Structure of the inner kinetochore CCAN complex assembled onto a centromeric nucleosome.

In eukaryotes, accurate chromosome segregation in mitosis and meiosis maintains genome stability and prevents aneuploidy. Kinetochores are large protein complexes that, by assembling onto specialized Cenp-A nucleosomes1,2, function to connect centromeric chromatin to microtubules of the mitotic spindle3,4. Whereas the centromeres of vertebrate chromosomes comprise millions of DNA base pairs and attach to multiple microtubules, the simple point centromeres of budding yeast are connected to individual microtubules5,6. All 16 budding yeast chromosomes assemble complete kinetochores using a single Cenp-A nucleosome (Cenp-ANuc), each of which is perfectly centred on its cognate centromere7-9. The inner and outer kinetochore modules are responsible for interacting with centromeric chromatin and microtubules, respectively. Here we describe the cryo-electron microscopy structure of the Saccharomyces cerevisiae inner kinetochore module, the constitutive centromere associated network (CCAN) complex, assembled onto a Cenp-A nucleosome (CCAN-Cenp-ANuc). The structure explains the interdependency of the constituent subcomplexes of CCAN and shows how the Y-shaped opening of CCAN accommodates Cenp-ANuc to enable specific CCAN subunits to contact the nucleosomal DNA and histone subunits. Interactions with the unwrapped DNA duplex at the two termini of Cenp-ANuc are mediated predominantly by a DNA-binding groove in the Cenp-L-Cenp-N subcomplex. Disruption of these interactions impairs assembly of CCAN onto Cenp-ANuc. Our data indicate a mechanism of Cenp-A nucleosome recognition by CCAN and how CCAN acts as a platform for assembly of the outer kinetochore to link centromeres to the mitotic spindle for chromosome segregation.

Detection of queuosine and queuosine precursors in tRNAs by direct RNA sequencing.

Queuosine (Q) is a complex tRNA modification found in bacteria and eukaryotes at position 34 of four tRNAs with a GUN anticodon, and it regulates the translational efficiency and fidelity of the respective codons that differ at the Wobble position. In bacteria, the biosynthesis of Q involves two precursors, preQ0 and preQ1, whereas eukaryotes directly obtain Q from bacterial sources. The study of queuosine has been challenging due to the limited availability of high-throughput methods for its detection and analysis. Here, we have employed direct RNA sequencing using nanopore technology to detect the modification of tRNAs with Q and Q precursors. These modifications were detected with high accuracy on synthetic tRNAs as well as on tRNAs extracted from Schizosaccharomyces pombe and Escherichia coli by comparing unmodified to modified tRNAs using the tool JACUSA2. Furthermore, we present an improved protocol for the alignment of raw sequence reads that gives high specificity and recall for tRNAs ex cellulo that, by nature, carry multiple modifications. Altogether, our results show that 7-deazaguanine-derivatives such as queuosine are readily detectable using direct RNA sequencing. This advancement opens up new possibilities for investigating these modifications in native tRNAs, furthering our understanding of their biological function.

A dual-purpose polymerase engineered for direct sequencing of pseudouridine and queuosine.

More than 170 posttranscriptional RNA modifications are so far known on both coding and noncoding RNA species. Within this group, pseudouridine (Ψ) and queuosine (Q) represent conserved RNA modifications with fundamental functional roles in regulating translation. Current detection methods of these modifications, which both are reverse transcription (RT)-silent, are mostly based on the chemical treatment of RNA prior to analysis. To overcome the drawbacks associated with indirect detection strategies, we have engineered an RT-active DNA polymerase variant called RT-KTq I614Y that produces error RT signatures specific for Ψ or Q without prior chemical treatment of the RNA samples. Combining this polymerase with next-generation sequencing techniques allows the direct identification of Ψ and Q sites of untreated RNA samples using a single enzymatic tool.

Functional integration of a semi-synthetic azido-queuosine derivative into translation and a tRNA modification circuit.

