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1.
Methods Enzymol ; 658: 251-275, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34517950

RESUMO

The application of in vitro kinetic tools has the potential to provide important insight into the molecular mechanisms of RNA modification enzymes. Utilizing quantitative biochemical approaches can reveal information about enzyme preferences for specific substrates that are relevant for understanding modification reactions in their biological contexts. Moreover, kinetic tools have been powerfully applied to identify and characterize roles for specific amino acid residues in catalysis, which can be essential information for understanding the molecular basis for human disease, as well as for targeting these enzymes for potential therapeutic interventions. RNA methyltransferases are a particularly interesting group of RNA modification enzymes because of the diversity in structure and mechanism that has been revealed among members of this group, even including some examples of enzymes that use entirely distinct reaction mechanisms to form identical methylated nucleotides in RNA. Yet, many questions remain unanswered about how these distinct catalytic strategies are facilitated by the relevant enzyme families. We have applied in vitro kinetic analysis to specifically focus on catalytically relevant ionizations in the context of tRNA methyltransferase reactions, by measuring rates under conditions of varied pH. This analysis can be applied broadly to RNA methyltransferases to expand our understanding of these important enzymes.


Assuntos
Metiltransferases , tRNA Metiltransferases , Catálise , Humanos , Cinética , Metiltransferases/metabolismo , RNA , RNA de Transferência , Especificidade por Substrato , tRNA Metiltransferases/metabolismo
2.
Methods Enzymol ; 658: 359-377, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34517954

RESUMO

A tRNA interacts with numerous proteins throughout its biogenesis and during translation, and a significant portion of these interacting proteins are involved in post-transcriptional modifications. While some of the modifying enzymes use relatively simple recognition elements for substrate recognition, many enzymes selectively modify a specific subset of tRNA species without obvious recognition rules. In this chapter we describe a semi-quantitative pull-down assay to study tRNA substrate specificity of modification enzymes, by using the yeast Saccharomyces cerevisiae m3C32 methyltransferase Trm140 as an example. We also discuss some overall considerations for a successful pull-down experiment, with a focus on practical applications of the dissociation constant KD between the protein and the tRNA and the off-rate.


Assuntos
Proteínas de Saccharomyces cerevisiae , tRNA Metiltransferases , RNA de Transferência/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Especificidade por Substrato , tRNA Metiltransferases/genética , tRNA Metiltransferases/metabolismo
3.
Biomolecules ; 11(6)2021 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-34207099

RESUMO

RNA methylation at the nitrogen sixth of adenosine (m6A, N6-methyladenosine) is the most abundant RNA modification which plays a crucial role in all RNA metabolic aspects. Recently, m6A modification has been assigned to mediate the biological processes of cancer cells, but their significance in HNSCC development is still poorly described. Thus, the main aim of this study was to globally quantify m6A modification by the mass spectrometry approach and determine the mRNA expression level of selected m6A RNA methyltransferase (METTL3), demethylase (FTO), and m6A readers (YTHDF2, YTHDC2) in 45 HNSCC patients and 4 cell lines (FaDu, Detroit 562, A-253 and SCC-15) using qPCR. In the results, we have not observed differences in the global amount of m6A modification and the mRNA level of the selected genes between the cancerous and paired-matched histopathologically unchanged tissues from 45 HNSCC patients. However, we have found a positive correlation between selected RNA methylation machinery genes expression and m6A abundance on total RNA and characterized the transcript level of those genes in the HNSCC cell lines. Moreover, the lack of global m6A differences between cancerous and histopathologically unchanged tissues suggests that m6A alterations in specific RNA sites may specifically influence HNSCC tumorigenesis.


Assuntos
RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Adenosina/análogos & derivados , Adenosina/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Dioxigenase FTO Dependente de alfa-Cetoglutarato/genética , Carcinogênese/genética , Feminino , Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/genética , Neoplasias de Cabeça e Pescoço/genética , Humanos , Masculino , Espectrometria de Massas/métodos , Metilação , Metiltransferases/genética , Metiltransferases/metabolismo , Pessoa de Meia-Idade , Polônia , RNA/genética , RNA Helicases/genética , Processamento Pós-Transcricional do RNA/genética , Processamento Pós-Transcricional do RNA/fisiologia , RNA Mensageiro/análise , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , tRNA Metiltransferases/metabolismo
4.
Nat Commun ; 12(1): 4644, 2021 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-34330903

