NT5C3A (cytosolic 5'-nucleotidase 3A; pyrimidine 5'-nucleotidase 1, P5'N-1/PN-I; UMP-CMP 5'-nucleotidase; also historically "p36") is a cytosolic, Mg2+-dependent hydrolase of the haloacid dehalogenase (HAD) superfamily that belongs to the pyrimidine 5'-nucleotidase family. It dephosphorylates pyrimidine (deoxy)ribonucleoside 5'-monophosphates to the corresponding nucleoside plus inorganic phosphate (EC 3.1.3.5), with a strong preference for pyrimidine over purine substrates; UMP and CMP are the physiological substrates and CMP is the preferred one in vitro. The enzyme uses a two-aspartate HAD catalytic mechanism (Asp88 nucleophile, Asp90 proton donor) with an active-site Mg2+ ion, and also exhibits an associated phosphotransferase activity that exchanges phosphate between pyrimidine monophosphates and pyrimidine nucleosides. NT5C3A is very highly expressed in reticulocytes and mature erythrocytes, where it degrades the pyrimidine ribonucleotides released by RNA breakdown during erythroid maturation so that the resulting nucleosides can be cleared from the cell; this makes it central to erythroid pyrimidine nucleotide catabolism. Loss-of-function variants cause an autosomal recessive hereditary non-spherocytic hemolytic anemia (pyrimidine 5'-nucleotidase deficiency) featuring marked basophilic stippling and intra-erythrocyte accumulation of pyrimidine nucleotides, and the enzyme is also implicated in the anemia of lead poisoning. An alternatively spliced, N-terminally distinct isoform (isoform 2/p36) is interferon-alpha-inducible and localizes to the endoplasmic reticulum.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005737 cytoplasm | IBA GO_REF:0000033 | ACCEPT | Summary: Phylogenetic (IBA) inference that NT5C3A is active in the cytoplasm. This matches the UniProt subcellular-location call for the soluble enzyme and experimental cytosol localization. Accept as a core localization (cytosol is the more specific term captured in the IDA annotation below). Supporting Evidence: file:human/NT5C3A/NT5C3A-uniprot.txt SUBCELLULAR LOCATION: Cytoplasm |
| GO:0008253 5'-nucleotidase activity | IBA GO_REF:0000033 | ACCEPT | Summary: Phylogenetic (IBA) inference of 5'-nucleotidase activity, the core molecular function of this pyrimidine 5'-nucleotidase. Directly supported by experimental enzymology on the purified/recombinant human enzyme. Accept as core. Supporting Evidence: file:human/NT5C3A/NT5C3A-uniprot.txt CMP seems to be the preferred substrate |
| GO:0000287 magnesium ion binding | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro-based electronic inference of magnesium ion binding. The enzyme is a HAD-family hydrolase whose activity is Mg2+-dependent, and the crystal structure shows Mg2+ coordinated at the active site (residues 88, 90, 277). Accept as a core catalytic cofactor requirement. Supporting Evidence: file:human/NT5C3A/NT5C3A-uniprot.txt BINDING 88 PMID:11795870 The activity is dependent on Mg(2+) |
| GO:0005737 cytoplasm | IEA GO_REF:0000120 | ACCEPT | Summary: Electronic (UniProt automated) annotation of cytoplasmic location, consistent with the curated UniProt subcellular-location call and experimental data. Accept as a core localization; cytosol (below) is the more specific term. Supporting Evidence: file:human/NT5C3A/NT5C3A-uniprot.txt SUBCELLULAR LOCATION: Cytoplasm |
| GO:0005783 endoplasmic reticulum | IEA GO_REF:0000044 | KEEP AS NON CORE | Summary: Electronic annotation of ER localization from UniProt Subcellular Location mapping. ER localization is specific to isoform 2 (the interferon-alpha-induced p36-like isoform bearing an N-terminal membrane segment), not the soluble cytosolic enzyme. Real but not the core location of the characterized nucleotidase; keep as non-core. Reason: Examined as a synthesis row: GO_REF:0000044 maps SL-0095 from the UniProt line "[Isoform 2]: Endoplasmic reticulum", which rests on the same p36 study as the IDA row (PMID:8557639). ORGANELLES ER check (2026-11-05). The displayed