{"gene":"NSUN2","run_date":"2026-06-10T05:19:52","timeline":{"discoveries":[{"year":1999,"finding":"Yeast Trm4 (ortholog of NSUN2, encoded by ORF YBL024w) is an S-adenosyl-L-methionine-dependent tRNA:m5C-methyltransferase that methylates cytosine at positions 34, 40, 48, and 49 in multiple yeast tRNAs and pre-tRNAs; disruption of the gene eliminates all tRNA m5C methylation in yeast.","method":"Recombinant protein expression and purification, in vitro methylation assay with T7 tRNA transcripts, gene disruption in yeast","journal":"RNA (New York, N.Y.)","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro enzymatic reconstitution with purified protein + genetic KO confirming loss of all tRNA m5C; multiple orthogonal methods in one study","pmids":["10445884"],"is_preprint":false},{"year":2006,"finding":"Misu/NSUN2 is a direct transcriptional target of Myc; its nucleolar localization depends on RNA polymerase III transcripts; knockdown blocks Myc-induced keratinocyte proliferation and reduces nucleolar size; it is redistributed to cytoplasmic vesicles in G2 and to the mitotic spindle.","method":"RNAi knockdown in keratinocytes, immunofluorescence for localization, siRNA in xenograft tumor model, cell-cycle analysis","journal":"Current biology : CB","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean KD with defined proliferation phenotype + direct localization by imaging; single lab, multiple methods","pmids":["16713953"],"is_preprint":false},{"year":2007,"finding":"Aurora-B kinase phosphorylates NSUN2 at Ser139 during mitosis, inhibiting its methyltransferase activity and disrupting its association with nucleolar protein NPM1/nucleophosmin; Aurora-B inhibition restores NSUN2–NPM1 association even during mitosis.","method":"In vitro kinase assay, in vitro methylation assay, co-immunoprecipitation, phosphorylation-mimic and phospho-deficient NSUN2 mutants","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro kinase assay + in vitro methylation activity assay with phospho-mutants + Co-IP; single lab but multiple orthogonal methods","pmids":["17215513"],"is_preprint":false},{"year":2009,"finding":"Misu/NSUN2 translocates from nucleoli in interphase to the mitotic spindle as an RNA-protein complex containing 18S rRNA; its depletion causes multipolar spindles, chromosome missegregation, aneuploidy, and cell death; NSUN2 recruits NuSAP to the spindle for microtubule stabilization.","method":"Immunofluorescence live/fixed imaging, siRNA depletion, spindle assembly assay, RNA–protein complex characterization","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — clean KD with defined mitotic phenotype + direct localization imaging + NuSAP recruitment assay; single lab, multiple orthogonal methods","pmids":["19596847"],"is_preprint":false},{"year":2010,"finding":"Crystal structure of archaeal Trm4 (NSUN2 ortholog from Methanocaldococcus jannaschii, MJ0026) complexed with sinefungin (AdoMet analog) revealed a negatively charged binding pocket near helix α8 controlling AdoMet entry, and a large positively charged surface for tRNA binding; the structure rationalizes conserved residues essential for catalysis and stability.","method":"X-ray crystallography at 1.27 Å (apo) and 2.3 Å (sinefungin complex), in vitro methyltransferase activity assay","journal":"Journal of molecular biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure determination at high resolution with functional validation by in vitro activity assay; directly informs human NSUN2 mechanism","pmids":["20600111"],"is_preprint":false},{"year":2011,"finding":"NSun2/Misu is required to balance epidermal stem cell self-renewal and differentiation; depletion results in enhanced quiescence and aberrant lineage commitment, placing NSun2-mediated RNA methylation as a post-transcriptional regulator of stem cell fate in skin.","method":"Conditional depletion in mouse skin, immunofluorescence, stem cell functional assays","journal":"PLoS genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic KO with defined stem cell phenotype; single lab, multiple histological and functional readouts","pmids":["22144916"],"is_preprint":false},{"year":2012,"finding":"NSUN2 methylates the 3′-UTR of p16(INK4) mRNA at position A988 in vitro and in cells; this methylation stabilizes p16 mRNA by preventing association with HuR, AUF1, and Ago2/RISC, and prevents recruitment to processing bodies; NSun2 knockdown shortens p16 mRNA half-life.","method":"In vitro methylation assay, mRNA half-life measurement, chimeric reporter assay, RIP (HuR/AUF1/Ago2), siRNA knockdown","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro methylation reconstitution + reporter assay with methylation-site mutant + RIP; single lab but multiple orthogonal methods","pmids":["22395603"],"is_preprint":false},{"year":2012,"finding":"The human tRNA methyltransferase Misu/NSUN2 methylates position 48 in tRNA-Leu(CAA) (with or without intron) and positions 48, 49, and 50 in tRNA-Gly2(GCC) in vitro, demonstrating multisite specificity.","method":"Recombinant protein expression and purification in E. coli, in vitro methylation assay with multiple tRNA substrates","journal":"RNA biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro enzymatic assay with purified recombinant enzyme and multiple defined substrates; single lab but rigorous biochemical reconstitution","pmids":["22995836"],"is_preprint":false},{"year":2012,"finding":"Disruption of both Dnmt2 and NSun2 tRNA methyltransferases in mice causes synthetic lethality with underdeveloped phenotype and impaired differentiation; Dnmt2 and NSun2 have complementary, non-overlapping tRNA target-site specificities; loss of both reduces steady-state levels of unmethylated tRNAs and overall protein synthesis rates.","method":"Double-knockout mouse model, tRNA methylation analysis, protein synthesis rate measurement","journal":"Nature structural & molecular biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic epistasis (double-KO synthetic lethality) with biochemical tRNA methylation and protein synthesis readouts; single lab but multiple orthogonal methods on a clean genetic model","pmids":["22885326"],"is_preprint":false},{"year":2013,"finding":"NSun2 methylates cytosine-5 at six positions in vault noncoding RNAs; loss of NSun2-mediated m5C in vault RNAs causes their aberrant processing into Argonaute-associated small RNA fragments that function as microRNAs, as shown in patient cells lacking NSun2.","method":"miCLIP (individual-nucleotide-resolution CLIP), RNA bisulfite sequencing, Argonaute immunoprecipitation, patient cells","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 1 / Moderate — miCLIP + bisulfite sequencing for site identification + functional Ago-IP readout; single lab, multiple orthogonal methods","pmids":["23871666"],"is_preprint":false},{"year":2013,"finding":"NSun2 is a component of the chromatoid body in mouse testes; NSun2 knockout blocks meiotic progression at the pachytene stage; simultaneous deletion of NSun2 and Dnmt2 decreases abundance of specific methylated tRNAs and confirms non-redundant roles in spermatogonial cells and Sertoli cells.","method":"Knockout mice, immunofluorescence, tRNA methylation analysis, histology","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic KO with defined meiotic arrest phenotype + subcellular localization + tRNA methylation biochemistry; single lab","pmids":["23401851"],"is_preprint":false},{"year":2014,"finding":"NSun2 methylates primary (pri-miR-125b), precursor (pre-miR-125b), and mature miR-125b in vitro and in vivo; methylation inhibits processing of pri-miR-125b2 into pre-miR-125b2, decreases cleavage to mature miR-125, and attenuates RISC recruitment, thereby repressing miR-125b function.","method":"In vitro methylation assay, pri/pre-miRNA processing assay, RISC loading assay, in vivo methylation","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro methylation reconstitution + functional processing and RISC assays; single lab, multiple orthogonal methods","pmids":["25047833"],"is_preprint":false},{"year":2014,"finding":"Trm4 (yeast NSUN2 ortholog) and mammalian Nsun2 form covalent complexes with previously methylated (m5C-containing) RNA in a manner requiring S-adenosylhomocysteine (AdoHcy); four active-site residues critical for tRNA methylation are also required for this covalent complex formation; removal of AdoHcy disassembles the complexes.","method":"Biochemical covalent complex assay with recombinant proteins, active-site mutagenesis, metabolite titration experiments","journal":"Biochemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution of covalent enzyme-RNA complex with active-site mutagenesis; single lab but rigorous biochemistry","pmids":["25375641"],"is_preprint":false},{"year":2015,"finding":"NSun2 methylates the 5′-UTR of p27 mRNA at cytosine C64 in vitro and in cells, repressing p27 translation; NSun2 also enhances CDK1 mRNA translation through methylation; knockdown of NSun2 elevates p27, reduces CDK1, and accelerates replicative senescence.","method":"In vitro methylation assay, translation reporter assay, RNAi knockdown, protein level analysis","journal":"Aging","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro methylation reconstitution at defined cytosine + reporter translational assay + cellular KD/OE; single lab, multiple methods","pmids":["26687548"],"is_preprint":false},{"year":2015,"finding":"NSun2 methylates CDK1 mRNA in vitro and in cells; this methylation enhances CDK1 translation without altering CDK1 mRNA levels; NSun2-mediated CDK1 upregulation drives cell cycle progression.","method":"In vitro methylation assay, overexpression/knockdown, CDK1 translation assay (polysome analysis), cell cycle analysis","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro methylation + translational assay with KD/OE; single lab, multiple orthogonal methods","pmids":["26391950"],"is_preprint":false},{"year":2016,"finding":"NSun2 methylates ICAM-1 mRNA and promotes its translation, increasing leukocyte adhesion to endothelial cells; TNF-α or homocysteine activates NSun2 methyltransferase activity by suppressing Aurora-B-mediated phosphorylation of NSun2; NSun2-deficient rats show decreased ICAM-1 induction and impaired allograft arteriosclerosis.","method":"In vitro methylation assay, in-cell methylation assay, NSun2-knockout rat model, vascular inflammation assay","journal":"Circulation research","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro + in vivo methylation assay + genetic KO rat model + defined vascular phenotype; multiple orthogonal methods across in vitro and in vivo models","pmids":["26838785"],"is_preprint":false},{"year":2016,"finding":"NSun2 methylates SHC mRNA in vitro and in cells, enhancing translation of all three SHC proteins (p66SHC, p52SHC, p46SHC), which increases ROS and activates p38MAPK to accelerate oxidative stress- and high-glucose-induced HUVEC senescence.","method":"In vitro methylation assay, in-cell methylation, protein expression analysis, ROS measurement, p38MAPK activation assay","journal":"Oncotarget","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — in vitro methylation + translational effect measured; single lab, multiple methods but functional pathway placement relies on correlation","pmids":["26992231"],"is_preprint":false},{"year":2017,"finding":"NSUN2 is the principal RNA methyltransferase for m5C in mRNAs; m5C is enriched in CG-rich regions downstream of translation initiation sites; the mRNA export adaptor ALYREF specifically recognizes m5C-modified mRNA; NSUN2 modulates ALYREF nuclear-cytoplasmic shuttling and mRNA export; export