RESUMEN
Mycobacterium tuberculosis transfer RNA (tRNA) terminal nucleotidyltransferase toxin, MenT3, incorporates nucleotides at the 3'-CCA end of tRNAs, blocking their aminoacylation and inhibiting protein synthesis. Here, we show that MenT3 most effectively adds CMPs to the 3'-CCA end of tRNA. The crystal structure of MenT3 in complex with CTP reveals a CTP-specific nucleotide-binding pocket. The 4-NH2 and the N3 and O2 atoms of cytosine in CTP form hydrogen bonds with the main-chain carbonyl oxygen of P120 and the side chain of R238, respectively. MenT3 expression in Escherichia coli selectively reduces the levels of seryl-tRNASers, indicating specific inactivation of tRNASers by MenT3. Consistently, MenT3 incorporates CMPs into tRNASer most efficiently, among the tested E. coli tRNA species. The longer variable loop unique to class II tRNASers is crucial for efficient CMP incorporation into tRNASer by MenT3. Replacing the variable loop of E. coli tRNAAla with the longer variable loop of M. tuberculosis tRNASer enables MenT3 to incorporate CMPs into the chimeric tRNAAla. The N-terminal positively charged region of MenT3 is required for CMP incorporation into tRNASer. A docking model of tRNA onto MenT3 suggests that an interaction between the N-terminal region and the longer variable loop of tRNASer facilitates tRNA substrate selection.
Asunto(s)
Mycobacterium tuberculosis , ARN de Transferencia , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/enzimología , Especificidad por Sustrato , ARN de Transferencia/metabolismo , ARN de Transferencia/química , Escherichia coli/genética , Escherichia coli/metabolismo , Toxinas Bacterianas/química , Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/genética , Modelos Moleculares , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Citidina/química , Citidina/metabolismo , Sitios de Unión , Cristalografía por Rayos X , ARN Nucleotidiltransferasas/metabolismo , ARN Nucleotidiltransferasas/química , ARN Nucleotidiltransferasas/genéticaRESUMEN
Recent developments in sequencing and bioinformatics have advanced our understanding of adenosine-to-inosine (A-to-I) RNA editing. Surprisingly, recent analyses have revealed the capability of adenosine deaminase acting on RNA (ADAR) to edit DNA:RNA hybrid strands. However, edited inosines in DNA remain largely unexplored. A precise biochemical method could help uncover these potentially rare DNA editing sites. We explore maleimide as a scaffold for inosine labeling. With fluorophore-conjugated maleimide, we were able to label inosine in RNA or DNA. Moreover, with biotin-conjugated maleimide, we purified RNA and DNA containing inosine. Our novel technique of inosine chemical labeling and affinity molecular purification offers substantial advantages and provides a versatile platform for further discovery of A-to-I editing sites in RNA and DNA.
Asunto(s)
Adenosina , Inosina , Edición de ARN , Inosina/química , Inosina/metabolismo , Adenosina/química , Adenosina/metabolismo , Adenosina/análogos & derivados , Desaminación , ADN/química , ADN/metabolismo , Maleimidas/química , Adenosina Desaminasa/metabolismo , Adenosina Desaminasa/química , ARN/química , ARN/metabolismo , Coloración y Etiquetado/métodos , Humanos , Colorantes Fluorescentes/química , Biotina/química , Biotina/metabolismoRESUMEN
In the hypothetical RNA world, ribozymes could have acted as modern aminoacyl-tRNA synthetases (ARSs) to charge tRNAs, thus giving rise to the peptide synthesis along with the evolution of a primitive translation apparatus. We previously reported a T-boxzyme, Tx2.1, which selectively charges initiator tRNA with N-biotinyl-phenylalanine (BioPhe) in situ in a Flexible In-vitro Translation (FIT) system to produce BioPhe-initiating peptides. Here, we performed in vitro selection of elongation-capable T-boxzymes (elT-boxzymes), using para-azido-l-phenylalanine (PheAZ) as an acyl-donor. We implemented a new strategy to enrich elT-boxzyme-tRNA conjugates that self-aminoacylated on the 3'-terminus selectively. One of them, elT32, can charge PheAZ onto tRNA in trans in response to its cognate anticodon. Further evolution of elT32 resulted in elT49, with enhanced aminoacylation activity. We have demonstrated the translation of a PheAZ-containing peptide in an elT-boxzyme-integrated FIT system, revealing that elT-boxzymes are able to generate the PheAZ-tRNA in response to the cognate anticodon in situ of a custom-made translation system. This study, together with Tx2.1, illustrates a scenario where a series of ribozymes could have overseen aminoacylation and co-evolved with a primitive RNA-based translation system.
