RESUMEN
The ability of chemical modifications of single nucleotides to alter the electrostatic charge, hydrophobic surface and base pairing of RNA molecules is exploited for the clinical use of stable artificial RNAs such as mRNA vaccines and synthetic small RNA molecules - to increase or decrease the expression of therapeutic proteins. Furthermore, naturally occurring biochemical modifications of nucleotides regulate RNA metabolism and function to modulate crucial cellular processes. Studies showing the mechanisms by which RNA modifications regulate basic cell functions in higher organisms have led to greater understanding of how aberrant RNA modification profiles can cause disease in humans. Together, these basic science discoveries have unravelled the molecular and cellular functions of RNA modifications, have provided new prospects for therapeutic manipulation and have led to a range of innovative clinical approaches.
Asunto(s)
Nucleótidos , ARN , Humanos , ARN/metabolismo , Procesamiento Postranscripcional del ARNRESUMEN
Mitochondria contain a specific translation machinery for the synthesis of mitochondria-encoded respiratory chain components. Mitochondrial tRNAs (mt-tRNAs) are also generated from the mitochondrial DNA and, similar to their cytoplasmic counterparts, are post-transcriptionally modified. Here, we find that the RNA methyltransferase METTL8 is a mitochondrial protein that facilitates 3-methyl-cytidine (m3C) methylation at position C32 of the mt-tRNASer(UCN) and mt-tRNAThr. METTL8 knockout cells show a reduction in respiratory chain activity, whereas overexpression increases activity. In pancreatic cancer, METTL8 levels are high, which correlates with lower patient survival and an enhanced respiratory chain activity. Mitochondrial ribosome profiling uncovered mitoribosome stalling on mt-tRNASer(UCN)- and mt-tRNAThr-dependent codons. Further analysis of the respiratory chain complexes using mass spectrometry revealed reduced incorporation of the mitochondrially encoded proteins ND6 and ND1 into complex I. The well-balanced translation of mt-tRNASer(UCN)- and mt-tRNAThr-dependent codons through METTL8-mediated m3C32 methylation might, therefore, facilitate the optimal composition and function of the mitochondrial respiratory chain.
Asunto(s)
Metiltransferasas/metabolismo , ARN Mitocondrial/química , ARN de Transferencia/química , Animales , Anticodón , Proliferación Celular , Codón , Citoplasma , ADN Mitocondrial/metabolismo , Transporte de Electrón , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Ratones , Mitocondrias/metabolismo , Membranas Mitocondriales , Proteínas Mitocondriales/química , Consumo de Oxígeno , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/mortalidad , Ribosomas/metabolismo , Regulación hacia ArribaRESUMEN
Queuosine (Q) is a modified nucleoside at the wobble position of specific tRNAs. In mammals, queuosinylation is facilitated by queuine uptake from the gut microbiota and is introduced into tRNA by the QTRT1-QTRT2 enzyme complex. By establishing a Qtrt1 knockout mouse model, we discovered that the loss of Q-tRNA leads to learning and memory deficits. Ribo-Seq analysis in the hippocampus of Qtrt1-deficient mice revealed not only stalling of ribosomes on Q-decoded codons, but also a global imbalance in translation elongation speed between codons that engage in weak and strong interactions with their cognate anticodons. While Q-dependent molecular and behavioral phenotypes were identified in both sexes, female mice were affected more severely than males. Proteomics analysis confirmed deregulation of synaptogenesis and neuronal morphology. Together, our findings provide a link between tRNA modification and brain functions and reveal an unexpected role of protein synthesis in sex-dependent cognitive performance.
