Glycolytic Enzymes Coalesce in G Bodies under Hypoxic Stress

Jin, Meiyan; Fuller, Gregory G.; Han, Ting; Yao, Yao; Alessi, Amelia F.; Freeberg, Mallory; Roach, Nathan; Moresco, James J.; Karnovsky, Alla; Baba, Misuzu; Yates, John R.; Gitler, Aaron D.; Inoki, Ken; Klionsky, Daniel J.; Kim, John K.

doi:10.1016/j.celrep.2017.06.082

Cited by 149 publications

(228 citation statements)

References 49 publications

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“…This revealed preferential interaction of Eno1 with the 3′ regions of cytoplasmic tRNAs, which are suitable RNAs to interact with to achieve an anti‐phase separation effect. Notably, several other glycolytic enzymes were reported to form phase‐separated, cytoplasmic “G bodies” following hypoxic stress (Miura et al , ; Jin et al , ). It seems possible that formation of these bodies might be regulated by protein–RNA interactions.…”

Section: Discussionmentioning

confidence: 99%

Defining the RNA interactome by total RNA ‐associated protein purification

Shchepachev

Bresson

Spanos

et al. 2019

Molecular Systems Biology

126

139

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The RNA binding proteome ( RBP ome) was previously investigated using UV crosslinking and purification of poly(A)‐associated proteins. However, most cellular transcripts are not polyadenylated. We therefore developed total RNA ‐associated protein purification ( TRAPP ) based on 254 nm UV crosslinking and purification of all RNA –protein complexes using silica beads. In a variant approach ( PAR ‐ TRAPP ), RNA s were labelled with 4‐thiouracil prior to 350 nm crosslinking. PAR ‐ TRAPP in yeast identified hundreds of RNA binding proteins, strongly enriched for canonical RBP s. In comparison, TRAPP identified many more proteins not expected to bind RNA , and this correlated strongly with protein abundance. Comparing TRAPP in yeast and E. coli showed apparent conservation of RNA binding by metabolic enzymes. Illustrating the value of total RBP purification, we discovered that the glycolytic enzyme enolase interacts with tRNA s. Exploiting PAR ‐ TRAPP to determine the effects of brief exposure to weak acid stress revealed specific changes in late 60S ribosome biogenesis. Furthermore, we identified the precise sites of crosslinking for hundreds of RNA –peptide conjugates, using iTRAPP , providing insights into potential regulation. We conclude that TRAPP is a widely applicable tool for RBP ome characterization.

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Section: Discussionmentioning

confidence: 99%

Defining the RNA interactome by total RNA ‐associated protein purification

Shchepachev

Bresson

Spanos

et al. 2019

Molecular Systems Biology

126

139

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“…RNA also regulates the nucleation and spatiotemporal distribution of membraneless organelles [62,63]. Even G-bodies, which are composed of proteins involved in glucose metabolism, require RNA for biogenesis [64]. RNA can also affect the material properties of protein droplets [48,60].…”

Section: Molecular Determinants Of Protein Phase Separation In Vitro mentioning

confidence: 99%

“…Second, although protein aggregates in disease are considered harmful, accumulating evidence suggests that they could be an initial rescue mechanism of the cell to sequester the more toxic protein oligomers [149,150]. Third, numerous assemblies function as storage granules, as they sequester proteins and other biomolecules under times of stress or quiescence for later reuse [64,151]. Fourth, by concentrating receptors and signaling molecules, a cell can amplify certain signaling pathways.…”

Section: Figurementioning

confidence: 99%

Protein Phase Separation: A New Phase in Cell Biology

Boeynaems

Alberti

Fawzi

et al. 2018

Trends in Cell Biology

1,613

1,517

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Cellular compartments and organelles organize biological matter. Most well-known organelles are separated by a membrane boundary from their surrounding milieu. There are also many so-called membraneless organelles and recent studies suggest that these organelles, which are supramolecular assemblies of proteins and RNA molecules, form via protein phase separation. Recent discoveries have shed light on the molecular properties, formation, regulation, and function of membraneless organelles. A combination of techniques from cell biology, biophysics, physical chemistry, structural biology, and bioinformatics are starting to help establish the molecular principles of an emerging field, thus paving the way for exciting discoveries, including novel therapeutic approaches for the treatment of age-related disorders.

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“…In yeast, the presence of G bodies correlates with accelerated glucose consumption, and impairing G body formation leads to the accumulation of upstream glycolytic metabolites (Jin et al, 2017). These data suggest that during hypoxic stress, when oxidative phosphorylation is inhibited, G bodies form to enhance the rate of glycolysis by concentrating glycolysis enzymes.…”

Section: Introductionmentioning

confidence: 95%

“…Recently, we and others demonstrated that glycolysis enzymes coalesce into membraneless cytoplasmic granules called glycolytic bodies (G bodies) in hypoxic stress in yeast, C. elegans, and mammalian cells (Jang et al, 2016;Jin et al, 2017;Miura et al, 2013).…”

Section: Introductionmentioning

confidence: 99%

RNA promotes phase separation of glycolysis enzymes into yeast G bodies in hypoxia

Fuller

Han

Freeberg

et al. 2019

Preprint

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AbstractIn hypoxic stress conditions, glycolysis enzymes assemble into singular cytoplasmic granules called glycolytic (G) bodies. Formation of G bodies in yeast is correlated with increased glucose consumption and cell survival. However, the physical properties and organizing principles that define G body formation are unclear. We demonstrate that glycolysis enzymes are non-canonical RNA binding proteins, sharing many common mRNA substrates that are also integral constituents of G bodies. Tethering a G body component, the beta subunit of the yeast phosphofructokinase, Pfk2, to nonspecific endoribonucleases reveals that RNA nucleates G body formation and subsequent maintenance of G body structural integrity. Consistent with a phase separation mechanism of G body formation, recruitment of glycolysis enzymes to G bodies relies on multivalent homotypic and heterotypic interactions. Furthermore, G bodies can fuse in live cells and are largely insensitive to 1,6-hexanediol treatment, consistent with a hydrogel-like state in its composition. Taken together, our results elucidate the biophysical nature of G bodies and demonstrate that RNA nucleates phase separation of the glycolysis machinery in response to hypoxic stress.

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Glycolytic Enzymes Coalesce in G Bodies under Hypoxic Stress

Cited by 149 publications

References 49 publications

Defining the RNA interactome by total RNA ‐associated protein purification

Defining the RNA interactome by total RNA ‐associated protein purification

Protein Phase Separation: A New Phase in Cell Biology

RNA promotes phase separation of glycolysis enzymes into yeast G bodies in hypoxia

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