Fig. 2: Uridine-derived ribose contributes to the pentose phosphate pathway and glycolysis.
From: Salvage of ribose from uridine or RNA supports glycolysis in nutrient-limited conditions
a, Schematic of a genome-wide CRISPR–Cas9 depletion screen comparing the proliferation of UPP1-FLAG-expressing K562 cells in sugar-free medium containing 10 mM glucose or uridine after 21 d (n = 2), in the absence of supplemental pyruvate and uridine. b, Gene-level analysis of a genome-wide CRISPR–Cas9 screen in glucose versus uridine reported as z-scores relative to non-cutting controls in glucose (zglu) and uridine (zu; n = 10,442 expressed genes, n = 2 replicates). c, Differential sensitivity of UPP1-FLAG-expressing K562 cells treated with the indicated sgRNAs targeting enzymes of upper glycolysis (n = 4, P < 8.7 × 10−11, P < 3.2 × 10−10, P < 6.4 × 10−5), the PPP (n = 4, P < 5.7 × 10−8, P < 7.6 × 10−8, P < 2.1 × 10−5, P < 6.3 × 10−7) or the salvage of uridine for pyrimidine synthesis (n = 3) in glucose versus uridine, expressed as the fold change of glucose and compared to control sgRNAs (n = 11). Data are shown as the mean ± s.e.m. after 4–5 d. P values were calculated using a two-sided Student’s t-test relative to control sgRNAs. Statistics were not adjusted for multiple comparisons. d, Lactate determination in medium containing 10 mM glucose, galactose or uridine (sugar-free) after 3 h (n = 3 replicate wells, P < 7.8 × 10−7). Data are shown as the mean ± s.e.m. with two-sided t-test relative to control cells. e, Labelling with 13C5-uridine ([1′,2′,3′,4′,5′-13C5]uridine; labelled carbon atoms in the ribose of uridine are indicated in magenta) and 13C5-uridine tracer analysis of representative intracellular metabolites from the PPP, glycolysis and the TCA cycle in control or UPP1-FLAG-expressing K562 cells (n = 3). Data are shown as the mean ± s.e.m. and are corrected for natural isotope abundance. f, 13C5-uridine tracer analysis of liver metabolites 30 min after intraperitoneal injection in overnight fasted mice with 0.4 g per kg body weight shown as the percentage of 13C-labelled intermediates compared to the total pool. Data are shown as the mean ± s.e.m. and are corrected for natural isotope abundance (n = 4 mice). g, Schematic of uridine-derived ribose catabolism integrating gene essentiality results in glucose versus uridine. Gln, glutamine; Asp, aspartate. All growth assays and metabolomics experiments included 4 mM (DMEM) l-glutamine and 10% dialysed FBS. a.u., arbitrary units.