RESUMO

Frameshifting of mRNA during translation provides a strategy to expand the coding repertoire of cells and viruses. How and where in the elongation cycle +1-frameshifting occurs remains poorly understood. We describe seven ~3.5-Å-resolution cryo-EM structures of 70S ribosome complexes, allowing visualization of elongation and translocation by the GTPase elongation factor G (EF-G). Four structures with a + 1-frameshifting-prone mRNA reveal that frameshifting takes place during translocation of tRNA and mRNA. Prior to EF-G binding, the pre-translocation complex features an in-frame tRNA-mRNA pairing in the A site. In the partially translocated structure with EF-G•GDPCP, the tRNA shifts to the +1-frame near the P site, rendering the freed mRNA base to bulge between the P and E sites and to stack on the 16S rRNA nucleotide G926. The ribosome remains frameshifted in the nearly post-translocation state. Our findings demonstrate that the ribosome and EF-G cooperate to induce +1 frameshifting during tRNA-mRNA translocation.


Assuntos
Mudança da Fase de Leitura do Gene Ribossômico/genética , Elongação Traducional da Cadeia Peptídica/genética , Fator G para Elongação de Peptídeos/genética , RNA Mensageiro/genética , RNA de Transferência/genética , Ribossomos/genética , Biocatálise , Microscopia Crioeletrônica , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , Conformação de Ácido Nucleico , Fator G para Elongação de Peptídeos/química , Fator G para Elongação de Peptídeos/metabolismo , Conformação Proteica , RNA Mensageiro/química , RNA Mensageiro/metabolismo , RNA Ribossômico 16S/química , RNA Ribossômico 16S/genética , RNA Ribossômico 16S/metabolismo , RNA de Transferência/química , RNA de Transferência/metabolismo , Ribossomos/metabolismo , Ribossomos/ultraestrutura , tRNA Metiltransferases/genética , tRNA Metiltransferases/metabolismo
5.
Aging (Albany NY) ; 13(12): 15833-15874, 2021 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-34139673

RESUMO

Cellular senescence may be a side effect of chemotherapy and other anti-cancer treatments that may promote inflammation and paracrine secondary senescence in healthy tissues. DNMT2/TRDMT1 methyltransferase is implicated in the regulation of cellular lifespan and DNA damage response (DDR). In the present study, the responses to senescence inducing concentrations of doxorubicin and etoposide in different cancer cells with DNMT2/TRDMT1 gene knockout were evaluated, namely changes in the cell cycle, apoptosis, autophagy, interleukin levels, genetic stability and DDR, and 5-mC and NSUN1-6 levels. Moreover, the effect of azacytidine post-treatment was considered. Diverse responses were revealed that was based on type of cancer cells (breast and cervical cancer, osteosarcoma and glioblastoma cells) and anti-cancer drugs. DNMT2/TRDMT1 gene knockout in drug-treated glioblastoma cells resulted in decreased number of apoptotic and senescent cells, IL-8 levels and autophagy, and increased number of necrotic cells, DNA damage and affected DDR compared to drug-treated glioblastoma cells with unmodified levels of DNMT2/TRDMT1. We suggest that DNMT2/TRDMT1 gene knockout in selected experimental settings may potentiate some adverse effects associated with chemotherapy-induced senescence.


Assuntos
Senescência Celular/genética , DNA (Citosina-5-)-Metiltransferases/genética , Doxorrubicina/farmacologia , Etoposídeo/farmacologia , Neoplasias/genética , Neoplasias/patologia , 5-Metilcitosina/metabolismo , Autofagia/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Citoplasma/metabolismo , Quebras de DNA de Cadeia Dupla , Técnicas de Inativação de Genes , Humanos , Estresse Oxidativo/efeitos dos fármacos , beta-Galactosidase/metabolismo , tRNA Metiltransferases/metabolismo
6.
Genes Cells ; 26(4): 203-218, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33527595

