RESUMO
Mosquitoes such as Aedes aegypti must consume a blood meal for the nutrients necessary for egg production. Several transcriptome and proteome changes occur post-blood meal that likely corresponds with codon usage alterations. Transfer RNA (tRNA) is the adapter molecule that reads messenger RNA codons to add the appropriate amino acid during protein synthesis. Chemical modifications to tRNA enhance codon decoding, improving the accuracy and efficiency of protein synthesis. Here, we examined tRNA modifications and transcripts associated with the blood meal and subsequent periods of vitellogenesis in A. aegypti. More specifically, we assessed tRNA transcript abundance and modification levels in the fat body at critical times post blood-feeding. Based on a combination of alternative codon usage and identification of particular modifications, we discovered that increased transcription of tyrosine tRNAs is likely critical during the synthesis of egg yolk proteins in the fat body following a blood meal. Altogether, changes in both the abundance and modification of tRNA are essential factors in the process of vitellogenin production after blood-feeding in mosquitoes.
RESUMO
Post-transcriptional modifications are added to ribosomal RNAs (rRNAs) to govern ribosome biogenesis and to fine-tune protein biosynthesis. In Escherichia coli and related bacteria, RlhA uniquely catalyzes formation of a 5-hydroxycytidine (ho5C) at position 2501 of 23S rRNA. However, the molecular and biological functions as well as the regulation of ho5C2501 modification remain unclear. We measured growth curves with the modification-deficient ΔrlhA strain and quantified the extent of the modification during different conditions by mass spectrometry and reverse transcription. The levels of ho5C2501 in E. coli ribosomes turned out to be highly dynamic and growth phase-dependent, with the most effective hydroxylation yields observed in the stationary phase. We demonstrated a direct effect of ho5C2501 on translation efficiencies in vitro and in vivo. High ho5C2501 levels reduced protein biosynthesis which however turned out to be beneficial for E. coli for adapting to oxidative stress. This functional advantage was small under optimal conditions or during heat or cold shock, but becomes pronounced in the presence of hydrogen peroxide. Taken together, these data provided first functional insights into the role of this unique 23S rRNA modification for ribosome functions and bacterial growth under oxidative stress.
Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , RNA Bacteriano/metabolismo , RNA Ribossômico 23S/metabolismo , Ribossomos/metabolismo , Estresse Oxidativo , Processamento Pós-Transcricional do RNARESUMO
Organisms from all domains of life invest a substantial amount of energy for the introduction of RNA modifications into nearly all transcripts studied to date. Instrumental analysis of RNA can focus on the modified residues and reveal the function of these epitranscriptomic marks. Here, we will review recent advances and breakthroughs achieved by NMR spectroscopy, sequencing, and mass spectrometry of the epitranscriptome.
Assuntos
Processamento Pós-Transcricional do RNA , RNA/genética , Animais , Epigênese Genética , Humanos , Espectrometria de Massas/métodos , Ressonância Magnética Nuclear Biomolecular/métodos , RNA/química , Análise de Sequência de RNA/métodos , TranscriptomaRESUMO
Nucleic acids undergo naturally occurring chemical modifications. Over 100 different modifications have been described and every position in the purine and pyrimidine bases can be modified; often the sugar is also modified. Despite recent progress, the mechanism for the biosynthesis of most modifications is not fully understood, owing, in part, to the difficulty associated with reconstituting enzyme activity in vitro. Whereas some modifications can be efficiently formed with purified components, others may require more intricate pathways. A model for modification interdependence, in which one modification is a prerequisite for another, potentially explains a major hindrance in reconstituting enzymatic activity in vitro. This model was prompted by the earlier discovery of tRNA cytosine-to-uridine editing in eukaryotes, a reaction that has not been recapitulated in vitro and the mechanism of which remains unknown. Here we show that cytosine 32 in the anticodon loop of Trypanosoma brucei tRNAThr is methylated to 3-methylcytosine (m3C) as a pre-requisite for C-to-U deamination. Formation of m3C in vitro requires the presence of both the T. brucei m3C methyltransferase TRM140 and the deaminase ADAT2/3. Once formed, m3C is deaminated to 3-methyluridine (m3U) by the same set of enzymes. ADAT2/3 is a highly mutagenic enzyme, but we also show that when co-expressed with the methyltransferase its mutagenicity is kept in check. This helps to explain how T. brucei escapes 'wholesale deamination' of its genome while harbouring both enzymes in the nucleus. This observation has implications for the control of another mutagenic deaminase, human AID, and provides a rationale for its regulation.