Substitution of the queuine nucleobase precursor preQ1 by an azide-containing derivative (azido-propyl-preQ1) led to incorporation of this clickable chemical entity into tRNA via transglycosylation in vitro as well as in vivo in Escherichia coli, Schizosaccharomyces pombe and human cells. The resulting semi-synthetic RNA modification, here termed Q-L1, was present in tRNAs on actively translating ribosomes, indicating functional integration into aminoacylation and recruitment to the ribosome. The azide moiety of Q-L1 facilitates analytics via click conjugation of a fluorescent dye, or of biotin for affinity purification. Combining the latter with RNAseq showed that TGT maintained its native tRNA substrate specificity in S. pombe cells. The semi-synthetic tRNA modification Q-L1 was also functional in tRNA maturation, in effectively replacing the natural queuosine in its stimulation of further modification of tRNAAsp with 5-methylcytosine at position 38 by the tRNA methyltransferase Dnmt2 in S. pombe. This is the first demonstrated in vivo integration of a synthetic moiety into an RNA modification circuit, where one RNA modification stimulates another. In summary, the scarcity of queuosinylation sites in cellular RNA, makes our synthetic q/Q system a 'minimally invasive' system for placement of a non-natural, clickable nucleobase within the total cellular RNA.

The kinetochore module Okp1CENP-Q/Ame1CENP-U is a reader for N-terminal modifications on the centromeric histone Cse4CENP-A.

Kinetochores are supramolecular assemblies that link centromeres to microtubules for sister chromatid segregation in mitosis. For this, the inner kinetochore CCAN/Ctf19 complex binds to centromeric chromatin containing the histone variant CENP-A, but whether the interaction of kinetochore components to centromeric nucleosomes is regulated by posttranslational modifications is unknown. Here, we investigated how methylation of arginine 37 (R37Me) and acetylation of lysine 49 (K49Ac) on the CENP-A homolog Cse4 from Saccharomyces cerevisiae regulate molecular interactions at the inner kinetochore. Importantly, we found that the Cse4 N-terminus binds with high affinity to the Ctf19 complex subassembly Okp1/Ame1 (CENP-Q/CENP-U in higher eukaryotes), and that this interaction is inhibited by R37Me and K49Ac modification on Cse4. In vivo defects in cse4-R37A were suppressed by mutations in OKP1 and AME1, and biochemical analysis of a mutant version of Okp1 showed increased affinity for Cse4. Altogether, our results demonstrate that the Okp1/Ame1 heterodimer is a reader module for posttranslational modifications on Cse4, thereby targeting the yeast CCAN complex to centromeric chromatin.

The ATAD2/ANCCA homolog Yta7 cooperates with Scm3HJURP to deposit Cse4CENP-A at the centromere in yeast.

The AAA+ ATPase and bromodomain factor ATAD2/ANCCA is overexpressed in many types of cancer, but how it contributes to tumorigenesis is not understood. Here, we report that the Saccharomyces cerevisiae homolog Yta7ATAD2 is a deposition factor for the centromeric histone H3 variant Cse4CENP-A at the centromere in yeast. Yta7ATAD2 regulates the levels of centromeric Cse4CENP-A in that yta7∆ causes reduced Cse4CENP-A deposition, whereas YTA7 overexpression causes increased Cse4CENP-A deposition. Yta7ATAD2 coimmunoprecipitates with Cse4CENP-A and is associated with the centromere, arguing for a direct role of Yta7ATAD2 in Cse4CENP-A deposition. Furthermore, increasing centromeric Cse4CENP-A levels by YTA7 overexpression requires the activity of Scm3HJURP, the centromeric nucleosome assembly factor. Importantly, Yta7ATAD2 interacts in vivo with Scm3HJURP, indicating that Yta7ATAD2 is a cochaperone for Scm3HJURP The absence of Yta7 causes defects in growth and chromosome segregation with mutations in components of the inner kinetochore (CTF19/CCAN, Mif2CENP-C, Cbf1). Since Yta7ATAD2 is an AAA+ ATPase and potential hexameric unfoldase, our results suggest that it may unfold the Cse4CENP-A histone and hand it over to Scm3HJURP for subsequent deposition in the centromeric nucleosome. Furthermore, our findings suggest that ATAD2 overexpression may enhance malignant transformation in humans by misregulating centromeric CENP-A levels, thus leading to defects in kinetochore assembly and chromosome segregation.

Queuosine-modified tRNAs confer nutritional control of protein translation.