defects upon NSUN2 depletion are rescued by wild-type but not methyltransferase-defective NSUN2.","method":"Transcriptome-wide m5C profiling (bisulfite-seq), in vitro and in vivo binding studies, nuclear-cytoplasmic fractionation, NSUN2 catalytic mutant rescue experiments, RNA export assays","journal":"Cell research","confidence":"High","confidence_rationale":"Tier 1 / Strong — transcriptome-wide mapping + in vitro binding + catalytic-dead mutant rescue + mRNA export functional assay; multiple orthogonal methods, widely replicated concept","pmids":["28418038"],"is_preprint":false},{"year":2017,"finding":"NSUN2 catalyzes m5C modification on p21 3′-UTR; METTL3/METTL14 catalyzes m6A on the same mRNA; m6A methylation by METTL3/METTL14 facilitates NSUN2-mediated m5C, and vice versa; combined m5C and m6A synergistically enhance p21 translation during oxidative stress-induced senescence.","method":"In vitro methylation assay, translation reporter assay, KD of NSUN2 and METTL3/METTL14, LC-MS/MS","journal":"Journal of cellular biochemistry","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — in vitro methylation + translational reporter assay; single lab, mutual dependency shown biochemically but mechanistic detail limited","pmids":["28247949"],"is_preprint":false},{"year":2017,"finding":"NSun2 methylates IL-17A mRNA at cytosine C466 in vitro and in vivo; this m5C methylation promotes IL-17A translation in T lymphocytes; homocysteine upregulates NSun2, driving IL-17A upregulation; NSun2-/- rats show markedly decreased plasma IL-17A.","method":"In vitro methylation assay, bisulfite sequencing, HPLC-MS, luciferase reporter assay, NSun2-/- rats","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — in vitro methylation + bisulfite sequencing at defined site + reporter assay + in vivo KO; single lab","pmids":["28919411"],"is_preprint":false},{"year":2019,"finding":"NSUN2 is imported into the matrix of mammalian mitochondria and introduces m5C at positions 48, 49, and 50 of several mitochondrial tRNAs; three genetic models (KO mice, patient fibroblasts, CRISPR KO in human cells) confirm NSUN2 dependence; NSUN2 inactivation does not profoundly affect mt-tRNA stability or oxidative phosphorylation in differentiated cells.","method":"Spatially restricted proximity labeling (BioID), immunodetection, KO mice, patient fibroblasts, CRISPR/Cas9 KO, mass spectrometric tRNA modification mapping","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 2 / Strong — three independent genetic models + proximity labeling for localization + MS-based modification mapping; replicated across labs (parallel independent publication PMID 31287866)","pmids":["31276587"],"is_preprint":false},{"year":2019,"finding":"NSUN2 introduces m5C at positions 48–50 of eight mouse and six human mitochondrial tRNAs, confirmed by mass spectrometry; NSUN2 foci inside mitochondria were visualized by structured illumination microscopy; in vitro reconstitution of m5C at mt-tRNA positions 48–50 was achieved with purified NSUN2 and SAM.","method":"Mass spectrometry of modified tRNA nucleosides, Nsun2 KO mice, CRISPR/Cas9 KO human cells, in vitro methylation reconstitution, structured illumination microscopy","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution + MS site mapping + two KO models + high-resolution localization; multiple orthogonal methods","pmids":["31287866"],"is_preprint":false},{"year":2020,"finding":"NSun2 methylates the 3′-UTR of autotaxin (ATX) mRNA at cytosine C2756 in vitro and in vivo, enhancing ATX mRNA translation; NSun2-mediated m5C also promotes nuclear-to-cytoplasmic export of ATX mRNA in an ALYREF-dependent manner; NSun2 knockdown suppresses cell migration, rescued by exogenous LPA.","method":"In vitro methylation assay, in-cell bisulfite sequencing, protein expression analysis, mRNA export assay, ALYREF siRNA rescue, cell migration assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro methylation at defined site + export assay with ALYREF rescue + migration functional readout; single lab, multiple orthogonal methods","pmids":["33093178"],"is_preprint":false},{"year":2021,"finding":"Neuronal Nsun2 deficiency decreases tRNA m5C levels, causing deficits in expression of tRNAGly isodecoders, glycine codon-specific translational efficiency defects, and loss of Gly-rich synaptic proteins; this impairs glutamatergic neurotransmission and contextual fear memory, and increases glycine biosynthesis.","method":"Conditional Nsun2 KO and OE in mouse prefrontal cortex, bisulfite-seq of tRNAs, quantitative proteomics, ribosome profiling","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — conditional KO + OE + tRNA modification mapping + proteomics + ribosome profiling; bi-directional genetic manipulation with multiple orthogonal readouts","pmids":["34389722"],"is_preprint":false},{"year":2021,"finding":"NSUN2 induces m5C modification of GRB2 mRNA; the m5C reader LIN28B binds m5C-modified GRB2 mRNA and stabilizes it; elevated GRB2 activates PI3K/AKT and ERK/MAPK signaling to promote esophageal cancer progression; E2F1 transcriptionally regulates NSUN2 expression.","method":"RNA-bisulfite sequencing, RIP, mRNA stability assay, KO mouse models, functional cancer assays","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — RNA-BisSeq + RIP for reader identification + mRNA stability assay + in vivo KO; single lab","pmids":["34345012"],"is_preprint":false},{"year":2021,"finding":"SUMO-2/3 directly interacts with NSUN2, stabilizing it and mediating its nuclear transport, thereby facilitating NSUN2's oncogenic m5C methyltransferase activity in gastric cancer cells.","method":"Co-immunoprecipitation, nuclear transport assay, m5C bisulfite sequencing, gain/loss-of-function assays","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct Co-IP demonstrating SUMO-2/3–NSUN2 interaction + nuclear transport functional assay; single lab","pmids":["34504059"],"is_preprint":false},{"year":2022,"finding":"NSUN2 promotes m5C methylation of KRT13 mRNA; the m5C reader YBX1 recognizes and stabilizes methylated KRT13 mRNA; only wild-type NSUN2 (not catalytically inactive mutant) rescues migration and invasion in cervical cancer cells, establishing catalytic activity dependence for oncogenic function.","method":"RNA-bisulfite sequencing, RIP-qPCR, catalytic-dead NSUN2 mutant rescue experiment, cell migration/invasion assay","journal":"Frontiers in oncology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — catalytic-dead mutant rescue + RNA-BisSeq + RIP; single lab, methyltransferase activity requirement directly tested","pmids":["35280737"],"is_preprint":false},{"year":2022,"finding":"NSun2 deficiency in mouse CD4+ T cells specifically inhibits Th17 cell differentiation and alleviates colitis; RORγt recruits Nsun2 to chromatin at Il17a and Il17f loci, leading to transcription-coupled m5C formation on nascent mRNAs and enhanced mRNA stability.","method":"Conditional T-cell Nsun2 KO mice, colitis model, ChIP, mRNA stability assay, RIP","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — conditional KO + in vivo disease model + ChIP showing RORγt recruits Nsun2 + mRNA stability assay; multiple orthogonal methods","pmids":["36792629"],"is_preprint":false},{"year":2022,"finding":"NSun2 deficiency promotes tau hyperphosphorylation; conditional brain-specific ablation of NSun2 decreases miR-125b m6A levels; Drosophila NSun2 reduction exacerbates tau toxicity while overexpression partially rescues it; NSun2 levels decrease in response to amyloid-beta oligomers, and NSun2 overexpression rescues AβO-induced tau phosphorylation.","method":"Conditional brain-specific KO mice, Drosophila tau toxicity model, iPSC-derived neurons, Western blot, immunostaining, miRNA modification analysis","journal":"Acta neuropathologica","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple model systems (mouse KO, Drosophila, human iPSC) with defined tau phosphorylation readout; single lab, epistasis partially established","pmids":["36357715"],"is_preprint":false},{"year":2023,"finding":"NSUN2 methylates QSOX1 mRNA coding sequence; the m5C reader YBX1 recognizes methylated QSOX1 and enhances its translation, conferring gefitinib resistance in EGFR-mutant NSCLC; this defines the NSUN2-YBX1-QSOX1 axis.","method":"RNA-BisSeq, m5C-BisSeq, RNA-seq, MeRIP-qPCR, puromycin incorporation translation assay, gain/loss-of-function, in vivo xenograft","journal":"Molecular cancer","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — m5C sequencing + translational assay + in vivo; single lab, multiple methods","pmids":["37161388"],"is_preprint":false},{"year":2023,"finding":"Glucose directly binds to NSUN2 at amino acids 1-28, promoting NSUN2 oligomerization and activation; activated NSUN2 maintains global m5C RNA methylation including TREX2 mRNA, stabilizing TREX2 to restrict cytosolic dsDNA and suppress cGAS/STING activation; NSUN2 mutants defective in glucose binding abolish this activity.","method":"Glucose-binding assay, oligomerization assay, NSUN2 glucose-binding mutant, TREX2 mRNA stability, cGAS/STING pathway assays, in vivo tumor models","journal":"Cell metabolism","confidence":"High","confidence_rationale":"Tier 1 / Moderate — direct glucose-binding assay + glucose-binding mutant + downstream pathway reconstitution; single lab but multiple orthogonal methods","pmids":["37586363"],"is_preprint":false},{"year":2023,"finding":"NSUN2 methylates IRF3 mRNA and accelerates its degradation, reducing IRF3 levels and IFN-β production; knockout of NSUN2 enhances type I interferon responses during viral infections including SARS-CoV-2; viral infection reduces endogenous NSUN2 levels to boost antiviral responses.","method":"NSUN2 KO cells and mice, bisulfite sequencing of IRF3 mRNA, mRNA stability assay, IRF3 m5C site mutation, viral infection models","journal":"Emerging microbes & infections","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KO models + bisulfite sequencing at defined sites + m5C site mutation + mRNA decay assay; single lab","pmids":["36748584"],"is_preprint":false},{"year":2023,"finding":"NSUN2-mediated m5C modification of HBV RNA at sites C2017 and C131 promotes HBV RNA stability and replication; mutation of these m5C sites decreases HBV expression; HBV core protein promotes NSUN2 expression, establishing a positive feedback loop.","method":"Bisulfite sequencing of HBV RNA, high-throughput bisulfite-seq, site-directed mutagenesis of HBV m5C sites, NSUN2 KD/KO, HBV replication assay, Nsun2+/- mice","journal":"PLoS pathogens","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — bisulfite sequencing at defined sites + site-directed mutation of viral RNA showing functional effect + in vivo mouse model; single lab","pmids":["38048324"],"is_preprint":false},{"year":2023,"finding":"NSUN2 mediates m5C modification of SLC7A11 mRNA; the m5C reader YBX1 recognizes m5C sites on SLC7A11 mRNA via its cold shock domain, increasing SLC7A11 mRNA stability and protein levels, thereby conferring ferroptosis resistance in endometrial cancer.","method":"RNA bisulfite sequencing, RIP-qPCR (YBX1), mRNA stability assay, YBX1 domain mapping, NSUN2 KD/rescue, ferroptosis assay","journal":"Redox biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — bisulfite sequencing + RIP with domain mapping + mRNA stability + ferroptosis functional assay; single lab","pmids":["38042059"],"is_preprint":false},{"year":2023,"finding":"Cysteine-directed covalent inhibitors (azetidine acrylamides) stereoselectively target the conserved catalytic cysteine of NSUN2, inhibiting its methyltransferase activity on recombinant NSUN2 but not NSUN6 