2.1.1.202	evolution	both the nucleotide position and percent methylation of tRNAs and rRNAs m5C sites are conserved across all species analysed	735864	
2.1.1.202	evolution	both the nucleotide position and percent methylation of tRNAs and rRNAs m5C sites are conserved across all species analysed, overview	735864	
2.1.1.202	evolution	both the nucleotide position and percent methylation of tRNAs and rRNAs m5C sites were conserved across all species analysed	735864	
2.1.1.202	evolution	Identification of m5C sites in nuclear, chloroplast and mitochondrial tRNAs. 39 cytosine 5-methylation sites are identified at 5 structural positions and are located in tRNA secondary structure at positions C38, C48, C49, C50 and C72, pattern of methylation in individual tRNA isodecoders, overview. Identification of single-nucleotide resolution of cytosine 5-methylation sites in non-coding ribosomal RNAs and transfer RNAs of all three subcellular transcriptomes across six diverse species. Both the nucleotide position and percent methylation of tRNAs and rRNAs cytosine 5-methylation sites are conserved across all species analysed, overview	735864	
2.1.1.202	evolution	phylogenetic tree of Trm4/ NSun2 homologues, overview. Enzyme Trm4a is responsible for in vivo methylation of C34 and C48 methylation, whereas Trm4b methylates C49 and C50, tRNAProCGG is the only tRNA methylated by both Trm4a and Trm4b. Schizosaccharomyces pombe shows an unusual separation of activities of the NSun2/Trm4 enzymes that are united in a single enzyme in other eukaryotes like humans, mice and Saccharomyces cerevisiae	-, 757846	
2.1.1.202	evolution	phylogenetic tree of Trm4/ NSun2 homologues, overview. In contrast to most other organisms, fission yeast Schizosaccharomyces pombe carries two Trm4/NSun2 homologues, Trm4a (SPAC17D4.04) and Trm4b (SPAC23C4.17). Enzyme Trm4a is responsible for in vivo methylation of C34 and C48 methylation, whereas Trm4b methylates C49 and C50, tRNAProCGG is the only tRNA methylated by both Trm4a and Trm4b. Schizosaccharomyces pombe shows an unusual separation of activities of the NSun2/Trm4 enzymes that are united in a single enzyme in other eukaryotes like humans, mice and Saccharomyces cerevisiae	-, 757846	
2.1.1.202	evolution	the enzyme belongs to the RsmF/YebU/NSUN2 family of cytosine 5-methylation-RNA methyltransferases utilizing two cysteines in their catalytic pocket	736856	
2.1.1.202	malfunction	autosomal-recessive loss of the NSUN2 gene is a causative link to intellectual disability disorders in humans. Loss of cytosine-5 methylation in vault RNAs causes aberrant processing into Argonaute-associated small RNA fragments that can function as microRNAs. Impaired processing of vault ncRNA may contribute to the etiology of NSun2-deficiency human disorders	735907	
2.1.1.202	malfunction	disruption of the ORF YBL024w leads to the complete absence of m5C in total yeast tRNA. No tRNA:m5C-methyltransferase activity towards all potential m5C methylation sites is detected in the extract of the disrupted yeast strain. The protein product of a single gene is responsible for complete m5C methylation of yeast tRNA	639471	
2.1.1.202	malfunction	exposure to oxidative stress efficiently repressed NSUN2, causing a reduction of methylation at specific tRNA sites. Loss of NSUN2 alters the biogenesis of tRNA-derived noncoding fragments (tRFs) in response to stress, leading to impaired regulation of protein synthesis. The intracellular accumulation of a specific subset of tRFs correlates with the dynamic repression of global protein synthesis	758103	
2.1.1.202	malfunction	exposure to oxidative stress efficiently repressed NSUN2, causing a reduction of methylation at specific tRNA sites. Loss of NSUN2 alters the biogenesis of tRNA-derived noncoding fragments (tRFs) in response to stress, leading to impaired regulation of protein synthesis. The intracellular accumulation of a specific subset of tRFs correlates with the dynamic repression of global protein synthesis. Disruption of the Nsun2 gene in mice causes global hypomethylation of tRNAs and a developmental growth retardation. The abnormal development of tissues including brain and skin is the result of impaired stem cell differentiation. The expression of NSUN2 is highly dynamic within tissues. For instance, NSUN2 is absent in quiescent stem cells in hair follicle bulges (BGs), steadily increases in progenitor cells in the hair germ, and is highest in the growing (anagen) hair bulb	758103	