Assuntos
Metiltransferases/metabolismo , Nucleosídeo Desaminases/metabolismo , Edição de RNA , RNA de Transferência de Treonina/química , RNA de Transferência de Treonina/metabolismo , Trypanosoma brucei brucei/enzimologia , Trypanosoma brucei brucei/genética , Anticódon/metabolismo , Sequência de Bases , Citosina/análogos & derivados , Citosina/metabolismo , Desaminação , Metilação , RNA de Transferência de Treonina/genética , Uridina/metabolismoRESUMO
Transfer RNAs (tRNAs) are key players in protein synthesis. To be fully active, tRNAs undergo extensive post-transcriptional modifications, including queuosine (Q), a hypermodified 7-deaza-guanosine present in the anticodon of several tRNAs in bacteria and eukarya. Here, molecular and biochemical approaches revealed that in the protozoan parasite Trypanosoma brucei, Q-containing tRNAs have a preference for the U-ending codons for asparagine, aspartate, tyrosine and histidine, analogous to what has been described in other systems. However, since a lack of tRNA genes in T. brucei mitochondria makes it essential to import a complete set from the cytoplasm, we surprisingly found that Q-modified tRNAs are preferentially imported over their unmodified counterparts. In turn, their absence from mitochondria has a pronounced effect on organellar translation and affects function. Although Q modification in T. brucei is globally important for codon selection, it is more so for mitochondrial protein synthesis. These results provide a unique example of the combined regulatory effect of codon usage and wobble modifications on protein synthesis; all driven by tRNA intracellular transport dynamics.
Assuntos
Mitocôndrias/genética , Conformação de Ácido Nucleico , Nucleosídeo Q/genética , RNA de Transferência/genética , Anticódon/genética , Núcleo Celular/genética , Núcleo Celular/ultraestrutura , Códon/genética , Citoplasma/genética , Citoplasma/ultraestrutura , Guanosina/genética , Biossíntese de Proteínas/genética , Processamento Pós-Transcricional do RNA/genética , RNA de Transferência/ultraestrutura , Trypanosoma brucei brucei/genéticaRESUMO
Every type of nucleic acid in cells undergoes programmed chemical post-transcriptional modification. Generally, modification enzymes use substrates derived from intracellular metabolism, one exception is queuine (q)/queuosine (Q), which eukaryotes obtain from their environment; made by bacteria and ultimately taken into eukaryotic cells via currently unknown transport systems. Here, we use a combination of molecular, cell biology and biophysical approaches to show that in Trypanosoma brucei tRNA Q levels change dynamically in response to concentration variations of a sub-set of amino acids in the growth media. Most significant were variations in tyrosine, which at low levels lead to increased Q content for all the natural tRNAs substrates of tRNA-guanine transglycosylase (TGT). Such increase results from longer nuclear dwell time aided by retrograde transport following cytoplasmic splicing. In turn high tyrosine levels lead to rapid decrease in Q content. Importantly, the dynamic changes in Q content of tRNAs have negligible effects on global translation or growth rate but, at least, in the case of tRNATyr it affected codon choice. These observations have implications for the occurrence of other tunable modifications important for 'normal' growth, while connecting the intracellular localization of modification enzymes, metabolites and tRNAs to codon selection and implicitly translational output.
