MOCS2 links nucleotide metabolism to nucleoli function
Year: 2021
Type: article
Abstract: 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. MOCS2 governs purine metabolism, pyrimidine metabolism, and nucleoli functions. (A) Diagram showing metabolic pathways linked to polyamine transfer in humans (details in Supplementary material). (B) Heatmap showing the top 50 metabolites, including undetermined metabolites (MP, multiple possibilities) in control and MOCS2-knockdown cells (n = 5), from a volcano plot in untargeted metabolomics analyzed by negative heated electrospray ionization. Results were normalized by protein concentration; the log scale is based on the normalized intensity. (C) Reduced mRNA N7G methylation levels in MOCS2-knockdown cells relative to those in control HEK293 cells were improved by supplementation of 50 or 200 µM SAMe or 50 µM sodium acetate in the culture media. (D) Polysome profiles of MOCS2- and XDH-knockdown and control HEK293 cells. (E) Gene Ontology terms clustered by biological process (BP) for significantly reduced transcripts (RPKM > 1) in polysomes (≥3) from XDH-knockdown vs. control cells were analyzed by RNA sequencing. (F) Nuclear extracts of indicated knockdown cells were used for NOP56 immunoprecipitations. MOCS2B protein levels were examined in cytoplasmic extracts. eIF3β and eIF4E were used as the loading control. We previously found that S-adenosylmethionine (SAMe) levels decreased, N1,N12-diacetylspermine levels increased, and the urea cycle was upregulated in MOCS2-knockdown 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). N1,N12-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 5′ cap in mRNA consists of N7-methylguanine (N7meG) connected by a 5′-to-5′ triphosphate bridge to the first mRNA nucleotide (Gao et al., 2000). The N7meG cap prevents mRNA degradation by exonucleases and influences translation (Gao et al., 2000). Therefore, we measured methylation levels of N7G in mRNA. Methylation of N7G 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 N7G in MOCS2-knockdown cells (Figure 1C). Hence, MOCS2-dependent SAMe levels and metabolism are required for mRNA processing. We sought to determine how MOCS2-dependent 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 MOCS2-knockdown, XDH-knockdown, and control HEK293 cells. Polysome numbers and sizes were smaller in MOCS2- and 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 XDH-knockdown cells based on Gene Ontology term analysis (Supplementary Figure S1B). XDH requires MOCS2 (Schwarz et al., 2009); therefore, MOCS2 expression is demanded by XDH. Interestingly, genes related to histone H2A K119 mono-ubiquitylation [H2AK119u1; ring finger protein 2 (RNF2)/RING1B, polycomb ring finger (BMI1), and polycomb group ring finger 5 (PCGF5)] significantly decreased in XDH-knockdown cells (Figure 1E). RING1B is a subunit of a polycomb repressive complex 1 (PRC1) and catalyzes H2AK119u1 (Almeida et al., 2017). BMI1 is another subunit of PRC1 (Almeida et al., 2017). PCGF5 forms the noncanonical PRC1 complex (hereafter referred to as vPRC1) with PCGF3 and initiates recruitment of vPRC1 and PRC2 via the long noncoding RNA (lncRNA) Xist. Xist mediates H2AK119u1 in the initiation of X-chromosome inactivation (XCI) (Almeida et al., 2017). In mice, deletion of the Xist dissociates the ectopic X-inactivation centre (Xic) from the perinucleolus, resulting in reactivation of the inactive X-chromosome (Xi) (Zhang et al., 2007). Polyamines are utilized in nucleolar RNA synthesis (Hernandez-Verdun et al., 2010). Polyamine-assisted nucleolar RNA production increases the size and activity of nucleoli and influences the organization of nucleoli and the nucleus (Hernandez-Verdun et al., 2010). Therefore, we examined whether MOCS2-dependent polyamine metabolism influences the features of the nucleolus associating with Xist functions. The majority of small nucleolar ribonucleoproteins (snoRNPs) are classified into two groups, box C/D and box H/ACA snoRNPs (Watkins and Bohnsack, 2012). Box C/D snoRNPs are highly expressed and form a complex with small nuclear ribonucleoprotein 13 (SNU13)/NHP2L1, NOP56 ribonucleoprotein, NOP58, and fibrillarin rRNA methyltransferase (Watkins and Bohnsack, 2012). We did not observe differences in the expression levels of box C/D snoRNPs between MOCS2/XDH-knockdown and control cells based on western blotting analysis of global expression of subunits and NOP56 immunoprecipitations (Figure 1F). However, PCGF5 protein levels were reduced, and the associations of RING1B and PCGF5 with box C/D snoRNPs observed in control cells were lost in MOCS2- and XDH-knockdown cells (Figure 1F). Hence, in MOCS2/XDH-knockdown cells, altered nucleoli and modified PRC1 expression lead to loss of the contact of nucleoli with PRC1/vPRC1. Together, MOCS2 and XDH mediate nucleotide metabolism and polyamine metabolism to regulate RNA synthesis and nucleoli functions. If MOCS2/XDH contributes to XCI, alteration of several metabolic pathways including the purine salvage pathway in MOCS2- and XDH-deficient cells may be the result of altered X-linked genes due to impaired nucleoli functions. [Supplementary material is available at Journal of Molecular Cell Biology online. We thank Dr Blake Ebner and Mr Troy Green for technical support. This research was supported by the Stowers Institute for Medical Research. T.S. and J.L.W. wrote the manuscript. T.S. designed and conducted the study. T.S., S.K.S., and T.J.G. performed the experiments. T.S., S.K.S., T.J.G., M.G., L.F., and J.L.W. analyzed the data.]
DOI: 10.1093/jmcb/mjab069
Institutions: Stowers Institute for Medical Research, University of Florida
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $2,180
OpenAlex ID: W3208098051