TET-mediated 5-methylcytosine oxidation in tRNA promotes translation
2020; Elsevier BV; Volume: 296; Linguagem: Inglês
10.1074/jbc.ra120.014226
ISSN1083-351X
AutoresHui Shen, Robert Jordan Ontiveros, Michael C. Owens, Monica Yun Liu, Uday Ghanty, Rahul M. Kohli, Kathy Fange Liu,
Tópico(s)Cancer-related gene regulation
ResumoOxidation of 5-methylcytosine (5mC) in DNA by the ten-eleven translocation (TET) family of enzymes is indispensable for gene regulation in mammals. More recently, evidence has emerged to support a biological function for TET-mediated m5C oxidation in messenger RNA. Here, we describe a previously uncharacterized role of TET-mediated m5C oxidation in transfer RNA (tRNA). We found that the TET-mediated oxidation product 5-hydroxylmethylcytosine (hm5C) is specifically enriched in tRNA inside cells and that the oxidation activity of TET2 on m5C in tRNAs can be readily observed in vitro. We further observed that hm5C levels in tRNA were significantly decreased in Tet2 KO mouse embryonic stem cells (mESCs) in comparison with wild-type mESCs. Reciprocally, induced expression of the catalytic domain of TET2 led to an obvious increase in hm5C and a decrease in m5C in tRNAs relative to uninduced cells. Strikingly, we also show that TET2-mediated m5C oxidation in tRNA promotes translation in vitro. These results suggest TET2 may influence translation through impacting tRNA methylation and reveal an unexpected role for TET enzymes in regulating multiple nodes of the central dogma. Oxidation of 5-methylcytosine (5mC) in DNA by the ten-eleven translocation (TET) family of enzymes is indispensable for gene regulation in mammals. More recently, evidence has emerged to support a biological function for TET-mediated m5C oxidation in messenger RNA. Here, we describe a previously uncharacterized role of TET-mediated m5C oxidation in transfer RNA (tRNA). We found that the TET-mediated oxidation product 5-hydroxylmethylcytosine (hm5C) is specifically enriched in tRNA inside cells and that the oxidation activity of TET2 on m5C in tRNAs can be readily observed in vitro. We further observed that hm5C levels in tRNA were significantly decreased in Tet2 KO mouse embryonic stem cells (mESCs) in comparison with wild-type mESCs. Reciprocally, induced expression of the catalytic domain of TET2 led to an obvious increase in hm5C and a decrease in m5C in tRNAs relative to uninduced cells. Strikingly, we also show that TET2-mediated m5C oxidation in tRNA promotes translation in vitro. These results suggest TET2 may influence translation through impacting tRNA methylation and reveal an unexpected role for TET enzymes in regulating multiple nodes of the central dogma. Along with the post-translational modifications of histone proteins, the direct, reversible methylation of cytosines in CG dinucleotides in DNA (called CpG sites) is one of several layers of regulatory information that determines chromatin state (1Smith Z.D. Meissner A. DNA methylation: roles in mammalian development.Nat. Rev. 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Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1.Science. 2009; 324: 930-935Crossref PubMed Scopus (3778) Google Scholar, 5He Y.F. Li B.Z. Li Z. Liu P. Wang Y. Tang Q. Ding J. Jia Y. Chen Z. Li L. Sun Y. Li X. Dai Q. Song C.X. Zhang K. et al.Tet-mediated formation of 5-carboxylcytosine and its excision by TDG in mammalian DNA.Science. 2011; 333: 1303-1307Crossref PubMed Scopus (1789) Google Scholar, 6Ito S. Shen L. Dai Q. Wu S.C. Collins L.B. Swenberg J.A. He C. Zhang Y. Tet proteins can convert 5-methylcytosine to 5-formylcytosine and 5-carboxylcytosine.Science. 