RESUMO

In fission yeast, siRNA generated by RNA interference (RNAi) factors plays critical roles in establishment and maintenance of heterochromatin. To achieve efficient siRNA synthesis, RNAi factors assemble on heterochromatin via association with Swi6, a homologue of heterochromatin protein 1 (HP1), and heterochromatic noncoding RNA (hncRNA) retained on chromatin. In addition, spliceosomes formed on hncRNA introns recruit RNAi factors to hncRNA and heterochromatin. Small nuclear RNAs, components of the spliceosome, have a trimethylguanosine (TMG) cap that is generated by Tgs1-dependent hypermethylation of the normal m7G cap; this cap is required for efficient splicing of some mRNAs in budding yeast and Drosophila. In this study, we found that loss of Tgs1 in fission yeast destabilizes centromeric heterochromatin. Tgs1 was required for Swi6-independent siRNA synthesis, as well as for the establishment of centromeric heterochromatin. Loss of Tgs1 affected the splicing efficiency of hncRNA introns in the absence of Swi6. Furthermore, some hncRNAs have a TMG cap, and we found that loss of Tgs1 diminished the chromatin binding of these hncRNAs. Together, these results suggest that the Tgs1-dependent TMG cap plays critical roles in establishment of heterochromatin by ensuring spliceosome-dependent recruitment of RNAi factors and regulating the binding between chromatin and hncRNA.


Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Heterocromatina/metabolismo , RNA Interferente Pequeno/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , tRNA Metiltransferases/metabolismo , Centrômero/metabolismo , Inativação Gênica , Íntrons/genética , Modelos Biológicos , Domínios Proteicos , Splicing de RNA/genética , RNA Antissenso/metabolismo , RNA Fúngico/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Schizosaccharomyces pombe/química , tRNA Metiltransferases/química
7.
Trends Genet ; 37(6): 498-500, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33622495

RESUMO

Recent studies have unequivocally confirmed the presence of 5-methylcytosine (m5C) in mammalian mRNAs while indicating significant functional roles for this internal base modification type. Here, a brief history of m5C epitranscriptome research and a discussion of the important ways in which the field may now progress is presented.


Assuntos
5-Metilcitosina/metabolismo , Técnicas Genéticas , RNA Mensageiro/metabolismo , tRNA Metiltransferases/metabolismo , Animais , Códon de Terminação , Humanos , Mamíferos/genética , Metilação , Transcriptoma
8.
Cell Death Dis ; 12(1): 61, 2021 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-33431792

RESUMO

Accumulating evidence has revealed that mitochondria dynamics and function regulation is essential for the successful mesenchymal stem cell (MSC) differentiation. In the present study, the researchers reported for the first time that Mtu1 defects are correlated with reduced osteogenic differentiation. Using in vitro cultured bone marrow MSCs and stromal cell line MS5, we demonstrated that depressed Mtu1 expression was associated with reduced 2-thiouridine modification of the U34 of mitochondrial tRNAGln, tRNAGlu, and tRNALys, which led to respiratory deficiencies and reduced mitochondrial ATP production, and finally suppressed osteogenic differentiation. As expected, these Mtu1-deficient mice exhibited obvious osteopenia. Therefore, our findings in this study provide new insights into the pathophysiology of osteopenia.


Assuntos
Proteínas Mitocondriais/metabolismo , Osteogênese/genética , tRNA Metiltransferases/metabolismo , Animais , Diferenciação Celular , Humanos , Camundongos
9.
EBioMedicine ; 63: 103195, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33418496

RESUMO

BACKGROUND: Pancreatic cancer (PC) is one of the most lethal solid malignancies in the world due to its excessive cell proliferation and aggressive metastatic features. Emerging evidences revealed the importance of posttranscriptional modifications of RNAs in PC progression. However, knowledge about the 5-methylcytosine (m5C) RNA modification in PC is still extremely limited. In this study, we attempted to explore the expression changes and clinical significances of 12 known m5C-related genes among PC patients. METHODS: A total of 362 normal and 382 tumor specimens from PC patients were examined for candidate m5C-related gene and protein expression by using quantitative PCR (qPCR) and immunohistochemistry (IHC). The proliferation rate of PC cells was detected by MTS assay. Xenograft mouse models were used to assess the role of NSUN6 in PC tumor formation. FINDINGS: Through analyzing the four Gene Expression Omnibus (GEO) databases, six m5C-related genes shown significant and consistent alterations were selected for further examination in our 3 independent PC cohorts. Finally, we identified the reduction of NSUN6 as a common feature of all PC sample sets examined. NSUN6 expression correlated with clinicopathologic parameters including T stage, and Ki67+ cell rate. Further assessing the transcriptional profiles of 50 PC tissues, we found biological processes associated with cell proliferation like cell cycle and G2M checkpoint were enriched in NSUN6 lower expression group. Helped by in vitro PC cell lines and in vivo xenograft mouse models, we confirmed the role of NSUN6 in regulating cell proliferation and PC tumor growth. Last but also importantly, we also show the good performance of NSUN6 in evaluating tumor recurrence and survival among PC patients. INTERPRETATION: Our data suggested that NSUN6 is an important factor involved in regulating cell proliferation of PC, and highlights the potential of novel m5C-based clinical modalities as a therapeutic approach in PC patients. FUNDING: This study was supported by the National Natural Science Foundation of China (Grant Nos. 81803014, 81802424, and 81802911).


