RESUMO
Following transcription, tRNAs undergo a series of processing and modification events to become functional adaptors in protein synthesis. Eukaryotes have also evolved intracellular transport systems whereby nucleus-encoded tRNAs may travel out and into the nucleus. In trypanosomes, nearly all tRNAs are also imported from the cytoplasm into the mitochondrion, which lacks tRNA genes. Differential subcellular localization of the cytoplasmic splicing machinery and a nuclear enzyme responsible for queuosine modification at the anticodon "wobble" position appear to be important quality control mechanisms for tRNATyr, the only intron-containing tRNA in T. brucei Since tRNA-guanine transglycosylase (TGT), the enzyme responsible for Q formation, cannot act on an intron-containing tRNA, retrograde nuclear transport is an essential step in maturation. Unlike maturation/processing pathways, the general mechanisms of tRNA stabilization and degradation in T. brucei are poorly understood. Using a combination of cellular and molecular approaches, we show that tRNATyr has an unusually short half-life. tRNATyr, and in addition tRNAAsp, also show the presence of slow-migrating bands during electrophoresis; we term these conformers: alt-tRNATyr and alt-tRNAAsp, respectively. Although we do not know the chemical or structural nature of these conformers, alt-tRNATyr has a short half-life resembling that of tRNATyr; the same is not true for alt-tRNAAsp We also show that RRP44, which is usually an exosome subunit in other organisms, is involved in tRNA degradation of the only intron-containing tRNA in T. brucei and is partly responsible for its unusually short half-life.
Assuntos
Trypanosoma brucei brucei , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismo , RNA de Transferência de Tirosina/química , Meia-Vida , RNA de Transferência de Ácido Aspártico/metabolismo , RNA de Transferência/químicaRESUMO
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.
Assuntos
Nucleosídeo Q , Schizosaccharomyces , 5-Metilcitosina/metabolismo , Azidas , Biotina/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Corantes Fluorescentes/metabolismo , Humanos , Nucleosídeo Q/química , RNA de Transferência/metabolismo , RNA de Transferência de Ácido Aspártico/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , tRNA Metiltransferases/metabolismoRESUMO
Dnmt2, a member of the DNA methyltransferase superfamily, catalyzes the formation of 5-methylcytosine at position 38 in the anticodon loop of tRNAs. Dnmt2 regulates many cellular biological processes, especially the production of tRNA-derived fragments and intergenerational transmission of paternal metabolic disorders to offspring. Moreover, Dnmt2 is closely related to human cancers. The tRNA substrates of mammalian Dnmt2s are mainly detected using bisulfite sequencing; however, we lack supporting biochemical data concerning their substrate specificity or recognition mechanism. Here, we deciphered the tRNA substrates of human DNMT2 (hDNMT2) as tRNAAsp(GUC), tRNAGly(GCC) and tRNAVal(AAC). Intriguingly, for tRNAAsp(GUC) and tRNAGly(GCC), G34 is the discriminator element; whereas for tRNAVal(AAC), the inosine modification at position 34 (I34), which is formed by the ADAT2/3 complex, is the prerequisite for hDNMT2 recognition. We showed that the C32U33(G/I)34N35 (C/U)36A37C38 motif in the anticodon loop, U11:A24 in the D stem, and the correct size of the variable loop are required for Dnmt2 recognition of substrate tRNAs. Furthermore, mammalian Dnmt2s possess a conserved tRNA recognition mechanism.