UniProt sequence is isoform 2 (Q9H0P0-4), whose 50-residue N-terminal extension (absent from isoforms 1, 3 and 4) begins with a hydrophobic stretch (about residues 14-32, VGAVASASVCALVAGVVLA) followed by basic residues; UniProt annotates no TM or signal anchor there. UniProt restricts the ER location to isoform 2 and ties the interferon-alpha induction of p36 to it (ECO:0000269, PMID:8557639), although its isoform list gives p36 as a synonym of isoform 3, which lacks the extension; which isoform p36 is remains unsettled. The ER evidence is endogenous, interferon-induced p36 co-localizing with the ER-resident marker PDI by immunofluorescence (PMID:8557639, abstract only), plus HPA antibody staining. That is a real, minor, isoform-restricted ER pool, so the root claim holds; the characterized pyrimidine 5'-nucleotidase activity is cytosolic, so the row is KEEP_AS_NON_CORE. No ER membrane row is added: whole-organelle co-staining cannot resolve membrane from lumen, the hydrophobic N-terminal segment is an unannotated prediction, and the isoform behind the ER signal is uncertain, so only one line points at the membrane. Supporting Evidence: file:human/NT5C3A/NT5C3A-uniprot.txt [Isoform 2]: Endoplasmic reticulum file:human/NT5C3A/NT5C3A-uniprot.txt Isoform 2 is induced by interferon alpha in Raji cells in association with lupus inclusions. {ECO:0000269|PubMed:8557639} PMID:8557639 p36 is a new interferon-alpha-induced protein that localizes in the endoplasmic reticulum |
| GO:0005829 cytosol | IEA GO_REF:0000117 | ACCEPT | Summary: ARBA machine-learning electronic annotation of cytosol localization. Correct and the most specific location term for the soluble enzyme; corroborated by the MGI IDA cytosol annotation. Accept as core. Supporting Evidence: file:human/NT5C3A/NT5C3A-uniprot.txt SUBCELLULAR LOCATION: Cytoplasm |
| GO:0008253 5'-nucleotidase activity | IEA GO_REF:0000120 | ACCEPT | Summary: Electronic annotation (UniProt automated, backed by InterPro/RHEA/EC:3.1.3.5) of 5'-nucleotidase activity. This is the gene's own correct core molecular function, also supported experimentally; accept (redundant with the EXP/IDA/IBA calls but not an over-annotation). Supporting Evidence: file:human/NT5C3A/NT5C3A-uniprot.txt EC=3.1.3.5 |
| GO:0005515 protein binding | IPI PMID:32296183 A reference map of the human binary protein interactome. | MARK AS OVER ANNOTATED | Summary: Bare "protein binding" from the HuRI systematic binary-interactome (yeast two-hybrid) screen; the interactor here is DESI1 (Q6ICB0), matching the UniProt INTERACTION block. Systematic high-throughput binary interaction that does not describe a specific molecular function; uninformative as an MF term. Do not use bare protein binding for the core function. Supporting Evidence: file:human/NT5C3A/NT5C3A-uniprot.txt Q9H0P0; Q6ICB0: DESI1 |
| GO:0005783 endoplasmic reticulum | IDA GO_REF:0000052 | KEEP AS NON CORE | Summary: Immunofluorescence (HPA) evidence of ER localization. Consistent with the interferon-inducible ER-localized isoform 2/p36; genuine but isoform-specific and not the location of the core cytosolic enzyme. Keep as non-core. Reason: HPA antibody immunofluorescence (GO_REF:0000052) is a whole-organelle assay that cannot resolve membrane from lumen or say which isoform it detects; under rule 2 the root term is what it showed, and it agrees with the independent p36/PDI co-staining. ORGANELLES ER check (2026-11-05). The displayed UniProt sequence is isoform 2 (Q9H0P0-4), whose 50-residue N-terminal extension (absent from isoforms 1, 3 and 4) begins with a hydrophobic stretch (about residues 14-32, VGAVASASVCALVAGVVLA) followed by basic residues; UniProt annotates no TM or signal anchor there. UniProt restricts the ER location to isoform 2 and ties the interferon-alpha induction of p36 to it (ECO:0000269, PMID:8557639), although its isoform list gives p36 as a synonym of isoform 3, which lacks the extension; which isoform p36 is remains unsettled. The ER evidence