RNA cytosine-5 methyltransferase, the human orthologue of yeast Trm4, and the enzyme responsible for most 5-methylcytosine in human RNA. NSUN2 uses S-adenosyl-L-methionine and a catalytic cysteine to methylate cytosines in transfer RNA: the wobble base C34 of intron-containing pre-tRNA-Leu(CAA), and C47-C50 in the variable loop of many cytoplasmic tRNAs. It is imported into the mitochondrial matrix, where it makes m5C at positions 48-50 of several mitochondrial tRNAs, and it is the sole enzyme that methylates C69 of the vault RNA VTRNA1.1, thereby directing its processing into small vault RNAs. The methylation it deposits protects tRNAs from stress-induced endonucleolytic cleavage, and loss of the enzyme alters translation, tRNA-fragment production and the differentiation of skin, hair follicle and neural stem cells; biallelic loss-of-function variants cause autosomal-recessive intellectual disability and a Dubowitz-like syndrome. NSUN2 is predominantly nuclear and nucleolar, with cytoplasmic and mitochondrial pools, and it has been detected in secreted exosomes. NSUN2 also methylates messenger RNA, and m5C in mRNA has been linked to nuclear export via ALYREF and to transcript stabilisation via YBX1; this substrate class is genuinely contested, because the mapping methods used to locate mRNA m5C sites - bisulfite sequencing, Aza-IP and miCLIP - agree poorly with one another, mRNA structure generates false-positive bisulfite signals, and the modification occurs at roughly one in several thousand cytosines, which leaves it unclear how the proposed reader proteins discriminate modified transcripts.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0009383 rRNA (cytosine-C5-)-methyltransferase activity | IBA GO_REF:0000033 | MARK AS OVER ANNOTATED | Summary: Phylogenetic transfer of rRNA C5 methyltransferase activity across the NSUN family node. Reason: The only donor named in the GOA WITH/FROM column besides the family node is mouse Nsun4 (MGI:MGI:1919431), whose characterised substrate is mitochondrial 12S rRNA. NSUN2 has a well-mapped and quite different substrate repertoire - cytoplasmic pre-tRNA C34, tRNA and mt-tRNA C47-C50, vault RNA C69, and mRNA - and no rRNA substrate has been reported for it in twenty years of work on the enzyme. This looks like a paralogue-specific function propagated too broadly rather than a genuine ancestral activity retained by NSUN2. Not removed outright, since the PAINT tree itself was not inspected and an ancestral rRNA-methylating activity for the family is plausible; marked as over-annotation of this particular gene. Propagation Review Root cause: PROPAGATION BAD Failure modes: WRONG ORTHOLOG OR PARALOG FUNCTIONAL DIVERGENCE Sources checked: MGI:MGI:1919431 · Nsun4 SUPPORTS SOURCE BUT NOT TARGET Donor named in the GOA WITH/FROM column is mouse Nsun4, the mitochondrial 12S rRNA m5C methyltransferase that partners MTERF4 in mitoribosomal large subunit assembly. That is a paralogue-specific substrate and complex; NSUN2 has no reported rRNA substrate. PANTHER:PTN000516076 · PTN000516076 UNRESOLVED Family-wide NSUN node. The PAINT tree was not inspected for this review, so no claim is made about where the IBD was placed; the objection rests only on the donor gene identity and on NSUN2-specific substrate data. |
| GO:0016428 tRNA (cytidine-N5)-methyltransferase activity | IBA GO_REF:0000033 | ACCEPT | Summary: Phylogenetic assertion that NSUN2 inherits the Trm4-type tRNA m5C methyltransferase activity of its family. Reason: Correct and core. The donor list spans mouse, fission yeast (two genes), budding yeast and C. elegans as well as NSUN2 itself, which is the expected pattern for a deeply conserved function: NSUN2 appearing in its own WITH/FROM simply reflects that its experimental annotation was one of the descendant evidences used to place the ancestral node, and the IBA adds the further statement that the activity is inherited rather than human-specific. Supporting Evidence: PMID:17071714 We identified a human orthologue of tRNA:m5C methyltransferase from Saccharomyces cerevisiae, which has been previously shown to catalyse the specific modification of C34 in the intron-containing yeast pre-tRNA Leu (CAA). |
| GO:0002127 tRNA wobble base cytosine methylation | IBA GO_REF:0000033 | ACCEPT | Summary: Methylation of the wobble cytosine C34 of tRNA, inherited from the Trm4 ancestor. Reason: Directly matched by human experimental work: m5C34 is introduced into intron-containing pre-tRNA-Leu(CAA) in HeLa extract, and the human enzyme complements a yeast trm4 deletion. Supporting Evidence: PMID:17071714 Using transcripts of intron-less and intron-containing human pre-tRNA Leu (CAA) genes as substrates, we have shown that m5C34 is introduced only in the intron-containing tRNA precursors when the substrates were incubated in the HeLa extract. |
| GO:0005634 nucleus | IBA GO_REF:0000033 | ACCEPT | Summary: NSUN2 is active in the nucleus, where pre-tRNA methylation occurs. Reason: Correct core location; NSUN2 is predominantly nuclear, with nucleolar concentration. Supporting Evidence: PMID:31287866 Although NSUN2 is predominantly localized to the nucleus and introduces m5C into cytoplasmic tRNAs and mRNAs, structured illumination microscopy clearly revealed NSUN2 foci inside mitochondria. |
| GO:0005737 cytoplasm | IBA GO_REF:0000033 | ACCEPT | Summary: NSUN2 is also active in the cytoplasm. Reason: Correct; a cytoplasmic pool is seen by immunofluorescence and NSUN2 methylates cytoplasmic tRNAs. |