Assuntos
5-Metilcitosina/metabolismo , Anticódon/metabolismo , DNA (Citosina-5-)-Metiltransferases/metabolismo , RNA de Transferência/metabolismo , Adenosina Desaminase/genética , Adenosina Desaminase/metabolismo , Animais , Anticódon/genética , Sequência de Bases , DNA (Citosina-5-)-Metiltransferases/química , DNA (Citosina-5-)-Metiltransferases/genética , Células HEK293 , Células HeLa , Humanos , Inosina/metabolismo , Camundongos , Modelos Moleculares , Células NIH 3T3 , Conformação de Ácido Nucleico , Ligação Proteica , RNA de Transferência/química , RNA de Transferência/genética , RNA de Transferência de Ácido Aspártico/química , RNA de Transferência de Ácido Aspártico/genética , RNA de Transferência de Ácido Aspártico/metabolismo , RNA de Transferência de Glicina/química , RNA de Transferência de Glicina/genética , RNA de Transferência de Glicina/metabolismo , RNA de Transferência de Valina/química , RNA de Transferência de Valina/genética , RNA de Transferência de Valina/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Especificidade por SubstratoRESUMO
In this report, we investigated the molecular mechanism underlying a deafness-associated m.7516delA mutation affecting the 5' end processing sites of mitochondrial tRNAAsp and tRNASer(UCN). An in vitro processing experiment demonstrated that m.7516delA mutation caused the aberrant 5' end processing of tRNASer(UCN) and tRNAAsp precursors, catalyzed by RNase P. Using cytoplasmic hybrids (cybrids) derived from one hearing-impaired Chinese family bearing the m.7516delA mutation and control, we demonstrated the asymmetrical effects of m.7516delA mutation on the processing of tRNAs in the heavy (H)-strand and light (L)-strand polycistronic transcripts. Specially, the m.7516delA mutation caused the decreased levels of tRNASer(UCN) and downstream five tRNAs, including tRNATyr from the L-strand transcripts and tRNAAsp from the H-strand transcripts. Strikingly, mutant cybrids exhibited the lower level of COX2 mRNA and accumulation of longer and uncleaved precursors of COX2 from the H-strand transcripts. Aberrant RNA metabolisms yielded variable reductions in the mitochondrial proteins, especially marked reductions in the levels of ND4, ND5, CO1, CO2 and CO3. The impairment of mitochondrial translation caused the proteostasis stress and respiratory deficiency, diminished ATP production and membrane potential, increased production of reactive oxygen species and promoted apoptosis. Our findings provide new insights into the pathophysiology of deafness arising from mitochondrial tRNA processing defects.
Assuntos
DNA Mitocondrial/genética , Surdez/genética , RNA Mensageiro/metabolismo , RNA de Transferência de Ácido Aspártico/metabolismo , RNA de Transferência de Serina/metabolismo , Apoptose , Linhagem Celular , Respiração Celular , Humanos , Potencial da Membrana Mitocondrial , Proteínas Mitocondriais/metabolismo , Mutação , Processamento Pós-Transcricional do RNA , Espécies Reativas de Oxigênio/metabolismoRESUMO
The nondiscriminating aspartyl-tRNA synthetase (ND-AspRS), found in many archaea and bacteria, covalently attaches aspartic acid to tRNAAsp and tRNAAsn generating a correctly charged Asp-tRNAAsp and an erroneous Asp-tRNAAsn . This relaxed tRNA specificity is governed by interactions between the tRNA and the enzyme. In an effort to assess the contributions of the anticodon-binding domain to tRNA specificity, we constructed two chimeric enzymes, Chimera-D and Chimera-N, by replacing the native anticodon-binding domain in the Helicobacter pylori ND-AspRS with that of a discriminating AspRS (Chimera-D) and an asparaginyl-tRNA synthetase (AsnRS, Chimera-N), both from Escherichia coli. Both chimeric enzymes showed similar secondary structure compared to wild-type (WT) ND-AspRS and maintained the ability to form dimeric complexes in solution. Although less catalytically active than WT, Chimera-D was more discriminating as it aspartylated tRNAAsp over tRNAAsn with a specificity ratio of 7.0 compared to 2.9 for the WT enzyme. In contrast, Chimera-N exhibited low catalytic activity toward tRNAAsp and was unable to aspartylate tRNAAsn . The observed catalytic activities for the two chimeras correlate with their heterologous toxicity when expressed in E. coli. Molecular dynamics simulations show a reduced hydrogen bond network at the interface between the anticodon-binding domain and the catalytic domain in Chimera-N compared to Chimera-D or WT, explaining its lower stability and catalytic activity.
