Adaptive Translatome Reprogramming in Response to Anaerobiosis

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: what are the translatome remodelers that reprogram protein output to activate key biochemical pathways. For our first study: we uncover a network of RNA-binding proteins (RBPs) that enhance 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 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 and confer hypoxia tolerance. RBP-controlled anaerobic metabolism induces a concurrent acidification of the extracellular milieu. For our second study we identified the ancient eIF5A as a pH-regulated translation factor that responds to hypoxia-induced acidosis. Proteomic 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, induce cellular dormancy and prevent acidosis-induced DNA damage. We suggest cells are equipped with translatome remodelers that control anaerobiosis.&nbsp;</p

Abstract A28: Oxygen-dependent translatome remodeling is controlled by eIF2α dephosphorylation

Abstract Introduction: Eukaryotic translation initiation factor 2α (eIF2α) is a core component of the cellular translation machinery. Alterations in eIF2α phosphorylation constitute a key adaptation during a variety of physiological stresses, including oxygen deprivation (hypoxia). We recently found that cancer cells respond to hypoxia through the systemic reprogramming of cap-dependent mRNA translation efficiencies by switching to the hypoxic eIF4F, eIF4FH. In the present study, we sought to elucidate the role(s) of eIF2α phosphorylation and dephosphorylation in this translatome remodeling process. In particular, the growth arrest and DNA damage-inducible protein GADD34 has been reported as an adaptive, stress-activated mediator of eIF2α dephosphorylation. We aimed to determine the role of GADD34 in the cellular hypoxic response. Findings: Our results indicate that hypoxia-induced eIF2α phosphorylation is a transient response with corresponding effects on translation efficiencies. Using next-generation mRNA sequencing (mRNA-seq), we reveal the dynamic profile of the translatome remodeling process during acute and chronic hypoxia. Our findings demonstrate that GADD34-mediated eIF2α dephosphorylation is required for hypoxic protein synthesis. GADD34 silencing results in high levels of eIF2α phosphorylation and severe global translational inhibition. Notably, GADD34 regulates the translation of hypoxia-inducible proteins in a hypoxia-inducible factor 2α (HIF-2α)-dependent manner. Finally, high throughput mass spectrometry (MS) analysis reveals several potential hypoxia-regulated GADD34 post-translational modifications (PTMs). Efforts are underway to validate and determine the effect(s) of these PTMs on GADD34 protein stability and/or activity. Conclusion: We demonstrate that GADD34-mediated eIF2α dephosphorylation regulates oxygen-dependent translatome reprogramming in cancer cells. This pathway represents an exciting potential target for cancer therapeutics. Citation Format: J.J. David Ho, Nathan C. Balukoff, Grissel Cervantes, Ayalivis De La Rosa, Stephen Lee. Oxygen-dependent translatome remodeling is controlled by eIF2α dephosphorylation. [abstract]. In: Proceedings of the AACR Special Conference on Translational Control of Cancer: A New Frontier in Cancer Biology and Therapy; 2016 Oct 27-30; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2017;77(6 Suppl):Abstract nr A28

RNA tailing machinery drives amyloidogenic phase transition

The RNA tailing machinery adds nucleotides to the 3'-end of RNA molecules that are implicated in various biochemical functions, including protein synthesis and RNA stability. Here, we report a role for the RNA tailing machinery as enzymatic modifiers of intracellular amyloidogenesis. A targeted RNA interference screen identified Terminal Nucleotidyl-transferase 4b (TENT4b/Papd5) as an essential participant in the amyloidogenic phase transition of nucleoli into solid-like Amyloid bodies. Full-length-and-mRNA sequencing uncovered starRNA, a class of unusually long untemplated RNA molecules synthesized by TENT4b. StarRNA consists of short rRNA fragments linked to long, linear mixed tails that operate as polyanionic stimulators of amyloidogenesis in cells and in vitro. Ribosomal intergenic spacer noncoding RNA (rIGSRNA) recruit TENT4b in intranucleolar foci to coordinate starRNA synthesis driving their amyloidogenic phase transition. The exoribonuclease RNA Exosome degrades starRNA and functions as a general suppressor of cellular amyloidogenesis. We propose that amyloidogenic phase transition is under tight enzymatic control by the RNA tailing and exosome axis

The CSN/COP9 Signalosome Regulates Synaptonemal Complex Assembly during Meiotic Prophase I of <i>Caenorhabditis elegans</i>

<div><p>The synaptonemal complex (SC) is a conserved protein structure that holds homologous chromosome pairs together throughout much of meiotic prophase I. It is essential for the formation of crossovers, which are required for the proper segregation of chromosomes into gametes. The assembly of the SC is likely to be regulated by post-translational modifications. The CSN/COP9 signalosome has been shown to act in many pathways, mainly via the ubiquitin degradation/proteasome pathway. Here we examine the role of the CSN/COP9 signalosome in SC assembly in the model organism <i>C. elegans</i>. Our work shows that mutants in three subunits of the CSN/COP9 signalosome fail to properly assemble the SC. In these mutants, SC proteins aggregate, leading to a decrease in proper pairing between homologous chromosomes. The reduction in homolog pairing also results in an accumulation of recombination intermediates and defects in repair of meiotic DSBs to form the designated crossovers. The effect of the CSN/COP9 signalosome mutants on synapsis and crossover formation is due to increased neddylation, as reducing neddylation in these mutants can partially suppress their phenotypes. We also find a marked increase in apoptosis in <i>csn</i> mutants that specifically eliminates nuclei with aggregated SC proteins. <i>csn</i> mutants exhibit defects in germline proliferation, and an almost complete pachytene arrest due to an inability to activate the MAPK pathway. The work described here supports a previously unknown role for the CSN/COP9 signalosome in chromosome behavior during meiotic prophase I.</p></div

Quantification of the lack of oocytes and fecundity test.