| GO:0006364 rRNA processing | IBA GO_REF:0000033 | MARK AS OVER ANNOTATED | Summary: rRNA processing propagated across the NSUN family node. Reason: Same propagation issue as GO:0009383: the named donor is mouse Nsun4, whose role is in mitochondrial ribosome biogenesis. NSUN2 is nucleolar, so a role in ribosome biogenesis is not absurd, but no NSUN2-dependent rRNA processing defect has been demonstrated, and the assertion here is inherited from a paralogue rather than grounded in NSUN2 data. Propagation Review Root cause: PROPAGATION BAD Failure modes: WRONG ORTHOLOG OR PARALOG FUNCTIONAL DIVERGENCE Sources checked: MGI:MGI:1919431 · Nsun4 SUPPORTS SOURCE BUT NOT TARGET Donor named in the GOA WITH/FROM column is mouse Nsun4, the mitochondrial 12S rRNA m5C methyltransferase that partners MTERF4 in mitoribosomal large subunit assembly. That is a paralogue-specific substrate and complex; NSUN2 has no reported rRNA substrate. PANTHER:PTN000516076 · PTN000516076 UNRESOLVED Family-wide NSUN node. The PAINT tree was not inspected for this review, so no claim is made about where the IBD was placed; the objection rests only on the donor gene identity and on NSUN2-specific substrate data. |
| GO:1902775 mitochondrial large ribosomal subunit assembly | IBA GO_REF:0000033 | MARK AS OVER ANNOTATED | Summary: Mitochondrial large ribosomal subunit assembly propagated across the NSUN family node. Reason: This is the documented function of NSUN4, which forms a complex with MTERF4 and is required for mitoribosome large-subunit assembly; the GOA WITH/FROM column names mouse Nsun4 as the donor. NSUN2 is genuinely imported into the mitochondrial matrix, but its demonstrated job there is m5C at positions 48-50 of mt-tRNAs, and its loss does not have a profound effect on mitochondrial translation or oxidative phosphorylation in differentiated cells (PMID:31276587) - which is what one would expect to be perturbed if NSUN2 were needed for large-subunit assembly. Propagation Review Root cause: PROPAGATION BAD Failure modes: WRONG ORTHOLOG OR PARALOG FUNCTIONAL DIVERGENCE Sources checked: MGI:MGI:1919431 · Nsun4 SUPPORTS SOURCE BUT NOT TARGET Donor named in the GOA WITH/FROM column is mouse Nsun4, the mitochondrial 12S rRNA m5C methyltransferase that partners MTERF4 in mitoribosomal large subunit assembly. That is a paralogue-specific substrate and complex; NSUN2 has no reported rRNA substrate. PANTHER:PTN000516076 · PTN000516076 UNRESOLVED Family-wide NSUN node. The PAINT tree was not inspected for this review, so no claim is made about where the IBD was placed; the objection rests only on the donor gene identity and on NSUN2-specific substrate data. Supporting Evidence: PMID:31276587 Finally, we show that inactivation of NSUN2 does not have a profound effect on mitochondrial tRNA stability and oxidative phosphorylation in differentiated cells. |
| GO:0062152 mRNA (cytidine-5-)-methyltransferase activity | IBA GO_REF:0000033 | KEEP AS NON CORE | Summary: Phylogenetic assertion of mRNA m5C methyltransferase activity, from a narrower node (PTN002736915) than the tRNA assertion. Reason: GOA carries both sides of this question and both are retained. The catalytic assignment itself is supported by evidence that does not depend on the contested mapping assays: RNA-mediated activity-based protein profiling traps NSUN2 covalently on 5-fluorocytidine-containing RNA recovered by oligo-dT capture, and nucleoside LC-MS/MS of polyA-selected RNA shows an 82% fall in m5C in NSUN2 knockout cells (PMID:34556860); the mRNA export phenotype is rescued by wild-type but not methyltransferase-dead NSUN2 (PMID:28418038); and in vitro methylation of a defined p16 3'UTR fragment was reported (PMID:22395603). The activity is therefore not removed - and could not be, since this term also carries an EXP code whose full text has not been read here. What is disputed is the reliability and biological weight of the mRNA m5C maps. The methods disagree with one another (very low overlap between Aza-IP, bisulfite sequencing and miCLIP), mRNA secondary structure shields cytosines from bisulfite and generates false positives, and m5C occurs at roughly one in several thousand cytosines in mRNA, which leaves it unexplained how the proposed readers discriminate modified transcripts (PMID:42587746). The authors of the chemoproteomic study themselves note that polyA fractions carry ~40-fold lower m5C than total RNA and that contaminating tRNA/rRNA nucleosides could contribute to the measured signal. Demoted to non-core rather than accepted as core: the deeply conserved, structurally and genetically nailed-down substrate class for this Trm4 orthologue is tRNA (and vault RNA and mitochondrial tRNA), whereas the mRNA activity is quantitatively minor, its site maps are unresolved, and the large disease literature built on single mRNA m5C sites rarely supplies orthogonal validation. |
| GO:0005576 extracellular region | IEA GO_REF:0000044 | MARK AS OVER ANNOTATED | Summary: Extracellular region, mapped from the UniProt subcellular location line that records detection in secreted exosomes. Reason: Reads as though a nuclear and cytoplasmic RNA methyltransferase were a secreted protein. The underlying observation - NSUN2 in exosomes from HEK293 cells - is captured more precisely by GO:0070062 extracellular exosome, which this gene also carries. |
| GO:0005730 nucleolus | IEA GO_REF:0000044 | ACCEPT | Summary: Nucleolar localisation mapped from the UniProt subcellular location vocabulary. Reason: Consistent with the direct immunofluorescence localisation of the human enzyme. |