Assuntos
Códon/metabolismo , Nucleosídeo Q/metabolismo , Nutrientes/metabolismo , RNA de Transferência/metabolismo , Trypanosoma brucei brucei/metabolismo , Aminoácidos/metabolismo , Cromatografia Líquida/métodos , Códon/genética , Guanina/análogos & derivados , Guanina/metabolismo , Pentosiltransferases/genética , Pentosiltransferases/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Splicing de RNA , RNA de Transferência/genética , RNA de Transferência de Tirosina/genética , RNA de Transferência de Tirosina/metabolismo , Espectrometria de Massas em Tandem/métodos , Trypanosoma brucei brucei/genética , Tirosina/metabolismoRESUMO
RNA abasic sites and the mechanisms involved in their regulation are mostly unknown; in contrast, DNA abasic sites are well-studied. We found surprisingly that, in yeast and human cells, RNA abasic sites are prevalent. When a base is lost from RNA, the remaining ribose is found as a closed-ring or an open-ring sugar with a reactive C1' aldehyde group. Using primary amine-based reagents that react with the aldehyde group, we uncovered evidence for abasic sites in nascent RNA, messenger RNA, and ribosomal RNA from yeast and human cells. Mass spectroscopic analysis confirmed the presence of RNA abasic sites. The RNA abasic sites were found to be coupled to R-loops. We show that human methylpurine DNA glycosylase cleaves N-glycosidic bonds on RNA and that human apurinic/apyrimidinic endonuclease 1 incises RNA abasic sites in RNA-DNA hybrids. Our results reveal that, in yeast and human cells, there are RNA abasic sites, and we identify a glycosylase that generates these sites and an AP endonuclease that processes them.
Assuntos
Sequência de Bases/genética , RNA/química , RNA/genética , Sítios de Ligação , DNA/química , Dano ao DNA/genética , DNA Glicosilases/metabolismo , Reparo do DNA/genética , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , Desoxirribonuclease I/metabolismo , Humanos , Nucleotídeos/genética , Estruturas R-Loop/genética , Saccharomyces cerevisiae/genética , Especificidade por Substrato , Leveduras/genéticaRESUMO
Higher-energy collisional dissociation (HCD) of modified ribonucleosides generates characteristic and highly reproducible nucleoside-specific tandem mass spectra (MS/MS). Here, we demonstrate the capability of HCD spectra in combination with spectral matching for the semi-automated characterization of ribonucleosides. This process involved the generation of an HCD spectral library and the establishment of a mass spectral network for rapid detection with high sensitivity and specificity in a retention time-independent fashion. Systematic spectral matching analysis of the MS/MS spectra of tRNA hydrolysates from different organisms has helped us to uncover evidence for the existence of novel ribonucleoside modifications such as s2Cm and OHyW-14. Such an untargeted label-free approach has the potential to be integrated with other methods, including those that use isotope labeling, to simplify the characterization of unknown modified ribonucleosides. These findings suggest the compilation of a universal spectral network, for the characterization of known and unknown ribonucleosides, could accelerate discoveries in the epitranscriptome.
Assuntos
Ribonucleosídeos , Espectrometria de Massas em Tandem , Marcação por Isótopo , Nucleosídeos , RNA de Transferência , Ribonucleosídeos/análise , Espectrometria de Massas em Tandem/métodosRESUMO
Knowledge of the cleavage specificity of ribonucleases is critical for their application in RNA modification mapping or RNA-protein binding studies. Here, we detail the cleavage specificity and efficiency of ribonuclease MC1 and cusativin using a customized RNA sequence that contained all dinucleotide combinations and homopolymer sequences. The sequencing of the oligonucleotide digestion products by a semi-quantitative liquid chromatography coupled with mass spectrometry (LC-MS) analysis documented as little as 0.5-1% cleavage levels for a given dinucleotide sequence combination. While RNase MC1 efficiently cleaved the [A/U/C]pU dinucleotide bond, no cleavage was observed for the GpU bond. Similarly, cusativin efficiently cleaved Cp[U/A/G] dinucleotide combinations along with UpA and [A/U]pU, suggesting a broader specificity of dinucleotide preferences. The molecular interactions between the substrate and active site as determined by the dinucleotide docking studies of protein models offered additional evidence and support for the observed substrate specificity. Targeted alteration of the key amino acid residues in the nucleotide-binding site confirms the utility of this in silico approach for the identification of key interactions. Taken together, the use of bioanalytical and computational approaches, involving LC-MS and ligand docking of tertiary structural models, can form a powerful combination to help explain the RNA cleavage behavior of RNases.