<p>A) Relative sizes of the pre-meiotic tips for wild-type and the <i>csn</i> mutants. The size of the mitotic zone is reduced in <i>csn</i> mutants. n = 10 for each strain p<0.0005 for wild-type vs. <i>csn-2</i>; p = 0.005 for wild-type vs. <i>csn-5</i> and p<0.05 for <i>csn-2</i> vs. <i>csn-5</i>; Mann Whitney Test B) Quantification of the number of gonads that contained oocytes in diakinesis for the <i>csn</i> mutants, *<i>p</i>_MW<0.0005 and **<i>p</i>_MW<0.005, Mann Whitney Test C) Top: the average number of oocytes in diakinesis for the <i>csn</i> mutants and the <i>csn</i> mutant, apoptosis checkpoint double mutants. Bottom: the average number of eggs laid for <i>csn</i> mutants and apoptosis checkpoint double mutants. <i>csn</i> mutants have a severe reduction in the number of oocytes and lay no eggs.</p

<i>csn</i> mutant genetically interact with the ubiquitination-neddylation pathway in the regulation of SC assembly and recombination.

<p>A–D) Quantification of SYP-1 aggregates. A and C are data from all gonad, while B and D is from late pachytene nuclei. Percent of nuclei with: no SYP-1 (black), linear SYP-1 (blue), aggregated SYP-1 (purple pink and red) and other (yellow), zones as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004757#pgen-1004757-g002" target="_blank">Figure 2A</a>, n nuclei scored for whole gonad <i>csn-2</i>: with pL4440 = 2023 with <i>ned-8(RNAi)</i> = 430, with <i>uba-1(RNAi)</i> = 1121, <i>csn-5</i>: with pL4440 = 2096, with <i>ned-8(RNAi)</i> = 441, with <i>uba-1(RNAi)</i> = 1014. E) Quantitative analysis of COSA-1 foci in late pachytene wild-type: Percent of nuclei with: zero (black) one (orange), two (red), three (pink) four (magenta), five (purple), six (blue) and seven (gray), n nuclei scored for pL4440 = 57, with <i>ned-8(RNAi)</i> = 47, with <i>uba-1(RNAi)</i> = 25, <i>csn-2</i>: with pL4440 = 168, with <i>ned-8(RNAi)</i> = 66, with <i>uba-1(RNAi)</i> = 126, <i>csn-5</i>: with pL4440 = 152, with <i>ned-8(RNAi)</i> = 47 with, <i>uba-1(RNAi)</i> = 83, pL4440 = empty vector control vs. <i>ned-8(RNAi)</i> on <i>csn-2</i> or <i>csn-5</i> p = 0.002, p<0.001, Fisher's Exact Test, pL4440 vs. <i>uba-1(RNAi)</i> on <i>csn-2</i> or <i>csn-5</i> p<0.001 Mann Whitney Test). F–H) Quantification of SYP-1 aggregates in zones of the gonad for the indicated genotypes, as performed in figure, number of total nuclei scored: wild-type 25 n = 641, <i>rfl-1</i> at 25 n = 1674, <i>cul-4</i> n = 542 2, I) Schematic representation of the pathway examined in the experiment.</p

Molecular Pathophysiology of Chronic Wounds: Current State and Future Directions

Venous leg ulcers, diabetic foot ulcers, and pressure ulcers are complex chronic wounds with multifactorial etiologies that are associated with high patient morbidity and mortality. Despite considerable progress in deciphering the pathologies of chronic wounds using "omics" approaches, considerable gaps in knowledge remain, and current therapies are often not efficacious. We provide a comprehensive overview of current understanding of the molecular mechanisms that impair healing and current knowledge on cell-specific dysregulation including keratinocytes, fibroblasts, immune cells, endothelial cells and their contributions to impaired reepithelialization, inflammation, angiogenesis, and tissue remodeling that characterize chronic wounds. We also provide a rationale for further elucidation of ulcer-specific pathologic processes that can be therapeutically targeted to shift chronic nonhealing to acute healing wounds

Oxygen-Sensitive Remodeling of Central Carbon Metabolism by Archaic eIF5B

Summary: The eukaryotic translation initiation factor 5B (eIF5B) is a homolog of IF2, an ancient translation factor that enables initiator methionine-tRNAiMet (met-tRNAiMet) 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-tRNAiMet in eukaryotes. We suggest that aerobic eukarya retained eIF5B/IF2 to remodel anaerobic pathways during episodes of oxygen deficiency. : Ho et al. employed MATRIX to demonstrate that eIF5B is an essential hypoxic translation factor that facilitates met-tRNAiMet delivery to ribosomes, serving as the hypoxic surrogate of the textbook eIF2. Aerobic eukarya likely retained eIF5B for the oxygen-dependent regulation of central carbon metabolism and hypoxic survival. Keywords: eIF5B, IF2, translation, carbon metabolism, glycolysis, evolution, hypoxia, MATRIX, ATF4, stres