SI-MOIRAI: a new method to identify and quantify the metabolic fate of nucleotides
SI-MOIRAI: a new method to identify and quantify the metabolic fate of nucleotides
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SI-MOIRAI：一种识别和量化核苷酸代谢命运的新方法
DOI：
10.1093/jb/mvab077
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发表时间：
2021
期刊：
影响因子：
--
通讯作者：
Sasaki Atsuo T
中科院分区：
文献类型：
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作者：
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
自100多年前发现核苷酸以来，广泛的研究揭示了核苷酸对体内平衡、健康和疾病的重要性。然而，目前还没有确定的方法来定量和准确地研究完整的核苷酸并入RNA和DNA。在此，我们报告了一种新的方法，稳定同位素测量流入核糖核酸指数（SI-MOIRAI），用于鉴定和定量核糖核苷酸及其前体的代谢命运。SI-MOIRAI以希腊命运女神命名，将稳定同位素标记通量法与质谱法相结合，能够在代谢稳定状态下将新合成的核糖核苷酸定量为r/m/tRNA。使用胶质母细胞瘤（GBM） U87MG细胞和患者来源的异种移植（PDX） GBM小鼠模型，SI-MOIRAI分析显示，新合成的GTP特别且不成比例地高度用于rRNA和tRNA的合成，而不是用于GBMin体外和体内mRNA的合成。此外，新合成的嘧啶核苷酸在RNA合成中的利用率明显低于新合成的嘌呤核苷酸。结果揭示了RNA合成中嘌呤和嘧啶代谢的离散途径和区室化的存在，证明了SI-MOIRAI揭示核苷酸生物学以前未知方面的能力。
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 GBMin vitroandin 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.