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

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.

Keywords: cancer metabolism; flux analysis; glioblastoma (GBM); mass spectrometry; metabolomics; nucleotide metabolism.

Graphical Abstract

Fig. 1.

A pathway map of purine and pyrimidine nucleotides synthesis. Purine and pyrimidine nucleotides are synthesized through the incorporation of a series of precursors such as glucose, glutamine, aspartic acid and glycine. PRPP is a precursor metabolite used in all nucleotide synthesis. The human gene names (derived from the UniProt database) of enzymes responsible for nucleotide synthesis are shown in blue. Abbreviation: PRPP (ribose 5-phosphate is converted to phosphoribosyl pyrophosphate), PRA (phosphoribosylamine), GAR (glycinamide ribonucleotide), FGAR (5ʹ-phosphoribosylformylglycinamide), FGAM (5ʹ-phosphoribosylformylglycinamidine), AIR (aminoimidazole ribotide), CAIR (carboxyaminoimidazole ribotide), SAICAR (5ʹ-phosphoribosyl-5ʹ-aminoimidazole-4ʹ-N-succinocarboxamide), AICAR (5ʹ-aminoimidazole-4ʹ-carboxamide 1-β-d-ribofuranoside), FAICAR (5ʹ-formamidoimidazole-4ʹ-carboxamide ribotide), IMP (inosine-5ʹ-monophosphate), XMP (xanthosine monophosphate), S-AMP (succinyl adenosine-5ʹ-monophosphate), AMP (adenosine 5ʹ-monophosphate).

Fig. 2.

Overview of SI-MOIRAI: tracking the anabolic trajectory and the metabolic fate of newly synthesized nucleotides. The SI-MOIRAI enables tracking the metabolic fate of ribonucleotide for RNA synthesis using a stable isotope tracer metabolite(s). A schematic diagram of the metabolic pathway of ¹³C₆-glucose is depicted as an example. ¹³C₆-glucose is incorporated into ribonucleotides, which are further incorporated into r/m/tRNAs. First, total RNA is extracted and purified from the cells or tissue after labelling with ¹³C₆-glucose agarose gel, followed by size separation into rRNA, mRNA and tRNA by gel electrophoresis. The isolated r/m/tRNAs are digested into mononucleotides and then nucleoside and subjected to MS analysis for quantification of ¹³C-labelled isotopomers.

Fig. 3.

A schematic diagram of glucose-derived carbon incorporation to purine and pyrimidine nucleotides. ¹³C₆-Glucose is metabolized to ribose and amino acids such as glycine and aspartic acid, formyl-THF, methylene-THF and carbonate ions (HCO3−). Nucleoside triphosphate (NTP) is synthesized on the basis of the metabolites of ¹³C₆-glucose. Biosynthesized purine nucleotides can be in the range of ¹³C_1∼10-ATP and ¹³C_1∼10-GTP, and that of pyrimidine nucleotides are ¹³C_1∼10-dTTP and ¹³C_1∼9-UTP.

Fig. 4.

A representative result of RNA purification and digestion steps in SI-MOIRAI method. (A) The purified RNA was electrophoresed by native-agarose gel and extracted from the band to obtain objective species of RNA. For example, 2–3 μg of rRNA, 0.4–0.6 μg of tRNA, 0.4–0.6 μg of mRNA could be obtained from 20 μg of total RNA, as shown in Fig. 4A (details are described in Experimental Procedure section). (B) The extracted RNAs are subjected to enzymatic digestion into mono-ribonucleotides by two enzymes, nuclease P1 and phosphodiesterase 1 (left). The digestion efficiency of the purified total RNA is verified by 4––16% native-poly-acrylamide gel electrophoresis (PAGE) (right). Abbreviation: nt (nucleotides).

Fig. 5.

Quantification of the nucleosides by LC–MS. LC–MS signal of the injected nucleoside standards and the SI-MOIRAI samples are shown. The detected peak areas are normalized by internal standards to calculate their absolute amount.

Fig. 6.

SI-MOIRAI analysis of the GBM tissue reveals the distinctive metabolic utility of each ribonucleotide for RNA synthesis. (A) Using the digested nucleosides from each RNA specimen (rRNA, tRNA, mRNA) of the GBM tissue, the ¹³C- and ¹²C-labelled nucleosides were measured by LC–MS and normalized by internal standards, and calculated for the absolute amounts from the standard peak area. The deduced ≥M + 3 nucleoside levels were summed up and then normalized against total signal peaks (sum up from M + 0 to all) of each nucleotide, respectively (N = 1). (B) The M + 5 labelled ribonucleotide amounts per 1 mg of each RNA species (N = 1). (C) The M + 6 labelled ribonucleotide amounts per 1 mg of total RNA (N = 1). (D) A relative ratio of nucleotide incorporation in each RNA species. The nucleotides incorporation to mRNA was under the detection sensitivity (N = 1). (E) A comparison of isotopomers of each labelled nucleoside (M + 0 to M + 6) (N = 1).

Fig. 7.

SI-MOIRAI analysis of U87MG cells treated with ¹³C₅,¹⁵N₂-glutamine tracer. SI-MOIRAI analysis of U87MG cells treated with ¹³C₅,¹⁵N₂-glutamine tracer for 6 h. The total RNA was separated into rRNA (A) and tRNA (B), followed by the enzymatic digestion into nucleosides and LC–MS analysis. The graph shows a relative ratio of ¹⁵N (M + 1, M + 2, or M + 3) labelled nucleoside amount compared to ¹⁴N (M + 0), respectively (N = 3).

Fig. 8.

A schematic diagram of the results of SI-MOIRAI revealing the in vivo metabolic tracking of ¹³C₆-glucose to RNA synthesis. Tracking of metabolic products by SI-MOIRAI in GBM tissue derived from the PDX GBM mouse infused with ¹³C-glucose revealed that in brain tumour cells, the metabolic integration of de novo pathway-derived GTP is increased in rRNA and tRNA synthesis, but not in mRNA, compared to that of ATP. Arrows in the de novo and salvage pathways indicate the reaction steps by metabolic enzymes. The initiator (¹³C-glucose), metabolic products (GTP and r/m/tRNAs) and metabolic processes tracked by SI-MOIRAI are indicated by orange texts and colored arrows.