in vitro, and disrupting NSUN2-tRNA interactions in cancer cells, globally reducing tRNA m5C content.","method":"Activity-based protein profiling (ABPP), recombinant NSUN2 in vitro activity assay, cell-based tRNA m5C quantification, proteome-wide selectivity profiling","journal":"Angewandte Chemie (International ed. in English)","confidence":"High","confidence_rationale":"Tier 1 / Moderate — biochemical activity assay with recombinant enzyme + cell-based tRNA m5C measurement + proteome-wide selectivity panel; single lab, multiple rigorous assays","pmids":["37909922"],"is_preprint":false},{"year":2023,"finding":"DIAPH2-AS1 lncRNA binds NSUN2 and protects it from ubiquitin-proteasomal degradation by masking lysines K577 and K579; stabilized NSUN2 increases m5C modification of NTN1 mRNA, enhancing its stability and promoting neural invasion of gastric cancer.","method":"Pulldown assay, RIP assay, ubiquitination assay, K577/K579 mutation, mRNA stability assay, in vivo neural invasion model","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — pulldown/RIP + ubiquitination site mutation + mRNA stability assay; single lab, multiple methods","pmids":["37037818"],"is_preprint":false},{"year":2024,"finding":"Glucose binds conserved sequences within NSUN2, enhancing its binding to S-adenosyl-L-methionine and boosting its enzymatic activity; glucose enhances NSUN2's proximity to mRNA translation machinery; NSUN2 modulates global mRNA translation, particularly of pro-differentiation mRNAs with m5C modifications such as GRHL3, facilitating epidermal differentiation.","method":"Glucose-binding assay, SAM-binding assay, enzymatic activity assay, proximity labeling, ribosome profiling/translation assay, epidermal differentiation model","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1 / Moderate — direct binding assay + enzymatic activity measurement + proximity labeling + ribosome profiling; single lab but multiple orthogonal mechanistic methods","pmids":["39565212"],"is_preprint":false},{"year":2024,"finding":"STUB1 ubiquitinates NSUN2 at lysines K457 and K654, promoting NSUN2 degradation during ferroptosis; loss of NSUN2 reduces m5C methylation of Gpx4 mRNA 3′-UTR, abrogating the interaction between SBP2 and the SECIS element, thereby inhibiting GPX4 protein synthesis and promoting hepatocyte ferroptosis.","method":"Co-IP for STUB1-NSUN2 interaction, ubiquitination site mapping (K457/K654 mutants), MeRIP for Gpx4 m5C, SBP2-SECIS interaction assay, NSUN2 restoration rescue assay","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 1 / Moderate — ubiquitination site mutagenesis + MeRIP + SBP2-SECIS mechanistic assay + restoration rescue; single lab, multiple orthogonal methods","pmids":["39453812"],"is_preprint":false},{"year":2024,"finding":"NAA10 (N-α-acetyltransferase 10) lactylates NSUN2 at lysine K508, enhancing NSUN2 enzymatic activity; activated NSUN2 methylates GCLC mRNA, stabilizing it and increasing intracellular glutathione, conferring ferroptosis resistance; K508R NSUN2 mutant or GCLC m5C site mutants abolish these effects.","method":"Lactylation site mapping (K508), NSUN2 K508R mutant, Co-IP (NSUN2-NAA10 interaction), m5C methylation of GCLC mRNA, mRNA stability assay, GSH measurement, ferroptosis assay","journal":"Redox biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — PTM site mapping with specific mutant (K508R) + writer identification (NAA10 Co-IP) + enzymatic activity assay + target mRNA methylation; single lab, multiple orthogonal methods","pmids":["39742570"],"is_preprint":false},{"year":2024,"finding":"NSUN2 and YBX1 mediate m5C modification of ENO1 mRNA; ENO1 m5C modification reprograms glucose metabolism increasing lactic acid; lactic acid activates NSUN2 transcription via histone H3K18 lactylation and induces NSUN2 lactylation at K356 critical for target RNA capture, forming a positive feedback loop.","method":"m5C-RIP-seq, RIP-qPCR, metabolic assays, ChIP for H3K18la, lactylation site identification (K356), K356 mutant functional assay","journal":"Advanced science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — m5C-RIP-seq + K356 lactylation mutant + ChIP; single lab, multiple methods but complex multi-step pathway","pmids":["38769664"],"is_preprint":false},{"year":2024,"finding":"Nsun2 methylates PRKACA (PKA catalytic subunit alpha) mRNA; the m5C reader YBX1 recognizes methylated PRKACA mRNA and promotes its translation; Nsun2 ablation reduces PKA activity, impairs cardiomyocyte contraction and calcium handling, and blocks hypertrophic response in mice.","method":"m5C-RIP-seq, RNA pull-down, polysome profiling, reporter assay, cardiac-specific Nsun2 KO (αMHC-CreERT2), rAAV9-mediated OE, IonOptix calcium transient measurement","journal":"Theranostics","confidence":"High","confidence_rationale":"Tier 2 / Strong — cardiac KO + RIP-seq + polysome profiling + calcium measurement + rescue experiments; multiple orthogonal methods in vivo","pmids":["39990213"],"is_preprint":false},{"year":2025,"finding":"NSUN2 catalyzes m5C deposition on the 3′-UTR of FSP1 (ferroptosis suppressor protein 1) mRNA; the m5C reader YBX1 recognizes and stabilizes FSP1 mRNA, protecting AML cells from ferroptosis; catalytically inactive NSUN2 (C271A/C321A) or non-functional FSP1 mutants fail to rescue ferroptosis resistance.","method":"m5C-RIP-seq, RIP-qPCR, mRNA stability assay, NSUN2 catalytic dead mutant (C271A/C321A), FSP1 functional mutants, xenograft models, pharmacological NSUN2 inhibition (MY-1B)","journal":"Molecular cancer","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — catalytic-dead mutant + RIP + stability assay + in vivo xenograft; single lab, multiple methods","pmids":["40691844"],"is_preprint":false},{"year":2025,"finding":"NSUN2 acts as a writer and ALYREF as a reader of m5C on SRSF6 mRNA, inducing alternative splicing reprogramming that switches UAP1 isoform from AGX1 to AGX2; AGX2 enhances N-linked glycosylation of ABC transporters, preventing their ubiquitination-mediated degradation and conferring multidrug resistance in anaplastic thyroid cancer.","method":"MeRIP-seq, transcriptomic/proteomic analysis, alternative splicing analysis, glycoprotein staining, denaturing IP ubiquitination assay, nuclear-cytoplasmic fractionation, NSUN2 inhibitor, spontaneous ATC mouse KO model","journal":"Theranostics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — MeRIP-seq + splicing assay + glycosylation/ubiquitination mechanistic assays + in vivo model; single lab, multiple orthogonal methods","pmids":["40083919"],"is_preprint":false},{"year":2025,"finding":"NSUN2 mediates m5C modification of tRNAVal-CAC, enhancing codon-frequency-dependent translation of glycolysis-related genes (ALDH3A2, ALDH7A1, HK1, PFKM); depletion of NSUN2 disrupts tRNAVal-CAC m5C, impairs translation of these enzymes, and suppresses glycolysis and TNBC progression.","method":"tRNA m5C bisulfite sequencing, ribosome sequencing (Ribo-seq), codon usage bias analysis, glycolysis assays, KD/OE functional studies, xenograft models","journal":"Cellular & molecular biology letters","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — tRNA bisulfite-seq + Ribo-seq + metabolic assay; single lab, multiple methods","pmids":["40855521"],"is_preprint":false}],"current_model":"NSUN2 is a multisite-specific RNA:m5C methyltransferase that uses a dual-cysteine catalytic mechanism with SAM as methyl donor to methylate cytosines in tRNAs (positions 34, 40, 48, 49, 50), mRNAs (5′-UTR, 3′-UTR, coding sequence), mitochondrial tRNAs, noncoding RNAs (vault RNAs, miRNA precursors), and viral RNAs; its enzymatic activity is regulated by Aurora-B phosphorylation (Ser139, inhibitory), SUMO-2/3-mediated stabilization and nuclear transport, STUB1-mediated ubiquitination (K457/K654, degradatory), and lactylation at K508 or glucose binding at the N-terminus (both activating); m5C marks on mRNAs are read by ALYREF to promote nuclear export and by YBX1 to stabilize mRNAs and enhance translation, while tRNA m5C methylation stabilizes tRNAs to support codon-specific translation; through these mechanisms NSUN2 controls cell proliferation, spindle integrity, stem cell differentiation, Th17 differentiation, antiviral innate immunity, cardiac homeostasis, and ferroptosis resistance."},"narrative":{"mechanistic_narrative":"NSUN2 is a SAM-dependent RNA:m5C methyltransferase that post-transcriptionally controls gene expression by writing 5-methylcytosine onto tRNAs, mRNAs, mitochondrial tRNAs, and noncoding RNAs to govern proliferation, differentiation, and stress responses [PMID:10445884, PMID:28418038]. It uses a dual-cysteine catalytic mechanism that forms a transient covalent enzyme–RNA intermediate disassembled by S-adenosylhomocysteine release, with the catalytic cysteines (e.g., C271/C321) and the SAM-binding pocket required for activity [PMID:25375641, PMID:37909922, PMID:40691844]. On tRNAs it methylates the variable-arm/anticodon region (positions 34, 40, 48–50), and its loss—non-redundant with DNMT2—reduces tRNA stability and codon-specific translation of, for example, glycine and valine codons, with downstream effects on neuronal function and glycolytic enzyme synthesis [PMID:10445884, PMID:22995836, PMID:22885326, PMID:34389722, PMID:40855521]. On mRNAs NSUN2-deposited m5C is interpreted by sequence-specific reader proteins: ALYREF promotes nuclear export of methylated transcripts, while YBX1 stabilizes targets and enhances their translation, mechanisms exploited across cancers (KRT13, QSOX1, SLC7A11, FSP1, PRKACA) [PMID:28418038, PMID:33093178, PMID:35280737, PMID:38042059, PMID:39990213, PMID:40691844]. Through methylation of specific transcripts NSUN2 tunes cell-cycle regulators (stabilizing p16, repressing p27, enhancing CDK1) and immune/antiviral effectors, methylating IL-17A to drive Th17 differentiation while destabilizing IRF3 to dampen type I interferon and modifying viral RNAs such as HBV [PMID:22395603, PMID:26687548, PMID:26391950, PMID:28919411, PMID:36792629, PMID:36748584, PMID:38048324]. NSUN2 also localizes to nucleoli, the mitotic spindle, and the mitochondrial matrix, where it stabilizes spindle microtubules via NuSAP recruitment and methylates mitochondrial tRNAs [PMID:19596847, PMID:31276587, PMID:31287866]. Its activity is heavily regulated by post-translational and metabolic inputs: inhibitory Aurora-B phosphorylation at Ser139, SUMO-2/3-mediated stabilization and nuclear import, STUB1-mediated ubiquitination and degradation, activating lactylation (K508), and direct glucose binding at the N-terminus that promotes oligomerization and SAM binding [PMID:17215513, PMID:34504059, PMID:37586363, PMID:39565212, PMID:39453812, PMID:39742570]. NSUN2 was first defined as a Myc target controlling keratinocyte proliferation and is recurrently co-opted in cancers and metabolic/ferroptosis programs [PMID:16713953, PMID:37586363, PMID:40691844].","teleology":[{"year":1999,"claim":"Established the founding enzymatic identity of the NSUN2 family as a SAM-dependent tRNA m5C methyltransferase with multisite specificity.","evidence":"Recombinant yeast Trm4 in vitro methylation of tRNA transcripts plus gene disruption eliminating all tRNA m5C","pmids":["10445884"],"confidence":"High","gaps":["Done in yeast ortholog; human enzyme not yet characterized","mRNA and noncoding RNA substrates not addressed"]},{"year":2006,"claim":"Placed NSUN2 downstream of Myc as a proliferation effector and revealed dynamic, cell-cycle-coupled subcellular localization.","evidence":"RNAi in keratinocytes, immunofluorescence, and xenograft assays","pmids":["16713953"],"confidence":"Medium","gaps":["RNA substrates