2.1.1.202	malfunction	in trm4a defective mutants, the cytosine 5-methylation profile is the same as wild-type, showing that TRM4A is not required for methylation of any of the detected tRNAs. In contrast for trm4b-1 and trm4b-2 mutants, a total of 18 sites have no detectable methylation and 7 sites have reduced methylation when compared to wild-type, the sites are corresponding to structural positions C48, C49, and C50. trdmt1/trm4b double mutants are hypersensitive to the antibiotic hygromycin B	735864	
2.1.1.202	malfunction	loss-of-function mutations in the NSUN2 gene in both mouse and human cause growth retardation and neurodevelopmental deficits including microcephaly, as well as defects in cognition and motor function. Loss of NSUN2-mediated methylation of tRNA increases their endonucleolytic cleavage by angiogenin, and 5' tRNA fragments accumulate in Nsun2-/- brains. Neural differentiation of NES cells is impaired by both NSUN2 depletion and the presence of angiogenin. Since repression of NSUN2 also inhibits neural cell migration toward the chemoattractant fibroblast growth factor 2, the impaired differentiation capacity in the absence of NSUN2 may be driven by the inability to efficiently respond to growth factors. Upper-layer neurons are decreased in Nsun2 knockout brains, phenotype, detailed overview	758477	
2.1.1.202	malfunction	loss-of-function mutations in the NSUN2 gene in both mouse and human cause growth retardation and neurodevelopmental deficits including microcephaly, as well as defects in cognition and motor function. Loss of NSUN2-mediated methylation of tRNA increases their endonucleolytic cleavage by angiogenin, and 5' tRNA fragments accumulate in Nsun2-/- brains. Neural differentiation of NES cells is impaired by both NSUN2 depletion and the presence of angiogenin. Since repression of NSUN2 also inhibits neural cell migration toward the chemoattractant fibroblast growth factor 2, the impaired differentiation capacity in the absence of NSUN2 may be driven by the inability to efficiently respond to growth factors. Upper-layer neurons are decreased in Nsun2 knockout brains, phenotype, detailed overview. In the developing Nsun2-/- mouse cerebral cortex, intermediate progenitors accumulate and upper-layer neurons decrease. .Loss of NSUN2-mediated methylation of tRNA increases their endonucleolytic cleavage by angiogenin, and 5' tRNA fragments accumulate in Nsun2 -/- brains	758477	
2.1.1.202	malfunction	mutations in TRM4B display defects in root development and decreased m5C peaks. TRM4B affects the transcript levels of the genes involved in root development, which is positively correlated with their mRNA stability and m5C levels	757702	
2.1.1.202	malfunction	NSUN2 is associated with Myc-induced proliferation of cancer cells, mitotic spindle stability, infertility in male mice, and the balance of selfrenewal and differentiation in skin stem cells. In humans NSUN2 mutations cause an autosomal recessive syndrome characterized by intellectual disability and mental retardation	736856	
2.1.1.202	malfunction	the absence of Trm4a, but not Trm4b, causes a mild resistance of Schizosaccharomyces pombe to calcium chloride. Absence of Trm4a, but not Trm4b, causes a mild resistance of Schizosaccharomyces pombe to calcium chloride. In trm4aDELTA mutants, the C34 methylation level drops to background levels (8%), whereas C49 methylation remains at 82%. m5C34 is at 94% in trm4bDELTA mutant, and m5C49 drops to 9%, thus confirming that Trm4a is responsible for m5C34 and Trm4b for m5C49 on tRNAProCGG in vivo	-, 757846	
2.1.1.202	malfunction	the absence of Trm4a, but not Trm4b, causes a mild resistance of Schizosaccharomyces pombe to calcium chloride. In trm4aDELTA mutants, the C34 methylation level drops to background levels (8%), whereas C49 methylation remains at 82%. m5C34 is at 94% in trm4bDELTA mutant, and m5C49 drops to 9%, thus confirming that Trm4a is responsible for m5C34 and Trm4b for m5C49 on tRNAProCGG in vivo	-, 757846	