Assuntos
Anticódon , Aspartato-tRNA Ligase/metabolismo , Escherichia coli/enzimologia , Helicobacter pylori/enzimologia , Aminoacil-RNA de Transferência/metabolismo , RNA de Transferência de Asparagina/metabolismo , RNA de Transferência de Ácido Aspártico/metabolismo , Sequência de Aminoácidos , Aspartato-tRNA Ligase/química , Aspartato-tRNA Ligase/genética , Sítios de Ligação , Biocatálise , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Helicobacter pylori/genética , Simulação de Dinâmica Molecular , Mutação , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Aminoacil-RNA de Transferência/química , Aminoacil-RNA de Transferência/genética , RNA de Transferência de Asparagina/química , RNA de Transferência de Ácido Aspártico/química , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
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.
Assuntos
Estresse do Retículo Endoplasmático , Alimentos Formulados , Nucleosídeo Q/metabolismo , Processamento Pós-Transcricional do RNA , RNA de Transferência de Ácido Aspártico/metabolismo , Resposta a Proteínas não Dobradas , Animais , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Células HCT116 , Células HeLa , Humanos , Camundongos , Nucleosídeo Q/genética , RNA de Transferência de Ácido Aspártico/genéticaRESUMO
Dnmt2 methylates cytosine at position 38 of tRNAAsp in a variety of eukaryotic organisms. A correlation between the presence of the hypermodified nucleoside queuosine (Q) at position 34 of tRNAAsp and the Dnmt2 dependent C38 methylation was recently found in vivo for S. pombe and D. discoideum. We demonstrate a direct effect of the Q-modification on the methyltransferase catalytic efficiency in vitro, as Vmax/K0.5 of purified S. pombe Dnmt2 shows an increase for in vitro transcribed tRNAAsp containing Q34 to 6.27 ∗ 10-3 s-1 µM-1 compared to 1.51 ∗ 10-3 s-1 µM-1 for the unmodified substrate. Q34tRNAAsp exhibits an only slightly increased affinity for Dnmt2 in comparison to unmodified G34tRNA. In order to get insight into the structural basis for the Q-dependency, the crystal structure of S. pombe Dnmt2 was determined at 1.7 Å resolution. It closely resembles the known structures of human and E. histolytica Dnmt2, and contains the entire active site loop. The interaction with tRNA was analyzed by means of mass-spectrometry using UV cross-linked Dnmt2-tRNA complex. These cross-link data and computational docking of Dnmt2 and tRNAAsp reveal Q34 positioned adjacent to the S-adenosylmethionine occupying the active site, suggesting that the observed increase of Dnmt2 catalytic efficiency by queuine originates from optimal positioning of the substrate molecules and residues relevant for methyl transfer.
Assuntos
DNA (Citosina-5-)-Metiltransferases/química , DNA (Citosina-5-)-Metiltransferases/metabolismo , Ativadores de Enzimas/metabolismo , Nucleosídeo Q/metabolismo , RNA de Transferência de Ácido Aspártico/metabolismo , Schizosaccharomyces/enzimologia , Schizosaccharomyces/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Espectrometria de Massas , Modelos Moleculares , Simulação de Acoplamento Molecular , Ligação Proteica , Conformação ProteicaRESUMO
In eukaryotes, cytosine methylation regulates diverse biological processes such as gene expression, development and maintenance of genomic integrity. However, cytosine methylation and its functions in pathogenic apicomplexan protozoans remain enigmatic. To address this, here we investigated the presence of cytosine methylation in the nucleic acids of the protozoan Plasmodium falciparum. Interestingly, P. falciparum has TRDMT1, a conserved homologue of DNA methyltransferase DNMT2. However, we found that TRDMT1 did not methylate DNA, in vitro. We demonstrate that TRDMT1 methylates cytosine in the endogenous aspartic acid tRNA of P. falciparum. Through RNA bisulfite sequencing, we mapped the position of 5-methyl cytosine in aspartic acid tRNA and found methylation only at C38 position. P. falciparum proteome has significantly higher aspartic acid content and a higher proportion of proteins with poly aspartic acid repeats than other apicomplexan pathogenic protozoans. Proteins with such repeats are functionally important, with significant roles in host-pathogen interactions. Therefore, TRDMT1 mediated C38 methylation of aspartic acid tRNA might play a critical role by translational regulation of important proteins and modulate the pathogenicity of the malarial parasite.