Asunto(s)
Anticodón , Biosíntesis de Proteínas , ARN Catalítico , Aminoacil-ARN de Transferencia , ARN Catalítico/metabolismo , ARN Catalítico/genética , Anticodón/genética , Aminoacil-ARN de Transferencia/metabolismo , Aminoacil-ARN de Transferencia/genética , Fenilalanina/metabolismo , Fenilalanina/análogos & derivados , Aminoacil-ARNt Sintetasas/metabolismo , Aminoacil-ARNt Sintetasas/genética , Aminoacilación de ARN de Transferencia , Aminoacilación , Extensión de la Cadena Peptídica de TranslaciónRESUMEN
Transfer RNA (tRNA) modifications are critical for protein synthesis. Queuosine (Q), a 7-deaza-guanosine derivative, is present in tRNA anticodons. In vertebrate tRNAs for Tyr and Asp, Q is further glycosylated with galactose and mannose to generate galQ and manQ, respectively. However, biogenesis and physiological relevance of Q-glycosylation remain poorly understood. Here, we biochemically identified two RNA glycosylases, QTGAL and QTMAN, and successfully reconstituted Q-glycosylation of tRNAs using nucleotide diphosphate sugars. Ribosome profiling of knockout cells revealed that Q-glycosylation slowed down elongation at cognate codons, UAC and GAC (GAU), respectively. We also found that galactosylation of Q suppresses stop codon readthrough. Moreover, protein aggregates increased in cells lacking Q-glycosylation, indicating that Q-glycosylation contributes to proteostasis. Cryo-EM of human ribosome-tRNA complex revealed the molecular basis of codon recognition regulated by Q-glycosylations. Furthermore, zebrafish qtgal and qtman knockout lines displayed shortened body length, implying that Q-glycosylation is required for post-embryonic growth in vertebrates.
Asunto(s)
ARN de Transferencia , Animales , Humanos , Ratas , Anticodón , Línea Celular , Codón , Glicosilación , Nucleósido Q/química , Nucleósido Q/genética , Nucleósido Q/metabolismo , ARN de Transferencia/química , ARN de Transferencia/metabolismo , Porcinos , Pez Cebra/metabolismo , Conformación de Ácido NucleicoRESUMEN
In the early stage of bacterial translation, peptidyl-tRNAs frequently dissociate from the ribosome (pep-tRNA drop-off) and are recycled by peptidyl-tRNA hydrolase. Here, we establish a highly sensitive method for profiling of pep-tRNAs using mass spectrometry, and successfully detect a large number of nascent peptides from pep-tRNAs accumulated in Escherichia coli pthts strain. Based on molecular mass analysis, we found about 20% of the peptides bear single amino-acid substitutions of the N-terminal sequences of E. coli ORFs. Detailed analysis of individual pep-tRNAs and reporter assay revealed that most of the substitutions take place at the C-terminal drop-off site and that the miscoded pep-tRNAs rarely participate in the next round of elongation but dissociate from the ribosome. These findings suggest that pep-tRNA drop-off is an active mechanism by which the ribosome rejects miscoded pep-tRNAs in the early elongation, thereby contributing to quality control of protein synthesis after peptide bond formation.