2.1.1.202	malfunction	tRNAs lacking m5C48/49/50 modifications are bound more tightly by angiogenin, leading to accumulation of 5' tRNA-derived small RNA fragments, which trigger cellular stress and are implicated in disease. Expression of catalytically inactive forms of NSUN2, but not NSUN1, NSUN5, or NSUN6, is reported to alter the total amount of m5C detected in the mRNA pool. svRNA4 acts analogously to a microRNA and a concomitant increase in the levels of the svRNA4 target mRNAs CACNG7 and CACNG8 is observed in NSUN2-/- cells. Loss of function mutations in NSUN2 underlie several neurodevelopmental disorders. A homozygous mutation in the NSUN2 gene that leads to the substitution of Gly679 for Arg (p.Gly679Arg) in the protein has been detected in individuals with autosomal-recessive intellectual disability. This amino acid substitution is suggested to impede NSUN2 function by preventing localization of the protein to its site of action in the nucleolus. NSUN2 has also been linked to Dubowitz syndrome, which is characterized by microcephaly, growth and mental retardation, eczema, and characteristic facial features. A homozygous mutation in the canonical splice acceptor of exon 6 leads to use of a cryptic splice donor, instability of the NSUN2 mRNA, a significant decrease in protein levels, and reduced methylation of NSUN2 target RNAs (m5C47/48 of tRNAAsp(GUC)	756866	
2.1.1.202	malfunction	yeast strains depleted of tRNAHis guanylyltransferase accumulate uncharged tRNAHis lacking the G-1 residue and subsequently accumulate additional 5-methylcytidine (m5C) at residues C48 and C50 of tRNAHis, due to the activity of the m5Cmethyltransferase Trm4. The increase in tRNAHis m5C levels does not require loss of Thg1, loss of G-1 of tRNAHis, or cell death but is associated with growth arrest following different stress conditions. Substantially increased tRNAHis m5C levels occur after temperature-sensitive strains are grown at nonpermissive temperature, and after wild-type strains are grown to stationary phase, starved for required amino acids, or treated with rapamycin. More modest accumulations of m5C in tRNAHis occur after starvation for glucose and after starvation for uracil. In virtually all cases examined, the additional m5C on tRNAHis occurs while cells are fully viable, and the increase is neither due to the GCN4 pathway, nor to increased Trm4 levels, phenotypes, overview. The increased amount of m5C is specific to tRNAHis. tRNAVal(AAC), which also normally has unmodified C48 and C50 residues adjacent to m5C49, has only marginally increased levels of m5C 7 h after temperature shift in the fcp1-1ts mutant	-, 737227	
2.1.1.202	metabolism	enzyme NSUN2 plays a central role in regulation of the stress respone pathway, detailed overview. tRNAs play multiple regulatory roles in the adaptation of protein synthesis to the cellular stress response	758103	
2.1.1.202	metabolism	enzyme NSUN2 plays a central role in regulation of the stress response pathway, detailed overview. tRNAs play multiple regulatory roles in the adaptation of protein synthesis to the cellular stress response	758103	
2.1.1.202	metabolism	formation of a covalent complex between dual-cysteine RNA:m5C methyltransferases and methylated RNA provides a unique means by which metabolic factors can influence RNA. By controlling the degree of formation of the enzyme-RNA covalent complex, S-adenosyl-L-homocysteine and pH are likely to influence the extent of m5C formation and the rate of release of methylated RNA from RNA:m5C methyltransferases. Metabolite-induced covalent complexes could plausibly affect the processing and function of m5C-containing RNAs	735680	
2.1.1.202	metabolism	m5C methylation of RNA is catalysed by the NOL1/NOP2/Sun domain (NSUN) RNA methyltransferase family, which includes NSUN1-7, as well as the DNA MTase homologue TRDMT1 (formerly DNMT2)	757867	
2.1.1.202	metabolism	on a global level, depletion, overexpression, or expression of catalytically inactive forms of NSUN2, but not NSUN1, NSUN5, or NSUN6, is reported to alter the total amount of m5C detected in the mRNA pool. Roles of m5C RNA methyltransferases in development and disease, overview	756866	
2.1.1.202	metabolism	the GCN4 pathway is not responsible for additional m5C levels on tRNAHis	-, 737227	
2.1.1.202	additional information	analysis of the catalytic mechanism of the RNA:m5C methyltransferase family, structure-function analysis of RNA:m5C methyltransferases, overview. Lys248, Asp323, Cys326 and Cys373 are the residues at the active site of hNSun6, they are strictly conserved in NSun6s and in the entire RNA:m5CMTase family, suggesting their conserved roles in catalysis. Multiple roles of Lys248 and Asp323. Motif IVCys (C326) plays a role in product release. In the hNSun6/tRNA complex, instead of nucleotide flipping, the acceptor region of tRNA undergoes a complicated conformational reconstitution for access to hNSun6	757846	