Assuntos
Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Suscetibilidade a Doenças , Neoplasias Pancreáticas/etiologia , Neoplasias Pancreáticas/metabolismo , tRNA Metiltransferases/genética , Animais , Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células , Biologia Computacional/métodos , Bases de Dados Genéticas , Modelos Animais de Doenças , Expressão Gênica , Perfilação da Expressão Gênica , Xenoenxertos , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Camundongos , Neoplasias Pancreáticas/diagnóstico , Neoplasias Pancreáticas/mortalidade , Prognóstico , Curva ROC , tRNA Metiltransferases/metabolismo
10.
Nucleic Acids Res ; 49(2): 1075-1093, 2021 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-33398350

RESUMO

Defects in the posttranscriptional modifications of mitochondrial tRNAs have been linked to human diseases, but their pathophysiology remains elusive. In this report, we investigated the molecular mechanism underlying a deafness-associated tRNAIle 4295A>G mutation affecting a highly conserved adenosine at position 37, 3' adjacent to the tRNA's anticodon. Primer extension and methylation activity assays revealed that the m.4295A>G mutation introduced a tRNA methyltransferase 5 (TRMT5)-catalyzed m1G37 modification of tRNAIle. Molecular dynamics simulations suggested that the m.4295A>G mutation affected tRNAIle structure and function, supported by increased melting temperature, conformational changes and instability of mutated tRNA. An in vitro processing experiment revealed that the m.4295A>G mutation reduced the 5' end processing efficiency of tRNAIle precursors, catalyzed by RNase P. We demonstrated that cybrid cell lines carrying the m.4295A>G mutation exhibited significant alterations in aminoacylation and steady-state levels of tRNAIle. The aberrant tRNA metabolism resulted in the impairment of mitochondrial translation, respiratory deficiency, decreasing membrane potentials and ATP production, increasing production of reactive oxygen species and promoting autophagy. These demonstrated the pleiotropic effects of m.4295A>G mutation on tRNAIle and mitochondrial functions. Our findings highlighted the essential role of deficient posttranscriptional modifications in the structure and function of tRNA and their pathogenic consequence of deafness.


Assuntos
Perda Auditiva Neurossensorial/genética , Mutação Puntual , RNA de Transferência de Isoleucina/genética , Trifosfato de Adenosina/biossíntese , Adulto , Proteínas Arqueais/metabolismo , Autofagia , Sequência de Bases , Linhagem Celular , DNA Mitocondrial/genética , Grupos Étnicos/genética , Feminino , Pleiotropia Genética , Perda Auditiva Neurossensorial/etnologia , Humanos , Isoleucina/metabolismo , Masculino , Herança Materna , Potencial da Membrana Mitocondrial , Methanocaldococcus/enzimologia , Metilação , Pessoa de Meia-Idade , Mitocôndrias/metabolismo , Simulação de Dinâmica Molecular , Conformação de Ácido Nucleico , Fosforilação Oxidativa , Linhagem , Biossíntese de Proteínas , Processamento Pós-Transcricional do RNA , Proteínas Recombinantes/metabolismo , Aminoacilação de RNA de Transferência , Adulto Jovem , tRNA Metiltransferases/metabolismo
11.
Methods Mol Biol ; 2192: 89-101, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33230768

RESUMO

Mitochondrial RNAs are modified posttranscriptionally. These modifications are required for proper functioning of RNA molecules, and thereby contribute to essential mitochondrial processes. Herein, we describe our latest mass spectrometry-based platform for analysis of posttranscriptional modifications of mitochondrial tRNAs, and measuring the in vitro activity of mitochondrial RNA-modifying enzymes.