2011; 333: 1300-1303Crossref PubMed Scopus (2178) Google Scholar). Biochemical assays suggest that one member of the TET family, TET2, works on both DNA and RNA as well (7DeNizio J.E. Liu M.Y. Leddin E.M. Cisneros G.A. Kohli R.M. Selectivity and promiscuity in TET-mediated oxidation of 5-methylcytosine in DNA and RNA.Biochemistry. 2019; 58: 411-421Crossref PubMed Scopus (13) Google Scholar), and recent findings have begun to reveal the biological function of TET-mediated oxidation in RNA. One study suggested that TET-mediated oxidation in mRNA promotes global protein synthesis in Drosophila (8Delatte B. Wang F. Ngoc L.V. Collignon E. Bonvin E. Deplus R. Calonne E. Hassabi B. Putmans P. Awe S. Wetzel C. Kreher J. Soin R. Creppe C. Limbach P.A. et al.RNA biochemistry. Transcriptome-wide distribution and function of RNA hydroxymethylcytosine.Science. 2016; 351: 282-285Crossref PubMed Scopus (216) Google Scholar). Several earlier studies have also shown that TET-mediated mRNA oxidation decreases stability. These effects may result from ADAR1-mediated repression of the target genes (9Shen Q.C. Zhang Q. Shi Y. Shi Q.Z. Jiang Y.Y. Gu Y. Li Z.Q. Li X. Zhao K. Wang C.M. Li N. Cao X.T. Tet2 promotes pathogen infection-induced myelopoiesis through mRNA oxidation.Nature. 2018; 554: 123-127Crossref PubMed Scopus (70) Google Scholar). Conversely, 5-methylcytosine in RNA (abbreviated as m5C) can also promote mRNA stability through specific m5C reader proteins (10Chen X. Li A. Sun B.F. Yang Y. Han Y.N. Yuan X. Chen R.X. Wei W.S. Liu Y.C. Gao C.C. Chen Y.S. Zhang M.M. Ma X.D. Liu Z.W. Luo J.H. et al.5-methylcytosine promotes pathogenesis of bladder cancer through stabilizing mRNAs.Nat. Cell Biol. 2019; 21: 978-990Crossref PubMed Scopus (102) Google Scholar, 11Yang Y. Wang L. Han X. Yang W.L. Zhang M. Ma H.L. Sun B.F. Li A. Xia J. Chen J. Heng J. Wu B. Chen Y.S. Xu J.W. Yang X. et al.RNA 5-methylcytosine facilitates the maternal-to-zygotic transition by preventing maternal mRNA decay.Mol. Cell. 2019; 75: 1188-1202Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). m5C oxidation has been postulated to disrupt the binding of these m5C-specific readers and thereby tune stability. While the abundance and occupancy of m5C sites in mRNA remain under investigation, m5C is highly abundant in tRNA. The majority of known functional roles of m5C in RNA species are also from the studies of m5C sites in tRNAs. In tRNA, m5C sites occur most often at the junction of the variable loop and the T stem-loop. The modification of three cytosines spanning positions 47 to 5-0 has been suggested to stabilize the tRNA structure (12Trixl L. Lusser A. The dynamic RNA modification 5-methylcytosine and its emerging role as an epitranscriptomic mark.Wiley Interdiscip. Rev. RNA. 2019; 10e1510Crossref PubMed Scopus (78) Google Scholar, 13Vare V.Y. Eruysal E.R. Narendran A. Sarachan K.L. Agris P.F. Chemical and conformational diversity of modified nucleosides affects tRNA structure and function.Biomolecules. 2017; 7: 29Crossref PubMed Scopus (72) Google Scholar). Cytosine 38 in the anticodon loop of the tRNA is another frequently modified site. m5C38 in mouse tRNAAsp has been shown to stimulate amino acid charging of the tRNA and to facilitate translation of poly-Asp–containing proteins (14Shanmugam R. Fierer J. Kaiser S. Helm M. Jurkowski T.P. Jeltsch A. Cytosine methylation of tRNA-Asp by DNMT2 has a role in translation of proteins containing poly-Asp sequences.Cell Discov. 2015; 1: 15010Crossref PubMed Scopus (31) Google Scholar). In addition, m5C38 can protect tRNAs from stress-induced endonuclease-mediated fragmentation (15Schaefer M. Pollex T. Hanna K. Tuorto F. Meusburger M. Helm M. Lyko F. RNA methylation by Dnmt2 protects transfer RNAs against stress-induced cleavage.Genes Dev. 2010; 24: 1590-1595Crossref PubMed Scopus (386) Google Scholar, 16Tuorto F. Liebers R. 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Helm M. tRNA stabilization by modified nucleotides.Biochemistry. 