Guanylate kinase 1 is an enzyme responsible for inosine accumulation in RNA of mammalian cells deficient in inosine triphosphatase

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Abstract
Inosine triphosphate (ITP) is a noncanonical nucleotide. Human cells possess inosine triphosphate pyrophosphatase (ITPA), and ITPA deficiency causes ITP accumulation within cells, leading to the onset of “Developmental and Epileptic Encephalopathy 35.” Two potential pathways for ITP biosynthesis have been proposed: i) oxidative deamination of the adenine base in adenosine triphosphate or ii) a two-step phosphorylation reaction of inosine monophosphate (IMP), an intermediate in the de novo synthesis of purine nucleotides. However, the mechanisms responsible for ITP biosynthesis in mammals remain unclear. In this study, using an Itpa knockout (KO) mouse cell line, we identified guanylate kinase 1 ( Guk1 ) as a responsible gene for inosine accumulation in RNA. In Itpa -KO/ Guk1 knockdown (KD) cell lines, Guk1 mRNA expression levels were positively correlated with the amount of inosine in RNA, most of which is incorporated during RNA synthesis in ITPA-deficient cells. The transient expression of human GUK1 in Itpa -KO/ Guk1 -KD mouse cells significantly increased inosine levels in RNA. Recombinant human GUK1 phosphorylates IMP into inosine diphosphate. Furthermore, in brain-specific Itpa -KO mice, Guk1 haploinsufficiency resulted in a significant reduction in inosine in cortical RNA and an extended lifespan. Thus, our results suggest that GUK1 is probably an enzyme responsible for ITP biosynthesis although the K m of GUK1 for IMP is very high in vitro.
Funding Information
  • Japan Society for the Promotion of Science (18K06960)
  • Japan Society for the Promotion of Science (21K06869)
  • Japan Society for the Promotion of Science (17H01391)
  • MEXT Cooperative Research Project Program (1)
  • Coalition of Universities for Research Excellence Program (JPMXP1323015486)
  • Medical Research Center Initiative for High Depth Omics (1)

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