| GO:0005737 cytoplasm | IEA GO_REF:0000120 | ACCEPT | Summary: Cytoplasmic localisation from combined automatic methods. Reason: Correct; agrees with the IDA and EXP cytoplasm annotations on this gene. |
| GO:0005739 mitochondrion | IEA GO_REF:0000044 | ACCEPT | Summary: Mitochondrial localisation from the UniProt subcellular location vocabulary. Reason: Supported by two independent 2019 studies using proximity labelling and structured illumination microscopy, and by the mt-tRNA substrate. |
| GO:0005819 spindle | IEA GO_REF:0000044 | KEEP AS NON CORE | Summary: Spindle localisation from the UniProt subcellular location vocabulary. Reason: NSUN2 has a reported mitotic spindle association, but this is not where the characterised RNA methylation chemistry happens and no spindle-associated substrate has been defined. Retained as a peripheral localisation. |
| GO:0006397 mRNA processing | IEA GO_REF:0000117 | MODIFY | Summary: GO:0006397 mRNA processing covers conversion of a primary transcript into mature mRNA - splicing, capping, polyadenylation. The cited work reports mRNA stabilisation and nuclear export instead. Reason: A term-choice problem rather than an evidence problem, so the annotations are modified rather than removed. PMID:22395603 reports that NSUN2 inhibits turnover of p16 mRNA, PMID:31358969 that NSUN2 with YBX1 stabilises HDGF mRNA, and PMID:28418038 that NSUN2-dependent m5C promotes ALYREF-mediated nuclear export. None of these is a pre-mRNA maturation step. The accurate terms are mRNA stabilization (GO:0048255) and regulation of mRNA export from nucleus (GO:0010793), the latter of which this gene already carries from PMID:28418038. Proposed replacements: mRNA stabilization regulation of mRNA export from nucleus |
| GO:0008168 methyltransferase activity | IEA GO_REF:0000002 | MODIFY | Summary: Generic methyltransferase activity from the InterPro family signature. Reason: Correct but far too general for a gene whose exact reaction, substrate and modified positions are established. The specific child is already annotated from experiment. Proposed replacements: tRNA (cytidine-N5)-methyltransferase activity |
| GO:0008173 RNA methyltransferase activity | IEA GO_REF:0000002 | MODIFY | Summary: RNA methyltransferase activity from the InterPro family signature. Reason: Correct but under-specific; replaced by the established reaction-level child. Note that for NSUN2, unlike its pseudoenzyme paralogue NSUN7, the family signature does correspond to a real catalytic activity. Proposed replacements: tRNA (cytidine-N5)-methyltransferase activity |
| GO:0016428 tRNA (cytidine-N5)-methyltransferase activity | IEA GO_REF:0000120 | ACCEPT | Summary: tRNA m5C methyltransferase activity from combined automatic methods. Reason: Agrees with the experimental annotations; core molecular function. |
| GO:0030488 tRNA methylation | IEA GO_REF:0000117 | ACCEPT | Summary: tRNA methylation, from an ARBA machine-learning model. Reason: Correct core process, independently supported by IDA on this gene. |
| GO:0062152 mRNA (cytidine-5-)-methyltransferase activity | IEA GO_REF:0000120 | KEEP AS NON CORE | Summary: mRNA m5C methyltransferase activity from combined automatic methods. Reason: GOA carries both sides of this question and both are retained. The catalytic assignment itself is supported by evidence that does not depend on the contested mapping assays: RNA-mediated activity-based protein profiling traps NSUN2 covalently on 5-fluorocytidine-containing RNA recovered by oligo-dT capture, and nucleoside LC-MS/MS of polyA-selected RNA shows an 82% fall in m5C in NSUN2 knockout cells (PMID:34556860); the mRNA export phenotype is rescued by wild-type but not methyltransferase-dead NSUN2 (PMID:28418038); and in vitro methylation of a defined p16 3'UTR fragment was reported (PMID:22395603). The activity is therefore not removed - and could not be, since this term also carries an EXP code whose full text has not been read here. What is disputed is the reliability and biological weight of the mRNA m5C maps. The methods disagree with one another (very low overlap between Aza-IP, bisulfite sequencing and miCLIP), mRNA secondary structure shields cytosines from bisulfite and generates false positives, and m5C occurs at roughly one in several thousand cytosines in mRNA, which leaves it unexplained how the proposed readers discriminate modified transcripts (PMID:42587746). The authors of the chemoproteomic study themselves note that polyA fractions carry ~40-fold lower m5C than total RNA and that contaminating tRNA/rRNA nucleosides could contribute to the measured signal. Demoted to non-core rather than accepted as core: the deeply conserved, structurally and genetically nailed-down substrate class for this Trm4 orthologue is tRNA (and vault RNA and mitochondrial tRNA), whereas the mRNA activity is quantitatively minor, its site maps are unresolved, and the large disease literature built on single mRNA m5C sites rarely supplies orthogonal validation. |
| GO:0005515 protein binding | IPI PMID:33961781 Dual proteome-scale networks reveal cell-specific remodeling... | REMOVE | Summary: Bare protein binding from a proteome-scale affinity-purification interactome survey. Reason: Per project curation guidance, protein binding carries no functional information and no partner is named by the term. Any biologically meaningful interaction of NSUN2 - for example with the m5C readers ALYREF and YBX1 - is better captured by the process terms for mRNA export and mRNA stability. No more informative molecular function is supportable from this evidence, so the row is removed rather than rewritten. Removal does not imply the reported interaction is false. |
