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- Title
SI-MOIRAI: a new method to identify and quantify the metabolic fate of nucleotides.
- Authors
Ikeda, Yoshiki; Hirayama, Akiyoshi; Kofuji, Satoshi; Hirota, Yoshihisa; Kamata, Ryo; Osaka, Natsuki; Fujii, Yuki; Sasaki, Mika; Ikeda, Satsuki; Smith, Eric P; Bachoo, Robert; Soga, Tomoyoshi; Sasaki, Atsuo T
- 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.
- Subjects
NUCLEOTIDES; PYRIMIDINE nucleotides; PURINE nucleotides; RNA; RIBONUCLEOTIDES; RNA metabolism
- Publication
Journal of Biochemistry, 2021, Vol 170, Issue 6, p699
- ISSN
0021-924X
- Publication type
Article
- DOI
10.1093/jb/mvab077