2010; 49: 4934-4944Crossref PubMed Scopus (273) Google Scholar, 25Squires J.E. Preiss T. Function and detection of 5-methylcytosine in eukaryotic RNA.Epigenomics. 2010; 2: 709-715Crossref PubMed Scopus (33) Google Scholar, 26Boccaletto P. Machnicka M.A. Purta E. Piatkowski P. Baginski B. Wirecki T.K. de Crecy-Lagard V. Ross R. Limbach P.A. Kotter A. Helm M. Bujnicki J.M. MODOMICS: a database of RNA modification pathways. 2017 update.Nucleic Acids Res. 2018; 46: D303-D307Crossref PubMed Scopus (673) Google Scholar). The m5C sites in RNA are installed by several methyltransferases, including tRNA aspartic acid MTase1 (TRDMT1), Dnmt2, NOP2/Sun domain protein 2 (NSUN2), Nsun3 (20Haag S. Sloan K.E. Ranjan N. Warda A.S. Kretschmer J. Blessing C. Hubner B. Seikowski J. Dennerlein S. Rehling P. Rodnina M.V. Hobartner C. Bohnsack M.T. NSUN3 and ABH1 modify the wobble position of mt-tRNAMet to expand codon recognition in mitochondrial translation.EMBO J. 2016; 35: 2104-2119Crossref PubMed Scopus (98) Google Scholar, 21Nakano S. Suzuki T. Kawarada L. Iwata H. Asano K. Suzuki T. NSUN3 methylase initiates 5-formylcytidine biogenesis in human mitochondrial tRNA(Met).Nat. Chem. Biol. 2016; 12: 546-551Crossref PubMed Scopus (80) Google Scholar), and Nsun6 (27Long T. Li J. Li H. Zhou M. Zhou X.L. Liu R.J. Wang E.D. Sequence-specific and shape-selective RNA recognition by the human RNA 5-methylcytosine methyltransferase NSun6.J. Biol. Chem. 2016; 291: 24293-24303Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). The biological significance of m5C in RNA is further emphasized by genetic studies. For instance, knockout of NSUN2 in mice causes male infertility and reduced growth (28Hussain S. Tuorto F. Menon S. Blanco S. Cox C. Flores J.V. Watt S. Kudo N.R. Lyko F. Frye M. The mouse cytosine-5 RNA methyltransferase NSun2 is a component of the chromatoid body and required for testis differentiation.Mol. Cell Biol. 2013; 33: 1561-1570Crossref PubMed Scopus (89) Google Scholar), while mutations in human NSUN2 are involved in intellectual disability (29Abbasi-Moheb L. Mertel S. Gonsior M. Nouri-Vahid L. Kahrizi K. Cirak S. Wieczorek D. Motazacker M.M. Esmaeeli-Nieh S. Cremer K. Weissmann R. Tzschach A. Garshasbi M. Abedini S.S. Najmabadi H. et al.Mutations in NSUN2 cause autosomal-recessive intellectual disability.Am. J. Hum. Genet. 2012; 90: 847-855Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). Lastly, DNMT2 deficiency has also been shown to affect polypeptide synthesis in humans (16Tuorto F. Liebers R. Musch T. Schaefer M. Hofmann S. Kellner S. Frye M. Helm M. Stoecklin G. Lyko F. RNA cytosine methylation by Dnmt2 and NSun2 promotes tRNA stability and protein synthesis.Nat. Struct. Mol. Biol. 2012; 19: 900-905Crossref PubMed Scopus (275) Google Scholar). Interestingly, cytoplasmic and mitochondrial tRNAs have been shown to carry f5C, an oxidation product of m5C. The alpha-ketoglutaric acid–dependent dioxygenase ALKBH1 has been shown to be involved in the biogenesis of f5C at the first position of the anticodon (position 34 of canonical tRNAs) in mitochondrial tRNAMet (20Haag S. Sloan K.E. Ranjan N. Warda A.S. Kretschmer J. Blessing C. Hubner B. Seikowski J. Dennerlein S. Rehling P. Rodnina M.V. Hobartner C. Bohnsack M.T. NSUN3 and ABH1 modify the wobble position of mt-tRNAMet to expand codon recognition in mitochondrial translation.EMBO J. 2016; 35: 2104-2119Crossref PubMed Scopus (98) Google Scholar, 30Kawarada L. Suzuki T. Ohira T. Hirata S. Miyauchi K. Suzuki T. ALKBH1 is an RNA dioxygenase responsible for cytoplasmic and mitochondrial tRNA modifications.Nucleic Acids Res. 2017; 45: 7401-7415Crossref PubMed Scopus (73) Google Scholar). f5C of mt-tRNAMet is important for the decoding of AUA methionine codons