Global protein translation as well as translation at the codon level can be regulated by tRNA modifications. In eukaryotes, levels of tRNA queuosinylation reflect the bioavailability of the precursor queuine, which is salvaged from the diet and gut microbiota. We show here that nutritionally determined Q-tRNA levels promote Dnmt2-mediated methylation of tRNA Asp and control translational speed of Q-decoded codons as well as at near-cognate codons. Deregulation of translation upon queuine depletion results in unfolded proteins that trigger endoplasmic reticulum stress and activation of the unfolded protein response, both in cultured human cell lines and in germ-free mice fed with a queuosine-deficient diet. Taken together, our findings comprehensively resolve the role of this anticodon tRNA modification in the context of native protein translation and describe a novel mechanism that links nutritionally determined modification levels to effective polypeptide synthesis and cellular homeostasis.

Queuosine salvage in fission yeast by Qng1-mediated hydrolysis to queuine.

Queuosine (Q) is a hypermodified 7-deaza-guanosine nucleoside that is found at position 34, also known as the wobble position, of tRNAs with a GUN anticodon, and Q ensures faithful translation of the respective C- and U-ending codons. While Q is present in tRNAs in most eukaryotes, only bacteria can synthesize it denovo. In contrast, eukaryotes rely on external sources like their food and the gut microbiome in order to Q-modify their tRNAs, and Q therefore can be regarded as a micronutrient. The eukaryotic tRNA guanine transglycosylase (eTGT) uses the base queuine (q) as a substrate to replace G34 by Q in the tRNAs. Eukaryotic cells can uptake both q and Q, raising the question how the Q nucleoside is converted to q for incorporation into the tRNAs. Here, we identified Qng1 (also termed Duf2419) as a queuosine nucleoside glycosylase in Schizosaccharomyces pombe. S. pombe cells with a deletion of qng1+ contained Q-modified tRNAs only when cultured in the presence of the nucleobase q, but not with the nucleoside Q, indicating that the cells are proficient at q incorporation, but not in Q hydrolysis. Furthermore, purified recombinant Qng1 hydrolyzed Q to q in vitro. Qng1 displays homology to DNA glycosylases and has orthologs across eukaryotes, including flies, mice and humans. Qng1 therefore plays an essential role in allowing eukaryotic cells to salvage Q from bacterial sources and to recycle Q from endogenous tRNAs.

Increased CG, CHG and CHH methylation at the cycloidea gene in the "Peloria" mutant of Linaria vulgaris.

Heritable DNA methylation variation is frequently observed in natural populations of plants, but is thought mostly to be functionally inconsequential. An exception to this is the "Peloria" mutant of Linaria vulgaris, which was originally described by Carl von Linné in 1744. A study in 1999 found that the Peloria phenotype is caused by an epiallele of the L. vulgaris cycloidea homolog Lcyc that showed increased levels of DNA methylation compared to wild-type. The DNA methylation results in silencing of Lcyc, which causes radial flower symmetry in the peloric mutant, whereas wild-type plants have flowers with bilateral symmetry. However, a detailed view of DNA methylation at Lcyc at the single-nucleotide level has not been available. In this study, we investigated DNA methylation at Lcyc and, as a control, at the LvHIRZ gene in wild-type and peloric plants of L. vulgaris using DNA bisulfite treatment coupled to next-generation sequencing. We found strong increases in CHG and CHH methylation at Lcyc, but not LvHIRZ, in Peloria. CG methylation was also increased, but wild-type Lcyc also showed moderate levels of CG methylation. Our results suggest that DNA methylation in all three sequence contexts has been maintained, and potentially transgenerationally inherited, in the peloric L. vulgaris population over decades or even centuries.

Queuine links translational control in eukaryotes to a micronutrient from bacteria.

In eukaryotes, the wobble position of tRNA with a GUN anticodon is modified to the 7-deaza-guanosine derivative queuosine (Q34), but the original source of Q is bacterial, since Q is synthesized by eubacteria and salvaged by eukaryotes for incorporation into tRNA. Q34 modification stimulates Dnmt2/Pmt1-dependent C38 methylation (m5C38) in the tRNAAsp anticodon loop in Schizosaccharomyces pombe. Here, we show by ribosome profiling in S. pombe that Q modification enhances the translational speed of the C-ending codons for aspartate (GAC) and histidine (CAC) and reduces that of U-ending codons for asparagine (AAU) and tyrosine (UAU), thus equilibrating the genome-wide translation of synonymous Q codons. Furthermore, Q prevents translation errors by suppressing second-position misreading of the glycine codon GGC, but not of wobble misreading. The absence of Q causes reduced translation of mRNAs involved in mitochondrial functions, and accordingly, lack of Q modification causes a mitochondrial defect in S. pombe. We also show that Q-dependent stimulation of Dnmt2 is conserved in mice. Our findings reveal a direct mechanism for the regulation of translational speed and fidelity in eukaryotes by a nutrient originating from bacteria.