Asunto(s)
Nucleósido Q , ARN de Transferencia , Femenino , Ratones , Animales , Nucleósido Q/genética , ARN de Transferencia/genética , ARN de Transferencia/metabolismo , Anticodón , Biosíntesis de Proteínas , Codón , Mamíferos/genéticaRESUMEN
Dihydrouridine (D), a prevalent and evolutionarily conserved base in the transcriptome, primarily resides in tRNAs and, to a lesser extent, in mRNAs. Notably, this modification is found at position 2449 in the Escherichia coli 23S rRNA, strategically positioned near the ribosome's peptidyl transferase site. Despite the prior identification, in E. coli genome, of three dihydrouridine synthases (DUS), a set of NADPH and FMN-dependent enzymes known for introducing D in tRNAs and mRNAs, characterization of the enzyme responsible for D2449 deposition has remained elusive. This study introduces a rapid method for detecting D in rRNA, involving reverse transcriptase-blockage at the rhodamine-labeled D2449 site, followed by PCR amplification (RhoRT-PCR). Through analysis of rRNA from diverse E. coli strains, harboring chromosomal or single-gene deletions, we pinpoint the yhiN gene as the ribosomal dihydrouridine synthase, now designated as RdsA. Biochemical characterizations uncovered RdsA as a unique class of flavoenzymes, dependent on FAD and NADH, with a complex structural topology. In vitro assays demonstrated that RdsA dihydrouridylates a short rRNA transcript mimicking the local structure of the peptidyl transferase site. This suggests an early introduction of this modification before ribosome assembly. Phylogenetic studies unveiled the widespread distribution of the yhiN gene in the bacterial kingdom, emphasizing the conservation of rRNA dihydrouridylation. In a broader context, these findings underscore nature's preference for utilizing reduced flavin in the reduction of uridines and their derivatives.
Asunto(s)
Escherichia coli , Escherichia coli/genética , Escherichia coli/metabolismo , ARN Ribosómico 23S/metabolismo , ARN Ribosómico 23S/genética , ARN Ribosómico 23S/química , Uridina/análogos & derivados , Uridina/metabolismo , Uridina/química , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/química , ARN Bacteriano/metabolismo , ARN Bacteriano/genética , ARN Bacteriano/químicaRESUMEN
Therapeutic fluoropyrimidines 5-fluorouracil (5-FU) and 5-fluorocytosine (5-FC) are in long use for treatment of human cancers and severe invasive fungal infections, respectively. 5-Fluorouridine triphosphate represents a bioactive metabolite of both drugs and is incorporated into target cells' RNA. Here we use the model fungus Saccharomyces cerevisiae to define fluorinated tRNA as a key mediator of 5-FU and 5-FC cytotoxicity when specific tRNA methylations are absent. tRNA methylation deficiency caused by loss of Trm4 and Trm8 was previously shown to trigger an RNA quality control mechanism resulting in partial destabilization of hypomodified tRNAValAAC. We demonstrate that, following incorporation into tRNA, fluoropyrimidines strongly enhance degradation of yeast tRNAValAAC lacking Trm4 and Trm8 dependent methylations. At elevated temperature, such effect occurs already in absence of Trm8 alone. Genetic approaches and quantification of tRNA modification levels reveal that enhanced fluoropyrimidine cytotoxicity results from additional, drug induced uridine modification loss and activation of tRNAValAAC decay involving the exonuclease Xrn1. These results suggest that inhibition of tRNA methylation may be exploited to boost therapeutic efficiency of 5-FU and 5-FC.
Asunto(s)
Flucitosina , Fluorouracilo , ARN de Transferencia , Saccharomyces cerevisiae , Exorribonucleasas/metabolismo , Exorribonucleasas/genética , Flucitosina/farmacología , Fluorouracilo/farmacología , Metilación , Estabilidad del ARN/efectos de los fármacos , ARN de Transferencia/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , ARNt Metiltransferasas/metabolismo , ARNt Metiltransferasas/genética , Uridina/metabolismoRESUMEN
Dihydrouridine (D) is a common modified base found predominantly in transfer RNA (tRNA). Despite its prevalence, the mechanisms underlying dihydrouridine biosynthesis, particularly in prokaryotes, have remained elusive. Here, we conducted a comprehensive investigation into D biosynthesis in Bacillus subtilis through a combination of genetic, biochemical, and epitranscriptomic approaches. Our findings reveal that B. subtilis relies on two FMN-dependent Dus-like flavoprotein homologs, namely DusB1 and DusB2, to introduce all D residues into its tRNAs. Notably, DusB1 exhibits multisite enzyme activity, enabling D formation at positions 17, 20, 20a and 47, while DusB2 specifically catalyzes D biosynthesis at positions 20 and 20a, showcasing a functional redundancy among modification enzymes. Extensive tRNA-wide D-mapping demonstrates that this functional redundancy impacts the majority of tRNAs, with DusB2 displaying a higher dihydrouridylation efficiency compared to DusB1. Interestingly, we found that BsDusB2 can function like a BsDusB1 when overexpressed in vivo and under increasing enzyme concentration in vitro. Furthermore, we establish the importance of the D modification for B. subtilis growth at suboptimal temperatures. Our study expands the understanding of D modifications in prokaryotes, highlighting the significance of functional redundancy in this process and its impact on bacterial growth and adaptation.