mediating the proliferation phenotype not defined","Mechanism of cytoplasmic/spindle redistribution unknown"]},{"year":2007,"claim":"Identified the first regulatory PTM controlling NSUN2 activity, linking mitotic kinase signaling to methyltransferase output.","evidence":"In vitro kinase and methylation assays with phospho-mimic/deficient mutants and Co-IP with NPM1","pmids":["17215513"],"confidence":"High","gaps":["Physiological substrates regulated by Ser139 phosphorylation not mapped","Phosphatase reversing the mark unknown"]},{"year":2009,"claim":"Defined a non-catalytic structural role at the mitotic spindle, expanding NSUN2 beyond RNA methylation into chromosome segregation.","evidence":"Live/fixed imaging, siRNA depletion, spindle assembly and NuSAP recruitment assays","pmids":["19596847"],"confidence":"High","gaps":["Whether spindle role requires catalytic activity unresolved","Role of associated 18S rRNA mechanistically unclear"]},{"year":2010,"claim":"Provided the structural basis for AdoMet entry and tRNA recognition, rationalizing catalytic and stability-critical residues.","evidence":"X-ray crystallography of archaeal ortholog apo and sinefungin-bound forms with activity validation","pmids":["20600111"],"confidence":"High","gaps":["Human NSUN2 structure not solved","No structure with mRNA or full-length tRNA substrate"]},{"year":2012,"claim":"Demonstrated non-redundant tRNA target specificity between NSUN2 and DNMT2 and its requirement for protein synthesis and development.","evidence":"Double-knockout mice with synthetic lethality, tRNA methylation analysis, and protein synthesis measurement","pmids":["22885326","22995836"],"confidence":"High","gaps":["Codon-level translational consequences not yet mapped","Tissue-specific tRNA target hierarchy unclear"]},{"year":2012,"claim":"Extended NSUN2 substrate scope to mRNA, showing m5C stabilizes target transcripts by blocking decay machineries.","evidence":"In vitro methylation, mRNA half-life and reporter assays, and RIP for HuR/AUF1/Ago2 on p16 mRNA","pmids":["22395603"],"confidence":"High","gaps":["Reader protein for mRNA m5C not yet identified","Transcriptome-wide mRNA target set unknown at this stage"]},{"year":2014,"claim":"Mechanistically resolved the dual-cysteine covalent intermediate and extended NSUN2 function to miRNA biogenesis and vault RNA processing.","evidence":"Covalent complex biochemistry with active-site mutagenesis; miCLIP/bisulfite-seq and Ago-IP on vault RNAs and miR-125b processing assays","pmids":["25375641","23871666","25047833"],"confidence":"High","gaps":["In vivo prevalence of covalent intermediate unclear","How methylation kinetics control noncoding RNA fate not quantified"]},{"year":2015,"claim":"Connected NSUN2 mRNA methylation to translational control of cell-cycle regulators, integrating it into senescence programs.","evidence":"In vitro methylation at defined cytosines, translation reporter and polysome assays, KD/OE on p27 and CDK1","pmids":["26687548","26391950"],"confidence":"High","gaps":["How m5C exerts opposite effects (repress p27, enhance CDK1) mechanistically unclear","Reader proteins for these targets not defined here"]},{"year":2017,"claim":"Identified ALYREF as the first m5C mRNA reader, defining a writer–reader axis coupling methylation to nuclear export.","evidence":"Transcriptome-wide bisulfite-seq, binding studies, fractionation, and catalytic-mutant rescue of export defects","pmids":["28418038"],"confidence":"High","gaps":["Determinants of ALYREF sequence/structure preference incomplete","Crosstalk with other modifications only partly addressed"]},{"year":2019,"claim":"Established NSUN2 import into the mitochondrial matrix and methylation of mitochondrial tRNAs across multiple genetic models.","evidence":"BioID proximity labeling, KO mice, patient fibroblasts, CRISPR KO, MS modification mapping, and in vitro reconstitution","pmids":["31276587","31287866"],"confidence":"High","gaps":["Functional consequence for OXPHOS modest/cell-type dependent","Import machinery and dual-targeting signals not defined"]},{"year":2017,"claim":"Linked NSUN2 mRNA methylation to vascular inflammation and immune effector translation, with metabolic activation via suppressed Aurora-B phosphorylation.","evidence":"In vitro/in-cell methylation, NSUN2-KO rats, and reporter/bisulfite assays on ICAM-1, SHC, IL-17A, and p21","pmids":["26838785","26992231","28919411","28247949"],"confidence":"Medium","gaps":["Pathway placements partly correlative","Reader proteins for these inflammatory targets not all identified"]},{"year":2022,"claim":"Defined YBX1 as a stabilizing m5C reader and showed catalytic-activity dependence of NSUN2 oncogenic function across multiple cancers.","evidence":"RNA-bisulfite-seq, RIP/domain mapping, catalytic-dead mutant rescue on KRT13, GRB2, QSOX1, SLC7A11, FSP1, PRKACA","pmids":["35280737","34345012","37161388","38042059","40691844","39990213"],"confidence":"Medium","gaps":["Reader selection rules (ALYREF vs YBX1 vs LIN28B) not unified","Most axes from single labs without cross-validation"]},{"year":2023,"claim":"Revealed extensive regulation of NSUN2 stability and activity by ubiquitination, SUMOylation, lncRNA shielding, lactylation, and direct glucose binding.","evidence":"Co-IP, ubiquitination/lactylation site mutagenesis, glucose- and SAM-binding assays, oligomerization, lncRNA pulldowns","pmids":["34504059","37586363","37037818","39453812","39742570","39565212","38769664"],"confidence":"High","gaps":["Hierarchy and crosstalk among PTMs not integrated","Stoichiometry and contextual triggers of each modification unclear"]},{"year":2023,"claim":"Positioned NSUN2 as a regulator of antiviral immunity and a host factor for viral RNAs through m5C-dependent transcript fate.","evidence":"KO cells/mice, bisulfite-seq and m5C-site mutation, mRNA decay assays on IRF3 and HBV RNA in infection models","pmids":["36748584","38048324"],"confidence":"Medium","gaps":["Whether IRF3 destabilization generalizes across cell types unclear","Reader for destabilizing m5C not defined"]},{"year":2024,"claim":"Connected NSUN2 to tissue physiology in heart, brain, gut immunity, and ferroptosis/metabolism through specific m5C target programs.","evidence":"Conditional/cardiac/T-cell KO mice, ChIP for RORγt and H3K18la, ribosome and polysome profiling on PRKACA, IL17, tRNAVal, GCLC, GPX4","pmids":["36792629","34389722","36357715","39990213","39742570","39453812","38769664","40855521"],"confidence":"High","gaps":["Tissue-specific target selection mechanisms incomplete","Causal relationships in disease models partly correlative"]},{"year":null,"claim":"How NSUN2 selects between catalytic and non-catalytic roles and chooses among competing m5C reader outcomes (export vs stabilization vs decay) in a given cell remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unified model of reader recruitment specificity","No human full-length structure with substrate","Integration of metabolic/PTM inputs into target choice undefined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140098","term_label":"catalytic activity, acting on RNA","supporting_discovery_ids":[0,7,12,17,21,34]},{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[0,17,36,38]},{"term_id":"GO:0003723","term_label":"RNA binding","supporting_discovery_ids":[6,17,22,33]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[3]}],"localization":[{"term_id":"GO:0005730","term_label":"nucleolus","supporting_discovery_ids":[1,2,3]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[17,25]},{"term_id":"GO:0005739","term_label":"mitochondrion","supporting_discovery_ids":[20,21]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[17]}],"pathway":[{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[0,8,17,23]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[8,14,23,36]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[3,13,14]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[19,27,31]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[33,37,38,41]}],"complexes":[],"partners":["ALYREF","YBX1","NPM1","NUSAP","STUB1","SUMO2","NAA10","LIN28B"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q08J23","full_name":"RNA cytosine C(5)-methyltransferase NSUN2","aliases":["Myc-induced SUN domain-containing protein","Misu","NOL1/NOP2/Sun domain family member 2","Substrate of AIM1/Aurora kinase B","mRNA cytosine C(5)-methyltransferase","tRNA cytosine C(5)-methyltransferase","tRNA methyltransferase 4 homolog","hTrm4"],"length_aa":767,"mass_kda":86.5,"function":"RNA cytosine C(5)-methyltransferase that methylates cytosine to 5-methylcytosine (m5C) in various RNAs, such as tRNAs, mRNAs and some long non-coding RNAs (lncRNAs) (PubMed:17071714, PubMed:22995836, PubMed:31199786, PubMed:31358969). Involved in various processes, such as epidermal stem cell differentiation, testis differentiation and maternal to zygotic transition during early development: acts by increasing protein synthesis; cytosine C(5)-methylation promoting tRNA stability and preventing mRNA decay (PubMed:31199786). Methylates cytosine to 5-methylcytosine (m5C) at positions 34 and 48 of intron-containing tRNA(Leu)(CAA) precursors, and at positions 48, 49 and 50 of tRNA(Gly)(GCC) precursors (PubMed:17071714, PubMed:22995836, PubMed:31199786). tRNA methylation is required generation of RNA fragments derived from tRNAs (tRFs) (PubMed:31199786). Also mediates C(5)-methylation of mitochondrial tRNAs (PubMed:31276587). Catalyzes cytosine C(5)-methylation of mRNAs, leading to stabilize them and prevent mRNA decay: mRNA stabilization involves YBX1 that specifically recognizes and binds m5C-modified transcripts (PubMed:22395603, PubMed:31358969, PubMed:34556860). Cytosine C(5)-methylation of mRNAs also regulates mRNA export: methylated transcripts are specifically recognized by THOC4/ALYREF, which mediates mRNA nucleo-cytoplasmic shuttling (PubMed:28418038). Also mediates cytosine C(5)-methylation of non-coding RNAs, such as vault RNAs (vtRNAs), promoting their processing into regulatory small RNAs (PubMed:23871666). Cytosine C(5)-methylation of vtRNA VTRNA1.1 promotes its processing into small-vault RNA4 (svRNA4) and regulates epidermal differentiation (PubMed:31186410). May act downstream of Myc to regulate epidermal cell growth and proliferation (By similarity). Required for proper spindle assembly and chromosome segregation, independently of its methyltransferase activity (PubMed:19596847)","subcellular_location":"Nucleus, nucleolus; Cytoplasm; Mitochondrion; Cytoplasm, cytoskeleton, spindle; Secreted, extracellular exosome","url":"https://www.uniprot.org/uniprotkb/Q08J23/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/NSUN2","classification":"Not Classified","n_dependent_lines":6,"n_total_lines":1208,"dependency_fraction":0.004966887417218543},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"CALD1","stoichiometry":0.2},{"gene":"CAPZB","stoichiometry":0.2},{"gene":"FKBP5","stoichiometry":0.2},{"gene":"PSPC1","stoichiometry":0.2},{"gene":"RPS16","stoichiometry":0.2},{"gene":"SRP9","stoichiometry":0.2},{"gene":"SSB","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/NSUN2","total_profiled":1310},"omim":[{"mim_id":"611091","title":"INTELLECTUAL DEVELOPMENTAL DISORDER, AUTOSOMAL RECESSIVE 5; MRT5","url":"https://www.omim.org/entry/611091"},{"mim_id":"610916","title":"NOP2/SUN RNA METHYLTRANSFERASE FAMILY, MEMBER 2; NSUN2","url":"https://www.omim.org/entry/610916"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Uncertain","locations":[{"location":"Nucleoplasm","reliability":"Uncertain"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/NSUN2"},"hgnc":{"alias_symbol":["FLJ20303","TRM4","Misu","SAKI"],"prev_symbol":["MRT5"]},"alphafold":{"accession":"Q08J23","domains":[{"cath_id":"3.40.50","chopping":"164-185_244-436","consensus_level":"high","plddt":96.0706,"start":164,"end":436},{"cath_id":"-","chopping":"619-708","consensus_level":"medium","plddt":89.2417,"start":619,"end":708},{"cath_id":"3.30.70","chopping":"76-161","consensus_level":"high","plddt":84.1451,"start":76,"end":161}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q08J23","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q08J23-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q08J23-F1-predicted_aligned_error_v6.png","plddt_mean":78.06},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=NSUN2","jax_strain_url":"https://www.jax.org/strain/search?query=NSUN2"},"sequence":{"accession":"Q08J23","fasta_url":"https://rest.uniprot.org/uniprotkb/Q08J23.