomic loci. Importantly, these genes exhibit differential EC enrichment of H3 and H4 acetylation in their promoter in ECs versus non-ECs. Interestingly, VEGFR-2 and VEGFR-1 show EC-enriched acetylation across broad intragenic regions and are up-regulated in non-ECs by histone deacetylase inhibition. It is unclear which histone acetyltransferases (KATs) are key to EC physiology. Depletion of KAT7 reduced VEGFR-2 expression and disrupted angiogenic potential. Microarray analysis of KAT7-depleted ECs identified 263 differentially regulated genes, many of which are key for growth and angiogenic potential. KAT7 inhibition in zebrafish embryos disrupted vessel formation and caused loss of circulatory integrity, especially hemorrhage, all of which were rescued with human KAT7. Notably, perturbed EC-enriched gene expression, especially the VEGFR-2 homologs, contributed to these vascular defects. Mechanistically, KAT7 participates in VEGFR-2 transcription by mediating RNA polymerase II binding, H3 lysine 14, and H4 acetylation in its intragenic region. Collectively, our findings support the importance of differential histone acetylation at both promoter and intragenic regions of EC genes and reveal a previously underappreciated role of KAT7 and intragenic histone acetylation in regulating VEGFR-2 and endothelial function., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

Endothelial cell (EC)-enriched protein coding genes, such as endothelial nitric oxide synthase (eNOS), define quintessential EC-specific physiologic functions. It is not clear whether long noncoding RNAs (lncRNAs) also define cardiovascular cell type-specific phenotypes, especially in the vascular endothelium. Here, we report the existence of a set of EC-enriched lncRNAs and define a role for s pliced- t ranscript e ndothelial- e nriched lncRNA (STEEL) in angiogenic potential, macrovascular/microvascular identity, and shear stress responsiveness. STEEL is expressed from the terminus of the HOXD locus and is transcribed antisense to HOXD transcription factors. STEEL RNA increases the number and integrity of de novo perfused microvessels in an in vivo model and augments angiogenesis in vitro. The STEEL RNA is polyadenylated, nuclear enriched, and has microvascular predominance. Functionally, STEEL regulates a number of genes in diverse ECs. Of interest, STEEL up-regulates both eNOS and the transcription factor Kruppel-like factor 2 (KLF2), and is subject to feedback inhibition by both eNOS and shear-augmented KLF2. Mechanistically, STEEL up-regulation of eNOS and KLF2 is transcriptionally mediated, in part, via interaction of chromatin-associated STEEL with the poly-ADP ribosylase, PARP1. For instance, STEEL recruits PARP1 to the KLF2 promoter. This work identifies a role for EC-enriched lncRNAs in the phenotypic adaptation of ECs to both body position and hemodynamic forces and establishes a newer role for lncRNAs in the transcriptional regulation of EC identity., Competing Interests: The authors declare no conflict of interest.

The eukaryotic translation initiation factor 5B (eIF5B) is a homolog of IF2, an ancient translation factor that enables initiator methionine-tRNAi Met (met-tRNAi Met ) loading on prokaryotic ribosomes. While it can be traced back to the last universal common ancestor, eIF5B is curiously dispensable in modern aerobic yeast and mammalian cells. Here, we show that eIF5B is an essential element of the cellular hypoxic cap-dependent protein synthesis machinery. System-wide interrogation of dynamic translation machineries by MATRIX (mass spectrometry analysis of active translation factors using ribosome density fractionation and isotopic labeling experiments) demonstrated augmented eIF5B activity in hypoxic translating ribosomes. Global translatome studies revealed central carbon metabolism, cellular hypoxic adaptation, and ATF4-mediated stress response as major eIF5B-dependent pathways. These primordial processes rely on eIF5B even in the presence of oxygen and active eIF2, the canonical recruiter of met-tRNAi Met in eukaryotes. We suggest that aerobic eukarya retained eIF5B/IF2 to remodel anaerobic pathways during episodes of oxygen deficiency., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

The eukaryotic translation initiation factor 4F (eIF4F) has become essentially synonymous with 5' cap-dependent mRNA translation. Recent studies demonstrate that cells assemble variants of eIF4F to produce adaptive, cap-dependent translatomes during physiological conditions that inhibit eIF4F. These findings challenge us to reassess classical perceptions of cellular translational pathways., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Protein concentrations evolve under greater evolutionary constraint than mRNA levels. Translation efficiency of mRNA represents the chief determinant of basal protein concentrations. This raises a fundamental question of how mRNA and protein levels are coordinated in dynamic systems responding to physiological stimuli. This report examines the contributions of mRNA abundance and translation efficiency to protein output in cells responding to oxygen stimulus. We show that changes in translation efficiencies, and not mRNA levels, represent the major mechanism governing cellular responses to [O2] perturbations. Two distinct cap-dependent protein synthesis machineries select mRNAs for translation: the normoxic eIF4F and the hypoxic eIF4F(H). O2-dependent remodeling of translation efficiencies enables cells to produce adaptive translatomes from preexisting mRNA pools. Differences in mRNA expression observed under different [O2] are likely neutral, given that they occur during evolution. We propose that mRNAs contain translation efficiency determinants for their triage by the translation apparatus on [O2] stimulus., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Heme oxygenases (HOs) -1 and -2 catalyze the breakdown of heme to release carbon monoxide, biliverdin, and ferrous iron, which may preserve cell function during oxidative stress. HO-1 levels decrease in endothelial cells exposed to hypoxia, whereas the effect of hypoxia on HO-2 expression is unknown. The current study was carried out to determine if hypoxia alters HO-2 protein levels in human endothelial cells and whether this enzyme plays a role in preserving their viability during hypoxic stress. Human umbilical vein endothelial cells (HUVECs), human aortic endothelial cells (HAECs), and human blood outgrowth endothelial cells were exposed to 21% or 1% O(2) for 48 or 16 h in the presence or absence of tumor necrosis factor-alpha (10 ng/ml) or H(2)O(2) (100 microm). In all three endothelial cell types HO-1 mRNA and protein levels were decreased following hypoxic incubation, whereas HO-2 protein levels were unaltered. In HUVECs HO-2 levels were maintained during hypoxia despite a 57% reduction in steady-state HO-2 mRNA level and a 43% reduction in total protein synthesis. Polysome profiling revealed increased HO-2 transcript association with polysomes during hypoxia consistent with enhanced translation of these transcripts. Importantly, inhibition of HO-2 expression by small interference RNA increased oxidative stress, exacerbated mitochondrial membrane depolarization, and enhanced caspase activation and apoptotic cell death in cells incubated under hypoxic but not normoxic conditions. These data indicate that HO-2 is important in maintaining endothelial viability and may preserve local regulation of vascular tone, thrombosis, and inflammatory responses during reductions in systemic oxygen delivery.