Asunto(s)
Bacillus subtilis , ARN de Transferencia , Uridina , Bacillus subtilis/enzimología , Bacillus subtilis/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , ARN Bacteriano/metabolismo , ARN Bacteriano/genética , ARN de Transferencia/metabolismo , ARN de Transferencia/genética , Uridina/metabolismo , Uridina/análogos & derivados , Expresión GénicaRESUMEN
N6-methyladenosine (m6A) is the most abundant mRNA modification in eukaryotes, playing crucial roles in multiple biological processes. m6A is catalyzed by the activity of methyltransferase-like 3 (Mettl3), which depends on additional proteins whose precise functions remain poorly understood. Here we identified Zc3h13 (zinc finger CCCH domain-containing protein 13)/Flacc [Fl(2)d-associated complex component] as a novel interactor of m6A methyltransferase complex components in Drosophila and mice. Like other components of this complex, Flacc controls m6A levels and is involved in sex determination in Drosophila We demonstrate that Flacc promotes m6A deposition by bridging Fl(2)d to the mRNA-binding factor Nito. Altogether, our work advances the molecular understanding of conservation and regulation of the m6A machinery.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas de Unión al ADN/metabolismo , Drosophila melanogaster/fisiología , Metiltransferasas/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Unión al ARN/metabolismo , Adenosina/metabolismo , Animales , Proteínas de Ciclo Celular , Línea Celular , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimología , Regulación del Desarrollo de la Expresión Génica , Metilación , Ratones , Células Madre Embrionarias de Ratones , Transporte de Proteínas , Precursores del ARN/genética , Empalme del ARN , Factores de Empalme de ARN , Procesos de Determinación del Sexo/genéticaRESUMEN
Methylation of carbon-5 of cytosines (m5 C) is a post-transcriptional nucleotide modification of RNA found in all kingdoms of life. While individual m5 C-methyltransferases have been studied, the impact of the global cytosine-5 methylome on development, homeostasis and stress remains unknown. Here, using Caenorhabditis elegans, we generated the first organism devoid of m5 C in RNA, demonstrating that this modification is non-essential. Using this genetic tool, we determine the localisation and enzymatic specificity of m5 C sites in the RNome in vivo. We find that NSUN-4 acts as a dual rRNA and tRNA methyltransferase in C. elegans mitochondria. In agreement with leucine and proline being the most frequently methylated tRNA isoacceptors, loss of m5 C impacts the decoding of some triplets of these two amino acids, leading to reduced translation efficiency. Upon heat stress, m5 C loss leads to ribosome stalling at UUG triplets, the only codon translated by an m5 C34-modified tRNA. This leads to reduced translation efficiency of UUG-rich transcripts and impaired fertility, suggesting a role of m5 C tRNA wobble methylation in the adaptation to higher temperatures.
Asunto(s)
5-Metilcitosina/metabolismo , Adaptación Fisiológica/genética , Caenorhabditis elegans/genética , Respuesta al Choque Térmico/genética , Procesamiento Postranscripcional del ARN/genética , Animales , Sistemas CRISPR-Cas/genética , Caenorhabditis elegans/fisiología , Citosina/química , Edición Génica , Calor , Leucina/química , Metiltransferasas/metabolismo , Mitocondrias/genética , Mitocondrias/metabolismo , Prolina/química , Biosíntesis de Proteínas/genética , Biosíntesis de Proteínas/fisiología , ARN/química , ARN/genética , Ribosomas/metabolismoRESUMEN
ConspectusAmong the many analytical methods applied to RNA modifications, a particularly pronounced surge has occurred in the past decade in the field of modification mapping. The occurrence of modifications such as m6A in mRNA, albeit known since the 1980s, became amenable to transcriptome-wide analyses through the advent of next-generation sequencing techniques in a rather sudden manner. The term "mapping" here refers to detection of RNA modifications in a sequence context, which has a dramatic impact on the interpretation of biological functions. As a consequence, an impressive number of mapping techniques were published, most in the perspective of what now has become known as "epitranscriptomics". While more and more different modifications were reported to occur in mRNA, conflicting reports and controversial results pointed to a number of technical and theoretical problems rooted in analytics, statistics, and reagents. Rather than finding the proverbial needle in a haystack, the tasks were to determine how many needles of what color in what size of a haystack one was looking at.As the authors of this Account, we think it important to outline the limitations of different mapping methods since many life scientists freshly entering the field confuse the accuracy and precision of modification mapping with that of normal sequencing, which already features numerous caveats by itself. Indeed, we propose here to qualify a specific mapping method by the size of the transcriptome that can be meaningfully analyzed with it.We here focus on high throughput sequencing by Illumina technology, referred to as RNA-Seq. We noted with interest the development of methods for modification detection by other high throughput sequencing platforms that act directly on RNA, e.g., PacBio SMRT and nanopore sequencing, but those are not considered here.In contrast to approaches relying on direct RNA sequencing, current Illumina RNA-Seq protocols require prior conversion of RNA into DNA. This conversion relies on reverse transcription (RT) to create cDNA; thereafter, the cDNA undergoes a sequencing-by-synthesis type of analysis. Thus, a particular behavior of RNA modified nucleotides during the RT-step is a prerequisite for their detection (and quantification) by deep sequencing, and RT properties have great influence on the detection efficiency and reliability. Moreover, the RT-step requires annealing of a synthetic primer, a prerequisite with a crucial impact on library preparation. Thus, all RNA-Seq protocols must feature steps for the introduction of primers, primer landing sites, or adapters on both the RNA 3'- and 5'-ends.