Asunto(s)
Escherichia coli , Aminoacil-ARN de Transferencia , Escherichia coli/genética , Escherichia coli/metabolismo , Aminoacil-ARN de Transferencia/metabolismo , Ribosomas/metabolismo , ARN de Transferencia/genética , ARN de Transferencia/química , Péptidos/química , Control de Calidad , Biosíntesis de ProteínasRESUMEN
The class 2 type V CRISPR effector Cas12 is thought to have evolved from the IS200/IS605 superfamily of transposon-associated TnpB proteins1. Recent studies have identified TnpB proteins as miniature RNA-guided DNA endonucleases2,3. TnpB associates with a single, long RNA (ωRNA) and cleaves double-stranded DNA targets complementary to the ωRNA guide. However, the RNA-guided DNA cleavage mechanism of TnpB and its evolutionary relationship with Cas12 enzymes remain unknown. Here we report the cryo-electron microscopy (cryo-EM) structure of Deinococcus radiodurans ISDra2 TnpB in complex with its cognate ωRNA and target DNA. In the structure, the ωRNA adopts an unexpected architecture and forms a pseudoknot, which is conserved among all guide RNAs of Cas12 enzymes. Furthermore, the structure, along with our functional analysis, reveals how the compact TnpB recognizes the ωRNA and cleaves target DNA complementary to the guide. A structural comparison of TnpB with Cas12 enzymes suggests that CRISPR-Cas12 effectors acquired an ability to recognize the protospacer-adjacent motif-distal end of the guide RNA-target DNA heteroduplex, by either asymmetric dimer formation or diverse REC2 insertions, enabling engagement in CRISPR-Cas adaptive immunity. Collectively, our findings provide mechanistic insights into TnpB function and advance our understanding of the evolution from transposon-encoded TnpB proteins to CRISPR-Cas12 effectors.
Asunto(s)
Proteínas Bacterianas , Microscopía por Crioelectrón , Elementos Transponibles de ADN , Deinococcus , Endodesoxirribonucleasas , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/ultraestructura , Proteínas Asociadas a CRISPR/química , Proteínas Asociadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , ADN/química , ADN/genética , ADN/metabolismo , ADN/ultraestructura , Elementos Transponibles de ADN/genética , ARN Guía de Sistemas CRISPR-Cas/química , ARN Guía de Sistemas CRISPR-Cas/genética , ARN Guía de Sistemas CRISPR-Cas/metabolismo , ARN Guía de Sistemas CRISPR-Cas/ultraestructura , Endodesoxirribonucleasas/química , Endodesoxirribonucleasas/metabolismo , Endodesoxirribonucleasas/ultraestructura , Deinococcus/enzimología , Deinococcus/genética , Especificidad por SustratoRESUMEN
Post-transcriptional modifications have critical roles in tRNA stability and function1-4. In thermophiles, tRNAs are heavily modified to maintain their thermal stability under extreme growth temperatures5,6. Here we identified 2'-phosphouridine (Up) at position 47 of tRNAs from thermophilic archaea. Up47 confers thermal stability and nuclease resistance to tRNAs. Atomic structures of native archaeal tRNA showed a unique metastable core structure stabilized by Up47. The 2'-phosphate of Up47 protrudes from the tRNA core and prevents backbone rotation during thermal denaturation. In addition, we identified the arkI gene, which encodes an archaeal RNA kinase responsible for Up47 formation. Structural studies showed that ArkI has a non-canonical kinase motif surrounded by a positively charged patch for tRNA binding. A knockout strain of arkI grew slowly at high temperatures and exhibited a synthetic growth defect when a second tRNA-modifying enzyme was depleted. We also identified an archaeal homologue of KptA as an eraser that efficiently dephosphorylates Up47 in vitro and in vivo. Taken together, our findings show that Up47 is a reversible RNA modification mediated by ArkI and KptA that fine-tunes the structural rigidity of tRNAs under extreme environmental conditions.