Assuntos
DNA (Citosina-5-)-Metiltransferases/química , Plasmodium falciparum/enzimologia , RNA de Protozoário/química , RNA de Transferência de Ácido Aspártico/química , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação , RNA de Protozoário/genética , RNA de Protozoário/metabolismo , RNA de Transferência de Ácido Aspártico/genética , RNA de Transferência de Ácido Aspártico/metabolismoRESUMO
Transfer RNAs (tRNAs) function in translational machinery and further serves as a source of short non-coding RNAs (ncRNAs). tRNA-derived ncRNAs show differential expression profiles and play roles in many biological processes beyond translation. Molecular mechanisms that shape and regulate their expression profiles are largely unknown. Here, we report the mechanism of biogenesis for tRNA-derived Piwi-interacting RNAs (td-piRNAs) expressed in Bombyx BmN4 cells. In the cells, two cytoplasmic tRNA species, tRNAAspGUC and tRNAHisGUG, served as major sources for td-piRNAs, which were derived from the 5'-part of the respective tRNAs. cP-RNA-seq identified the two tRNAs as major substrates for the 5'-tRNA halves as well, suggesting a previously uncharacterized link between 5'-tRNA halves and td-piRNAs. An increase in levels of the 5'-tRNA halves, induced by BmNSun2 knockdown, enhanced the td-piRNA expression levels without quantitative change in mature tRNAs, indicating that 5'-tRNA halves, not mature tRNAs, are the direct precursors for td-piRNAs. For the generation of tRNAHisGUG-derived piRNAs, BmThg1l-mediated nucleotide addition to -1 position of tRNAHisGUG was required, revealing an important function of BmThg1l in piRNA biogenesis. Our study advances the understanding of biogenesis mechanisms and the genesis of specific expression profiles for tRNA-derived ncRNAs.
Assuntos
Proteínas Argonautas/genética , Bombyx/genética , Proteínas de Insetos/genética , RNA Interferente Pequeno/genética , RNA de Transferência de Ácido Aspártico/genética , RNA de Transferência de Histidina/genética , Animais , Proteínas Argonautas/metabolismo , Sequência de Bases , Bombyx/crescimento & desenvolvimento , Bombyx/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Células Germinativas/crescimento & desenvolvimento , Células Germinativas/metabolismo , Proteínas de Insetos/metabolismo , Conformação de Ácido Nucleico , RNA Interferente Pequeno/metabolismo , RNA de Transferência de Ácido Aspártico/metabolismo , RNA de Transferência de Histidina/metabolismoRESUMO
The RNase P family comprises structurally diverse endoribonucleases ranging from complex ribonucleoproteins to single polypeptides. We show that the organellar (AtPRORP1) and the two nuclear (AtPRORP2,3) single-polypeptide RNase P isoenzymes from Arabidopsis thaliana confer viability to Escherichia coli cells with a lethal knockdown of its endogenous RNA-based RNase P. RNA-Seq revealed that AtPRORP1, compared with bacterial RNase P or AtPRORP3, cleaves several precursor tRNAs (pre-tRNAs) aberrantly in E. coli. Aberrant cleavage by AtPRORP1 was mainly observed for pre-tRNAs that can form short acceptor-stem extensions involving G:C base pairs, including tRNAAsp(GUC), tRNASer(CGA) and tRNAHis. However, both AtPRORP1 and 3 were defective in processing of E. coli pre-tRNASec carrying an acceptor stem expanded by three G:C base pairs. Instead, pre-tRNASec was degraded, suggesting that tRNASec is dispensable for E. coli under laboratory conditions. AtPRORP1, 2 and 3 are also essentially unable to process the primary transcript of 4.5S RNA, a hairpin-like non-tRNA substrate processed by E. coli RNase P, indicating that PRORP enzymes have a narrower, more tRNA-centric substrate spectrum than bacterial RNA-based RNase P enzymes. The cells' viability also suggests that the essential function of the signal recognition particle can be maintained with a 5Î-extended 4.5S RNA.