Asunto(s)
Secuenciación de Nucleótidos de Alto Rendimiento , ARN , ADN Complementario/genética , Reproducibilidad de los Resultados , ARN/genética , ARN Mensajero/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodosRESUMEN
One mechanism of particular interest to regulate mRNA fate post-transcriptionally is mRNA modification. Especially the extent of m1A mRNA methylation is highly discussed due to methodological differences. However, one single m1A site in mitochondrial ND5 mRNA was unanimously reported by different groups. ND5 is a subunit of complex I of the respiratory chain. It is considered essential for the coupling of oxidation and proton transport. Here we demonstrate that this m1A site might be involved in the pathophysiology of Alzheimer's disease (AD). One of the pathological hallmarks of this neurodegenerative disease is mitochondrial dysfunction, mainly induced by Amyloid ß (Aß). Aß mainly disturbs functions of complex I and IV of the respiratory chain. However, the molecular mechanism of complex I dysfunction is still not fully understood. We found enhanced m1A methylation of ND5 mRNA in an AD cell model as well as in AD patients. Formation of this m1A methylation is catalyzed by increased TRMT10C protein levels, leading to translation repression of ND5. As a consequence, here demonstrated for the first time, TRMT10C induced m1A methylation of ND5 mRNA leads to mitochondrial dysfunction. Our findings suggest that this newly identified mechanism might be involved in Aß-induced mitochondrial dysfunction.
Asunto(s)
Adenosina , Enfermedad de Alzheimer , Péptidos beta-Amiloides , Complejo I de Transporte de Electrón , Mitocondrias , ARN Mensajero , Humanos , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/genética , ARN Mensajero/metabolismo , Adenosina/metabolismo , Mitocondrias/metabolismo , Metilación , Complejo I de Transporte de Electrón/metabolismo , Complejo I de Transporte de Electrón/genética , Péptidos beta-Amiloides/metabolismo , Masculino , Femenino , Anciano , Metiltransferasas/metabolismo , Metiltransferasas/genética , Anciano de 80 o más Años , Proteínas Mitocondriales/metabolismo , Proteínas Mitocondriales/genéticaRESUMEN
Epstein-Barr virus (EBV) expresses two highly abundant noncoding RNAs called EBV-encoded RNA 1 (EBER1) and EBER2, which are preserved in all clinical isolates of EBV, thus underscoring their essential function in the viral life cycle. Recent epitranscriptomics studies have uncovered a vast array of distinct RNA modifications within cellular as well as viral noncoding RNAs that are instrumental in executing their function. Here we show that EBER2 is marked by pseudouridylation, and by using HydraPsiSeq the modification site was mapped to a single nucleotide within the 3' region of EBER2. The writer enzyme was identified to be the snoRNA-dependent pseudouridine synthase Dyskerin, which is the catalytic subunit of H/ACA small nucleolar ribonucleoprotein complexes, and is guided to EBER2 by SNORA22. Similar to other noncoding RNAs for which pseudouridylation has a positive effect on RNA stability, loss of EBER2 pseudouridylation results in a decrease in RNA levels. Furthermore, pseudouridylation of EBER2 is required for the prolific accumulation of progeny viral genomes, suggesting that this single modification in EBER2 is important for efficient viral lytic replication. Taken together, our findings add to the list of RNA modifications that are essential for noncoding RNAs to implement their physiological roles.