Division of labour: tRNA methylation by the NSun2 tRNA methyltransferases Trm4a and Trm4b in fission yeast.

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. In contrast to most other organisms, fission yeast Schizosaccharomyces pombe carries two Trm4/NSun2 homologs, Trm4a (SPAC17D4.04) and Trm4b (SPAC23C4.17). Here, we have employed tRNA methylome analysis to determine the dependence of cytosine-5 methylation (m5C) tRNA methylation in vivo on the two enzymes. Remarkably, 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. Conversely, Trm4b methylates C49 and C50, which both lie in the TΨC-stem. Thus, S. pombe show 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. Furthermore, in vitro activity assays showed that Trm4a displays intron-dependent methylation of C34, whereas Trm4b activity is independent of the intron. The absence of Trm4a, but not Trm4b, causes a mild resistance of S. pombe to calcium chloride.

Structural basis for the role of the Sir3 AAA+ domain in silencing: interaction with Sir4 and unmethylated histone H3K79.

The silent information regulator 2/3/4 (Sir2/3/4) complex is required for gene silencing at the silent mating-type loci and at telomeres in Saccharomyces cerevisiae. Sir3 is closely related to the origin recognition complex 1 subunit and consists of an N-terminal bromo-adjacent homology (BAH) domain and a C-terminal AAA(+) ATPase-like domain. Here, through a combination of structure biology and exhaustive mutagenesis, we identified unusual, silencing-specific features of the AAA(+) domain of Sir3. Structural analysis of the putative nucleotide-binding pocket in this domain reveals a shallow groove that would preclude nucleotide binding. Mutation of this site has little effect on Sir3 function in vivo. In contrast, several surface regions are shown to be necessary for the Sir3 silencing function. Interestingly, the Sir3 AAA(+) domain is shown here to bind chromatin in vitro in a manner sensitive to histone H3K79 methylation. Moreover, an exposed loop on the surface of this Sir3 domain is found to interact with Sir4. In summary, the unique folding of this conserved Sir3 AAA(+) domain generates novel surface regions that mediate Sir3-Sir4 and Sir3-nucleosome interactions, both being required for the proper assembly of heterochromatin in living cells.

Widespread colocalization of the Drosophila histone acetyltransferase homolog MYST5 with DREF and insulator proteins at active genes.

MYST family histone acetyltransferases play important roles in gene regulation. Here, we have characterized the Drosophila MYST histone acetyltransferase (HAT) encoded by cg1894, whose closest homolog is Drosophila MOF, and which we have termed MYST5. We found it localized to a large number of interbands as well as to the telomeres of polytene chromosomes, and it showed strong colocalization with the interband protein Z4/Putzig and RNA polymerase II. Accordingly, genome-wide location analysis by ChIP-seq showed co-occurrence of MYST5 with the Z4-interacting partner Chriz/Chromator. Interestingly, MYST5 bound to the promoter of actively transcribed genes, and about half of MYST5 sites colocalized with the transcription factor DNA replication-related element-binding factor (DREF), indicating a role for MYST5 in gene expression. Moreover, we observed substantial overlap of MYST5 binding with that of the insulator proteins CP190, dCTCF, and BEAF-32, which mediate the organization of the genome into functionally distinct topological domains. Altogether, our data suggest a broad role for MYST5 both in gene-specific transcriptional regulation and in the organization of the genome into chromatin domains, with the two roles possibly being functionally interconnected.

A role for CENP-A/Cse4 phosphorylation on serine 33 in deposition at the centromere.