Assuntos
Proteínas de Escherichia coli/genética , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Precursores de RNA/genética , Ribonuclease P/genética , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Pareamento de Bases , Sequência de Bases , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Teste de Complementação Genética , Viabilidade Microbiana , Conformação de Ácido Nucleico , Precursores de RNA/metabolismo , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA de Transferência de Ácido Aspártico/genética , RNA de Transferência de Ácido Aspártico/metabolismo , RNA de Transferência de Histidina/genética , RNA de Transferência de Histidina/metabolismo , RNA de Transferência de Serina/genética , RNA de Transferência de Serina/metabolismo , Ribonuclease P/deficiência , Ribonuclease P/metabolismo , TransgenesRESUMO
The N-terminal anticodon-binding domain of the nondiscriminating aspartyl-tRNA synthetase (ND-AspRS) plays a crucial role in the recognition of both tRNAAsp and tRNAAsn. Here, the first X-ray crystal structure of the N-terminal domain of this enzyme (ND-AspRS1-104) from the human-pathogenic bacterium Helicobacter pylori is reported at 2.0â Å resolution. The apo form of H. pylori ND-AspRS1-104 shares high structural similarity with the N-terminal anticodon-binding domains of the discriminating aspartyl-tRNA synthetase (D-AspRS) from Escherichia coli and ND-AspRS from Pseudomonas aeruginosa, allowing recognition elements to be proposed for tRNAAsp and tRNAAsn. It is proposed that a long loop (Arg77-Lys90) in this H. pylori domain influences its relaxed tRNA specificity, such that it is classified as nondiscriminating. A structural comparison between D-AspRS from E. coli and ND-AspRS from P. aeruginosa suggests that turns E and F (78GAGL81 and 83NPKL86) in H. pylori ND-AspRS play a crucial role in anticodon recognition. Accordingly, the conserved Pro84 in turn F facilitates the recognition of the anticodons of tRNAAsp (34GUC36) and tRNAAsn (34GUU36). The absence of the amide H atom allows both C and U bases to be accommodated in the tRNA-recognition site.
Assuntos
Anticódon/química , Aspartato-tRNA Ligase/química , Proteínas de Bactérias/química , Helicobacter pylori/química , RNA de Transferência de Asparagina/química , RNA de Transferência de Ácido Aspártico/química , Sequência de Aminoácidos , Anticódon/metabolismo , Apoproteínas/química , Apoproteínas/genética , Apoproteínas/metabolismo , Aspartato-tRNA Ligase/genética , Aspartato-tRNA Ligase/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/enzimologia , Escherichia coli/genética , Expressão Gênica , Helicobacter pylori/enzimologia , Modelos Moleculares , Plasmídeos/química , Plasmídeos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Pseudomonas aeruginosa/enzimologia , Pseudomonas aeruginosa/genética , RNA de Transferência de Asparagina/genética , RNA de Transferência de Asparagina/metabolismo , RNA de Transferência de Ácido Aspártico/genética , RNA de Transferência de Ácido Aspártico/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia Estrutural de ProteínaRESUMO
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.
Assuntos
DNA (Citosina-5-)-Metiltransferases/metabolismo , Guanina/análogos & derivados , Micronutrientes/metabolismo , RNA de Transferência/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Dictyostelium/enzimologia , Guanina/metabolismo , Metilação , Pentosiltransferases/metabolismo , RNA de Transferência de Ácido Aspártico/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismoRESUMO
Methylation of tRNA is an important post-transcriptional modification and aberrations in tRNA modification has been implicated in cancer. The DNMT2 protein methylates C38 of tRNA-Asp and it has a role in cellular physiology and stress response and its expression levels are altered in cancer tissues. Here we studied whether DNMT2 somatic mutations found in cancer tissues affect the activity of the enzyme. We have generated 13 DNMT2 variants and purified the corresponding proteins. All proteins were properly folded as determined by circular dichroism spectroscopy. We tested their RNA methylation activity using in vitro generated tRNA-Asp. One of the mutations (E63K) caused a twofold increase in activity, while two of them led to a strong (over fourfold) decrease in activity (G155S and L257V). Two additional mutant proteins were almost inactive (R371H and G155V). The strong effect of some of the somatic cancer mutations on DNMT2 activity suggests that these mutations have a functional role in tumorigenesis.