Asunto(s)
Archaea , ARN de Transferencia , Termotolerancia , Archaea/genética , Ambientes Extremos , Fosforilación , Procesamiento Postranscripcional del ARN , ARN de Archaea/química , ARN de Archaea/metabolismo , ARN de Transferencia/química , ARN de Transferencia/metabolismo , UridinaRESUMEN
Contact-dependent growth inhibition is a mechanism of interbacterial competition mediated by delivery of the C-terminal toxin domain of CdiA protein (CdiA-CT) into neighboring bacteria. The CdiA-CT of enterohemorrhagic Escherichia coli EC869 (CdiA-CTEC869) cleaves the 3'-acceptor regions of specific tRNAs in a reaction that requires the translation factors Tu/Ts and GTP. Here, we show that CdiA-CTEC869 has an intrinsic ability to recognize a specific sequence in substrate tRNAs, and Tu:Ts complex promotes tRNA cleavage by CdiA-CTEC869. Uncharged and aminoacylated tRNAs (aa-tRNAs) were cleaved by CdiA-CTEC869 to the same extent in the presence of Tu/Ts, and the CdiA-CTEC869:Tu:Ts:tRNA(aa-tRNA) complex formed in the presence of GTP. CdiA-CTEC869 interacts with domain II of Tu, thereby preventing the 3'-moiety of tRNA to bind to Tu as in canonical Tu:GTP:aa-tRNA complexes. Superimposition of the Tu:GTP:aa-tRNA structure onto the CdiA-CTEC869:Tu structure suggests that the 3'-portion of tRNA relocates into the CdiA-CTEC869 active site, located on the opposite side to the CdiA-CTEC869 :Tu interface, for tRNA cleavage. Thus, CdiA-CTEC869 is recruited to Tu:GTP:Ts, and CdiA-CT:Tu:GTP:Ts recognizes substrate tRNAs and cleaves them. Tu:GTP:Ts serves as a reaction scaffold that increases the affinity of CdiA-CTEC869 for substrate tRNAs and induces a structural change of tRNAs for efficient cleavage by CdiA-CTEC869.
Asunto(s)
Escherichia coli Enterohemorrágica , Proteínas de Escherichia coli , Escherichia coli Enterohemorrágica/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Inhibidores de Crecimiento , Guanosina Trifosfato/metabolismo , Proteínas de la Membrana/metabolismo , Factor Tu de Elongación Peptídica/metabolismo , ARN de Transferencia/metabolismo , Aminoacil-ARN de TransferenciaRESUMEN
Bacterial toxin-antitoxin modules contribute to the stress adaptation, persistence, and dormancy of bacteria for survival under environmental stresses and are involved in bacterial pathogenesis. In Salmonella Typhimurium, the Gcn5-related N-acetyltransferase toxin TacT reportedly acetylates the α-amino groups of the aminoacyl moieties of several aminoacyl-tRNAs, inhibits protein synthesis, and promotes persister formation during the infection of macrophages. Here, we show that TacT exclusively acetylates Gly-tRNAGlyin vivo and in vitro. The crystal structure of the TacT:acetyl-Gly-tRNAGly complex and the biochemical analysis reveal that TacT specifically recognizes the discriminator U73 and G71 in tRNAGly, a combination that is only found in tRNAGly isoacceptors, and discriminates tRNAGly from other tRNA species. Thus, TacT is a Gly-tRNAGly-specific acetyltransferase toxin. The molecular basis of the specific aminoacyl-tRNA acetylation by TacT provides advanced information for the design of drugs targeting Salmonella.
Asunto(s)
Acetiltransferasas/metabolismo , Toxinas Bacterianas/metabolismo , Aminoacil-ARN de Transferencia/metabolismo , ARN de Transferencia de Glicerina/metabolismo , Salmonella typhimurium/enzimología , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismo , Acetilación , Acetiltransferasas/química , Antitoxinas/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Toxinas Bacterianas/química , ADN Bacteriano , Procesamiento Proteico-Postraduccional , Infecciones por Salmonella/microbiología , Salmonella typhimurium/químicaRESUMEN
Transfer RNA[Ser]Sec carries multiple post-transcriptional modifications. The A37G mutation in tRNA[Ser]Sec abrogates isopentenylation of base 37 and has a profound effect on selenoprotein expression in mice. Patients with a homozygous pathogenic p.R323Q variant in tRNA-isopentenyl-transferase (TRIT1) show a severe neurological disorder, and hence we wondered whether selenoprotein expression was impaired. Patient fibroblasts with the homozygous p.R323Q variant did not show a general decrease in selenoprotein expression. However, recombinant human TRIT1R323Q had significantly diminished activities towards several tRNA substrates in vitro. We thus engineered mice conditionally deficient in Trit1 in hepatocytes and neurons. Mass-spectrometry revealed that hypermodification of U34 to mcm5Um occurs independently of isopentenylation of A37 in tRNA[Ser]Sec. Western blotting and 75Se metabolic labeling showed only moderate effects on selenoprotein levels and 75Se incorporation. A detailed analysis of Trit1-deficient liver using ribosomal profiling demonstrated that UGA/Sec re-coding was moderately affected in Selenop, Txnrd1, and Sephs2, but not in Gpx1. 2'O-methylation of U34 in tRNA[Ser]Sec depends on FTSJ1, but does not affect UGA/Sec re-coding in selenoprotein translation. Taken together, our results show that a lack of isopentenylation of tRNA[Ser]Sec affects UGA/Sec read-through but differs from a A37G mutation.