Assuntos
Ribonuclease Pancreático , Ribonucleases , Domínio Catalítico , Endorribonucleases , RNA , Clivagem do RNA , Ribonuclease Pancreático/metabolismo , Ribonucleases/metabolismo , Especificidade por SubstratoRESUMO
The tRNA m1R9 methyltransferase (Trm10) family is conserved throughout Eukarya and Archaea. Despite the presence of a single Trm10 gene in Archaea and most single-celled eukaryotes, metazoans encode up to three homologs of Trm10. Several disease states correlate with a deficiency in the human homolog TRMT10A, despite the presence of another cytoplasmic enzyme, TRMT10B. Here we investigate these phenomena and demonstrate that human TRMT10A (hTRMT10A) and human TRMT10B (hTRMT10B) are not biochemically redundant. In vitro activity assays with purified hTRMT10A and hTRMT10B reveal a robust activity for hTRMT10B as a tRNAAsp-specific m1A9 methyltransferase and suggest that it is the relevant enzyme responsible for this newly discovered m1A9 modification in humans. Moreover, a comparison of the two cytosolic enzymes with multiple tRNA substrates exposes the enzymes' distinct substrate specificities, and suggests that hTRMT10B exhibits a restricted selectivity hitherto unseen in the Trm10 enzyme family. Single-turnover kinetics and tRNA binding assays highlight further differences between the two enzymes and eliminate overall tRNA affinity as a primary determinant of substrate specificity for either enzyme. These results increase our understanding of the important biology of human tRNA modification systems, which can aid in understanding the molecular basis for diseases in which their aberrant function is increasingly implicated.
Assuntos
Metiltransferases/metabolismo , Isoformas de Proteínas/metabolismo , tRNA Metiltransferases/metabolismo , Catálise , Humanos , Cinética , Especificidade por SubstratoRESUMO
Assembly of 30S ribosomal subunits from their protein and RNA components requires extensive refolding of the 16S rRNA and is assisted by 10-20 assembly factors in bacteria. We probed the structures of 30S assembly intermediates in E. coli cells, using a synchrotron X-ray beam to generate hydroxyl radical in the cytoplasm. Widespread differences between mature and pre-30S complexes in the absence of assembly factors RbfA and RimM revealed global reorganization of RNA-protein interactions prior to maturation of the 16S rRNA and showed how RimM reduces misfolding of the 16S 3' domain during transcription in vivo. Quantitative (14)N/(15)N mass spectrometry of affinity-purified pre-30S complexes confirmed the absence of tertiary assembly proteins and showed that N-terminal acetylation of proteins S18 and S5 correlates with correct folding of the platform and central pseudoknot. Our results indicate that cellular factors delay specific RNA folding steps to ensure the quality of assembly.
Assuntos
Escherichia coli/metabolismo , RNA Ribossômico 16S/metabolismo , Subunidades Ribossômicas Menores de Bactérias/metabolismo , Acetilação , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Deleção de Genes , Sequências Repetidas Invertidas , Modelos Moleculares , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Conformação de Ácido Nucleico , Multimerização Proteica , Processamento de Proteína Pós-Traducional , Clivagem do RNA/efeitos da radiação , Dobramento de RNA , RNA Ribossômico 16S/química , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Transcrição GênicaRESUMO
Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has become the gold-standard technique to study RNA and its various modifications. While most research on RNA nucleosides has been focused on their biological roles, discovery of new modifications remains of interest. With state-of-the-art technology, the presence of artifacts can confound the identification of new modifications. Here, we report the characterization of a non-natural mcm5 isoC ribonucleoside in S. cerevisiae total tRNA hydrolysate by higher-energy collisional dissociation (HCD)-based fingerprints and isotope labeling of RNA. Its discovery revealed a class of amino/imino ribonucleoside artifacts that are generated during RNA hydrolysis under ammonium-buffered mild basic conditions. We then identified digestion conditions that can reduce or eliminate their formation. These finding and method enhancements will improve the accurate detection of new RNA modifications.
