MOCS2 links nucleotide metabolism to nucleoli function.

Dear Editor, Three polyamines, putrescine, spermidine, and spermine, are tightly regulated and are synthesized from arginine, ornithine, and methionine (Miller-Fleming et al., 2015; Figure 1A). Polyamine levels are increased in the red blood cells of amyotrophic lateral sclerosis and Parkinson’s disease (PD) patients (Miller-Fleming et al., 2015). Sulfur amino acid catabolism regulated by the molybdopterin synthase associating complex prevents onset of early events in PD and Alzheimer’s disease (Suganuma et al., 2018). MPT synthase, a heterodimer of MOCS2A and MOCS2B encoded by MOCS2, is essential for the biosynthesis of molybdenum cofactor (Moco), which is required for the activity of xanthine dehydrogenase (XDH) (Schwarz et al., 2009; Suganuma et al., 2018). XDH is the single transcribed form of xanthine oxidoreductase, which catalyzes the terminal two reactions in purine degradation, i.e. oxidation of hypoxanthine to xanthine and oxidation of xanthine to uric acid (Schwarz et al., 2009; Figure 1A). Defects in any step of Moco biosynthesis lead to Moco deficiency (MoCD), characterized by rapid progression of neurodegeneration and lethality in early childhood (Schwarz et al., 2009). Notably, increases in the levels of inosine, hypoxanthine, and xanthine have been found in both MoCD and Huntington’s disease (Toczek et al., 2016; Suganuma et al., 2018). Therefore, links between nucleotide metabolism and polyamine metabolism may contribute to the pathogenesis of these diseases. We previously found that S-adenosylmethionine (SAMe) levels decreased, N,Ndiacetylspermine levels increased, and the urea cycle was upregulated in MOCS2knockdown vs. control HEK293 cells (Suganuma et al., 2018; Figure 1A). SAMe serves as a methyl donor in reactions catalyzed by methyltransferases and is consumed in polyamine synthesis following decarboxylation to dcSAM (Figure 1A). N,N-diacetylspermine is the catabolite of acetylated spermine and spermidine (Miller-Fleming et al., 2015). Reduction of SAMe levels in MOCS2-knockdown cells might result from increased SAMe consumption for polyamine synthesis and the methionine salvage pathway. We also found reduced guanine levels and global acetylation levels in MOCS2-knockdown cells (Suganuma et al., 2018). In the current study, we found that levels of deoxyinosine decreased in MOCS2-knockdown cells through global metabolomics (Figure 1B). Purine catabolism generates purine bases guanine and hypoxanthine mediated by XDH. Hence, MOCS2 is required for purine metabolism. Importantly, uridine levels decreased in MOCS2-knockdown cells. Uridine is derived from uridine monophosphate by de novo synthesis and is an RNA catabolite (Figure 1B inset). The 50 cap in mRNA consists of N-methylguanine (NmeG) connected by a 50-to-50 triphosphate bridge to the first mRNA nucleotide (Gao et al., 2000). The NmeG cap prevents mRNA degradation by exonucleases and influences translation (Gao et al., 2000). Therefore, we measured methylation levels of NG in mRNA. Methylation of NG decreased in MOCS2-knockdown cells compared to control cells (Figure 1C). Supplementation of SAMe and sodium acetate (as acetyl-CoA) in the media recovered and improved the decreased methylation levels of NG in MOCS2-knockdown cells (Figure 1C). Hence, MOCS2-dependent SAMe levels and metabolism are required for mRNA processing. We sought to determine how MOCS2dependent metabolism and mRNA processing influence cellular processes. Polyamine is used in the synthesis of hypusine, an amino acid involved in a unique post-translational modification of the eukaryotic translation initiation factor 5A (eIF5A) (Miller-Fleming et al., 2015; Figure 1A). Hypusination of eIF5A is critical for peptidyl-tRNA hydrolysis and translation termination (Schuller et al., 2017). Thus, we examined the effects of MOCS2 knockdown on translation. Since polysomes are equated with the translationally active mRNA pool, we monitored polysome profiles in MOCS2knockdown, XDH-knockdown, and control HEK293 cells. Polysome numbers and sizes were smaller in MOCS2and XDH-knockdown cells than in control cells, indicating that MOCS2 and XDH are important for mRNA translation (Figure 1D). We used RNA sequencing to further analyze total RNA from polysomes ( 3). Transcripts of only 444 genes and 539 genes were significantly changed in polysomes from MOCS2-knockdown cells and XDH-knockdown cells, respectively, indicating that MOCS2 and XDH are crucial for translation of these genes (Supplementary Figure S1A). MOCS2 transcript levels were significantly reduced (RPKM>1) in polysomes of XDHknockdown cells based on Gene