Asunto(s)
Transferasas Alquil y Aril/genética , ARN de Transferencia/metabolismo , Selenoproteínas/metabolismo , Transferasas Alquil y Aril/metabolismo , Animales , Línea Celular , Cisteína/metabolismo , Hepatocitos/metabolismo , Humanos , Hígado/metabolismo , Ratones , Neuronas/metabolismo , Fosfotransferasas/genética , Fosfotransferasas/metabolismo , Biosíntesis de Proteínas/genética , ARN de Transferencia/genética , Ribosomas/metabolismo , Selenio/metabolismo , Selenocisteína/genética , Selenoproteína P/genética , Selenoproteínas/genéticaRESUMEN
The tRNA pool determines the efficiency, throughput, and accuracy of translation. Previous studies have identified dynamic changes in the tRNA (transfer RNA) supply and mRNA (messenger RNA) demand during cancerous proliferation. Yet dynamic changes may also occur during physiologically normal proliferation, and these are less well characterized. We examined the tRNA and mRNA pools of T cells during their vigorous proliferation and differentiation upon triggering their antigen receptor. We observed a global signature of switch in demand for codons at the early proliferation phase of the response, accompanied by corresponding changes in tRNA expression levels. In the later phase, upon differentiation, the response of the tRNA pool relaxed back to the basal level, potentially restraining excessive proliferation. Sequencing of tRNAs allowed us to evaluate their diverse base-modifications. We found that two types of tRNA modifications, wybutosine and ms2t6A, are reduced dramatically during T cell activation. These modifications occur in the anticodon loops of two tRNAs that decode "slippery codons," which are prone to ribosomal frameshifting. Attenuation of these frameshift-protective modifications is expected to increase the potential for proteome-wide frameshifting during T cell proliferation. Indeed, human cell lines deleted of a wybutosine writer showed increased ribosomal frameshifting, as detected with an HIV gag-pol frameshifting site reporter. These results may explain HIV's specific tropism toward proliferating T cells since it requires ribosomal frameshift exactly on the corresponding codon for infection. The changes in tRNA expression and modifications uncover a layer of translation regulation during T cell proliferation and expose a potential tradeoff between cellular growth and translation fidelity.
Asunto(s)
Activación de Linfocitos , ARN de Transferencia/metabolismo , Linfocitos T/inmunología , Proliferación Celular/genética , Codón , Mutación del Sistema de Lectura , Humanos , Procesamiento Postranscripcional del ARN , Linfocitos T/citologíaRESUMEN
RelA-SpoT Homolog (RSH) enzymes control bacterial physiology through synthesis and degradation of the nucleotide alarmone (p)ppGpp. We recently discovered multiple families of small alarmone synthetase (SAS) RSH acting as toxins of toxin-antitoxin (TA) modules, with the FaRel subfamily of toxSAS abrogating bacterial growth by producing an analog of (p)ppGpp, (pp)pApp. Here we probe the mechanism of growth arrest used by four experimentally unexplored subfamilies of toxSAS: FaRel2, PhRel, PhRel2, and CapRel. Surprisingly, all these toxins specifically inhibit protein synthesis. To do so, they transfer a pyrophosphate moiety from ATP to the tRNA 3' CCA. The modification inhibits both tRNA aminoacylation and the sensing of cellular amino acid starvation by the ribosome-associated RSH RelA. Conversely, we show that some small alarmone hydrolase (SAH) RSH enzymes can reverse the pyrophosphorylation of tRNA to counter the growth inhibition by toxSAS. Collectively, we establish RSHs as RNA-modifying enzymes.