Assuntos
Espectrometria de Massas/métodos , Mitocôndrias/metabolismo , Processamento Pós-Transcricional do RNA/fisiologia , RNA Mitocondrial/química , RNA Mitocondrial/metabolismo , RNA de Transferência/química , RNA de Transferência/metabolismo , Biocatálise , Humanos , Conformação de Ácido Nucleico , Nucleosídeos/química , RNA Mitocondrial/isolamento & purificação , RNA Ribossômico 16S/química , RNA Ribossômico 16S/metabolismo , RNA de Transferência/isolamento & purificação , tRNA Metiltransferases/metabolismo
12.
Nucleic Acids Res ; 49(2): 1006-1022, 2021 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-33330931

RESUMO

The highly abundant N6-methyladenosine (m6A) RNA modification affects most aspects of mRNA function, yet the precise function of the rarer 5-methylcytidine (m5C) remains largely unknown. Here, we map m5C in the human transcriptome using methylation-dependent individual-nucleotide resolution cross-linking and immunoprecipitation (miCLIP) combined with RNA bisulfite sequencing. We identify NSUN6 as a methyltransferase with strong substrate specificity towards mRNA. NSUN6 primarily targeted three prime untranslated regions (3'UTR) at the consensus sequence motif CTCCA, located in loops of hairpin structures. Knockout and rescue experiments revealed enhanced mRNA and translation levels when NSUN6-targeted mRNAs were methylated. Ribosome profiling further demonstrated that NSUN6-specific methylation correlated with translation termination. While NSUN6 was dispensable for mouse embryonic development, it was down-regulated in human tumours and high expression of NSUN6 indicated better patient outcome of certain cancer types. In summary, our study identifies NSUN6 as a methyltransferase targeting mRNA, potentially as part of a quality control mechanism involved in translation termination fidelity.


Assuntos
Citidina/análogos & derivados , Processamento Pós-Transcricional do RNA , RNA Mensageiro/metabolismo , tRNA Metiltransferases/metabolismo , Regiões 3' não Traduzidas , Animais , Sequência de Bases , Linhagem Celular Tumoral , Uso do Códon , Sequência Consenso , Citidina/metabolismo , Células-Tronco Embrionárias , Técnicas de Inativação de Genes , Genes Reporter , Células HEK293 , Humanos , Imunoprecipitação , Metilação , Camundongos , Camundongos Knockout , Mutagênese Sítio-Dirigida , RNA Mensageiro/genética , Transcriptoma , tRNA Metiltransferases/deficiência
13.
Nucleic Acids Res ; 48(21): 12310-12325, 2020 12 02.
Artigo em Inglês | MEDLINE | ID: mdl-33166396

RESUMO

The Mtq2-Trm112 methyltransferase modifies the eukaryotic translation termination factor eRF1 on the glutamine side chain of a universally conserved GGQ motif that is essential for release of newly synthesized peptides. Although this modification is found in the three domains of life, its exact role in eukaryotes remains unknown. As the deletion of MTQ2 leads to severe growth impairment in yeast, we have investigated its role further and tested its putative involvement in ribosome biogenesis. We found that Mtq2 is associated with nuclear 60S subunit precursors, and we demonstrate that its catalytic activity is required for nucleolar release of pre-60S and for efficient production of mature 5.8S and 25S rRNAs. Thus, we identify Mtq2 as a novel ribosome assembly factor important for large ribosomal subunit formation. We propose that Mtq2-Trm112 might modify eRF1 in the nucleus as part of a quality control mechanism aimed at proof-reading the peptidyl transferase center, where it will subsequently bind during translation termination.


Assuntos
Regulação Fúngica da Expressão Gênica , Metiltransferases/genética , Biogênese de Organelas , Fatores de Terminação de Peptídeos/genética , Subunidades Ribossômicas Maiores de Eucariotos/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , tRNA Metiltransferases/genética , Sítios de Ligação , Biocatálise , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Metiltransferases/química , Metiltransferases/metabolismo , Modelos Moleculares , Terminação Traducional da Cadeia Peptídica , Fatores de Terminação de Peptídeos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , RNA Ribossômico/biossíntese , RNA Ribossômico/genética , RNA Ribossômico 5,8S/biossíntese , RNA Ribossômico 5,8S/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Especificidade por Substrato , tRNA Metiltransferases/química , tRNA Metiltransferases/metabolismo
14.
PLoS One ; 15(11): e0242737, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33253256