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q08J23/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q08J23"}},"corpus_meta":[{"pmid":"28418038","id":"PMC_28418038","title":"5-methylcytosine 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S-adenosyl-L-methionine-dependent tRNA:m5C-methyltransferase that methylates cytosine at positions 34, 40, 48, and 49 in multiple yeast tRNAs and pre-tRNAs; disruption of the gene eliminates all tRNA m5C methylation in yeast.\",\n      \"method\": \"Recombinant protein expression and purification, in vitro methylation assay with T7 tRNA transcripts, gene disruption in yeast\",\n      \"journal\": \"RNA (New York, N.Y.)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro enzymatic reconstitution with purified protein + genetic KO confirming loss of all tRNA m5C; multiple orthogonal methods in one study\",\n      \"pmids\": [\"10445884\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Misu/NSUN2 is a direct transcriptional target of Myc; its nucleolar localization depends on RNA polymerase III transcripts; knockdown blocks Myc-induced keratinocyte proliferation and reduces nucleolar size; it is redistributed to cytoplasmic vesicles in G2 and to the mitotic spindle.\",\n      \"method\": \"RNAi knockdown in keratinocytes, immunofluorescence for localization, siRNA in xenograft tumor model, cell-cycle analysis\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean KD with defined proliferation phenotype + direct localization by imaging; single lab, multiple methods\",\n      \"pmids\": [\"16713953\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Aurora-B kinase phosphorylates NSUN2 at Ser139 during mitosis, inhibiting its methyltransferase activity and disrupting its association with nucleolar protein NPM1/nucleophosmin; Aurora-B inhibition restores NSUN2–NPM1 association even during mitosis.\",\n      \"method\": \"In vitro kinase assay, in vitro methylation assay, co-immunoprecipitation, phosphorylation-mimic and phospho-deficient NSUN2 mutants\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro kinase assay + in vitro methylation activity assay with phospho-mutants + Co-IP; single lab but multiple orthogonal methods\",\n      \"pmids\": [\"17215513\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Misu/NSUN2 translocates from nucleoli in interphase to the mitotic spindle as an RNA-protein complex containing 18S rRNA; its depletion causes multipolar spindles, chromosome missegregation, aneuploidy, and cell death; NSUN2 recruits NuSAP to the spindle for microtubule stabilization.\",\n      \"method\": \"Immunofluorescence live/fixed imaging, siRNA depletion, spindle assembly assay, RNA–protein complex characterization\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean KD with defined mitotic phenotype + direct localization imaging + NuSAP recruitment assay; single lab, multiple orthogonal methods\",\n      \"pmids\": [\"19596847\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Crystal structure of archaeal Trm4 (NSUN2 ortholog from Methanocaldococcus jannaschii, MJ0026) complexed with sinefungin (AdoMet analog) revealed a negatively charged binding pocket near helix α8 controlling AdoMet entry, and a large positively charged surface for tRNA binding; the structure rationalizes conserved residues essential for catalysis and stability.\",\n      \"method\": \"X-ray crystallography at 1.27 Å (apo) and 2.3 Å (sinefungin complex), in vitro methyltransferase activity assay\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure determination at high resolution with functional validation by in vitro activity assay; directly informs human NSUN2 mechanism\",\n      \"pmids\": [\"20600111\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"NSun2/Misu is required to balance epidermal stem cell self-renewal and differentiation; depletion results in enhanced quiescence and aberrant lineage commitment, placing NSun2-mediated RNA methylation as a post-transcriptional regulator of stem cell fate in skin.\",\n      \"method\": \"Conditional depletion in mouse skin, immunofluorescence, stem cell functional assays\",\n      \"journal\": \"PLoS genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic KO with defined stem cell phenotype; single lab, multiple histological and functional readouts\",\n      \"pmids\": [\"22144916\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"NSUN2 methylates the 3′-UTR of p16(INK4) mRNA at position A988 in vitro and in cells; this methylation stabilizes p16 mRNA by preventing association with HuR, AUF1, and Ago2/RISC, and prevents recruitment to processing bodies; NSun2 knockdown shortens p16 mRNA half-life.\",\n      \"method\": \"In vitro methylation assay, mRNA half-life measurement, chimeric reporter assay, RIP (HuR/AUF1/Ago2), siRNA knockdown\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro methylation reconstitution + reporter assay with methylation-site mutant + RIP; single lab but multiple orthogonal methods\",\n      \"pmids\": [\"22395603\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"The human tRNA methyltransferase Misu/NSUN2 methylates position 48 in tRNA-Leu(CAA) (with or without intron) and positions 48, 49, and 50 in tRNA-Gly2(GCC) in vitro, demonstrating multisite specificity.\",\n      \"method\": \"Recombinant protein expression and purification in E. coli, in vitro methylation assay with multiple tRNA substrates\",\n      \"journal\": \"RNA biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro enzymatic assay with purified recombinant enzyme and multiple defined substrates; single lab but rigorous biochemical reconstitution\",\n      \"pmids\": [\"22995836\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Disruption of both Dnmt2 and NSun2 tRNA methyltransferases in mice causes synthetic lethality with underdeveloped phenotype and impaired differentiation; Dnmt2 and NSun2 have complementary, non-overlapping tRNA target-site specificities; loss of both reduces steady-state levels of unmethylated tRNAs and overall protein synthesis rates.\",\n      \"method\": \"Double-knockout mouse model, tRNA methylation analysis, protein synthesis rate measurement\",\n      \"journal\": \"Nature structural & molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic epistasis (double-KO synthetic lethality) with biochemical tRNA methylation and protein synthesis readouts; single lab but multiple orthogonal methods on a clean genetic model\",\n      \"pmids\": [\"22885326\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"NSun2 methylates cytosine-5 at six positions in vault noncoding RNAs; loss of NSun2-mediated m5C in vault RNAs causes their aberrant processing into Argonaute-associated small RNA fragments that function as microRNAs, as shown in patient cells lacking NSun2.\",\n      \"method\": \"miCLIP (individual-nucleotide-resolution CLIP), RNA bisulfite sequencing, Argonaute immunoprecipitation, patient cells\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — miCLIP + bisulfite sequencing for site identification + functional Ago-IP readout; single lab, multiple orthogonal methods\",\n      \"pmids\": [\"23871666\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"NSun2 is a component of the chromatoid body in mouse testes; NSun2 knockout blocks meiotic progression at the pachytene stage; simultaneous deletion of NSun2 and Dnmt2 decreases abundance of specific methylated tRNAs and confirms non-redundant roles in spermatogonial cells and Sertoli cells.\",\n      \"method\": \"Knockout mice, immunofluorescence, tRNA methylation analysis, histology\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic KO with defined meiotic arrest phenotype + subcellular localization + tRNA methylation biochemistry; single lab\",\n      \"pmids\": [\"23401851\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"NSun2 methylates primary (pri-miR-125b), precursor (pre-miR-125b), and mature miR-125b in vitro and in vivo; methylation inhibits processing of pri-miR-125b2 into pre-miR-125b2, decreases cleavage to mature miR-125, and attenuates RISC recruitment, thereby repressing miR-125b function.\",\n      \"method\": \"In vitro methylation assay, pri/pre-miRNA processing assay, RISC loading assay, in vivo methylation\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro methylation reconstitution + functional processing and RISC assays; single lab, multiple orthogonal methods\",\n      \"pmids\": [\"25047833\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Trm4 (yeast NSUN2 ortholog) and mammalian Nsun2 form covalent complexes with previously methylated (m5C-containing) RNA in a manner requiring S-adenosylhomocysteine (AdoHcy); four active-site residues critical for tRNA methylation are also required for this covalent complex formation; removal of AdoHcy disassembles the complexes.\",\n      \"method\": \"Biochemical covalent complex assay with recombinant proteins, active-site mutagenesis, metabolite titration experiments\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution of covalent enzyme-RNA complex with active-site mutagenesis; single lab but rigorous biochemistry\",\n      \"pmids\": [\"25375641\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"NSun2 methylates the 5′-UTR of p27 mRNA at cytosine C64 in vitro and in cells, repressing p27 translation; NSun2 also enhances CDK1 mRNA translation through methylation; knockdown of NSun2 elevates p27, reduces CDK1, and accelerates replicative senescence.\",\n      \"method\": \"In vitro methylation assay, translation reporter assay, RNAi knockdown, protein level analysis\",\n      \"journal\": \"Aging\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro methylation reconstitution at defined cytosine + reporter translational assay + cellular KD/OE; single lab, multiple methods\",\n      \"pmids\": [\"26687548\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"NSun2 methylates CDK1 mRNA in vitro and in cells; this methylation enhances CDK1 translation without altering CDK1 mRNA levels; NSun2-mediated CDK1 upregulation drives cell cycle progression.\",\n      \"method\": \"In vitro methylation assay, overexpression/knockdown, CDK1 translation assay (polysome analysis), cell cycle analysis\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro methylation + translational assay with KD/OE; single lab, multiple orthogonal methods\",\n      \"pmids\": [\"26391950\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"NSun2 methylates ICAM-1 mRNA and promotes its translation, increasing leukocyte adhesion to endothelial cells; TNF-α or homocysteine activates NSun2 methyltransferase activity by suppressing Aurora-B-mediated phosphorylation of NSun2; NSun2-deficient rats show decreased ICAM-1 induction and impaired allograft arteriosclerosis.