Asunto(s)
Toxinas Bacterianas/metabolismo , Guanosina Pentafosfato/metabolismo , Ligasas/metabolismo , ARN de Transferencia/metabolismo , Toxinas Bacterianas/genética , Toxinas Bacterianas/farmacología , Bacilos Grampositivos Asporogénicos/química , Bacilos Grampositivos Asporogénicos/metabolismo , Guanosina Pentafosfato/química , Ligasas/química , Ligasas/genética , Fosforilación/efectos de los fármacos , Biosíntesis de Proteínas/efectos de los fármacos , Biosíntesis de Proteínas/fisiología , Inhibidores de la Síntesis de la Proteína/farmacología , Pirofosfatasas , Ribosomas/metabolismoRESUMEN
Nuclear stress bodies (nSBs) are nuclear membraneless organelles formed around stress-inducible HSATIII architectural long noncoding RNAs (lncRNAs). nSBs repress splicing of hundreds of introns during thermal stress recovery, which are partly regulated by CLK1 kinase phosphorylation of temperature-dependent Ser/Arg-rich splicing factors (SRSFs). Here, we report a distinct mechanism for this splicing repression through protein sequestration by nSBs. Comprehensive identification of RNA-binding proteins revealed HSATIII association with proteins related to N6 -methyladenosine (m6 A) RNA modification. 11% of the first adenosine in the repetitive HSATIII sequence were m6 A-modified. nSBs sequester the m6 A writer complex to methylate HSATIII, leading to subsequent sequestration of the nuclear m6 A reader, YTHDC1. Sequestration of these factors from the nucleoplasm represses m6 A modification of pre-mRNAs, leading to repression of m6 A-dependent splicing during stress recovery phase. Thus, nSBs serve as a common platform for regulation of temperature-dependent splicing through dual mechanisms employing two distinct ribonucleoprotein modules with partially m6 A-modified architectural lncRNAs.
Asunto(s)
Proteínas del Tejido Nervioso/genética , Factores de Empalme de ARN/genética , Empalme del ARN , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Adenosina/análogos & derivados , Adenosina/metabolismo , Núcleo Celular/genética , Células HeLa , Humanos , Proteínas del Tejido Nervioso/metabolismo , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Proteínas Tirosina Quinasas/genética , Factores de Empalme de ARN/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Secuencias Repetitivas de Ácidos Nucleicos , Factores de Empalme Serina-Arginina/genética , Factores de Empalme Serina-Arginina/metabolismo , TemperaturaRESUMEN
Toxin-antitoxin systems in bacteria contribute to stress adaptation, dormancy, and persistence. AtaT, a type-II toxin in enterohemorrhagic E. coli, reportedly acetylates the α-amino group of the aminoacyl-moiety of initiator Met-tRNAfMet, thus inhibiting translation initiation. Here, we show that AtaT has a broader specificity for aminoacyl-tRNAs than initially claimed. AtaT efficiently acetylates Gly-tRNAGly, Trp-tRNATrp, Tyr-tRNATyr and Phe-tRNAPhe isoacceptors, in addition to Met-tRNAfMet, and inhibits global translation. AtaT interacts with the acceptor stem of tRNAfMet, and the consecutive G-C pairs in the bottom-half of the acceptor stem are required for acetylation. Consistently, tRNAGly, tRNATrp, tRNATyr and tRNAPhe also possess consecutive G-C base-pairs in the bottom halves of their acceptor stems. Furthermore, misaminoacylated valyl-tRNAfMet and isoleucyl-tRNAfMet are not acetylated by AtaT. Therefore, the substrate selection by AtaT is governed by the specific acceptor stem sequence and the properties of the aminoacyl-moiety of aminoacyl-tRNAs.