Centromeres are the sites of assembly of the kinetochore, which connect the chromatids to the microtubules for sister chromatid segregation during cell division. Centromeres are characterized by the presence of the histone H3 variant CENP-A (termed Cse4 in Saccharomyces cerevisiae). Here, we investigated the function of serine 33 phosphorylation of Cse4 (Cse4-S33ph) in S. cerevisiae, which lies within the essential N-terminal domain (END) of the extended Cse4 N-terminus. Significantly, we identified histone H4-K5, 8, 12R to cause a temperature-sensitive growth defect with mutations in Cse4-S33 and sensitivity to nocodazole and hydroxyurea. Furthermore, the absence of Cse4-S33ph reduced the levels of Cse4 at centromeric sequences, suggesting that Cse4 deposition is defective in the absence of S33 phosphorylation. We furthermore identified synthetic genetic interactions with histone H2A-E57A and H2A-L66A, which both cause a reduced interaction with the histone chaperone FACT and reduced H2A/H2B levels in chromatin, again supporting the notion that a combined defect of H2A/H2B and Cse4 deposition causes centromeric defects. Altogether, our data highlight the importance of correct histone deposition in building a functional centromeric nucleosome and suggests a role for Cse4-S33ph in this process.

Defects in methylation of arginine 37 on CENP-A/Cse4 are compensated by the ubiquitin ligase complex Ubr2/Mub1.

The kinetochore, a supramolecular protein complex, provides the physical connection between chromatin and the microtubule and ensures correct chromosome segregation during mitosis. Centromeric regions are marked by the presence of the histone H3 variant CENP-A. Cse4, the CENP-A homologue from Saccharomyces cerevisiae, is methylated on arginine 37 in its N-terminus (R37), and the absence of methylation (cse4-R37A) causes synthetic genetic defects in combination with mutations or deletions in genes encoding components of the Ctf19/CCAN complex and with the CDEI binding protein Cbf1. Here, we report that the absence of the E3 ubiquitin ligase Ubr2 as well as its adaptor protein Mub1 suppresses the defects caused by the absence of Cse4-R37 methylation. Ubr2 is known to regulate the levels of the MIND complex component Dsn1 via ubiquitination and proteasome-mediated degradation. Accordingly, we found that overexpression of DSN1 also led to suppression of Cse4 methylation defects. Altogether, our data indicate that the absence of R37 methylation reduces the recruitment of kinetochore proteins to centromeric chromatin, and that this can be compensated for by stabilising the outer kinetochore protein Dsn1.

Dynamic modulation of Dnmt2-dependent tRNA methylation by the micronutrient queuine.

Dnmt2 enzymes are cytosine-5 methyltransferases that methylate C38 of several tRNAs. We report here that the activities of two Dnmt2 homologs, Pmt1 from Schizosaccharomyces pombe and DnmA from Dictyostelium discoideum, are strongly stimulated by prior queuosine (Q) modification of the substrate tRNA. In vivo tRNA methylation levels were stimulated by growth of cells in queuine-containing medium; in vitro Pmt1 activity was enhanced on Q-containing RNA; and queuine-stimulated in vivo methylation was abrogated by the absence of the enzyme that inserts queuine into tRNA, eukaryotic tRNA-guanine transglycosylase. Global analysis of tRNA methylation in S. pombe showed a striking selectivity of Pmt1 for tRNA(Asp) methylation, which distinguishes Pmt1 from other Dnmt2 homologs. The present analysis also revealed a novel Pmt1- and Q-independent tRNA methylation site in S. pombe, C34 of tRNA(Pro). Notably, queuine is a micronutrient that is scavenged by higher eukaryotes from the diet and gut microflora. This work therefore reveals an unanticipated route by which the environment can modulate tRNA modification in an organism.

A novel sirtuin 2 (SIRT2) inhibitor with p53-dependent pro-apoptotic activity in non-small cell lung cancer.

Sirtuin 2 (SIRT2) is an NAD(+)-dependent protein deacetylase whose targets include histone H4 lysine 16, p53, and α-tubulin. Because deacetylation of p53 regulates its effect on apoptosis, pharmacological inhibition of SIRT2-dependent p53 deacetylation is of great therapeutic interest for the treatment of cancer. Here, we have identified two structurally related compounds, AEM1 and AEM2, which are selective inhibitors of SIRT2 (IC50 values of 18.5 and 3.8 µM, respectively), but show only weak effects on other sirtuins such as SIRT1, SIRT3, and yeast Sir2. Interestingly, both compounds sensitized non-small cell lung cancer cell lines toward the induction of apoptosis by the DNA-damaging agent etoposide. Importantly, this sensitization was dependent on the presence of functional p53, thus establishing a link between SIRT2 inhibition by these compounds and p53 activation. Further, treatment with AEM1 and AEM2 led to elevated levels of p53 acetylation and to increased expression of CDKN1A, which encodes the cell cycle regulator p21(WAF1), as well as the pro-apoptotic genes PUMA and NOXA, three transcriptional targets of p53. Altogether, our data suggest that inhibition of SIRT2 by these compounds causes increased activation of p53 by decreasing SIRT2-dependent p53 deacetylation. These compounds thus provide a good opportunity for lead optimization and drug development to target p53-proficient cancers.