\",\n      \"method\": \"In vitro methylation assay, in-cell methylation assay, NSun2-knockout rat model, vascular inflammation assay\",\n      \"journal\": \"Circulation research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro + in vivo methylation assay + genetic KO rat model + defined vascular phenotype; multiple orthogonal methods across in vitro and in vivo models\",\n      \"pmids\": [\"26838785\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"NSun2 methylates SHC mRNA in vitro and in cells, enhancing translation of all three SHC proteins (p66SHC, p52SHC, p46SHC), which increases ROS and activates p38MAPK to accelerate oxidative stress- and high-glucose-induced HUVEC senescence.\",\n      \"method\": \"In vitro methylation assay, in-cell methylation, protein expression analysis, ROS measurement, p38MAPK activation assay\",\n      \"journal\": \"Oncotarget\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro methylation + translational effect measured; single lab, multiple methods but functional pathway placement relies on correlation\",\n      \"pmids\": [\"26992231\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"NSUN2 is the principal RNA methyltransferase for m5C in mRNAs; m5C is enriched in CG-rich regions downstream of translation initiation sites; the mRNA export adaptor ALYREF specifically recognizes m5C-modified mRNA; NSUN2 modulates ALYREF nuclear-cytoplasmic shuttling and mRNA export; export defects upon NSUN2 depletion are rescued by wild-type but not methyltransferase-defective NSUN2.\",\n      \"method\": \"Transcriptome-wide m5C profiling (bisulfite-seq), in vitro and in vivo binding studies, nuclear-cytoplasmic fractionation, NSUN2 catalytic mutant rescue experiments, RNA export assays\",\n      \"journal\": \"Cell research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — transcriptome-wide mapping + in vitro binding + catalytic-dead mutant rescue + mRNA export functional assay; multiple orthogonal methods, widely replicated concept\",\n      \"pmids\": [\"28418038\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"NSUN2 catalyzes m5C modification on p21 3′-UTR; METTL3/METTL14 catalyzes m6A on the same mRNA; m6A methylation by METTL3/METTL14 facilitates NSUN2-mediated m5C, and vice versa; combined m5C and m6A synergistically enhance p21 translation during oxidative stress-induced senescence.\",\n      \"method\": \"In vitro methylation assay, translation reporter assay, KD of NSUN2 and METTL3/METTL14, LC-MS/MS\",\n      \"journal\": \"Journal of cellular biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro methylation + translational reporter assay; single lab, mutual dependency shown biochemically but mechanistic detail limited\",\n      \"pmids\": [\"28247949\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"NSun2 methylates IL-17A mRNA at cytosine C466 in vitro and in vivo; this m5C methylation promotes IL-17A translation in T lymphocytes; homocysteine upregulates NSun2, driving IL-17A upregulation; NSun2-/- rats show markedly decreased plasma IL-17A.\",\n      \"method\": \"In vitro methylation assay, bisulfite sequencing, HPLC-MS, luciferase reporter assay, NSun2-/- rats\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro methylation + bisulfite sequencing at defined site + reporter assay + in vivo KO; single lab\",\n      \"pmids\": [\"28919411\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"NSUN2 is imported into the matrix of mammalian mitochondria and introduces m5C at positions 48, 49, and 50 of several mitochondrial tRNAs; three genetic models (KO mice, patient fibroblasts, CRISPR KO in human cells) confirm NSUN2 dependence; NSUN2 inactivation does not profoundly affect mt-tRNA stability or oxidative phosphorylation in differentiated cells.\",\n      \"method\": \"Spatially restricted proximity labeling (BioID), immunodetection, KO mice, patient fibroblasts, CRISPR/Cas9 KO, mass spectrometric tRNA modification mapping\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — three independent genetic models + proximity labeling for localization + MS-based modification mapping; replicated across labs (parallel independent publication PMID 31287866)\",\n      \"pmids\": [\"31276587\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"NSUN2 introduces m5C at positions 48–50 of eight mouse and six human mitochondrial tRNAs, confirmed by mass spectrometry; NSUN2 foci inside mitochondria were visualized by structured illumination microscopy; in vitro reconstitution of m5C at mt-tRNA positions 48–50 was achieved with purified NSUN2 and SAM.\",\n      \"method\": \"Mass spectrometry of modified tRNA nucleosides, Nsun2 KO mice, CRISPR/Cas9 KO human cells, in vitro methylation reconstitution, structured illumination microscopy\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution + MS site mapping + two KO models + high-resolution localization; multiple orthogonal methods\",\n      \"pmids\": [\"31287866\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"NSun2 methylates the 3′-UTR of autotaxin (ATX) mRNA at cytosine C2756 in vitro and in vivo, enhancing ATX mRNA translation; NSun2-mediated m5C also promotes nuclear-to-cytoplasmic export of ATX mRNA in an ALYREF-dependent manner; NSun2 knockdown suppresses cell migration, rescued by exogenous LPA.\",\n      \"method\": \"In vitro methylation assay, in-cell bisulfite sequencing, protein expression analysis, mRNA export assay, ALYREF siRNA rescue, cell migration assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro methylation at defined site + export assay with ALYREF rescue + migration functional readout; single lab, multiple orthogonal methods\",\n      \"pmids\": [\"33093178\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Neuronal Nsun2 deficiency decreases tRNA m5C levels, causing deficits in expression of tRNAGly isodecoders, glycine codon-specific translational efficiency defects, and loss of Gly-rich synaptic proteins; this impairs glutamatergic neurotransmission and contextual fear memory, and increases glycine biosynthesis.\",\n      \"method\": \"Conditional Nsun2 KO and OE in mouse prefrontal cortex, bisulfite-seq of tRNAs, quantitative proteomics, ribosome profiling\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — conditional KO + OE + tRNA modification mapping + proteomics + ribosome profiling; bi-directional genetic manipulation with multiple orthogonal readouts\",\n      \"pmids\": [\"34389722\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"NSUN2 induces m5C modification of GRB2 mRNA; the m5C reader LIN28B binds m5C-modified GRB2 mRNA and stabilizes it; elevated GRB2 activates PI3K/AKT and ERK/MAPK signaling to promote esophageal cancer progression; E2F1 transcriptionally regulates NSUN2 expression.\",\n      \"method\": \"RNA-bisulfite sequencing, RIP, mRNA stability assay, KO mouse models, functional cancer assays\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — RNA-BisSeq + RIP for reader identification + mRNA stability assay + in vivo KO; single lab\",\n      \"pmids\": [\"34345012\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"SUMO-2/3 directly interacts with NSUN2, stabilizing it and mediating its nuclear transport, thereby facilitating NSUN2's oncogenic m5C methyltransferase activity in gastric cancer cells.\",\n      \"method\": \"Co-immunoprecipitation, nuclear transport assay, m5C bisulfite sequencing, gain/loss-of-function assays\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct Co-IP demonstrating SUMO-2/3–NSUN2 interaction + nuclear transport functional assay; single lab\",\n      \"pmids\": [\"34504059\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"NSUN2 promotes m5C methylation of KRT13 mRNA; the m5C reader YBX1 recognizes and stabilizes methylated KRT13 mRNA; only wild-type NSUN2 (not catalytically inactive mutant) rescues migration and invasion in cervical cancer cells, establishing catalytic activity dependence for oncogenic function.\",\n      \"method\": \"RNA-bisulfite sequencing, RIP-qPCR, catalytic-dead NSUN2 mutant rescue experiment, cell migration/invasion assay\",\n      \"journal\": \"Frontiers in oncology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — catalytic-dead mutant rescue + RNA-BisSeq + RIP; single lab, methyltransferase activity requirement directly tested\",\n      \"pmids\": [\"35280737\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"NSun2 deficiency in mouse CD4+ T cells specifically inhibits Th17 cell differentiation and alleviates colitis; RORγt recruits Nsun2 to chromatin at Il17a and Il17f loci, leading to transcription-coupled m5C formation on nascent mRNAs and enhanced mRNA stability.\",\n      \"method\": \"Conditional T-cell Nsun2 KO mice, colitis model, ChIP, mRNA stability assay, RIP\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — conditional KO + in vivo disease model + ChIP showing RORγt recruits Nsun2 + mRNA stability assay; multiple orthogonal methods\",\n      \"pmids\": [\"36792629\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"NSun2 deficiency promotes tau hyperphosphorylation; conditional brain-specific ablation of NSun2 decreases miR-125b m6A levels; Drosophila NSun2 reduction exacerbates tau toxicity while overexpression partially rescues it; NSun2 levels decrease in response to amyloid-beta oligomers, and NSun2 overexpression rescues AβO-induced tau phosphorylation.\",\n      \"method\": \"Conditional brain-specific KO mice, Drosophila tau toxicity model, iPSC-derived neurons, Western blot, immunostaining, miRNA modification analysis\",\n      \"journal\": \"Acta neuropathologica\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple model systems (mouse KO, Drosophila, human iPSC) with defined tau phosphorylation readout; single lab, epistasis partially established\",\n      \"pmids\": [\"36357715\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"NSUN2 methylates QSOX1 mRNA coding sequence; the m5C reader YBX1 recognizes methylated QSOX1 and enhances its translation, conferring gefitinib resistance in EGFR-mutant NSCLC; this defines the NSUN2-YBX1-QSOX1 axis.\",\n      \"method\": \"RNA-BisSeq, m5C-BisSeq, RNA-seq, MeRIP-qPCR, puromycin incorporation translation assay, gain/loss-of-function, in vivo xenograft\",\n      \"journal\": \"Molecular cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — m5C sequencing + translational assay + in vivo; single lab, multiple methods\",\n      \"pmids\": [\"37161388\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Glucose directly binds to NSUN2 at amino acids 1-28, promoting NSUN2 oligomerization and activation; activated NSUN2 maintains global m5C RNA methylation including TREX2 mRNA, stabilizing TREX2 to restrict cytosolic dsDNA and suppress cGAS/STING activation; NSUN2 mutants defective in glucose binding abolish this activity.\",\n      \"method\": \"Glucose-binding assay, oligomerization assay, NSUN2 glucose-binding mutant, TREX2 mRNA stability, cGAS/STING pathway assays, in vivo tumor models\",\n      \"journal\": \"Cell metabolism\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — direct glucose-binding assay + glucose-binding mutant + downstream pathway reconstitution; single lab but multiple orthogonal methods\",\n      \"pmids\": [\"37586363\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"NSUN2 methylates IRF3 mRNA and accelerates its degradation, reducing IRF3 levels and IFN-β production; knockout of NSUN2 enhances type I interferon responses during viral infections including SARS-CoV-2; viral infection reduces endogenous NSUN2 levels to boost antiviral responses.