is endogenous, interferon-induced p36 co-localizing with the ER-resident marker PDI by immunofluorescence (PMID:8557639, abstract only), plus HPA antibody staining. That is a real, minor, isoform-restricted ER pool, so the root claim holds; the characterized pyrimidine 5'-nucleotidase activity is cytosolic, so the row is KEEP_AS_NON_CORE. No ER membrane row is added: whole-organelle co-staining cannot resolve membrane from lumen, the hydrophobic N-terminal segment is an unannotated prediction, and the isoform behind the ER signal is uncertain, so only one line points at the membrane. Supporting Evidence: file:human/NT5C3A/NT5C3A-uniprot.txt [Isoform 2]: Endoplasmic reticulum PMID:8557639 p36 is a new interferon-alpha-induced protein that localizes in the endoplasmic reticulum |
| GO:0008253 5'-nucleotidase activity | EXP PMID:15968458 Evidence for essential catalytic determinants for human eryt... | ACCEPT | Summary: Direct experimental characterization of the recombinant human enzyme, which dephosphorylates pyrimidine nucleoside monophosphates (CMP preferred) and whose catalytic determinants were defined by site-directed mutagenesis. This is the core, defining molecular function. Accept. Supporting Evidence: PMID:15968458 dephosphorylation of pyrimidine nucleoside monophosphates |
| GO:0005739 mitochondrion | HTP PMID:34800366 Quantitative high-confidence human mitochondrial proteome an... | KEEP AS NON CORE | Summary: High-throughput spatial/mitochondrial-proteome assignment (FlyBase-curated HTP). The characterized enzyme is cytosolic; this is most likely a low-level co-fractionation hit rather than a genuine functional mitochondrial pool. Keep as non-core rather than removing an HTP experimental-type annotation. Supporting Evidence: file:human/NT5C3A/NT5C3A-uniprot.txt SUBCELLULAR LOCATION: Cytoplasm |
| GO:0005829 cytosol | IDA PMID:11795870 Human erythrocyte pyrimidine 5'-nucleotidase, PN-I. | ACCEPT | Summary: Direct assay of the purified soluble ("cytoplasmic") enzyme from human erythrocytes, curated by MGI as active in the cytosol. This is the core location of the active enzyme. Accept. Supporting Evidence: file:human/NT5C3A/NT5C3A-uniprot.txt SUBCELLULAR LOCATION: Cytoplasm |
| GO:0006248 CMP catabolic process | IDA PMID:11795870 Human erythrocyte pyrimidine 5'-nucleotidase, PN-I. | ACCEPT | Summary: Experimental (IDA) involvement in CMP catabolism: the enzyme hydrolyzes CMP (its preferred pyrimidine substrate) to cytidine plus phosphate. This is a core biological process for the enzyme. Accept. Supporting Evidence: file:human/NT5C3A/NT5C3A-uniprot.txt CMP seems to be the preferred substrate PMID:11795870 PN-I is specific for pyrimidine |
| GO:0006249 dCMP catabolic process | IDA PMID:11795870 Human erythrocyte pyrimidine 5'-nucleotidase, PN-I. | KEEP AS NON CORE | Summary: Experimental (IDA) involvement in dCMP catabolism. The enzyme acts on pyrimidine deoxyribonucleotide monophosphates as well as ribonucleotides, though it prefers ribonucleotides. A genuine but secondary/less-preferred activity; keep as non-core relative to the UMP/CMP (ribonucleotide) core. Supporting Evidence: PMID:11795870 PN-I is specific for pyrimidine |
| GO:0008253 5'-nucleotidase activity | IDA PMID:11795870 Human erythrocyte pyrimidine 5'-nucleotidase, PN-I. | ACCEPT | Summary: Direct assay of the purified/recombinant human erythrocyte enzyme establishing pyrimidine 5'-nucleotidase (5'-nucleotidase) activity, Mg2+-dependent, specific for pyrimidine nucleoside monophosphates. Core molecular function. Accept. Supporting Evidence: PMID:11795870 PN-I is specific for pyrimidine |
| GO:0046050 UMP catabolic process | IDA PMID:11795870 Human erythrocyte pyrimidine 5'-nucleotidase, PN-I. | ACCEPT | Summary: Experimental (IDA) involvement in UMP catabolism. Dephosphorylation of UMP (a principal pyrimidine ribonucleotide released during erythroid RNA turnover) to uridine plus phosphate is a core physiological process for this enzyme. Accept. Supporting Evidence: PMID:11795870 PN-I is specific for pyrimidine |