A link between Sas2-mediated H4 K16 acetylation, chromatin assembly in S-phase by CAF-I and Asf1, and nucleosome assembly by Spt6 during transcription.

The histone acetyltransferase Sas2 is part of the SAS-I complex and acetylates lysine 16 of histone H4 (H4 K16Ac) in the genome of Saccharomyces cerevisiae. Sas2-mediated H4 K16Ac is strongest over the coding region of genes with low expression. However, it is unclear how Sas2-mediated acetylation is incorporated into chromatin. Our previous work has shown physical interactions of SAS-I with the histone chaperones CAF-I and Asf1, suggesting a link between SAS-I-mediated acetylation and chromatin assembly. Here, we find that Sas2-dependent H4 K16Ac in bulk histones requires passage of the cells through the S-phase of the cell cycle, and the rate of increase in H4 K16Ac depends on both CAF-I and Asf1, whereas steady-state levels and genome-wide distribution of H4 K16Ac show only mild changes in their absence. Furthermore, H4 K16Ac is deposited in chromatin at genes upon repression, and this deposition requires the histone chaperone Spt6, but not CAF-I, Asf1, HIR or Rtt106. Altogether, our data indicate that Spt6 controls H4 K16Ac levels by incorporating K16-unacetylated H4 in strongly transcribed genes. Upon repression, Spt6 association is decreased, resulting in less deposition of K16-unacetylated H4 and therefore in a concomitant increase of H4 K16Ac that is recycled during transcription.

Methylation of CenH3 arginine 37 regulates kinetochore integrity and chromosome segregation.

Centromeres of eukaryotic chromosomes mark the site for kinetochore formation and microtubule attachment and are essential for accurate chromosome segregation. Although centromere identity is defined by the presence of the histone H3 variant CenH3/centromere protein A (CENP-A), little is known about how epigenetic modifications on CenH3 might regulate kinetochore assembly and centromere function. Here we show that CENP-A from Saccharomyces cerevisiae, termed Cse4, is methylated on arginine 37 (R37) and that this methylation regulates the recruitment of kinetochore components to centromeric sequences. The absence of Cse4 R37 methylation caused a growth defect in cells lacking the centromere binding factor Cbf1 and synthetic lethality when combined with mutations in components of the Ctf19 linker complex that connects the inner kinetochore to microtubule-binding proteins. The cells showed a cell-cycle arrest in G2/M phase and defects in plasmid and chromosome segregation. Furthermore, the levels of Mtw1/MIND (Mtw1 including Nnf1-Nsl1-Dsn1) and Ctf19 components at the centromere, but not of Cse4 itself, were reduced in the absence of Cse4 R37 methylation, thus showing that this modification regulates the recruitment of linker components to the centromere. Altogether, our data identify a unique regulatory principle on centromeric chromatin by posttranslational modification of the amino terminus of CenH3.

Pmt1, a Dnmt2 homolog in Schizosaccharomyces pombe, mediates tRNA methylation in response to nutrient signaling.

The fission yeast Schizosaccharomyces pombe carries a cytosine 5-methyltransferase homolog of the Dnmt2 family (termed pombe methyltransferase 1, Pmt1), but contains no detectable DNA methylation. Here, we found that Pmt1, like other Dnmt2 homologs, has in vitro methylation activity on cytosine 38 of tRNA(Asp) and, to a lesser extent, of tRNA(Glu), despite the fact that it contains a non-consensus residue in catalytic motif IV as compared with its homologs. In vivo tRNA methylation also required Pmt1. Unexpectedly, however, its in vivo activity showed a strong dependence on the nutritional status of the cell because Pmt1-dependent tRNA methylation was induced in cells grown in the presence of peptone or with glutamate as a nitrogen source. Furthermore, this induction required the serine/threonine kinase Sck2, but not the kinases Sck1, Pka1 or Tor1 and was independent of glucose signaling. Taken together, this work reveals a novel connection between nutrient signaling and tRNA methylation that thus may link tRNA methylation to processes downstream of nutrient signaling like ribosome biogenesis and translation initiation.