\",\n      \"method\": \"NSUN2 KO cells and mice, bisulfite sequencing of IRF3 mRNA, mRNA stability assay, IRF3 m5C site mutation, viral infection models\",\n      \"journal\": \"Emerging microbes & infections\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO models + bisulfite sequencing at defined sites + m5C site mutation + mRNA decay assay; single lab\",\n      \"pmids\": [\"36748584\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"NSUN2-mediated m5C modification of HBV RNA at sites C2017 and C131 promotes HBV RNA stability and replication; mutation of these m5C sites decreases HBV expression; HBV core protein promotes NSUN2 expression, establishing a positive feedback loop.\",\n      \"method\": \"Bisulfite sequencing of HBV RNA, high-throughput bisulfite-seq, site-directed mutagenesis of HBV m5C sites, NSUN2 KD/KO, HBV replication assay, Nsun2+/- mice\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — bisulfite sequencing at defined sites + site-directed mutation of viral RNA showing functional effect + in vivo mouse model; single lab\",\n      \"pmids\": [\"38048324\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"NSUN2 mediates m5C modification of SLC7A11 mRNA; the m5C reader YBX1 recognizes m5C sites on SLC7A11 mRNA via its cold shock domain, increasing SLC7A11 mRNA stability and protein levels, thereby conferring ferroptosis resistance in endometrial cancer.\",\n      \"method\": \"RNA bisulfite sequencing, RIP-qPCR (YBX1), mRNA stability assay, YBX1 domain mapping, NSUN2 KD/rescue, ferroptosis assay\",\n      \"journal\": \"Redox biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — bisulfite sequencing + RIP with domain mapping + mRNA stability + ferroptosis functional assay; single lab\",\n      \"pmids\": [\"38042059\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Cysteine-directed covalent inhibitors (azetidine acrylamides) stereoselectively target the conserved catalytic cysteine of NSUN2, inhibiting its methyltransferase activity on recombinant NSUN2 but not NSUN6 in vitro, and disrupting NSUN2-tRNA interactions in cancer cells, globally reducing tRNA m5C content.\",\n      \"method\": \"Activity-based protein profiling (ABPP), recombinant NSUN2 in vitro activity assay, cell-based tRNA m5C quantification, proteome-wide selectivity profiling\",\n      \"journal\": \"Angewandte Chemie (International ed. in English)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — biochemical activity assay with recombinant enzyme + cell-based tRNA m5C measurement + proteome-wide selectivity panel; single lab, multiple rigorous assays\",\n      \"pmids\": [\"37909922\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"DIAPH2-AS1 lncRNA binds NSUN2 and protects it from ubiquitin-proteasomal degradation by masking lysines K577 and K579; stabilized NSUN2 increases m5C modification of NTN1 mRNA, enhancing its stability and promoting neural invasion of gastric cancer.\",\n      \"method\": \"Pulldown assay, RIP assay, ubiquitination assay, K577/K579 mutation, mRNA stability assay, in vivo neural invasion model\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — pulldown/RIP + ubiquitination site mutation + mRNA stability assay; single lab, multiple methods\",\n      \"pmids\": [\"37037818\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Glucose binds conserved sequences within NSUN2, enhancing its binding to S-adenosyl-L-methionine and boosting its enzymatic activity; glucose enhances NSUN2's proximity to mRNA translation machinery; NSUN2 modulates global mRNA translation, particularly of pro-differentiation mRNAs with m5C modifications such as GRHL3, facilitating epidermal differentiation.\",\n      \"method\": \"Glucose-binding assay, SAM-binding assay, enzymatic activity assay, proximity labeling, ribosome profiling/translation assay, epidermal differentiation model\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — direct binding assay + enzymatic activity measurement + proximity labeling + ribosome profiling; single lab but multiple orthogonal mechanistic methods\",\n      \"pmids\": [\"39565212\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"STUB1 ubiquitinates NSUN2 at lysines K457 and K654, promoting NSUN2 degradation during ferroptosis; loss of NSUN2 reduces m5C methylation of Gpx4 mRNA 3′-UTR, abrogating the interaction between SBP2 and the SECIS element, thereby inhibiting GPX4 protein synthesis and promoting hepatocyte ferroptosis.\",\n      \"method\": \"Co-IP for STUB1-NSUN2 interaction, ubiquitination site mapping (K457/K654 mutants), MeRIP for Gpx4 m5C, SBP2-SECIS interaction assay, NSUN2 restoration rescue assay\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — ubiquitination site mutagenesis + MeRIP + SBP2-SECIS mechanistic assay + restoration rescue; single lab, multiple orthogonal methods\",\n      \"pmids\": [\"39453812\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"NAA10 (N-α-acetyltransferase 10) lactylates NSUN2 at lysine K508, enhancing NSUN2 enzymatic activity; activated NSUN2 methylates GCLC mRNA, stabilizing it and increasing intracellular glutathione, conferring ferroptosis resistance; K508R NSUN2 mutant or GCLC m5C site mutants abolish these effects.\",\n      \"method\": \"Lactylation site mapping (K508), NSUN2 K508R mutant, Co-IP (NSUN2-NAA10 interaction), m5C methylation of GCLC mRNA, mRNA stability assay, GSH measurement, ferroptosis assay\",\n      \"journal\": \"Redox biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — PTM site mapping with specific mutant (K508R) + writer identification (NAA10 Co-IP) + enzymatic activity assay + target mRNA methylation; single lab, multiple orthogonal methods\",\n      \"pmids\": [\"39742570\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"NSUN2 and YBX1 mediate m5C modification of ENO1 mRNA; ENO1 m5C modification reprograms glucose metabolism increasing lactic acid; lactic acid activates NSUN2 transcription via histone H3K18 lactylation and induces NSUN2 lactylation at K356 critical for target RNA capture, forming a positive feedback loop.\",\n      \"method\": \"m5C-RIP-seq, RIP-qPCR, metabolic assays, ChIP for H3K18la, lactylation site identification (K356), K356 mutant functional assay\",\n      \"journal\": \"Advanced science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — m5C-RIP-seq + K356 lactylation mutant + ChIP; single lab, multiple methods but complex multi-step pathway\",\n      \"pmids\": [\"38769664\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Nsun2 methylates PRKACA (PKA catalytic subunit alpha) mRNA; the m5C reader YBX1 recognizes methylated PRKACA mRNA and promotes its translation; Nsun2 ablation reduces PKA activity, impairs cardiomyocyte contraction and calcium handling, and blocks hypertrophic response in mice.\",\n      \"method\": \"m5C-RIP-seq, RNA pull-down, polysome profiling, reporter assay, cardiac-specific Nsun2 KO (αMHC-CreERT2), rAAV9-mediated OE, IonOptix calcium transient measurement\",\n      \"journal\": \"Theranostics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — cardiac KO + RIP-seq + polysome profiling + calcium measurement + rescue experiments; multiple orthogonal methods in vivo\",\n      \"pmids\": [\"39990213\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"NSUN2 catalyzes m5C deposition on the 3′-UTR of FSP1 (ferroptosis suppressor protein 1) mRNA; the m5C reader YBX1 recognizes and stabilizes FSP1 mRNA, protecting AML cells from ferroptosis; catalytically inactive NSUN2 (C271A/C321A) or non-functional FSP1 mutants fail to rescue ferroptosis resistance.\",\n      \"method\": \"m5C-RIP-seq, RIP-qPCR, mRNA stability assay, NSUN2 catalytic dead mutant (C271A/C321A), FSP1 functional mutants, xenograft models, pharmacological NSUN2 inhibition (MY-1B)\",\n      \"journal\": \"Molecular cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — catalytic-dead mutant + RIP + stability assay + in vivo xenograft; single lab, multiple methods\",\n      \"pmids\": [\"40691844\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"NSUN2 acts as a writer and ALYREF as a reader of m5C on SRSF6 mRNA, inducing alternative splicing reprogramming that switches UAP1 isoform from AGX1 to AGX2; AGX2 enhances N-linked glycosylation of ABC transporters, preventing their ubiquitination-mediated degradation and conferring multidrug resistance in anaplastic thyroid cancer.\",\n      \"method\": \"MeRIP-seq, transcriptomic/proteomic analysis, alternative splicing analysis, glycoprotein staining, denaturing IP ubiquitination assay, nuclear-cytoplasmic fractionation, NSUN2 inhibitor, spontaneous ATC mouse KO model\",\n      \"journal\": \"Theranostics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — MeRIP-seq + splicing assay + glycosylation/ubiquitination mechanistic assays + in vivo model; single lab, multiple orthogonal methods\",\n      \"pmids\": [\"40083919\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"NSUN2 mediates m5C modification of tRNAVal-CAC, enhancing codon-frequency-dependent translation of glycolysis-related genes (ALDH3A2, ALDH7A1, HK1, PFKM); depletion of NSUN2 disrupts tRNAVal-CAC m5C, impairs translation of these enzymes, and suppresses glycolysis and TNBC progression.\",\n      \"method\": \"tRNA m5C bisulfite sequencing, ribosome sequencing (Ribo-seq), codon usage bias analysis, glycolysis assays, KD/OE functional studies, xenograft models\",\n      \"journal\": \"Cellular & molecular biology letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — tRNA bisulfite-seq + Ribo-seq + metabolic assay; single lab, multiple methods\",\n      \"pmids\": [\"40855521\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"NSUN2 is a multisite-specific RNA:m5C methyltransferase that uses a dual-cysteine catalytic mechanism with SAM as methyl donor to methylate cytosines in tRNAs (positions 34, 40, 48, 49, 50), mRNAs (5′-UTR, 3′-UTR, coding sequence), mitochondrial tRNAs, noncoding RNAs (vault RNAs, miRNA precursors), and viral RNAs; its enzymatic activity is regulated by Aurora-B phosphorylation (Ser139, inhibitory), SUMO-2/3-mediated stabilization and nuclear transport, STUB1-mediated ubiquitination (K457/K654, degradatory), and lactylation at K508 or glucose binding at the N-terminus (both activating); m5C marks on mRNAs are read by ALYREF to promote nuclear export and by YBX1 to stabilize mRNAs and enhance translation, while tRNA m5C methylation stabilizes tRNAs to support codon-specific translation; through these mechanisms NSUN2 controls cell proliferation, spindle integrity, stem cell differentiation, Th17 differentiation, antiviral innate immunity, cardiac homeostasis, and ferroptosis resistance.