RESUMO

Posttranscriptional modification of tRNA is critical for efficient protein translation and proper cell growth, and defects in tRNA modifications are often associated with human disease. Although most of the enzymes required for eukaryotic tRNA modifications are known, many of these enzymes have not been identified and characterized in several model multicellular eukaryotes. Here, we present two related approaches to identify the genes required for tRNA modifications in multicellular organisms using primer extension assays with fluorescent oligonucleotides. To demonstrate the utility of these approaches we first use expression of exogenous genes in yeast to experimentally identify two TRM1 orthologs capable of forming N2,N2-dimethylguanosine (m2,2G) on residue 26 of cytosolic tRNA in the model plant Arabidopsis thaliana. We also show that a predicted catalytic aspartate residue is required for function in each of the proteins. We next use RNA interference in cultured Drosophila melanogaster cells to identify the gene required for m2,2G26 formation on cytosolic tRNA. Additionally, using these approaches we experimentally identify D. melanogaster gene CG10050 as the corresponding ortholog of human DTWD2, which encodes the protein required for formation of 3-amino-3-propylcarboxyuridine (acp3U) on residue 20a of cytosolic tRNA. We further show that A. thaliana gene AT2G41750 can form acp3U20b on an A. thaliana tRNA expressed in yeast cells, and that the aspartate and tryptophan residues in the DXTW motif of this protein are required for modification activity. These results demonstrate that these approaches can be used to study tRNA modification enzymes.


Assuntos
Proteínas de Arabidopsis , Citosol/enzimologia , Proteínas de Drosophila , RNA de Transferência , tRNA Metiltransferases , Animais , Arabidopsis/enzimologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , RNA de Transferência/genética , RNA de Transferência/metabolismo , tRNA Metiltransferases/genética , tRNA Metiltransferases/metabolismo
15.
Nucleic Acids Res ; 48(19): 11068-11082, 2020 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-33035335

RESUMO

tRNAs play a central role during the translation process and are heavily post-transcriptionally modified to ensure optimal and faithful mRNA decoding. These epitranscriptomics marks are added by largely conserved proteins and defects in the function of some of these enzymes are responsible for neurodevelopmental disorders and cancers. Here, we focus on the Trm11 enzyme, which forms N2-methylguanosine (m2G) at position 10 of several tRNAs in both archaea and eukaryotes. While eukaryotic Trm11 enzyme is only active as a complex with Trm112, an allosteric activator of methyltransferases modifying factors (RNAs and proteins) involved in mRNA translation, former studies have shown that some archaeal Trm11 proteins are active on their own. As these studies were performed on Trm11 enzymes originating from archaeal organisms lacking TRM112 gene, we have characterized Trm11 (AfTrm11) from the Archaeoglobus fulgidus archaeon, which genome encodes for a Trm112 protein (AfTrm112). We show that AfTrm11 interacts directly with AfTrm112 similarly to eukaryotic enzymes and that although AfTrm11 is active as a single protein, its enzymatic activity is strongly enhanced by AfTrm112. We finally describe the first crystal structures of the AfTrm11-Trm112 complex and of Trm11, alone or bound to the methyltransferase inhibitor sinefungin.


Assuntos
Proteínas Arqueais , Archaeoglobus fulgidus/enzimologia , RNA Arqueal/metabolismo , RNA de Transferência/metabolismo , tRNA Metiltransferases , Proteínas Arqueais/química , Proteínas Arqueais/metabolismo , Modelos Moleculares , Estrutura Molecular , Ligação Proteica , Conformação Proteica , Processamento de Proteína Pós-Traducional , tRNA Metiltransferases/química , tRNA Metiltransferases/metabolismo
16.
EMBO J ; 39(23): e105364, 2020 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-33128823

RESUMO

Reversible infantile respiratory chain deficiency (RIRCD) is a rare mitochondrial myopathy leading to severe metabolic disturbances in infants, which recover spontaneously after 6-months of age. RIRCD is associated with the homoplasmic m.14674T>C mitochondrial DNA mutation; however, only ~ 1/100 carriers develop the disease. We studied 27 affected and 15 unaffected individuals from 19 families and found additional heterozygous mutations in nuclear genes interacting with mt-tRNAGlu including EARS2 and TRMU in the majority of affected individuals, but not in healthy carriers of m.14674T>C, supporting a digenic inheritance. Our transcriptomic and proteomic analysis of patient muscle suggests a stepwise mechanism where first, the integrated stress response associated with increased FGF21 and GDF15 expression enhances the metabolism modulated by serine biosynthesis, one carbon metabolism, TCA lipid oxidation and amino acid availability, while in the second step mTOR activation leads to increased mitochondrial biogenesis. Our data suggest that the spontaneous recovery in infants with digenic mutations may be modulated by the above described changes. Similar mechanisms may explain the variable penetrance and tissue specificity of other mtDNA mutations and highlight the potential role of amino acids in improving mitochondrial disease.


