Fig. 4: UMP is the key metabolite in PYR-1-mediated PCD.
A Schematic of the de novo pyrimidine synthesis pathway (black) and the salvage pathway (blue), as adapted from WormFlux (https://wormflux.umassmed.edu/). These pathways converge at UMP, a central molecule in nucleotide metabolism. UMP can be further converted into derivatives such as UTP and CTP (orange), which serve as essential precursors for synthesizing sugars, RNA, and phospholipids. UTP and CTP can also be transformed into dUTP, dCTP, and dTTP, which are crucial for DNA synthesis. B Knockdown of the de novo pyrimidine synthesis pathway leads to a bulged tail phenotype in the grp-1(gm350) background. Animals of the indicated genotype were injected with dsRNA targeting dhod-1, umps-1, R12E2.11 or umps-1 plus R12E2.11. The percentages of animals displaying the bulged tail phenotype or extra hyp8/9 (arIs99[Pdpy-7::2xnls::yfp]) are shown as mean ± SD (n = 50, from three independent experiments). C, D Schematic of the UMPS-1 protein with and without the OMPDC domain (C). CRISPR-mediated editing was performed in both wild-type and grp-1(gm350) worms to create genotypes harboring the truncated OMPDC domain. The percentages of animals displaying the bulged tail phenotype or extra hyp8/9 (arIs99[Pdpy-7::2xnls::yfp]) for indicated genotypes are shown as mean ± SD (n = 30, from three independent experiments) (D). E, F In grp-1(gm350) mutants, animals were injected with dsRNA targeting C29F7.3, F40F8.1, C29F7.3 plus F40F8.1, ndk-1, or ctps-1 to inhibit the conversion of UMP to UDP, UTP, or CTP (orange highlighted region in Fig. 4A) (E). In C47B2.2(tm2030); grp-1(gm350) mutants, animal were injected with dsRNA targeting Y10G11A.1, F25B5.3, Y10G11A.1 plus F25B5.3, or upp-1 to block UMP conversion into uridine and uracil (blue highlighted region in Fig. 4A) (F). The percentages of animals with the bulged tail phenotype are shown as mean ± SD (n ≥ 30, from three independent experiments). G, H The effect of uridine or uracil supplementation at different concentrations was tested in pyr-1(tp12); grp-1(gm350) (G) and grp-1(gm350) umps-1(ΔOMPDC) (H) mutants. The percentages of animals displaying the bulged tail phenotype or extra hyp8/9 (arIs99[Pdpy-7::2xnls::yfp]) are shown as mean ± SD (n ≥ 30, from at least three independent experiments). I, J In pyr-1(tp12); grp-1(gm350) mutants, dsRNA targeting upp-1, C47B2.2, or both was used to block uracil conversion to uridine or UMP (I). In pyr-1(tp12); grp-1(gm350) and pyr-1(tp12); grp-1(gm350); B0001.4(tm2740) mutants, dsRNA targeting C47B2.2, F19B6.2, or both was used to block uracil conversion to UMP (J). The percentages of animals with the bulged tail phenotype or extra hyp8/9 (arIs99[Pdpy-7::2xnls::yfp]) are presented as mean ± SD (n ≥ 30, from at least three independent experiments). In all histograms, * indicates P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 (one-way ANOVA with Tukey’s multiple comparisons test). ns indicates no statistical difference (P > 0.05).