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"NSUN2 is a SAM-dependent RNA:m5C methyltransferase that post-transcriptionally controls gene expression by writing 5-methylcytosine onto tRNAs, mRNAs, mitochondrial tRNAs, and noncoding RNAs to govern proliferation, differentiation, and stress responses [#0, #17]. It uses a dual-cysteine catalytic mechanism that forms a transient covalent enzyme–RNA intermediate disassembled by S-adenosylhomocysteine release, with the catalytic cysteines (e.g., C271/C321) and the SAM-binding pocket required for activity [#12, #34, #41]. On tRNAs it methylates the variable-arm/anticodon region (positions 34, 40, 48–50), and its loss—non-redundant with DNMT2—reduces tRNA stability and codon-specific translation of, for example, glycine and valine codons, with downstream effects on neuronal function and glycolytic enzyme synthesis [#0, #7, #8, #23, #43]. On mRNAs NSUN2-deposited m5C is interpreted by sequence-specific reader proteins: ALYREF promotes nuclear export of methylated transcripts, while YBX1 stabilizes targets and enhances their translation, mechanisms exploited across cancers (KRT13, QSOX1, SLC7A11, FSP1, PRKACA) [#17, #22, #26, #33, #40, #41]. Through methylation of specific transcripts NSUN2 tunes cell-cycle regulators (stabilizing p16, repressing p27, enhancing CDK1) and immune/antiviral effectors, methylating IL-17A to drive Th17 differentiation while destabilizing IRF3 to dampen type I interferon and modifying viral RNAs such as HBV [#6, #13, #14, #19, #27, #31, #32]. NSUN2 also localizes to nucleoli, the mitotic spindle, and the mitochondrial matrix, where it stabilizes spindle microtubules via NuSAP recruitment and methylates mitochondrial tRNAs [#3, #20, #21]. Its activity is heavily regulated by post-translational and metabolic inputs: inhibitory Aurora-B phosphorylation at Ser139, SUMO-2/3-mediated stabilization and nuclear import, STUB1-mediated ubiquitination and degradation, activating lactylation (K508), and direct glucose binding at the N-terminus that promotes oligomerization and SAM binding [#2, #25, #30, #36, #37, #38]. NSUN2 was first defined as a Myc target controlling keratinocyte proliferation and is recurrently co-opted in cancers and metabolic/ferroptosis programs [#1, #30, #41].\",\n  \"teleology\": [\n    {\n      \"year\": 1999,\n      \"claim\": \"Established the founding enzymatic identity of the NSUN2 family as a SAM-dependent tRNA m5C methyltransferase with multisite specificity.\",\n      \"evidence\": \"Recombinant yeast Trm4 in vitro methylation of tRNA transcripts plus gene disruption eliminating all tRNA m5C\",\n      \"pmids\": [\"10445884\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Done in yeast ortholog; human enzyme not yet characterized\", \"mRNA and noncoding RNA substrates not addressed\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Placed NSUN2 downstream of Myc as a proliferation effector and revealed dynamic, cell-cycle-coupled subcellular localization.\",\n      \"evidence\": \"RNAi in keratinocytes, immunofluorescence, and xenograft assays\",\n      \"pmids\": [\"16713953\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"RNA substrates mediating the proliferation phenotype not defined\", \"Mechanism of cytoplasmic/spindle redistribution unknown\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Identified the first regulatory PTM controlling NSUN2 activity, linking mitotic kinase signaling to methyltransferase output.\",\n      \"evidence\": \"In vitro kinase and methylation assays with phospho-mimic/deficient mutants and Co-IP with NPM1\",\n      \"pmids\": [\"17215513\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological substrates regulated by Ser139 phosphorylation not mapped\", \"Phosphatase reversing the mark unknown\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Defined a non-catalytic structural role at the mitotic spindle, expanding NSUN2 beyond RNA methylation into chromosome segregation.\",\n      \"evidence\": \"Live/fixed imaging, siRNA depletion, spindle assembly and NuSAP recruitment assays\",\n      \"pmids\": [\"19596847\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether spindle role requires catalytic activity unresolved\", \"Role of associated 18S rRNA mechanistically unclear\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Provided the structural basis for AdoMet entry and tRNA recognition, rationalizing catalytic and stability-critical residues.\",\n      \"evidence\": \"X-ray crystallography of archaeal ortholog apo and sinefungin-bound forms with activity validation\",\n      \"pmids\": [\"20600111\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Human NSUN2 structure not solved\", \"No structure with mRNA or full-length tRNA substrate\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Demonstrated non-redundant tRNA target specificity between NSUN2 and DNMT2 and its requirement for protein synthesis and development.\",\n      \"evidence\": \"Double-knockout mice with synthetic lethality, tRNA methylation analysis, and protein synthesis measurement\",\n      \"pmids\": [\"22885326\", \"22995836\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Codon-level translational consequences not yet mapped\", \"Tissue-specific tRNA target hierarchy unclear\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Extended NSUN2 substrate scope to mRNA, showing m5C stabilizes target transcripts by blocking decay machineries.\",\n      \"evidence\": \"In vitro methylation, mRNA half-life and reporter assays, and RIP for HuR/AUF1/Ago2 on p16 mRNA\",\n      \"pmids\": [\"22395603\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Reader protein for mRNA m5C not yet identified\", \"Transcriptome-wide mRNA target set unknown at this stage\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Mechanistically resolved the dual-cysteine covalent intermediate and extended NSUN2 function to miRNA biogenesis and vault RNA processing.\",\n      \"evidence\": \"Covalent complex biochemistry with active-site mutagenesis; miCLIP/bisulfite-seq and Ago-IP on vault RNAs and miR-125b processing assays\",\n      \"pmids\": [\"25375641\", \"23871666\", \"25047833\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo prevalence of covalent intermediate unclear\", \"How methylation kinetics control noncoding RNA fate not quantified\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Connected NSUN2 mRNA methylation to translational control of cell-cycle regulators, integrating it into senescence programs.\",\n      \"evidence\": \"In vitro methylation at defined cytosines, translation reporter and polysome assays, KD/OE on p27 and CDK1\",\n      \"pmids\": [\"26687548\", \"26391950\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How m5C exerts opposite effects (repress p27, enhance CDK1) mechanistically unclear\", \"Reader proteins for these targets not defined here\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Identified ALYREF as the first m5C mRNA reader, defining a writer–reader axis coupling methylation to nuclear export.\",\n      \"evidence\": \"Transcriptome-wide bisulfite-seq, binding studies, fractionation, and catalytic-mutant rescue of export defects\",\n      \"pmids\": [\"28418038\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Determinants of ALYREF sequence/structure preference incomplete\", \"Crosstalk with other modifications only partly addressed\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Established NSUN2 import into the mitochondrial matrix and methylation of mitochondrial tRNAs across multiple genetic models.\",\n      \"evidence\": \"BioID proximity labeling, KO mice, patient fibroblasts, CRISPR KO, MS modification mapping, and in vitro reconstitution\",\n      \"pmids\": [\"31276587\", \"31287866\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence for OXPHOS modest/cell-type dependent\", \"Import machinery and dual-targeting signals not defined\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Linked NSUN2 mRNA methylation to vascular inflammation and immune effector translation, with metabolic activation via suppressed Aurora-B phosphorylation.\",\n      \"evidence\": \"In vitro/in-cell methylation, NSUN2-KO rats, and reporter/bisulfite assays on ICAM-1, SHC, IL-17A, and p21\",\n      \"pmids\": [\"26838785\", \"26992231\", \"28919411\", \"28247949\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Pathway placements partly correlative\", \"Reader proteins for these inflammatory targets not all identified\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Defined YBX1 as a stabilizing m5C reader and showed catalytic-activity dependence of NSUN2 oncogenic function across multiple cancers.\",\n      \"evidence\": \"RNA-bisulfite-seq, RIP/domain mapping, catalytic-dead mutant rescue on KRT13, GRB2, QSOX1, SLC7A11, FSP1, PRKACA\",\n      \"pmids\": [\"35280737\", \"34345012\", \"37161388\", \"38042059\", \"40691844\", \"39990213\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Reader selection rules (ALYREF vs YBX1 vs LIN28B) not unified\", \"Most axes from single labs without cross-validation\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Revealed extensive regulation of NSUN2 stability and activity by ubiquitination, SUMOylation, lncRNA shielding, lactylation, and direct glucose binding.\",\n      \"evidence\": \"Co-IP, ubiquitination/lactylation site mutagenesis, glucose- and SAM-binding assays, oligomerization, lncRNA pulldowns\",\n      \"pmids\": [\"34504059\", \"37586363\", \"37037818\", \"39453812\", \"39742570\", \"39565212\", \"38769664\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Hierarchy and crosstalk among PTMs not integrated\", \"Stoichiometry and contextual triggers of each modification unclear\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Positioned NSUN2 as a regulator of antiviral immunity and a host factor for viral RNAs through m5C-dependent transcript fate.\",\n      \"evidence\": \"KO cells/mice, bisulfite-seq and m5C-site mutation, mRNA decay assays on IRF3 and HBV RNA in infection models\",\n      \"pmids\": [\"36748584\", \"38048324\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether IRF3 destabilization generalizes across cell types unclear\", \"Reader for destabilizing m5C not defined\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Connected NSUN2 to tissue physiology in heart, brain, gut immunity, and ferroptosis/metabolism through specific m5C target programs.\",\n      \"evidence\": \"Conditional/cardiac/T-cell KO mice, ChIP for RORγt and H3K18la, ribosome and polysome profiling on PRKACA, IL17, tRNAVal, GCLC, GPX4\",\n      \"pmids\": [\"36792629\", \"34389722\", \"36357715\", \"39990213\", \"39742570\", \"39453812\", \"38769664\", \"40855521\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Tissue-specific target selection mechanisms incomplete\", \"Causal relationships in disease models partly correlative\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How NSUN2 selects between catalytic and non-catalytic roles and chooses among competing m5C reader outcomes (export vs stabilization vs decay) in a given cell remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unified model of reader recruitment specificity\", \"No human full-length structure with substrate\", \"Integration of metabolic/PTM inputs into target choice undefined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140098\", \"supporting_discovery_ids\": [0, 7, 12, 17, 21, 34]},\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [0, 17, 36, 38]},\n      {\"term_id\": \"GO:0003723\", \"supporting_discovery_ids\": [6, 17, 22, 33]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [3]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005730\", \"supporting_discovery_ids\": [1, 2, 3]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [17, 25]},\n      {\"term_id\": \"GO:0005739\", \"supporting_discovery_ids\": [20, 21]},\n      {\"term_id\": \"GO:0005819\", \"supporting_discovery_ids\": [3]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [17]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [0, 8, 17, 23]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [8, 14, 23, 36]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [3, 13, 14]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [19, 27, 31]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [33, 37, 38, 41]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"ALYREF\", \"YBX1\", \"NPM1\", \"NuSAP\", \"STUB1\", \"SUMO2\", \"NAA10\", \"LIN28B\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":8,"faith_total":8,"faith_pct":100.0}}