Assuntos
Doenças Mitocondriais/genética , Doenças Mitocondriais/metabolismo , Miopatias Mitocondriais/genética , Miopatias Mitocondriais/metabolismo , Adolescente , Linhagem Celular , DNA Mitocondrial/genética , Feminino , Expressão Gênica , Humanos , Lactente , Masculino , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Mutação , Linhagem , Proteômica , Músculo Quadríceps/metabolismo , tRNA Metiltransferases/genética , tRNA Metiltransferases/metabolismo
17.
Proc Natl Acad Sci U S A ; 117(34): 20785-20793, 2020 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-32778592

RESUMO

Transfer RNA (tRNA) activity is tightly regulated to provide a physiological protein translation, and tRNA chemical modifications control its function in a complex with ribosomes and messenger RNAs (mRNAs). In this regard, the correct hypermodification of position G37 of phenylalanine-tRNA, adjacent to the anticodon, is critical to prevent ribosome frameshifting events. Here we report that the tRNA-yW Synthesizing Protein 2 (TYW2) undergoes promoter hypermethylation-associated transcriptional silencing in human cancer, particularly in colorectal tumors. The epigenetic loss of TYW2 induces guanosine hypomodification in phenylalanine-tRNA, an increase in -1 ribosome frameshift events, and down-regulation of transcripts by mRNA decay, such as of the key cancer gene ROBO1. Importantly, TYW2 epigenetic inactivation is linked to poor overall survival in patients with early-stage colorectal cancer, a finding that could be related to the observed acquisition of enhanced migration properties and epithelial-to-mesenchymal features in the colon cancer cells that harbor TYW2 DNA methylation-associated loss. These findings provide an illustrative example of how epigenetic changes can modify the epitranscriptome and further support a role for tRNA modifications in cancer biology.


Assuntos
Neoplasias do Colo/genética , Mudança da Fase de Leitura do Gene Ribossômico , RNA de Transferência/genética , Ribossomos/genética , tRNA Metiltransferases/deficiência , Adulto , Idoso , Anticódon/genética , Anticódon/metabolismo , Linhagem Celular Tumoral , Neoplasias do Colo/enzimologia , Neoplasias do Colo/metabolismo , Ilhas de CpG , Epigênese Genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Conformação de Ácido Nucleico , Fenilalanina/genética , Fenilalanina/metabolismo , Regiões Promotoras Genéticas , Biossíntese de Proteínas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Transferência/metabolismo , Ribossomos/metabolismo , tRNA Metiltransferases/genética , tRNA Metiltransferases/metabolismo
18.
PLoS Genet ; 16(8): e1008893, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32841241

RESUMO

All tRNAs are extensively modified, and modification deficiency often results in growth defects in the budding yeast Saccharomyces cerevisiae and neurological or other disorders in humans. In S. cerevisiae, lack of any of several tRNA body modifications results in rapid tRNA decay (RTD) of certain mature tRNAs by the 5'-3' exonucleases Rat1 and Xrn1. As tRNA quality control decay mechanisms are not extensively studied in other eukaryotes, we studied trm8Δ mutants in the evolutionarily distant fission yeast Schizosaccharomyces pombe, which lack 7-methylguanosine at G46 (m7G46) of their tRNAs. We report here that S. pombe trm8Δ mutants are temperature sensitive primarily due to decay of tRNATyr(GUA) and that spontaneous mutations in the RAT1 ortholog dhp1+ restored temperature resistance and prevented tRNA decay, demonstrating conservation of the RTD pathway. We also report for the first time evidence linking the RTD and the general amino acid control (GAAC) pathways, which we show in both S. pombe and S. cerevisiae. In S. pombe trm8Δ mutants, spontaneous GAAC mutations restored temperature resistance and tRNA levels, and the trm8Δ temperature sensitivity was precisely linked to GAAC activation due to tRNATyr(GUA) decay. Similarly, in the well-studied S. cerevisiae trm8Δ trm4Δ RTD mutant, temperature sensitivity was closely linked to GAAC activation due to tRNAVal(AAC) decay; however, in S. cerevisiae, GAAC mutations increased tRNA loss and exacerbated temperature sensitivity. A similar exacerbated growth defect occurred upon GAAC mutation in S. cerevisiae trm8Δ and other single modification mutants that triggered RTD. Thus, these results demonstrate a conserved GAAC activation coincident with RTD in S. pombe and S. cerevisiae, but an opposite impact of the GAAC response in the two organisms. We speculate that the RTD pathway and its regulation of the GAAC pathway is widely conserved in eukaryotes, extending to other mutants affecting tRNA body modifications.