Assuntos
Nucleosídeos/química , RNA/análise , Compostos de Sulfidrila/química , Aminação , Cromatografia Líquida de Alta Pressão , Hidrólise , Marcação por Isótopo , RNA/metabolismo , RNA de Transferência/química , RNA de Transferência/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Espectrometria de Massas em TandemRESUMO
Archaeosine (G+) is a structurally complex modified nucleoside found quasi-universally in the tRNA of Archaea and located at position 15 in the dihydrouridine loop, a site not modified in any tRNA outside the Archaea G+ is characterized by an unusual 7-deazaguanosine core structure with a formamidine group at the 7-position. The location of G+ at position 15, coupled with its novel molecular structure, led to a hypothesis that G+ stabilizes tRNA tertiary structure through several distinct mechanisms. To test whether G+ contributes to tRNA stability and define the biological role of G+, we investigated the consequences of introducing targeted mutations that disrupt the biosynthesis of G+ into the genome of the hyperthermophilic archaeon Thermococcus kodakarensis and the mesophilic archaeon Methanosarcina mazei, resulting in modification of the tRNA with the G+ precursor 7-cyano-7-deazaguansine (preQ0) (deletion of arcS) or no modification at position 15 (deletion of tgtA). Assays of tRNA stability from in vitro-prepared and enzymatically modified tRNA transcripts, as well as tRNA isolated from the T. kodakarensis mutant strains, demonstrate that G+ at position 15 imparts stability to tRNAs that varies depending on the overall modification state of the tRNA and the concentration of magnesium chloride and that when absent results in profound deficiencies in the thermophily of T. kodakarensisIMPORTANCE Archaeosine is ubiquitous in archaeal tRNA, where it is located at position 15. Based on its molecular structure, it was proposed to stabilize tRNA, and we show that loss of archaeosine in Thermococcus kodakarensis results in a strong temperature-sensitive phenotype, while there is no detectable phenotype when it is lost in Methanosarcina mazei Measurements of tRNA stability show that archaeosine stabilizes the tRNA structure but that this effect is much greater when it is present in otherwise unmodified tRNA transcripts than in the context of fully modified tRNA, suggesting that it may be especially important during the early stages of tRNA processing and maturation in thermophiles. Our results demonstrate how small changes in the stability of structural RNAs can be manifested in significant biological-fitness changes.
Assuntos
Guanosina/análogos & derivados , Methanosarcina/metabolismo , RNA Arqueal/genética , RNA de Transferência/genética , Thermococcus/metabolismo , Guanosina/metabolismo , Methanosarcina/química , Methanosarcina/genética , Estabilidade de RNA , RNA Arqueal/química , RNA Arqueal/metabolismo , RNA de Transferência/química , RNA de Transferência/metabolismo , Thermococcus/química , Thermococcus/genéticaRESUMO
Post-transcriptional chemical modifications of (t)RNA molecules are crucial in fundamental biological processes, such as translation. Despite their biological importance and accumulating evidence linking them to various human diseases, technical challenges have limited their detection and accurate quantification. Here, we present a sensitive capillary nanoflow liquid chromatography mass spectrometry (nLC-MS) pipeline for quantitative high-resolution analysis of ribonucleoside modifications from complex biological samples. We evaluated two porous graphitic carbon (PGC) materials and one end-capped C18 reference material as stationary phases for reversed-phase separation. We found that these matrices have complementing retention and separation characteristics, including the capability to separate structural isomers. PGC and C18 matrices yielded excellent signal-to-noise ratios in nLC-MS while differing in the separation capability and sensitivity for various nucleosides. This emphasizes the need for tailored LC-MS setups for optimally detecting as many nucleoside modifications as possible. Detection ranges spanning up to six orders of magnitude enable the analysis of individual ribonucleosides down to femtomol concentrations. Furthermore, normalizing the obtained signal intensities to a stable isotope labeled spike-in enabled direct comparison of ribonucleoside levels between different samples. In conclusion, capillary columns coupled to nLC-MS constitute a powerful and sensitive tool for quantitative analysis of modified ribonucleosides in complex biological samples. This setup will be invaluable for further unraveling the intriguing and multifaceted biological roles of RNA modifications.
Assuntos
Cromatografia Líquida , Espectrometria de Massas , Ribonucleosídeos/análise , Ribonucleosídeos/química , Cromatografia Líquida/métodos , Grafite/química , Humanos , Espectrometria de Massas/métodos , RNA Bacteriano , RNA Fúngico , RNA de Transferência/química , Ribonucleosídeos/isolamento & purificação , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em TandemRESUMO
Locating ribonucleoside modifications within an RNA sequence requires digestion of the RNA into oligoribonucleotides of amenable size for subsequent analysis by LC-MS (liquid chromatography-mass spectrometry). This approach, widely referred to as RNA modification mapping, is facilitated through ribonucleases (RNases) such as T1 (guanosine-specific), U2 (purine-selective) and A (pyrimidine-specific) among others. Sequence coverage by these enzymes depends on positioning of the recognized nucleobase (such as guanine or purine or pyrimidine) in the sequence and its ribonucleotide composition. Using E. coli transfer RNA (tRNA) and ribosomal RNA (rRNA) as model samples, we demonstrate the ability of complementary nucleobase-specific ribonucleases cusativin (C-specific) and MC1 (U-specific) to generate digestion products that facilitate confident mapping of modifications in regions such as G-rich and pyrimidine-rich segments of RNA, and to distinguish C to U sequence differences. These enzymes also increase the number of oligonucleotide digestion products that are unique to a specific RNA sequence. Further, with these additional RNases, multiple modifications can be localized with high confidence in a single set of experiments with minimal dependence on the individual tRNA abundance in a mixture. The sequence overlaps observed with these complementary digestion products and that of RNase T1 improved sequence coverage to 75% or above. A similar level of sequence coverage was also observed for the 2904 nt long 23S rRNA indicating their utility has no dependence on RNA size. Wide-scale adoption of these additional modification mapping tools could help expedite the characterization of modified RNA sequences to understand their structural and functional role in various living systems.