Asunto(s)
Acetiltransferasas/metabolismo , Escherichia coli Enterohemorrágica/metabolismo , Proteínas de Escherichia coli/metabolismo , Aminoacil-ARN de Transferencia/metabolismo , Aminoacilación de ARN de Transferencia , Acetilación , Acetiltransferasas/química , Acetiltransferasas/genética , Secuencia de Bases , Dominio Catalítico , Cristalografía por Rayos X , Escherichia coli Enterohemorrágica/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Modelos Moleculares , Conformación de Ácido Nucleico , Conformación Proteica , Aminoacil-ARN de Transferencia/química , Aminoacil-ARN de Transferencia/genéticaRESUMEN
Mitochondria generate most cellular energy via oxidative phosphorylation. Twenty-two species of mitochondrial (mt-)tRNAs encoded in mtDNA translate essential subunits of the respiratory chain complexes. mt-tRNAs contain post-transcriptional modifications introduced by nuclear-encoded tRNA-modifying enzymes. They are required for deciphering genetic code accurately, as well as stabilizing tRNA. Loss of tRNA modifications frequently results in severe pathological consequences. Here, we perform a comprehensive analysis of post-transcriptional modifications of all human mt-tRNAs, including 14 previously-uncharacterized species. In total, we find 18 kinds of RNA modifications at 137 positions (8.7% in 1575 nucleobases) in 22 species of human mt-tRNAs. An up-to-date list of 34 genes responsible for mt-tRNA modifications are provided. We identify two genes required for queuosine (Q) formation in mt-tRNAs. Our results provide insight into the molecular mechanisms underlying the decoding system and could help to elucidate the molecular pathogenesis of human mitochondrial diseases caused by aberrant tRNA modifications.
Asunto(s)
Procesamiento Postranscripcional del ARN , ARN Mitocondrial/química , ARN de Transferencia/química , Femenino , Código Genético , Células HEK293 , Células HeLa , Humanos , Espectrometría de Masas , Mitocondrias/metabolismo , Mitocondrias/patología , Enfermedades Mitocondriales/genética , Enfermedades Mitocondriales/patología , Estructura Molecular , Nucleósido Q/biosíntesis , Nucleósido Q/química , Fosforilación Oxidativa , Placenta , Embarazo , ARN Mitocondrial/aislamiento & purificación , ARN Mitocondrial/metabolismo , ARN de Transferencia/aislamiento & purificación , ARN de Transferencia/metabolismo , RNA-SeqRESUMEN
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.
Asunto(s)
Neoplasias del Colon/genética , Sistema de Lectura Ribosómico , ARN de Transferencia/genética , Ribosomas/genética , ARNt Metiltransferasas/deficiencia , Adulto , Anciano , Anticodón/genética , Anticodón/metabolismo , Línea Celular Tumoral , Neoplasias del Colon/enzimología , Neoplasias del Colon/metabolismo , Islas de CpG , Epigénesis Genética , Femenino , Humanos , Masculino , Persona de Mediana Edad , Conformación de Ácido Nucleico , Fenilalanina/genética , Fenilalanina/metabolismo , Regiones Promotoras Genéticas , Biosíntesis de Proteínas , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN de Transferencia/metabolismo , Ribosomas/metabolismo , ARNt Metiltransferasas/genética , ARNt Metiltransferasas/metabolismoRESUMEN
Escherichia coli ItaT toxin reportedly acetylates the α-amino group of the aminoacyl-moiety of Ile-tRNAIle specifically, using acetyl-CoA as an acetyl donor, thereby inhibiting protein synthesis. The mechanism of the substrate specificity of ItaT had remained elusive. Here, we present functional and structural analyses of E. coli ItaT, which revealed the mechanism of ItaT recognition of specific aminoacyl-tRNAs for acetylation. In addition to Ile-tRNAIle, aminoacyl-tRNAs charged with hydrophobic residues, such as Val-tRNAVal and Met-tRNAMet, were acetylated by ItaT in vivo. Ile-tRNAIle, Val-tRNAVal and Met-tRNAMet were acetylated by ItaT in vitro, while aminoacyl-tRNAs charged with other hydrophobic residues, such as Ala-tRNAAla, Leu-tRNALeu and Phe-tRNAPhe, were less efficiently acetylated. A comparison of the structures of E. coli ItaT and the protein N-terminal acetyltransferase identified the hydrophobic residues in ItaT that possibly interact with the aminoacyl moiety of aminoacyl-tRNAs. Mutations of the hydrophobic residues of ItaT reduced the acetylation activity of ItaT toward Ile-tRNAIlein vitro, as well as the ItaT toxicity in vivo. Altogether, the size and shape of the hydrophobic pocket of ItaT are suitable for the accommodation of the specific aminoacyl-moieties of aminoacyl-tRNAs, and ItaT has broader specificity toward aminoacyl-tRNAs charged with certain hydrophobic amino acids.
