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

Abstract

<jats:title>Abstract</jats:title><jats:p>In 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.</jats:p>

RNA Granules: A View from the RNA Perspective

“…In vitro, a small amount of RNA initially promotes protein droplet formation and afterwards begins dissolving the droplets once the concentration of the RNA reaches a threshold [ 76 , 77 ]. RNAs change physical properties of condensates (i.e., viscosity), augment granule assembly in vitro and in vivo [ 13 , 44 , 67 , 76 , 77 , 78 , 79 , 80 , 81 , 82 , 83 , 84 ], determine the three dimensional structure of granules in an RNA concentration-dependent manner [ 49 , 85 ], and can even instruct the morphology of granules by their ability to engage in intermolecular RNA-RNA interactions [ 86 ].…”

The RNA regulatory programs that govern lymphocyte development and function

“…Glycolytic enzymes are often enriched in protein:RNA interactome analyses and can be found within cytoplasmic RNA granules in yeast (Figure 3(b,c)) (Baltz et al, 2012; Beckmann et al, 2015; Castello et al, 2012; Fuller et al, 2020; Matia‐Gonzalez et al, 2015). Recent characterization of the ribosome‐associated interactome identified PKM2, LDHA, and ALDOA as being part of a macromolecular complex that controls mRNA location, stability, and translation (Simsek et al, 2017).…”

“…Cellular stress causes phase separation (Protter & Parker, 2016; Riback et al, 2017). This can be associated to the transient termination of highly energetic processes for the cell (e.g., glycolysis and translation) (Anderson & Kedersha, 2009; Fuller et al, 2020) and the storage of different proteins and RNAs that will be used during later cell recovery after the resolution of the cellular stress (Amen & Kaganovich, 2020; Standart & Weil, 2018). Activation of B lymphocytes with mitogens also enables cytoplasmic RNA granules assembly (Diaz‐Munoz et al, 2017).…”

The role of liquid–liquid phase separation in regulating enzyme activity

“…Recent discussions have addressed the ability of metabolic enzymes to phase separate under nutrient deprivation [31][32][33]. Additional works by Fuller, et al [34] Jin, et al [35] and Kohnhorst, et al [36] expanded on our understanding of glycolytic (G) bodies (or glucosomes as they have also been called), i.e., membraneless condensates which form under hypoxic stress to co-localize glycolytic enzymes and enhance the rate of glycolysis. G bodies were identified to alter glycolysis rates by measuring the levels of glycolytic metabolites in wild-type cells and in cells that lacked Snf1p, a conserved AMP-activated protein kinase found to be vital for G body formation [35].…”