| GO:0046074 dTMP catabolic process | IDA PMID:11795870 Human erythrocyte pyrimidine 5'-nucleotidase, PN-I. | KEEP AS NON CORE | Summary: Experimental (IDA) involvement in dTMP catabolism, reflecting activity toward pyrimidine deoxyribonucleotide monophosphates. A genuine but less-preferred (deoxyribonucleotide) activity relative to the UMP/CMP ribonucleotide core; keep as non-core. Supporting Evidence: PMID:11795870 PN-I is specific for pyrimidine |
| GO:0046079 dUMP catabolic process | IDA PMID:11795870 Human erythrocyte pyrimidine 5'-nucleotidase, PN-I. | KEEP AS NON CORE | Summary: Experimental (IDA) involvement in dUMP catabolism, reflecting activity toward pyrimidine deoxyribonucleotide monophosphates. Genuine but less-preferred (deoxyribonucleotide) activity relative to the UMP/CMP ribonucleotide core; keep as non-core. Supporting Evidence: PMID:11795870 PN-I is specific for pyrimidine |
| GO:0051607 defense response to virus | IDA PMID:21478870 A diverse range of gene products are effectors of the type I... | KEEP AS NON CORE | Summary: Annotation derived from a large interferon-stimulated-gene (ISG) overexpression antiviral screen. NT5C3A (as an ISG/p36) was tested but was a weak/non-effector: when expressed after infection its effect on HCV replication was less than 20%, and it was not among the confirmed strong antiviral hits. This is at most a peripheral, interferon-context role and not a core evolved function of the nucleotidase; keep as non-core. Supporting Evidence: PMID:21478870 less than 20% (DDIT4, NT5C3, IFI44L, MAP3K14, IRF2) |
| GO:0005829 cytosol | TAS Reactome:R-HSA-109449 | ACCEPT | Summary: Reactome traceable-author-statement placing the enzyme in the cytosol, within the "(d)CMP, TMP, or (d)UMP + H2O => ... + orthophosphate" reaction of pyrimidine catabolism. Correct core location. Accept. Supporting Evidence: file:human/NT5C3A/NT5C3A-uniprot.txt SUBCELLULAR LOCATION: Cytoplasm |
| GO:0000215 tRNA 2'-phosphotransferase activity | TAS PMID:10942414 Human erythrocyte pyrimidine 5-nucleotidase, PN-I, is identi... | MARK AS OVER ANNOTATED | Summary: tRNA 2'-phosphotransferase activity (the tRNA-splicing 2'-phosphate-removing activity, e.g. of TRPT1) does not match this pyrimidine 5'-nucleotidase. The cited paper is about PN-I/p36 identity and pyrimidine 5'-nucleotidase, and makes no mention of tRNA 2'-phosphotransferase; this appears to be a legacy mis-annotation, likely conflating the enzyme's pyrimidine-nucleotide phosphotransferase activity with the unrelated tRNA 2'-phosphotransferase. Not experimental (TAS); flag as over-annotation rather than a correct function. Proposed replacements: 5'-nucleotidase activity Supporting Evidence: PMID:10942414 We conclude that p36 and PN-I are identical proteins |
| GO:0000215 tRNA 2'-phosphotransferase activity | NAS PMID:9428647 Pyrimidine nucleotidases from human erythrocyte possess phos... | MARK AS OVER ANNOTATED | Summary: Non-traceable author statement of tRNA 2'-phosphotransferase activity. The cited paper instead reports that erythrocyte pyrimidine nucleotidases possess a pyrimidine-specific phosphotransferase activity exchanging phosphate between pyrimidine mononucleotides and pyrimidine nucleosides - not tRNA 2'-phosphotransferase activity. The GO term does not match the described biochemistry; flag as over-annotation. The associated activity is better captured as the enzyme's 5'-nucleotidase / pyrimidine phosphotransferase function. Proposed replacements: 5'-nucleotidase activity Supporting Evidence: PMID:9428647 pyrimidine-specific phosphotransferase activity |
| GO:0000287 magnesium ion binding | NAS PMID:11795870 Human erythrocyte pyrimidine 5'-nucleotidase, PN-I. | ACCEPT | Summary: Author statement that enzyme activity is magnesium-dependent. Corroborated by the crystal structure showing active-site Mg2+ coordination. Correct core catalytic cofactor. Accept. Supporting Evidence: PMID:11795870 The activity is dependent on Mg(2+) |