| GO:0016428 tRNA (cytidine-N5)-methyltransferase activity | EXP PMID:17071714 Identification of human tRNA:m5C methyltransferase catalysin... | ACCEPT | Summary: Experimental demonstration that the human Trm4 orthologue forms m5C34 in intron-containing pre-tRNA-Leu(CAA). Reason: This is the founding evidence for the core molecular function, with substrate specificity defined at single-nucleotide resolution and cross-species complementation of a yeast trm4 deletion. Supporting Evidence: PMID:17071714 Using transcripts of intron-less and intron-containing human pre-tRNA Leu (CAA) genes as substrates, we have shown that m5C34 is introduced only in the intron-containing tRNA precursors when the substrates were incubated in the HeLa extract. |
| GO:0005737 cytoplasm | EXP PMID:31276587 NSUN2 introduces 5-methylcytosines in mammalian mitochondria... | ACCEPT | Summary: Experimental localisation of NSUN2 including a cytoplasmic pool, in the study that traced its import into mitochondria. Reason: Correct, if general. |
| GO:0062152 mRNA (cytidine-5-)-methyltransferase activity | EXP PMID:34556860 Activity-based RNA-modifying enzyme probing reveals DUS3L-me... | KEEP AS NON CORE | Summary: Chemoproteomic profiling that traps m5C methyltransferases covalently on 5-fluorocytidine-containing RNA and recovers NSUN2 from an oligo-dT-selected fraction, with an 82% loss of polyA m5C on knockout. Reason: GOA carries both sides of this question and both are retained. The catalytic assignment itself is supported by evidence that does not depend on the contested mapping assays: RNA-mediated activity-based protein profiling traps NSUN2 covalently on 5-fluorocytidine-containing RNA recovered by oligo-dT capture, and nucleoside LC-MS/MS of polyA-selected RNA shows an 82% fall in m5C in NSUN2 knockout cells (PMID:34556860); the mRNA export phenotype is rescued by wild-type but not methyltransferase-dead NSUN2 (PMID:28418038); and in vitro methylation of a defined p16 3'UTR fragment was reported (PMID:22395603). The activity is therefore not removed - and could not be, since this term also carries an EXP code whose full text has not been read here. What is disputed is the reliability and biological weight of the mRNA m5C maps. The methods disagree with one another (very low overlap between Aza-IP, bisulfite sequencing and miCLIP), mRNA secondary structure shields cytosines from bisulfite and generates false positives, and m5C occurs at roughly one in several thousand cytosines in mRNA, which leaves it unexplained how the proposed readers discriminate modified transcripts (PMID:42587746). The authors of the chemoproteomic study themselves note that polyA fractions carry ~40-fold lower m5C than total RNA and that contaminating tRNA/rRNA nucleosides could contribute to the measured signal. Demoted to non-core rather than accepted as core: the deeply conserved, structurally and genetically nailed-down substrate class for this Trm4 orthologue is tRNA (and vault RNA and mitochondrial tRNA), whereas the mRNA activity is quantitatively minor, its site maps are unresolved, and the large disease literature built on single mRNA m5C sites rarely supplies orthogonal validation. Supporting Evidence: PMID:34556860 Consistent with previous RNA bisulfite sequencing and m5C mass spectrometry that have implicated NSUN2 as the major mRNA m5C-forming enzyme16, 17, we found an 82% reduction in mRNA m5C levels upon NSUN2 knockout PMID:34556860 We have employed a rigorous polyA mRNA purification, however since m5C is ~40-fold higher in total RNA than polyA-RNA, and comparably reduced in each sample upon NSUN2 KO (Figure 3b and 3c), we cannot exclude the possibility that contaminating tRNA/rRNA nucleosides may contribute to the measured m5C content. |
| GO:0005739 mitochondrion | HTP PMID:34800366 Quantitative high-confidence human mitochondrial proteome an... | ACCEPT | Summary: Presence in a high-confidence quantitative human mitochondrial proteome. Reason: Consistent with the two dedicated 2019 studies showing matrix import and mt-tRNA methylation; a high-throughput code here corroborates rather than substitutes for them. |
| GO:0006397 mRNA processing | IDA PMID:22395603 The tRNA methyltransferase NSun2 stabilizes p16INK⁴ mRNA by ... | MODIFY | Summary: GO:0006397 mRNA processing covers conversion of a primary transcript into mature mRNA - splicing, capping, polyadenylation. The cited work reports mRNA stabilisation and nuclear export instead. Reason: A term-choice problem rather than an evidence problem, so the annotations are modified rather than removed. PMID:22395603 reports that NSUN2 inhibits turnover of p16 mRNA, PMID:31358969 that NSUN2 with YBX1 stabilises HDGF mRNA, and PMID:28418038 that NSUN2-dependent m5C promotes ALYREF-mediated nuclear export. None of these is a pre-mRNA maturation step. The accurate terms are mRNA stabilization (GO:0048255) and regulation of mRNA export from nucleus (GO:0010793), the latter of which this gene already carries from PMID:28418038. Proposed replacements: mRNA stabilization regulation of mRNA export from nucleus |