Assuntos
Endorribonucleases/metabolismo , RNA não Traduzido/metabolismo , Ribonucleases/metabolismo , Sequência de Bases , Cromatografia Líquida de Alta Pressão , Escherichia coli/genética , RNA Ribossômico/análise , RNA Ribossômico/metabolismo , RNA de Transferência/análise , RNA de Transferência/química , RNA de Transferência/metabolismo , Espectrometria de Massas em TandemRESUMO
Research into post-transcriptional processing and modification of RNA continues to speed forward, as their ever-emerging role in the regulation of gene expression in biological systems continues to unravel. Liquid chromatography tandem mass spectrometry (LC-MS/MS) has proven for over two decades to be a powerful ally in the elucidation of RNA modification identity and location, but the technique has not proceeded without its own unique technical challenges. The throughput of LC-MS/MS modification mapping experiments continues to be impeded by tedious and time-consuming spectral interpretation, particularly during for the analysis of complex RNA samples. RNAModMapper was recently developed as a tool to improve the interpretation and annotation of LC-MS/MS data sets from samples containing post-transcriptionally modified RNAs. Here, we delve deeper into the methodology and practice of RNAModMapper to provide greater insight into its utility, and remaining hurdles, in current RNA modification mapping experiments.
Assuntos
Cromatografia Líquida/estatística & dados numéricos , Oligorribonucleotídeos/análise , Processamento Pós-Transcricional do RNA , RNA de Transferência de Fenilalanina/análise , Software , Espectrometria de Massas em Tandem/estatística & dados numéricos , Fosfatase Alcalina/metabolismo , Interpretação Estatística de Dados , Oligorribonucleotídeos/química , Oligorribonucleotídeos/metabolismo , RNA de Transferência de Fenilalanina/química , RNA de Transferência de Fenilalanina/metabolismo , Ribonuclease T1/metabolismo , Saccharomyces cerevisiae , Análise de Sequência de RNA/estatística & dados numéricosRESUMO
Active tRNAs are extensively post-transcriptionally modified, particularly at the wobble position 34 and the position 37 on the 3'-side of the anticodon. The 5-carboxy-methoxy modification of U34 (cmo5U34) is present in Gram-negative tRNAs for six amino acids (Ala, Ser, Pro, Thr, Leu and Val), four of which (Ala, Ser, Pro and Thr) have a terminal methyl group to form 5-methoxy-carbonyl-methoxy-uridine (mcmo5U34) for higher reading-frame accuracy. The molecular basis for the selective terminal methylation is not understood. Many cmo5U34-tRNAs are essential for growth and cannot be substituted for mutational analysis. We show here that, with a novel genetic approach, we have created and isolated mutants of Escherichia coli tRNAPro and tRNAVal for analysis of the selective terminal methylation. We show that substitution of G35 in the anticodon of tRNAPro inactivates the terminal methylation, whereas introduction of G35 to tRNAVal confers it, indicating that G35 is a major determinant for the selectivity. We also show that, in tRNAPro, the terminal methylation at U34 is dependent on the primary m1G methylation at position 37 but not vice versa, indicating a hierarchical ranking of modifications between positions 34 and 37. We suggest that this hierarchy provides a mechanism to ensure top performance of a tRNA inside of cells.