Assuntos
DNA (Citosina-5-)-Metiltransferases/química , Mutação de Sentido Incorreto , Proteínas de Neoplasias/química , Neoplasias/enzimologia , Processamento Pós-Transcricional do RNA , RNA de Transferência de Ácido Aspártico/química , Substituição de Aminoácidos , Animais , Catálise , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Humanos , Metilação , Camundongos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias/genética , RNA de Transferência de Ácido Aspártico/genética , RNA de Transferência de Ácido Aspártico/metabolismoRESUMO
In most organisms, the widely conserved 1-methyl-adenosine58 (m1A58) tRNA modification is catalyzed by an S-adenosyl-L-methionine (SAM)-dependent, site-specific enzyme TrmI. In archaea, TrmI also methylates the adjacent adenine 57, m1A57 being an obligatory intermediate of 1-methyl-inosine57 formation. To study this multi-site specificity, we used three oligoribonucleotide substrates of Pyrococcus abyssi TrmI (PabTrmI) containing a fluorescent 2-aminopurine (2-AP) at the two target positions and followed the RNA binding kinetics and methylation reactions by stopped-flow and mass spectrometry. PabTrmI did not modify 2-AP but methylated the adjacent target adenine. 2-AP seriously impaired the methylation of A57 but not A58, confirming that PabTrmI methylates efficiently the first adenine of the A57A58A59 sequence. PabTrmI binding provoked a rapid increase of fluorescence, attributed to base unstacking in the environment of 2-AP. Then, a slow decrease was observed only with 2-AP at position 57 and SAM, suggesting that m1A58 formation triggers RNA release. A model of the protein-tRNA complex shows both target adenines in proximity of SAM and emphasizes no major tRNA conformational change except base flipping during the reaction. The solvent accessibility of the SAM pocket is not affected by the tRNA, thereby enabling S-adenosyl-L-homocysteine to be replaced by SAM without prior release of monomethylated tRNA.
Assuntos
Adenina/metabolismo , Proteínas Arqueais/metabolismo , RNA de Transferência de Ácido Aspártico/metabolismo , tRNA Metiltransferases/metabolismo , 2-Aminopurina/metabolismo , Proteínas Arqueais/química , Sequência de Bases , Modelos Moleculares , Pyrococcus abyssi/enzimologia , RNA de Transferência de Ácido Aspártico/química , S-Adenosilmetionina/metabolismo , Especificidade por Substrato , tRNA Metiltransferases/químicaRESUMO
Dnmt2 enzymes are conserved in eukaryotes, where they methylate C38 of tRNA-Asp with high activity. Here, the activity of one of the very few prokaryotic Dnmt2 homologs from Geobacter species (GsDnmt2) was investigated. GsDnmt2 was observed to methylate tRNA-Asp from flies and mice. Unexpectedly, it had only a weak activity toward its matching Geobacter tRNA-Asp, but methylated Geobacter tRNA-Glu with good activity. In agreement with this result, we show that tRNA-Glu is methylated in Geobacter while the methylation is absent in tRNA-Asp. The activities of Dnmt2 enzymes from Homo sapiens, Drosophila melanogaster, Schizosaccharomyces pombe and Dictyostelium discoideum for methylation of the Geobacter tRNA-Asp and tRNA-Glu were determined showing that all these Dnmt2s preferentially methylate tRNA-Asp. Hence, the GsDnmt2 enzyme has a swapped transfer ribonucleic acid (tRNA) specificity. By comparing the different tRNAs, a characteristic sequence pattern was identified in the variable loop of all preferred tRNA substrates. An exchange of two nucleotides in the variable loop of murine tRNA-Asp converted it to the corresponding variable loop of tRNA-Glu and led to a strong reduction of GsDnmt2 activity. Interestingly, the same loss of activity was observed with human DNMT2, indicating that the variable loop functions as a specificity determinant in tRNA recognition of Dnmt2 enzymes.
