SI-MOIRAI: A new method to identify and quantify the metabolic fate of nucleotides

Yoshiki Ikeda, Akiyoshi Hirayama, Satoshi Kofuji, Yoshihisa Hirota, Ryo Kamata, Natsuki Osaka, Yuki Fujii, Mika Sasaki, Satsuki Ikeda, Eric P. Smith, Robert Bachoo, Tomoyoshi Soga, Atsuo T. Sasaki

Research output: Contribution to journalArticlepeer-review

Abstract

Since the discovery of nucleotides over 100 years ago, extensive studies have revealed the importance of nucleotides for homeostasis, health and disease. However, there remains no established method to investigate quantitatively and accurately intact nucleotide incorporation into RNA and DNA. Herein, we report a new method, Stable-Isotope Measure Of Influxed Ribonucleic Acid Index (SI-MOIRAI), for the identification and quantification of the metabolic fate of ribonucleotides and their precursors. SI-MOIRAI, named after Greek goddesses of fate, combines a stable isotope-labelling flux assay with mass spectrometry to enable quantification of the newly synthesized ribonucleotides into r/m/tRNA under a metabolic stationary state. Using glioblastoma (GBM) U87MG cells and a patient-derived xenograft (PDX) GBM mouse model, SI-MOIRAI analyses showed that newly synthesized GTP was particularly and disproportionally highly utilized for rRNA and tRNA synthesis but not for mRNA synthesis in GBM in vitro and in vivo. Furthermore, newly synthesized pyrimidine nucleotides exhibited a significantly lower utilization rate for RNA synthesis than newly synthesized purine nucleotides. The results reveal the existence of discrete pathways and compartmentalization of purine and pyrimidine metabolism designated for RNA synthesis, demonstrating the capacity of SI-MOIRAI to reveal previously unknown aspects of nucleotide biology.

Original languageEnglish (US)
Pages (from-to)699-711
Number of pages13
JournalJournal of Biochemistry
Volume170
Issue number6
DOIs
StatePublished - Dec 1 2021

Keywords

  • cancer metabolism
  • flux analysis
  • glioblastoma (GBM)
  • mass spectrometry
  • metabolomics
  • nucleotide metabolism

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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