Assuntos
Exorribonucleases/metabolismo , Processamento Pós-Transcricional do RNA , Estabilidade de RNA , RNA de Transferência/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , tRNA Metiltransferases/metabolismo , Aminoácidos/metabolismo , Evolução Molecular , Exorribonucleases/genética , RNA de Transferência/metabolismo , Schizosaccharomyces , Proteínas de Schizosaccharomyces pombe/genética , tRNA Metiltransferases/genética
19.
Nucleic Acids Res ; 48(14): 8099-8112, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32602532

RESUMO

Translational frameshift errors are often deleterious to the synthesis of functional proteins and could therefore be promoted therapeutically to kill bacteria. TrmD (tRNA-(N(1)G37) methyltransferase) is an essential tRNA modification enzyme in bacteria that prevents +1 errors in the reading frame during protein translation and represents an attractive potential target for the development of new antibiotics. Here, we describe the application of a structure-guided fragment-based drug discovery approach to the design of a new class of inhibitors against TrmD in Mycobacterium abscessus. Fragment library screening, followed by structure-guided chemical elaboration of hits, led to the rapid development of drug-like molecules with potent in vitro TrmD inhibitory activity. Several of these compounds exhibit activity against planktonic M. abscessus and M. tuberculosis as well as against intracellular M. abscessus and M. leprae, indicating their potential as the basis for a novel class of broad-spectrum mycobacterial drugs.


Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , RNA de Transferência/metabolismo , tRNA Metiltransferases/antagonistas & inibidores , Antibacterianos/química , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Descoberta de Drogas/métodos , Inibidores Enzimáticos/química , Simulação de Acoplamento Molecular , Mycobacterium abscessus/efeitos dos fármacos , Mycobacterium abscessus/enzimologia , Mycobacterium leprae/efeitos dos fármacos , Mycobacterium leprae/enzimologia , Ligação Proteica , tRNA Metiltransferases/química , tRNA Metiltransferases/metabolismo
20.
EMBO Rep ; 21(8): e50095, 2020 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-32558197

RESUMO

tRNA modifications at the anti-codon loop are critical for accurate decoding. FTSJ1 was hypothesized to be a human tRNA 2'-O-methyltransferase. tRNAPhe (GAA) from intellectual disability patients with mutations in ftsj1 lacks 2'-O-methylation at C32 and G34 (Cm32 and Gm34). However, the catalytic activity, RNA substrates, and pathogenic mechanism of FTSJ1 remain unknown, owing, in part, to the difficulty in reconstituting enzymatic activity in vitro. Here, we identify an interacting protein of FTSJ1, WDR6. For the first time, we reconstitute the 2'-O-methylation activity of the FTSJ1-WDR6 complex in vitro, which occurs at position 34 of specific tRNAs with m1 G37 as a prerequisite. We find that modifications at positions 32, 34, and 37 are interdependent and occur in a hierarchical order in vivo. We also show that the translation efficiency of the UUU codon, but not the UUC codon decoded by tRNAPhe (GAA), is reduced in ftsj1 knockout cells. Bioinformatics analysis reveals that almost 40% of the high TTT-biased genes are related to brain/nervous functions. Our data potentially enhance our understanding of the relationship between FTSJ1 and nervous system development.


Assuntos
Deficiência Intelectual , Códon , Humanos , Deficiência Intelectual/genética , Metilação , Metiltransferases/genética , Metiltransferases/metabolismo , Proteínas Nucleares/metabolismo , RNA de Transferência/genética , RNA de Transferência/metabolismo , tRNA Metiltransferases/genética , tRNA Metiltransferases/metabolismo
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