Assuntos
Anticódon/genética , Conformação de Ácido Nucleico , RNA de Transferência de Prolina/genética , RNA de Transferência/genética , Sequência de Bases , Códon/genética , Escherichia coli/genética , Metilação , RNA Bacteriano/genética , Uridina/análogos & derivados , Uridina/genéticaRESUMO
Protein synthesis is a complex and highly coordinated process requiring many different protein factors as well as various types of nucleic acids. All translation machinery components require multiple maturation events to be functional. These include post-transcriptional and post-translational modification steps and methylations are the most frequent among these events. In eukaryotes, Trm112, a small protein (COG2835) conserved in all three domains of life, interacts and activates four methyltransferases (Bud23, Trm9, Trm11 and Mtq2) that target different components of the translation machinery (rRNA, tRNAs, release factors). To clarify the function of Trm112 in archaea, we have characterized functionally and structurally its interaction network using Haloferax volcanii as model system. This led us to unravel that methyltransferases are also privileged Trm112 partners in archaea and that this Trm112 network is much more complex than anticipated from eukaryotic studies. Interestingly, among the identified enzymes, some are functionally orthologous to eukaryotic Trm112 partners, emphasizing again the similarity between eukaryotic and archaeal translation machineries. Other partners display some similarities with bacterial methyltransferases, suggesting that Trm112 is a general partner for methyltransferases in all living organisms.
Assuntos
Proteínas Arqueais/fisiologia , Proteínas de Bactérias/fisiologia , Haloferax volcanii/enzimologia , Processamento Pós-Transcricional do RNA , tRNA Metiltransferases/fisiologia , Proteínas de Bactérias/genética , Cristalografia por Raios X , Conjuntos de Dados como Assunto , Ativação Enzimática , Células Eucarióticas/enzimologia , Evolução Molecular , Holoenzimas/fisiologia , Imunoprecipitação , Espectrometria de Massas , Metilação , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Mapeamento de Interação de Proteínas , Proteômica , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Especificidade da Espécie , tRNA Metiltransferases/deficiência , tRNA Metiltransferases/genéticaRESUMO
MODOMICS is a database of RNA modifications that provides comprehensive information concerning the chemical structures of modified ribonucleosides, their biosynthetic pathways, the location of modified residues in RNA sequences, and RNA-modifying enzymes. In the current database version, we included the following new features and data: extended mass spectrometry and liquid chromatography data for modified nucleosides; links between human tRNA sequences and MINTbase - a framework for the interactive exploration of mitochondrial and nuclear tRNA fragments; new, machine-friendly system of unified abbreviations for modified nucleoside names; sets of modified tRNA sequences for two bacterial species, updated collection of mammalian tRNA modifications, 19 newly identified modified ribonucleosides and 66 functionally characterized proteins involved in RNA modification. Data from MODOMICS have been linked to the RNAcentral database of RNA sequences. MODOMICS is available at http://modomics.genesilico.pl.
Assuntos
Bases de Dados Genéticas , RNA/química , RNA/metabolismo , Ribonucleosídeos/química , Ribonucleosídeos/metabolismo , Cromatografia Líquida , Humanos , Espectrometria de Massas , RNA de Transferência/química , RNA de Transferência/metabolismo , Terminologia como AssuntoRESUMO
Tertiary sequence motifs encode interactions between RNA helices that create the three-dimensional structures of ribosomal subunits. A Right Angle motif at the junction between 16S helices 5 and 6 (J5/6) is universally conserved amongst small subunit rRNAs and forms a stable right angle in minimal RNAs. J5/6 does not form a right angle in the mature ribosome, suggesting that this motif encodes a metastable structure needed for ribosome biogenesis. In this study, J5/6 mutations block 30S ribosome assembly and 16S maturation in Escherichia coli. Folding assays and in-cell X-ray footprinting showed that J5/6 mutations favor an assembly intermediate of the 16S 5' domain and prevent formation of the central pseudoknot. Quantitative mass spectrometry revealed that mutant pre-30S ribosomes lack protein uS12 and are depleted in proteins uS5 and uS2. Together, these results show that impaired folding of the J5/6 right angle prevents the establishment of inter-domain interactions, resulting in global collapse of the 30S structure observed in electron micrographs of mutant pre-30S ribosomes. We propose that the J5/6 motif is part of a spine of RNA helices that switch conformation at distinct stages of assembly, linking peripheral domains with the 30S active site to ensure the integrity of 30S biogenesis.