Asunto(s)
Acetiltransferasas/química , Toxinas Bacterianas/química , Proteínas de Escherichia coli/química , Aminoacilación de ARN de Transferencia , Acetiltransferasas/genética , Acetiltransferasas/metabolismo , Secuencias de Aminoácidos , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Escherichia coli , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Mutación , Aminoacil-ARN de Transferencia/química , Aminoacil-ARN de Transferencia/metabolismo , Especificidad por SustratoRESUMEN
We have recently developed an in vitro yeast reconstituted translation system, which is capable of synthesizing long polypeptides. Utilizing the system, we examined the role of eIF5A and its hypusine modification in translating polyproline sequence within long open reading frames. We found that polyproline motif inserted at the internal position of the protein arrests translation exclusively at low Mg2+ concentrations, and peptidylpolyproline-tRNA intrinsically destabilizes 80S ribosomes. We demonstrate that unmodified eIF5A essentially resolves such ribosome stalling; however, the hypusine modification drastically stimulates ability of eIF5A to rescue polyproline-mediated ribosome stalling and is particularly important for the efficient translation of the N-terminal or long internal polyproline motifs.
Asunto(s)
Biosíntesis de Péptidos , Factores de Iniciación de Péptidos/metabolismo , Péptidos/metabolismo , Proteínas de Unión al ARN/metabolismo , Saccharomyces cerevisiae/metabolismo , Factores de Iniciación de Péptidos/genética , Péptidos/química , Proteínas de Unión al ARN/genética , Ribosomas/metabolismo , Factor 5A Eucariótico de Iniciación de TraducciónRESUMEN
Modification of tRNA anticodons plays a critical role in ensuring accurate translation. N4-acetylcytidine (ac4C) is present at the anticodon first position (position 34) of bacterial elongator tRNAMet. Herein, we identified Bacillus subtilis ylbM (renamed tmcAL) as a novel gene responsible for ac4C34 formation. Unlike general acetyltransferases that use acetyl-CoA, TmcAL activates an acetate ion to form acetyladenylate and then catalyzes ac4C34 formation through a mechanism similar to tRNA aminoacylation. The crystal structure of TmcAL with an ATP analog reveals the molecular basis of ac4C34 formation. The ΔtmcAL strain displayed a cold-sensitive phenotype and a strong genetic interaction with tilS that encodes the enzyme responsible for synthesizing lysidine (L) at position 34 of tRNAIle to facilitate AUA decoding. Mistranslation of the AUA codon as Met in the ΔtmcAL strain upon tilS repression suggests that ac4C34 modification of tRNAMet and L34 modification of tRNAIle act cooperatively to prevent misdecoding of the AUA codon.
Asunto(s)
Acetatos/química , Bacillus subtilis/genética , ARN de Transferencia/química , Adenosina Trifosfato/química , Bacillus subtilis/metabolismo , Proteínas Bacterianas/genética , Catálisis , Dominio Catalítico , Codón de Terminación , Cristalografía por Rayos X , Análisis Mutacional de ADN , Escherichia coli/metabolismo , Mutación , Mycoplasma/metabolismo , Conformación de Ácido Nucleico , Fenotipo , Mutación Puntual , Biosíntesis de Proteínas , Dominios Proteicos , Proteínas Recombinantes/metabolismoRESUMEN
It has been generally thought that tRNA modifications are stable and static, and their frequencies are rarely regulated. N6-threonylcarbamoyladenosine (t6A) occurs at position 37 of five mitochondrial (mt-)tRNA species. We show that YRDC and OSGEPL1 are responsible for t6A37 formation, utilizing L-threonine, ATP, and CO2/bicarbonate as substrates. OSGEPL1-knockout cells exhibit respiratory defects and reduced mitochondrial translation. We find low level of t6A37 in mutant mt-tRNA isolated from the MERRF-like patient's cells, indicating that lack of t6A37 results in pathological consequences. Kinetic measurements of t6A37 formation reveal that the Km value of CO2/bicarbonate is extremely high (31 mM), suggesting that CO2/bicarbonate is a rate-limiting factor for t6A37 formation. Consistent with this, we observe a low frequency of t6A37 in mt-tRNAs isolated from human cells cultured without bicarbonate. These findings indicate that t6A37 is regulated by sensing intracellular CO2/bicarbonate concentration, implying that mitochondrial translation is modulated in a codon-specific manner under physiological conditions.