Assuntos
Proteínas de Bactérias/metabolismo , Geobacter/enzimologia , RNA de Transferência de Ácido Glutâmico/metabolismo , tRNA Metiltransferases/metabolismo , Animais , Humanos , Metilação , Camundongos , Conformação de Ácido Nucleico , RNA de Transferência de Ácido Aspártico/química , RNA de Transferência de Ácido Aspártico/metabolismo , RNA de Transferência de Ácido Glutâmico/química , Especificidade por SubstratoRESUMO
Protein synthesis must rapidly and repeatedly discriminate between a single correct and many incorrect aminoacyl-tRNAs. We have attempted to measure the frequencies of all possible missense errors by tRNA , tRNA and tRNA . The most frequent errors involve three types of mismatched nucleotide pairs, Uâ¢U, Uâ¢C, or Uâ¢G, all of which can form a noncanonical base pair with geometry similar to that of the canonical Uâ¢A or Câ¢G Watson-Crick pairs. Our system is sensitive enough to measure errors at other potential mismatches that occur at frequencies as low as 1 in 500,000 codons. The ribosome appears to discriminate this efficiently against any pair with non-Watson-Crick geometry. This extreme accuracy may be necessary to allow discrimination against the errors involving near Watson-Crick pairing.
Assuntos
Pareamento Incorreto de Bases/fisiologia , Mutação de Sentido Incorreto , Biossíntese de Proteínas/fisiologia , Ribossomos/fisiologia , Substituição de Aminoácidos , Pareamento de Bases/fisiologia , Escherichia coli/genética , Escherichia coli/metabolismo , Mutagênese/fisiologia , Mutação de Sentido Incorreto/fisiologia , Conformação de Ácido Nucleico , Organismos Geneticamente Modificados , RNA de Transferência de Ácido Aspártico/metabolismo , RNA de Transferência de Ácido Glutâmico/metabolismo , RNA de Transferência de Tirosina/metabolismo , beta-Galactosidase/química , beta-Galactosidase/genética , beta-Galactosidase/metabolismoRESUMO
Although the DNA methyltransferase 2 family is highly conserved during evolution and recent reports suggested a dual specificity with stronger activity on transfer RNA (tRNA) than DNA substrates, the biological function is still obscure. We show that the Dictyostelium discoideum Dnmt2-homologue DnmA is an active tRNA methyltransferase that modifies C38 in tRNA(Asp(GUC)) in vitro and in vivo. By an ultraviolet-crosslinking and immunoprecipitation approach, we identified further DnmA targets. This revealed specific tRNA fragments bound by the enzyme and identified tRNA(Glu(CUC/UUC)) and tRNA(Gly(GCC)) as new but weaker substrates for both human Dnmt2 and DnmA in vitro but apparently not in vivo. Dnmt2 enzymes form transient covalent complexes with their substrates. The dynamics of complex formation and complex resolution reflect methylation efficiency in vitro. Quantitative PCR analyses revealed alterations in dnmA expression during development, cell cycle and in response to temperature stress. However, dnmA expression only partially correlated with tRNA methylation in vivo. Strikingly, dnmA expression in the laboratory strain AX2 was significantly lower than in the NC4 parent strain. As expression levels and binding of DnmA to a target in vivo are apparently not necessarily accompanied by methylation, we propose an additional biological function of DnmA apart from methylation.