| GO:0006397 mRNA processing | IDA PMID:28418038 5-methylcytosine promotes mRNA export - NSUN2 as the methylt... | MODIFY | Summary: GO:0006397 mRNA processing covers conversion of a primary transcript into mature mRNA - splicing, capping, polyadenylation. The cited work reports mRNA stabilisation and nuclear export instead. Reason: A term-choice problem rather than an evidence problem, so the annotations are modified rather than removed. PMID:22395603 reports that NSUN2 inhibits turnover of p16 mRNA, PMID:31358969 that NSUN2 with YBX1 stabilises HDGF mRNA, and PMID:28418038 that NSUN2-dependent m5C promotes ALYREF-mediated nuclear export. None of these is a pre-mRNA maturation step. The accurate terms are mRNA stabilization (GO:0048255) and regulation of mRNA export from nucleus (GO:0010793), the latter of which this gene already carries from PMID:28418038. Proposed replacements: mRNA stabilization regulation of mRNA export from nucleus |
| GO:0006397 mRNA processing | IDA PMID:31358969 5-methylcytosine promotes pathogenesis of bladder cancer thr... | MODIFY | Summary: GO:0006397 mRNA processing covers conversion of a primary transcript into mature mRNA - splicing, capping, polyadenylation. The cited work reports mRNA stabilisation and nuclear export instead. Reason: A term-choice problem rather than an evidence problem, so the annotations are modified rather than removed. PMID:22395603 reports that NSUN2 inhibits turnover of p16 mRNA, PMID:31358969 that NSUN2 with YBX1 stabilises HDGF mRNA, and PMID:28418038 that NSUN2-dependent m5C promotes ALYREF-mediated nuclear export. None of these is a pre-mRNA maturation step. The accurate terms are mRNA stabilization (GO:0048255) and regulation of mRNA export from nucleus (GO:0010793), the latter of which this gene already carries from PMID:28418038. Proposed replacements: mRNA stabilization regulation of mRNA export from nucleus |
| GO:0006400 tRNA modification | TAS Reactome:R-HSA-6785438 | ACCEPT | Summary: Reactome statement that NSUN2 methylates C40 and C48-C50 of tRNA-Gly(GCC). Reason: Correct, and the specific child GO:0030488 tRNA methylation is also annotated to this gene. |
| GO:0016428 tRNA (cytidine-N5)-methyltransferase activity | IDA PMID:34556860 Activity-based RNA-modifying enzyme probing reveals DUS3L-me... | ACCEPT | Summary: Mechanism-based covalent capture of NSUN2 on 5-fluorocytidine RNA, confirming the catalytic cysteine chemistry; NSUN2 knockout removes 76% of m5C from total RNA. Reason: Core molecular function, supported by an assay that depends on catalysis. Supporting Evidence: PMID:34556860 We also found that NSUN2 installs the majority of m5C sites on total RNA (76% reduction upon NSUN2 KO) (Figure 3b), likely reflective of abundant tRNA m5C sites |
| GO:0005739 mitochondrion | IDA PMID:31276587 NSUN2 introduces 5-methylcytosines in mammalian mitochondria... | ACCEPT | Summary: Proximity labelling and immunodetection place NSUN2 in the mitochondrial matrix. Reason: Well-supported second location where NSUN2 methylates mt-tRNAs. Supporting Evidence: PMID:31276587 Here we employ spatially restricted proximity labelling and immunodetection to demonstrate that NSUN2 is imported into the matrix of mammalian mitochondria. |
| GO:0005739 mitochondrion | IDA PMID:31287866 Mammalian NSUN2 introduces 5-methylcytidines into mitochondr... | ACCEPT | Summary: Structured illumination microscopy shows NSUN2 foci inside mitochondria. Reason: Independent confirmation of the mitochondrial pool, from a different laboratory in the same year. Supporting Evidence: PMID:31287866 Although NSUN2 is predominantly localized to the nucleus and introduces m5C into cytoplasmic tRNAs and mRNAs, structured illumination microscopy clearly revealed NSUN2 foci inside mitochondria. |
| GO:0010793 regulation of mRNA export from nucleus | IDA PMID:28418038 5-methylcytosine promotes mRNA export - NSUN2 as the methylt... | KEEP AS NON CORE | Summary: NSUN2-deposited m5C is read by ALYREF and promotes nuclear export of mRNA; the defect caused by NSUN2 depletion is rescued by wild-type but not catalytically dead NSUN2. Reason: The catalytically dead rescue is the right control and makes this one of the better-supported mRNA m5C phenotypes, so the annotation is kept. It is placed as non-core for the same reason as the mRNA methyltransferase activity itself: it is downstream of a quantitatively minor substrate class whose site maps remain contested, and a reader operating at roughly one m5C per several thousand cytosines is mechanistically unexplained (PMID:42587746). The core of this gene is tRNA modification. Supporting Evidence: PMID:28418038 Dysregulation of ALYREF-mediated mRNA export upon NSUN2 depletion could be restored by reconstitution of wild-type but not methyltransferase-defective NSUN2. |
| GO:0016428 tRNA (cytidine-N5)-methyltransferase activity | IDA PMID:31287866 Mammalian NSUN2 introduces 5-methylcytidines into mitochondr... | ACCEPT | Summary: m5C at positions 48-50 of mitochondrial tRNAs reconstituted in vitro with purified NSUN2 and SAM, and absent from Nsun2 knockout mouse and human cells. Reason: The strongest single piece of evidence for the core molecular function: a defined enzyme, a defined substrate, mapped product positions, and loss of the product on gene knockout. Supporting Evidence: PMID:31287866 In addition, we successfully reconstituted m5C at positions 48-50 of mt-tRNA in vitro with NSUN2 protein in the presence of S-adenosylmethionine. |
| GO:0030488 tRNA methylation | IDA PMID:31287866 Mammalian NSUN2 introduces 5-methylcytidines into mitochondr... | ACCEPT | Summary: NSUN2-dependent m5C mapped by mass spectrometry in eight mouse and six human mt-tRNAs. Reason: Core biological process. Supporting Evidence: PMID:31287866 In this study, we took advantage of mass spectrometric analysis to map 5-methylcytidine (m5C) at positions 48-50 in eight mouse and six human mt-tRNAs. |