during mitochondrial translation (31Takemoto C. Spremulli L.L. Benkowski L.A. Ueda T. Yokogawa T. Watanabe K. Unconventional decoding of the AUA codon as methionine by mitochondrial tRNAMet with the anticodon f5CAU as revealed with a mitochondrial in vitro translation system.Nucleic Acids Res. 2009; 37: 1616-1627Crossref PubMed Scopus (63) Google Scholar). Additionally, ALKBH1 had also been shown to catalyze the formation of 5-hydroxymethyl-2-O-methylcytidine (hm5Cm) and 5-formyl-2-O-methylcytidine (f5Cm) at the same position in cytoplasmic tRNALeu (32Huber S.M. van Delft P. Tanpure A. Miska E.A. Balasubramanian S. 2'-O-Methyl-5-hydroxymethylcytidine: a second oxidative derivative of 5-methylcytidine in RNA.J. Am. Chem. Soc. 2017; 139: 1766-1769Crossref PubMed Scopus (17) Google Scholar). ALKBH1-catalyzed oxidation reactions are important for translation and mitochondrial function. However, m5C oxidation in tRNALeu and mitochondrial tRNAiMet is specifically carried out by ALKBH1, not the TET enzymes (30Kawarada L. Suzuki T. Ohira T. Hirata S. Miyauchi K. Suzuki T. ALKBH1 is an RNA dioxygenase responsible for cytoplasmic and mitochondrial tRNA modifications.Nucleic Acids Res. 2017; 45: 7401-7415Crossref PubMed Scopus (73) Google Scholar). Whether TET-mediated m5C oxidation occurs on tRNA species as well as their biological functions are not fully understood. All these previous studies have highlighted the importance of the reversible regulation of m5C in tRNA. Here, we investigated whether TET2 can catalyze tRNA m5C oxidation and the potential biological function from this oxidation reaction. To study whether TET enzymes catalyze oxidation on m5C in other RNA species, we first quantified the levels of hm5C (the first m5C oxidation product, Fig. 1A) in several major RNA species. We extracted total RNA, 18S rRNA, 28S rRNA, polyadenylated RNA (poly(A)-RNA), and tRNA from mouse embryonic stem cells (mESCs) and HEK 293T cells (Fig. S1, A–D). The extracted RNAs were then degraded to single nucleosides before being analyzed via triple quadrupole liquid chromatography and tandem mass spectrometry (LC-MS/MS). The LC-MS/MS results show that among these different RNA species, hm5C is more significantly enriched in tRNAs in both mESCs and HEK293T cells (Fig. 1, B–C, Fig. S1E, and Fig. S2). In light of recent discoveries that TET2 may be responsible for modifying RNA in cells (33He C. Sidoli S. Warneford-Thomson R. Tatomer D.C. Wilusz J.E. Garcia B.A. Bonasio R. High-resolution mapping of RNA-binding regions in the nuclear proteome of embryonic stem cells.Mol. Cell. 2016; 64: 416-430Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar), we focused on the role of TET2 in accounting for the observed enrichment of hm5C in tRNA. To investigate if m5C can be oxidized by TET2, we conducted in vitro oxidation assays. The activity of a purified TET2 truncation variant (TET2-CS, previously crystallized (34Hu L.L. Li Z. Cheng J.D. Rao Q.H. Gong W. Liu M.J. Shi Y.J.G. Zhu J.Y. Wang P. Xu Y.H. Crystal structure of TET2-DNA complex: insight into TET-mediated 5mC oxidation.Cell. 2013; 155: 1545-1555Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar), Fig. 2A) was first confirmed on a 5mC-containing ssDNA oligo (Fig. S3, A–C). We then incubated tRNAs purified from HEK293T cells either with TET2-CS or in buffer alone. To investigate whether tRNA structure has an impact in TET-mediated oxidation, we used both native and denatured tRNAs in this biochemical oxidation reaction. Analysis of the oxidation products via LC-MS/MS clearly shows that TET2-CS is capable of oxidizing m5C to hm5C in tRNA in vitro (Fig. 2, B–C), while the further oxidation products, f5C and ca5C, were not detected (Fig. S3, D–E). However, in the presence of EDTA (an iron chelator), TET2-CS cannot