| GO:0005737 cytoplasm | IDA PMID:8557639 Purification, microsequencing, and immunolocalization of p36... | ACCEPT | Summary: Immunolocalization of p36 (= NT5C3A, isoform 2). In this study p36 was shown to localize to the endoplasmic reticulum; the broader "cytoplasm" term is consistent with the cytosolic soluble enzyme. Accept as a (general) core cytoplasmic localization. Supporting Evidence: file:human/NT5C3A/NT5C3A-uniprot.txt SUBCELLULAR LOCATION: Cytoplasm |
| GO:0005783 endoplasmic reticulum | IDA PMID:8557639 Purification, microsequencing, and immunolocalization of p36... | KEEP AS NON CORE | Summary: Immunofluorescence localization of interferon-alpha-induced p36 (isoform 2) to the ER, where it co-localizes with protein disulfide isomerase and lupus inclusions. Genuine but isoform-2-specific; not the location of the core cytosolic nucleotidase. Keep as non-core. Reason: Direct evidence on the endogenous, interferon-alpha-induced protein in Raji cells, co-localizing with the ER-resident marker PDI; the curator's IDA is kept under rule 2, since immunofluorescence cannot resolve membrane from lumen. ORGANELLES ER check (2026-11-05). The displayed UniProt sequence is isoform 2 (Q9H0P0-4), whose 50-residue N-terminal extension (absent from isoforms 1, 3 and 4) begins with a hydrophobic stretch (about residues 14-32, VGAVASASVCALVAGVVLA) followed by basic residues; UniProt annotates no TM or signal anchor there. UniProt restricts the ER location to isoform 2 and ties the interferon-alpha induction of p36 to it (ECO:0000269, PMID:8557639), although its isoform list gives p36 as a synonym of isoform 3, which lacks the extension; which isoform p36 is remains unsettled. The ER evidence is endogenous, interferon-induced p36 co-localizing with the ER-resident marker PDI by immunofluorescence (PMID:8557639, abstract only), plus HPA antibody staining. That is a real, minor, isoform-restricted ER pool, so the root claim holds; the characterized pyrimidine 5'-nucleotidase activity is cytosolic, so the row is KEEP_AS_NON_CORE. No ER membrane row is added: whole-organelle co-staining cannot resolve membrane from lumen, the hydrophobic N-terminal segment is an unannotated prediction, and the isoform behind the ER signal is uncertain, so only one line points at the membrane. Supporting Evidence: PMID:8557639 p36 is a new interferon-alpha-induced protein that localizes PMID:8557639 by immunofluorescence microscopy to co-localize with the endoplasmic reticulum resident protein, protein disulfide isomerase |
| GO:0006213 pyrimidine nucleoside metabolic process | NAS PMID:10942414 Human erythrocyte pyrimidine 5-nucleotidase, PN-I, is identi... | KEEP AS NON CORE | Summary: Author statement placing the enzyme in pyrimidine nucleoside/nucleotide metabolism. The term is correct in spirit but general; the enzyme's role is more precisely captured by the specific pyrimidine nucleotide catabolic-process terms (UMP/CMP catabolic process). Keep as a valid but non-core, broader-than-necessary process annotation. Supporting Evidence: PMID:10942414 Erythrocyte maturation is accompanied by RNA degradation |
| GO:0008253 5'-nucleotidase activity | IDA PMID:10942414 Human erythrocyte pyrimidine 5-nucleotidase, PN-I, is identi... | ACCEPT | Summary: Direct assay of recombinant PN-I (shown to be identical to p36/NT5C3A) demonstrating a fully active pyrimidine 5'-nucleotidase. Core molecular function. Accept. Supporting Evidence: PMID:10942414 expressed in Escherichia coli, yielding a fully active |
| GO:0008253 5'-nucleotidase activity | NAS PMID:12930399 Molecular characterization of six unrelated Italian patients... | ACCEPT | Summary: Author statement of pyrimidine 5'-nucleotidase activity in the context of P5ND patient characterization. Correct core molecular function. Accept. Supporting Evidence: PMID:12930399 accumulation of pyrimidine nucleotides within the erythrocytes |
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