| GO:0036416 tRNA stabilization | ISS GO_REF:0000024 | ACCEPT | Summary: m5C deposited by NSUN2 protects tRNAs from stress-induced endonucleolytic cleavage. Reason: A direct consequence of the core catalytic activity rather than a separate role, and consistent with the loss of C47/C48 methylation on tRNA-Asp in NSUN2-null patient cells. |
| GO:0048820 hair follicle maturation | ISS GO_REF:0000024 | KEEP AS NON CORE | Summary: Hair follicle maturation, transferred from the mouse Nsun2 knockout skin phenotype. Reason: A real organismal phenotype, but a distal consequence of tRNA hypomodification in a particular stem-cell compartment rather than an activity of the protein. |
| GO:0062152 mRNA (cytidine-5-)-methyltransferase activity | IDA PMID:22395603 The tRNA methyltransferase NSun2 stabilizes p16INK⁴ mRNA by ... | KEEP AS NON CORE | Summary: In vitro methylation of the p16 3'UTR by NSun2, with knockdown shortening and overexpression lengthening the p16 mRNA half-life. Reason: GOA carries both sides of this question and both are retained. The catalytic assignment itself is supported by evidence that does not depend on the contested mapping assays: RNA-mediated activity-based protein profiling traps NSUN2 covalently on 5-fluorocytidine-containing RNA recovered by oligo-dT capture, and nucleoside LC-MS/MS of polyA-selected RNA shows an 82% fall in m5C in NSUN2 knockout cells (PMID:34556860); the mRNA export phenotype is rescued by wild-type but not methyltransferase-dead NSUN2 (PMID:28418038); and in vitro methylation of a defined p16 3'UTR fragment was reported (PMID:22395603). The activity is therefore not removed - and could not be, since this term also carries an EXP code whose full text has not been read here. What is disputed is the reliability and biological weight of the mRNA m5C maps. The methods disagree with one another (very low overlap between Aza-IP, bisulfite sequencing and miCLIP), mRNA secondary structure shields cytosines from bisulfite and generates false positives, and m5C occurs at roughly one in several thousand cytosines in mRNA, which leaves it unexplained how the proposed readers discriminate modified transcripts (PMID:42587746). The authors of the chemoproteomic study themselves note that polyA fractions carry ~40-fold lower m5C than total RNA and that contaminating tRNA/rRNA nucleosides could contribute to the measured signal. Demoted to non-core rather than accepted as core: the deeply conserved, structurally and genetically nailed-down substrate class for this Trm4 orthologue is tRNA (and vault RNA and mitochondrial tRNA), whereas the mRNA activity is quantitatively minor, its site maps are unresolved, and the large disease literature built on single mRNA m5C sites rarely supplies orthogonal validation. Supporting Evidence: PMID:22395603 In vitro methylation assays show that NSun2 methylates the p16 3'UTR at A988. |
| GO:0062152 mRNA (cytidine-5-)-methyltransferase activity | IDA PMID:28418038 5-methylcytosine promotes mRNA export - NSUN2 as the methylt... | KEEP AS NON CORE | Summary: NSUN2 identified as the main enzyme forming m5C in mRNA, with transcriptome-wide maps and a catalytically dead rescue control. Reason: GOA carries both sides of this question and both are retained. The catalytic assignment itself is supported by evidence that does not depend on the contested mapping assays: RNA-mediated activity-based protein profiling traps NSUN2 covalently on 5-fluorocytidine-containing RNA recovered by oligo-dT capture, and nucleoside LC-MS/MS of polyA-selected RNA shows an 82% fall in m5C in NSUN2 knockout cells (PMID:34556860); the mRNA export phenotype is rescued by wild-type but not methyltransferase-dead NSUN2 (PMID:28418038); and in vitro methylation of a defined p16 3'UTR fragment was reported (PMID:22395603). The activity is therefore not removed - and could not be, since this term also carries an EXP code whose full text has not been read here. What is disputed is the reliability and biological weight of the mRNA m5C maps. The methods disagree with one another (very low overlap between Aza-IP, bisulfite sequencing and miCLIP), mRNA secondary structure shields cytosines from bisulfite and generates false positives, and m5C occurs at roughly one in several thousand cytosines in mRNA, which leaves it unexplained how the proposed readers discriminate modified transcripts (PMID:42587746). The authors of the chemoproteomic study themselves note that polyA fractions carry ~40-fold lower m5C than total RNA and that contaminating tRNA/rRNA nucleosides could contribute to the measured signal. Demoted to non-core rather than accepted as core: the deeply conserved, structurally and genetically nailed-down substrate class for this Trm4 orthologue is tRNA (and vault RNA and mitochondrial tRNA), whereas the mRNA activity is quantitatively minor, its site maps are unresolved, and the large disease literature built on single mRNA m5C sites rarely supplies orthogonal validation. Supporting Evidence: PMID:28418038 Moreover, m5C formation in mRNAs is mainly catalyzed by the RNA methyltransferase NSUN2, and m5C is specifically recognized by the mRNA export adaptor ALYREF as shown by in vitro and in vivo studies. |