lead to decreased m5C and increased hm5C in tRNA (Fig. S4). Given that we observed that the enrichment of hm5C in tRNA and that TET2 is capable of generating this modification in vitro, we sought to study if TET2-mediated oxidation generates tRNA hm5C in cells. To this end, we quantified m5C and hm5C levels in tRNAs extracted from wild-type and Tet2 KO mESCs (Fig. 3). The results show that Tet2 KO leads to a significant decrease of hm5C in tRNA. We also observed a noticeable, but not significant, increase of m5C in tRNA in Tet2 KO mESCs in comparison with the wild-type mESCs (Fig. 3, A–B and Fig. S5A). Although the expression of TET2 in mESCs leads to the starkly obvious increases of hm5C in tRNAs, we observed a low level of hm5C in tRNA. These results suggest that TET2 does not demethylate all m5C sites in tRNAs. It is possible that TET2 works with other demethylase enzymes such as ALKBH1 (30Kawarada L. Suzuki T. Ohira T. Hirata S. Miyauchi K. Suzuki T. ALKBH1 is an RNA dioxygenase responsible for cytoplasmic and mitochondrial tRNA modifications.Nucleic Acids Res. 2017; 45: 7401-7415Crossref PubMed Scopus (73) Google Scholar) to work on individual subsets of tRNAs. To further confirm that TET2 is responsible for tRNA m5C oxidation in cells, we first constructed a doxycycline-inducible expression system for the TET2 catalytic domain (TET2-CD) in HEK293T cell line (35Khandelia P. Yap K. Makeyev E.V. Streamlined platform for short hairpin RNA interference and transgenesis in cultured mammalian cells.Proc. Natl. Acad. Sci. U. S. A. 2011; 108: 12799-12804Crossref PubMed Scopus (49) Google Scholar). Using this expression system, TET2-CD expression can be titrated to near-physiological expression levels, thereby avoiding any potential artifacts caused by overexpression (Fig. 2A and Fig. S5, B–C). We then validated that this increase in TET2-CD expression resulted in an increase in TET2-CD activity. To do this, we performed dot-blot assays using anti-5mC and anti-5hmC antibodies to further validate our LC-MS/MS findings (Fig. S5D). In addition, we quantified and compared 5mC and 5hmC levels in DNA in our cells before and after doxycycline treatment using LC-MS/MS. The results show that induced expression of TET2-CD leads to decreased levels of 5mC and increased 5hmC levels in DNA (Fig. S5, E–F). These results were consistent with the results from the dot-blot assay and together collectively suggested that our doxycycline-inducible system can successfully induce the expression of functional TET2-CD. After we validated the inducible cell line, we next quantified m5C and hm5C levels in tRNA before and after doxycycline treatment. The LC-MS/MS results show that induced expression of the catalytic domain of TET2 leads to significantly increased hm5C levels in tRNA (Fig. 3, C–D) with no obvious change of m5C in tRNA (Fig. S5G). We reasoned that it is due to the higher level of m5C in comparison with hm5C in tRNA; also TET2 is not the only tRNA m5C demethylase. In contrast, the parental HEK293T cell line (which we used to construct the inducible cell line) did not show any change of both m5C and hm5C levels before and after doxycycline treatment (Fig. 3D and Fig. S5, G–H). Together with the results from Tet2 KO mESCs, these results revealed that TET2-mediated m5C oxidation occurs on tRNA inside cells. After observing tRNA as a target for TET-mediated oxidation, we investigated the possible biological consequences of TET2-mediated oxidation on m5C in tRNA. Given that tRNA modifications can tune translational efficiency (36Nedialkova D.D. Leidel S.A. Optimization of codon translation rates via tRNA modifications maintains proteome integrity.Cell. 2015; 161: 1606-1618Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar), it is possible that TET-mediated oxidation