2.1.1.202	additional information	catalytic mechanism of the enzyme, comparison of NSUN family enzymes, overview. The NSUN family enzymes use the cysteine located in amino acid motif VI for the nucleophilic attack on carbon 6 of the target cytosine in RNA. In all seven human NSUN variants, the catalytic cysteine is preceded by threonine. Hydrogen bonding with the backbone carbonyl of proline and the aspartate side chain in motif IV orients the base in the active site and assists bond formation by transient protonation of the endocyclic N3 of cytidine. The activated nucleobase then accepts a methyl group from the properly positioned SAM cofactor, resulting in the formation of a carbon-carbon bond and generation of S-adenosylhomocysteine (SAH). To complete the reaction, the covalently bound methylated RNA has to be released from the protein. This elimination is assisted by the cysteine located in motif IV of NSUN proteins. This cysteine is located next to the conserved proline and acts as a base to deprotonate the tetrahedral carbon and initiate the elimination reaction that restores the unsaturated m5C heterocycle	756866	
2.1.1.202	additional information	development of m5C-responsive probes, as a strategy for discriminating RNA and DNA m5C methyltransferase activity in cells (cf. EC 2.1.1.37), i.e. the m5C-switchable probe strategy, method development and evaluation, overview. The m5C-probe contains a 5'-terminal fluorescent nucleotide PC (2'-O-methyl 6-phenylpyrrolocytidine), which lights up spontaneously in response to m5C-induced terminal sugar pucker switch. When the probe is unmethylated, pC is able to base-pair with guanine in the complementary strand and stack strongly with its adjacent base. This results in efficient quenching of pC fluorescence through photoinduced electron transfer. m5C methylation of the probe by RNA:m5C MTase (e.g. NSUN2), however, is expected to trigger a C2'-endo to C3'-endo sugar pucker switch in PC and, since the sugar ring pucker defines the glycosidic bond angle, such a change in sugar puckering will also convert the orientation of PC base from axial to equatorial. This, in turn, disrupts its base-pairing and base-stacking interactions, leading to fluorescence activation. Schematic representation of 2'-OMe RNA probes and their methylated counterparts. The composition of the probes is confirmed through MALDI-TOF mass spectrometric analysis	757867	
2.1.1.202	additional information	four active-site residues critical for Trm4p-mediated tRNA methylation are also required for the formation of the denaturant-resistant complexes with m5C-containing RNA	735680	
2.1.1.202	additional information	tRNAHis m5C levels are unusually responsive to yeast growth conditions	-, 737227	
2.1.1.202	physiological function	cytoplasmic transfer RNAs are methylated by NSUN2, NSUN6, and DNMT2. NSUN6 specifically methylates C72 of particular tRNAs. U73, which has been termed the discriminator base, is critical for substrate recognition by NSUN6, and a flexible base pair (A:U or U:A) at positions 2:71 as well as a rigid base pair (C:G or G:C) formed between positions 3:70 are preferred. The binding pocket of human NSUN6 specifically accommodates U73. Roles of m5C RNA methyltransferases in development and disease, overview	756866	
2.1.1.202	physiological function	cytoplasmic transfer RNAs are methylated by NSUN2, NSUN6, and DNMT2. Structural differences in PhNSUN6 enable the archaeal enzyme to bind tRNAs containing either U73 or G73, thereby broadening its target spectrum compared to its human homologue enzyme, comparison of NSUN family enzymes, overview. The NSUN family enzymes use the cysteine located in amino acid motif VI for the nucleophilic attack on carbon 6 of the target cytosine in RNA. In all seven human NSUN variants, the catalytic cysteine is preceded by threonine. Hydrogen bonding with the backbone carbonyl of proline and the aspartate side chain in motif IV orients the base in the active site and assists bond formation by transient protonation of the endocyclic N3 of cytidine. The activated nucleobase then accepts a methyl group from the properly positioned SAM cofactor, resulting in the formation of a carbon-carbon bond and generation of S-adenosylhomocysteine (SAH). To complete the reaction, the covalently bound methylated RNA has to be released from the protein. This elimination is assisted by the cysteine located in motif IV of NSUN proteins. This cysteine is located next to the conserved proline and acts as a base to deprotonate the tetrahedral carbon and initiate the elimination reaction that restores the unsaturated m5C heterocycle. m5C72 is reported to promote the thermal stability of PhtRNAs. Roles of m5C RNA methyltransferases in development and disease, overview	-, 756866	