| GO:0062152 mRNA (cytidine-5-)-methyltransferase activity | IDA PMID:31358969 5-methylcytosine promotes pathogenesis of bladder cancer thr... | KEEP AS NON CORE | Summary: Single-nucleotide-resolution mRNA m5C landscape in bladder cancer, with NSUN2 as writer and YBX1 as reader on the HDGF 3'UTR. Reason: GOA carries both sides of this question and both are retained. The catalytic assignment itself is supported by evidence that does not depend on the contested mapping assays: RNA-mediated activity-based protein profiling traps NSUN2 covalently on 5-fluorocytidine-containing RNA recovered by oligo-dT capture, and nucleoside LC-MS/MS of polyA-selected RNA shows an 82% fall in m5C in NSUN2 knockout cells (PMID:34556860); the mRNA export phenotype is rescued by wild-type but not methyltransferase-dead NSUN2 (PMID:28418038); and in vitro methylation of a defined p16 3'UTR fragment was reported (PMID:22395603). The activity is therefore not removed - and could not be, since this term also carries an EXP code whose full text has not been read here. What is disputed is the reliability and biological weight of the mRNA m5C maps. The methods disagree with one another (very low overlap between Aza-IP, bisulfite sequencing and miCLIP), mRNA secondary structure shields cytosines from bisulfite and generates false positives, and m5C occurs at roughly one in several thousand cytosines in mRNA, which leaves it unexplained how the proposed readers discriminate modified transcripts (PMID:42587746). The authors of the chemoproteomic study themselves note that polyA fractions carry ~40-fold lower m5C than total RNA and that contaminating tRNA/rRNA nucleosides could contribute to the measured signal. Demoted to non-core rather than accepted as core: the deeply conserved, structurally and genetically nailed-down substrate class for this Trm4 orthologue is tRNA (and vault RNA and mitochondrial tRNA), whereas the mRNA activity is quantitatively minor, its site maps are unresolved, and the large disease literature built on single mRNA m5C sites rarely supplies orthogonal validation. Supporting Evidence: PMID:31358969 Moreover, NSUN2 and YBX1 are demonstrated to drive UCB pathogenesis by targeting the m5C methylation site in the HDGF 3' untranslated region. |
| GO:0070062 extracellular exosome | IDA PMID:28341602 Cytosolic YB-1 and NSUN2 are the only proteins recognizing s... | KEEP AS NON CORE | Summary: NSUN2 detected in exosomes secreted by HEK293 cells, where it binds a specific RNA motif. Reason: The observation is real but peripheral; it says where some of the protein ends up, not what the enzyme does. The accompanying motif-binding result is better expressed as RNA binding, which this gene already carries. Supporting Evidence: PMID:28341602 Both these proteins are found in exosomes secreted by HEK293 cells. |
| GO:2000736 regulation of stem cell differentiation | ISS GO_REF:0000024 | KEEP AS NON CORE | Summary: Regulation of stem cell differentiation, transferred from mouse. Reason: Genuine but downstream: NSUN2 loss perturbs differentiation through altered tRNA methylation, translation and stress-induced tRNA fragments, not through a dedicated regulatory activity. |
| GO:0005654 nucleoplasm | TAS Reactome:R-HSA-6785438 | ACCEPT | Summary: Nucleoplasmic location asserted by the Reactome tRNA modification reaction. Reason: Consistent with the nuclear localisation of the enzyme and of pre-tRNA methylation. |
| GO:0003723 RNA binding | HDA PMID:22658674 Insights into RNA biology from an atlas of mammalian mRNA-bi... | ACCEPT | Summary: Recovery of NSUN2 in mRNA interactome capture experiments. Reason: Unlike bare protein binding, RNA binding is informative here: it is the substrate-recognition function of an RNA-modifying enzyme, and NSUN2 also binds RNA in contexts where it is not obviously catalysing, such as the exosomal motif recognition reported in PMID:28341602. |
| GO:0003723 RNA binding | HDA PMID:22681889 The mRNA-bound proteome and its global occupancy profile on ... | ACCEPT | Summary: Recovery of NSUN2 in mRNA interactome capture experiments. Reason: Unlike bare protein binding, RNA binding is informative here: it is the substrate-recognition function of an RNA-modifying enzyme, and NSUN2 also binds RNA in contexts where it is not obviously catalysing, such as the exosomal motif recognition reported in PMID:28341602. |
| GO:0005730 nucleolus | IDA PMID:17071714 Identification of human tRNA:m5C methyltransferase catalysin... | ACCEPT | Summary: Immunolocalisation of the human Trm4 orthologue to the nucleolus. Reason: Correct and specific primary location. |
| GO:0005737 cytoplasm | IDA PMID:17071714 Identification of human tRNA:m5C methyltransferase catalysin... | ACCEPT | Summary: A cytoplasmic pool of the enzyme was also seen. Reason: Correct, if general. |
| GO:0016428 tRNA (cytidine-N5)-methyltransferase activity | IDA PMID:17071714 Identification of human tRNA:m5C methyltransferase catalysin... | ACCEPT | Summary: Direct assay of m5C34 formation on human pre-tRNA-Leu(CAA). Reason: Core molecular function. |
| GO:0030488 tRNA methylation | IDA PMID:17071714 Identification of human tRNA:m5C methyltransferase catalysin... | ACCEPT | Summary: tRNA methylation demonstrated directly for the human enzyme. Reason: Core biological process. |
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Download this section (compressed HTML)Q: Is NSUN2-dependent m5C in mRNA a physiologically significant modification in its own right, or largely the low-level off-target activity of an enzyme whose real substrate is tRNA? The nucleoside mass-spectrometry evidence for NSUN2-dependent m5C in polyA RNA is real, but polyA fractions carry about forty-fold less m5C than total RNA and the authors of that study cannot exclude contaminating tRNA and rRNA nucleosides. What level of polyA purity, or what orthogonal single-molecule method, would settle this?