of tRNAs could affect translation. To investigate this hypothesis, we utilized a rabbit reticulocyte-based in vitro translation system to measure the production of active luciferase protein from a fixed amount of luciferase mRNA (Fig. 4A). To probe how TET2-mediated oxidation of tRNAs affects translation, we first extracted tRNAs from either wild-type or Tet2 KO mESCs. We then spiked-in increasing amounts (100, 300, 500, and 1000 ng) of purified tRNAs into separate translation reactions along with the luciferase mRNA (uncapped in vitro-transcribed RNA containing a 30-base poly(A) tail from Promega) and measured luciferase activity. The results show that tRNAs originating from wild-type mESC cells lead to significant increase of luciferase activity. In contrast, spiking in tRNAs extracted from the Tet2 KO cells did not lead to increased luciferase signals (Fig. 4B). We also performed the in vitro translation assays using tRNAs extracted from TET2-inducible expression cells before and after administration of doxycycline. The results show that tRNAs originating from cells after doxycycline addition lead to an obvious increase of luciferase signal whereas the tRNAs from untreated cells elicit a relatively lower luciferase signal (Fig. 4C). In this in vitro translation reaction, we can rule out the possibility that TET2 proteins bind to the luciferase mRNA to promote translation since the reaction system only contains supplemented tRNAs, luciferase mRNA, and reticulocytes. Furthermore, we studied whether the expression of TET2 leads to increased translation inside cells. To this end, we quantified protein synthesis in TET2-induced and uninduced conditions using puromycin incorporation followed by Western blot analysis. The results showed that the expression of TET2 did not lead to an obvious impact on overall translation (Fig. S6A). It would be insightful to investigate the impact of TET2-mediated tRNA oxidation on specific transcripts in future studies. We also sought to identify the specific tRNA targets of TET2. Since NSUN2 is one of the most studied tRNA m5C methyltransferase enzymes, we examined whether TET2 works on the m5C-containing tRNA targets of NSUN2 (16Tuorto F. Liebers R. Musch T. Schaefer M. Hofmann S. Kellner S. Frye M. Helm M. Stoecklin G. Lyko F. RNA cytosine methylation by Dnmt2 and NSun2 promotes tRNA stability and protein synthesis.Nat. Struct. Mol. Biol. 2012; 19: 900-905Crossref PubMed Scopus (275) Google Scholar). As shown in updated Figure S6B, we quantified the levels of m5C and hm5C levels in four tRNAs extracted from Tet2 KO and wild-type mESCs. The results showed that Tet2 KO leads to a significant increase of hm5C and a noticed decrease of m5C level in tRNAGly (Fig. S6B). The effects of TET2-mediated oxidation are not obvious on the other three tRNAs including tRNAAsp, tRNAVal, and tRNALeu (Fig. S6B). These results collectively suggested that tRNAGly is a possible target of TET2. The presence of an appreciable level of hm5C in cellular RNA and the involvement of the TET family of enzymes in producing this modification support the hypothesis that the function of TET enzymes is not restricted to epigenetic regulation at the DNA level. Our results suggest that TET2 can oxidize m5C and generate hm5C on tRNAs in mammalian cells. We show that hm5C is particularly abundant in tRNAs in comparison with other major RNA species including rRNA and poly(A)-RNAs. This suggests that tRNA is possibly a major RNA target of TET2. Our finding that TET2 can oxidize m5C on tRNA inside cells raises questions regarding the localization and timing of this activity. Given that TET2 is known to localize to the nucleus (37Arioka Y. Watanabe A. Saito K. Yamada Y. Activation-induced cytidine deaminase alters the subcellular localization of Tet