Assuntos
Dictyostelium/enzimologia , Proteínas de Protozoários/metabolismo , tRNA Metiltransferases/metabolismo , Ciclo Celular , Resposta ao Choque Frio , DNA (Citosina-5-)-Metiltransferases/metabolismo , Dictyostelium/genética , Dictyostelium/crescimento & desenvolvimento , Regulação da Expressão Gênica , Humanos , Metilação , Proteínas de Protozoários/genética , RNA de Transferência de Ácido Aspártico/química , RNA de Transferência de Ácido Aspártico/metabolismo , RNA de Transferência de Ácido Glutâmico/metabolismo , RNA de Transferência de Glicina/metabolismo , Transcrição Gênica , tRNA Metiltransferases/genéticaRESUMO
Nucleotides of 28S rRNA involved in binding of the human 80S ribosome with acceptor ends of the A site and the P site tRNAs were determined using two complementary approaches, namely, cross-linking with application of tRNA(Asp) analogues substituted with 4-thiouridine in position 75 or 76 and hydroxyl radical footprinting with the use of the full sized tRNA and the tRNA deprived of the 3'-terminal trinucleotide CCA. In general, these 28S rRNA nucleotides are located in ribosomal regions homologous to the A, P and E sites of the prokaryotic 50S subunit. However, none of the approaches used discovered interactions of the apex of the large rRNA helix 80 with the acceptor end of the P site tRNA typical with prokaryotic ribosomes. Application of the results obtained to available atomic models of 50S and 60S subunits led us to a conclusion that the A site tRNA is actually present in both A/A and A/P states and the P site tRNA in the P/P and P/E states. Thus, the present study gives a biochemical confirmation of the data on the structure and dynamics of the mammalian ribosomal pretranslocation complex obtained with application of cryo-electron microscopy and single-molecule FRET [Budkevich et al., 2011]. Moreover, in our study, particular sets of 28S rRNA nucleotides involved in oscillations of tRNAs CCA-termini between their alternative locations in the mammalian 80S ribosome are revealed.
Assuntos
Biossíntese de Proteínas , RNA de Transferência de Ácido Aspártico/química , Ribossomos/química , Tiouridina/química , Animais , Pareamento de Bases , Sítios de Ligação , Reagentes de Ligações Cruzadas , Microscopia Crioeletrônica , Transferência Ressonante de Energia de Fluorescência , Humanos , Sequências Repetidas Invertidas/genética , Modelos Moleculares , Mutação , Conformação de Ácido Nucleico , RNA Ribossômico 28S , RNA de Transferência de Ácido Aspártico/genética , RNA de Transferência de Ácido Aspártico/metabolismo , Ribossomos/genética , Ribossomos/metabolismo , Tiouridina/metabolismoRESUMO
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.
Assuntos
DNA (Citosina-5-)-Metiltransferases/metabolismo , RNA de Transferência/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/enzimologia , tRNA Metiltransferases/metabolismo , Citosina/metabolismo , Metilação , Nitrogênio/metabolismo , Proteínas Serina-Treonina Quinases/fisiologia , RNA de Transferência de Ácido Aspártico/química , RNA de Transferência de Ácido Aspártico/metabolismo , RNA de Transferência de Ácido Glutâmico/metabolismo , Proteínas de Schizosaccharomyces pombe/fisiologia , Transdução de SinaisRESUMO
Many mammalian mitochondrial aminoacyl-tRNA synthetases are of bacterial-type and share structural domains with homologous bacterial enzymes of the same specificity. Despite this high similarity, synthetases from bacteria are known for their inability to aminoacylate mitochondrial tRNAs, while mitochondrial enzymes do aminoacylate bacterial tRNAs. Here, the reasons for non-aminoacylation by a bacterial enzyme of a mitochondrial tRNA have been explored. A mutagenic analysis performed on in vitro transcribed human mitochondrial tRNA(Asp) variants tested for their ability to become aspartylated by Escherichia coli aspartyl-tRNA synthetase, reveals that full conversion cannot be achieved on the basis of the currently established tRNA/synthetase recognition rules. Integration of the full set of aspartylation identity elements and stabilization of the structural tRNA scaffold by restoration of D- and T-loop interactions, enable only a partial gain in aspartylation efficiency. The sequence context and high structural instability of the mitochondrial tRNA are additional features hindering optimal adaptation of the tRNA to the bacterial enzyme. Our data support the hypothesis that non-aminoacylation of mitochondrial tRNAs by bacterial synthetases is linked to the large sequence and structural relaxation of the organelle encoded tRNAs, itself a consequence of the high rate of mitochondrial genome divergence.