2.1.1.202	physiological function	cytosine-5 methylation in RNA is mediated by a large protein family of conserved RNA:m5C-methyltransferases. NSUN2 is one member of this family and methylates the vast majority of tRNAs as well as a small number of other non-coding (ncRNAs) and coding RNAs (cRNAs). The correct deposition of m5C into RNAs is essential for normal development. Cytosine-5 RNA methylation regulates neural stem cell differentiation and motility. The correct deposition of m5C into RNAs is essential for normal development	758477	
2.1.1.202	physiological function	enzymes of the cytosine-5 RNA methyltransferase Trm4/NSun2 family methylate tRNAs at C48 and C49 in multiple tRNAs, as well as C34 and C40 in selected tRNAs. Trm4a is responsible for all C48 methylation, which lies in the tRNA variable loop, as well as for C34 in tRNALeuCAA and tRNAProCGG, which are at the anticodon wobble position. Trm4a displays intron-dependent methylation of C34	-, 757846	
2.1.1.202	physiological function	Enzymes of the cytosine-5 RNA methyltransferase Trm4/NSun2 family methylate tRNAs at C48 and C49 in multiple tRNAs, as well as C34 and C40 in selected tRNAs. Trm4b methylates C49 and C50, which both lie in the TPsiC-stem. Trm4b activity of methylation of C34 is independent of the intron	-, 757846	
2.1.1.202	physiological function	no evidence for a major role of NSun2 or NSun2-mediated cytosine-5 methylation in mRNA stability	735907	
2.1.1.202	physiological function	post-transcriptional methylation of RNA cytosine residues to 5-methylcytosine (m5C) is an important modification that regulates RNA metabolism	735864	
2.1.1.202	physiological function	post-transcriptional methylation of RNA cytosine residues to 5-methylcytosine is an important modification that regulates RNA metabolism. Nuclear tRNA methylation requires two evolutionarily conserved methyltransferases, TRDMT1 and TRM4B	735864	
2.1.1.202	physiological function	the cytosine-5 RNA methyltransferase NSUN2 is a sensor for external stress stimuli. NSUN2-driven RNA methylation is functionally required to adapt cell cycle progression to the early stress response. The nucleolus, where NSUN2 resides, can act as a stress sensor. Dynamic changes of site-specific Methylcytosine (m5C) levels require NSUN2. m5C is required to balance anabolic and catabolic pathways during the stress response	758103	
2.1.1.202	physiological function	the enzyme is responsible for complete m5C methylation of yeast tRNA	639471	
2.1.1.202	physiological function	the m5C-48/49/50 modifications installed by NSUN2 cluster within the variable loop at the junction with the T-stem. A Levitt pair interaction between C48 and G15 in the D-loop is critical for formation of the characteristic L-shaped tertiary fold of most tRNAs. NSUN2-mediated methylations within the variable loop have also been shown to protect tRNAs against stress-induced, angiogenin-mediated endonucleolytic cleavage. During the maturation of the cytoplasmic tRNALeu(CAA), an m5C34 modification is installed by NSUN2 (cf. EC 2.1.1.203). Cytoplasmic transfer RNAs are methylated by NSUN2, NSUN6, and DNMT2	756866	
2.1.1.202	physiological function	TRM4B encodes a potential mRNA m5C methyltransferase (RCMT). 5-methylcytosine (m5C) is a DNA modification predominantly reported in abundant non-coding RNAs in both prokaryotes and eukaryotes. tRNA-specific methyltransferase 4B (TRM4B) serves as a potential mRNA m5C methyltransferase. Transcriptome-wide profiling of m5C RNA in Arabidopsis thaliana is made by applying m5C RNA immunoprecipitation followed by a deepsequencing approach (m5C-RIP-seq). LC-MS/MS and dot blot analyses reveal a dynamic pattern of m5C mRNA modification in various tissues and at different developmental stages. m5C-RIP-seq analysis identified 6045m5C peaks in 4465 expressed genes in young seedlings. m5C is enriched in coding sequences with two peaks located immediately after start codons and before stop codons, and is associated with mRNAs with low translation activity. An RNA (cytosine-5)-methyltransferase, tRNA-specific methyltransferase 4B (TRM4B), exhibits m5C RNA methyltransferase activity positively correlated with transcript levels of genes in root development. m5C in mRNA is an epitranscriptome marker in Arabidopsis thaliana, and the regulation of this modification is an integral part of gene regulatory networks underlying plant development	757702