Suggested experts: Ralph E Kleiner, Michaela Frye
Q: Which of the thousands of reported mRNA m5C sites survive a method-agnostic test? Aza-IP, bisulfite sequencing and miCLIP overlap very poorly, and mRNA secondary structure produces false-positive bisulfite signals. Is there a consensus high-confidence site set for NSUN2, and what is its size?
Suggested experts: Alexey A Malygin
Q: If m5C occurs at roughly one in several thousand cytosines in mRNA, and ALYREF and YBX1 bind m5C-containing regions only a few fold better than unmodified ones, how can a reader-based mechanism achieve specificity in vivo? Does the reader model require additional co-recruitment, or should the phenotypes attributed to single mRNA m5C sites be re-examined?
Suggested experts: Alexey A Malygin, Yun-Gui Yang
Q: Does NSUN2 have any role in ribosomal RNA or in mitochondrial ribosome assembly, as its IBA annotations assert? These were propagated from mouse Nsun4, a paralogue with a distinct substrate and complex, and NSUN2 loss does not appreciably impair mitochondrial translation. Should the PAINT node for these terms be moved?
Q: How much of the NSUN2 loss-of-function phenotype in human intellectual disability is attributable to tRNA hypomodification and stress-induced tRNA fragments, and how much, if any, to loss of mRNA or vault RNA methylation? A separation-of-function allele would be the cleanest test.
Suggested experts: Michaela Frye, Joseph G Gleeson
Experiment: Apply direct RNA nanopore sequencing, or another method that does not require chemical conversion or antibody enrichment, to matched wild-type and NSUN2 knockout human cells, with a catalytically dead NSUN2 add-back as a third arm. Intersect the resulting site calls with published bisulfite, Aza-IP and miCLIP sets and report the size of the reproducible core. Include synthetic spike-ins of known modification stoichiometry to calibrate false-positive and false-negative rates.
Hypothesis: NSUN2-dependent m5C sites in mRNA are far fewer than current maps report, and only a small high-confidence subset is reproducible across methods.
Type: Direct RNA sequencing
Experiment: Quantify m5C by LC-MS/MS in polyA RNA purified to increasing stringency (successive oligo-dT rounds, then size selection, then depletion with tRNA- and rRNA-specific probes), measuring residual tRNA and rRNA in each fraction by qPCR, and plot m5C against measured contamination to extrapolate the true mRNA value.
Hypothesis: The mRNA m5C signal measured by nucleoside mass spectrometry in polyA fractions is partly contributed by contaminating tRNA and rRNA.
Type: Quantitative mass spectrometry
Experiment: For a small number of prominent claimed sites (for example HDGF and IL1B), introduce a silent or minimally disruptive C-to-U or C-to-A substitution at the endogenous locus by base editing, and test whether transcript stability and the downstream phenotype change as predicted, with NSUN2 knockout as a positive control. This replaces enzyme-level perturbation, which affects thousands of transcripts, with site-level perturbation.
Hypothesis: Individual mRNA m5C sites proposed to drive disease phenotypes are functional at that site.
Type: Base editing and phenotyping
Experiment: Screen NSUN2 variants, guided by the tRNA-bound structure, for alleles that retain tRNA C34 and C48-C50 methylation while losing mRNA methylation (or the reverse), validating each by nucleoside LC-MS/MS on separated RNA fractions. Introduce any separation-of-function allele into cells and mice and ask which arm accounts for the differentiation and neurodevelopmental phenotypes.
Hypothesis: The tRNA and mRNA functions of NSUN2 can be genetically separated.
Type: Structure-guided mutagenesis and mouse genetics
Experiment: Test directly for NSUN2-dependent rRNA m5C: map m5C in cytoplasmic and mitochondrial rRNA by mass spectrometry in wild-type and NSUN2 knockout cells, and assay mitoribosome assembly by sucrose-gradient profiling in the same cells, using NSUN4 knockout as the positive control.
Hypothesis: The rRNA-related IBA annotations on NSUN2 are paralogue leakage from NSUN4.
Type: Mass spectrometry and ribosome profiling
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