family proteins.PLoS One. 2012; 7e45031Crossref PubMed Scopus (30) Google Scholar), it is likely that the oxidation of tRNAs is performed in that compartment as a step in pre-tRNA maturation following transcription and m5C installation by NSUN2/DNMT2 (16Tuorto F. Liebers R. Musch T. Schaefer M. Hofmann S. Kellner S. Frye M. Helm M. Stoecklin G. Lyko F. RNA cytosine methylation by Dnmt2 and NSun2 promotes tRNA stability and protein synthesis.Nat. Struct. Mol. Biol. 2012; 19: 900-905Crossref PubMed Scopus (275) Google Scholar). These nascent pre-tRNAs would likely present accessible m5C sites, some of which may become occluded once fully folded and mature. While pre-tRNAs are the most likely primary tRNA substrate of the TET2-CD in our system, this is not the only possibility. Mature tRNAs are known to be transported into the nucleus via a retrograde importation mechanism (38Takano A. Endo T. Yoshihisa T. tRNA actively shuttles between the nucleus and cytosol in yeast.Science. 2005; 309: 140-142Crossref PubMed Scopus (129) Google Scholar, 39Shaheen H.H. Hopper A.K. Retrograde movement of tRNAs from the cytoplasm to the nucleus in Saccharomyces cerevisiae.Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 11290-11295Crossref PubMed Scopus (133) Google Scholar, 40Shaheen H.H. Horetsky R.L. Kimball S.R. Murthi A. Jefferson L.S. Hopper A.K. Retrograde nuclear accumulation of cytoplasmic tRNA in rat hepatoma cells in response to amino acid deprivation.Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 8845-8850Crossref PubMed Scopus (74) Google Scholar); thus, it is also possible that mature tRNAs are a substrate of the TET2-CD. As our results suggest, the decrease of m5C levels in tRNA upon expression of the TET2-CD is reproducible yet not significant while the increase of hm5C in tRNA is significant, (Fig. 3, C–D and Fig. S7G). This implies that only a subset of tRNAs and possibly only specific but not all m5C sites in tRNAs are the targets of the TET2-CD in our system, a hypothesis which is supported by the previous findings of ALKBH1-mediated tRNA m5C oxidation (30Kawarada L. Suzuki T. Ohira T. Hirata S. Miyauchi K. Suzuki T. ALKBH1 is an RNA dioxygenase responsible for cytoplasmic and mitochondrial tRNA modifications.Nucleic Acids Res. 2017; 45: 7401-7415Crossref PubMed Scopus (73) Google Scholar). Furthermore, whether TET1 and TET3 can oxidize m5C in tRNAs is unknown. Given that TET3 resides in both the cell nucleus and the cytoplasm (41Zhang Q. Liu X. Gao W. Li P. Hou J. Li J. Wong J. Differential regulation of the ten-eleven translocation (TET) family of dioxygenases by O-linked beta-N-acetylglucosamine transferase (OGT).J. Biol. Chem. 2014; 289: 5986-5996Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar), cytoplasmic TET3 might have optimal accessibility to mature tRNAs. It is possible that tRNA m5C sites are under special and temporal regulation. Along the tRNA maturation process, it is possible that several demethylase enzymes can work on different tRNA m5C sites in both the nucleus and the cytoplasm to facilitate proper tRNA biogenesis and regulation. In addition, it is known that reprogramming of m5C34 in tRNA facilitates translation of specific ribosomal proteins upon oxidative stress to sustain life (42Chan C.T. Pang Y.L. Deng W. Babu I.R. Dyavaiah M. Begley T.J. Dedon P.C. Reprogramming of tRNA modifications controls the oxidative stress response by codon-biased translation of proteins.Nat. Commun. 2012; 3: 937Crossref PubMed Scopus (254) Google Scholar). The dynamic regulation of the individual sites of m5C in tRNAs by distinct enzymes may represent another layer of translational control, especially when faced with different types of environmental stresses. This work provi
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