Asunto(s)
Infecciones por Virus de Epstein-Barr , Herpesvirus Humano 4 , Humanos , Herpesvirus Humano 4/genética , ARN Viral/genética , ARN no Traducido/genética , Estabilidad del ARN , Replicación Viral/genéticaRESUMEN
Transfer RNAs acquire a large plethora of chemical modifications. Among those, modifications of the anticodon loop play important roles in translational fidelity and tRNA stability. Four human wobble U-containing tRNAs obtain 5-methoxycarbonylmethyluridine (mcm5U34) or 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U34), which play a role in decoding. This mark involves a cascade of enzymatic activities. The last step is mediated by alkylation repair homolog 8 (ALKBH8). In this study, we performed a transcriptome-wide analysis of the repertoire of ALKBH8 RNA targets. Using a combination of HITS-CLIP and RIP-seq analyses, we uncover ALKBH8-bound RNAs. We show that ALKBH8 targets fully processed and CCA modified tRNAs. Our analyses uncovered the previously known set of wobble U-containing tRNAs. In addition, both our approaches revealed ALKBH8 binding to several other types of noncoding RNAs, in particular C/D box snoRNAs.
Asunto(s)
Secuenciación de Inmunoprecipitación de Cromatina , ARN de Transferencia , Humanos , ARN de Transferencia/genética , ARN de Transferencia/metabolismo , Anticodón , ARN no Traducido/genética , Homólogo 8 de AlkB ARNt Metiltransferasa/genéticaRESUMEN
SUMMARY: Oxford Nanopore Technologies' (ONT) sequencing platform offers an excellent opportunity to perform real-time analysis during sequencing. This feature allows for early insights into experimental data and accelerates a potential decision-making process for further analysis, which can be particularly relevant in the clinical context. Although some tools for the real-time analysis of DNA-sequencing data already exist, there is currently no application available for differential transcriptome data analysis designed for scientists or physicians with limited bioinformatics knowledge. Here, we introduce NanopoReaTA, a user-friendly real-time analysis toolbox for RNA-sequencing data from ONT. Sequencing results from a running or finished experiment are processed through an R Shiny-based graphical user interface with an integrated Nextflow pipeline for whole transcriptome or gene-specific analyses. NanopoReaTA provides visual snapshots of a sequencing run in progress, thus enabling interactive sequencing and rapid decision making that could also be applied to clinical cases. AVAILABILITY AND IMPLEMENTATION: Github https://github.com/AnWiercze/NanopoReaTA; Zenodo https://doi.org/10.5281/zenodo.8099825.
Asunto(s)
Nanoporos , Programas Informáticos , Perfilación de la Expresión Génica/métodos , Transcriptoma , Análisis de Secuencia de ARN/métodosRESUMEN
Synthetic mRNA has recently moved into the focus of therapeutic and vaccination efforts. Incorporation of modified nucleotides during in vitro transcription can improve translation and attenuate immunogenicity, but is limited to triphosphate nucleotides which are accepted by RNA polymerases, and their incorporation is either random or complete. In contrast, site-specific modification, herein termed 'point modification' in analogy to point mutations, holds significant technical challenge. We developed fundamental techniques for isolation of long, translatable and internally point-modified mRNAs. Enabling concepts include three-way-one-pot splint ligations, and isolation of mRNA by real-time elution from agarose gels. The use of blue light permitted visualization of mRNA in pre-stained gels without the photochemical damage associated with the use of hard UV-radiation. This allowed visualization of the mRNA through its migration in the agarose gel, which in turn, was a prerequisite for its recovery by electroelution into precast troughs. Co-eluting agarose particles were quantified and found to not be detrimental to mRNA translation in vitro. Translation of EGFP-coding mRNA into functional protein was quantified by incorporation of 35S-labelled methionine and by in-gel EGFP fluorescence. This enabled the functional analysis of point modifications, specifically of ribose methylations in the middle of a 1371 nt long mRNA.
Asunto(s)
Ingeniería Genética , Nucleótidos , Metilación , Nucleótidos/metabolismo , ARN Mensajero/síntesis química , ARN Mensajero/genética , Sefarosa , Ingeniería Genética/métodosRESUMEN
A plethora of modified nucleotides extends the chemical and conformational space for natural occurring RNAs. tRNAs constitute the class of RNAs with the highest modification rate. The extensive modification modulates their overall stability, the fidelity and efficiency of translation. However, the impact of nucleotide modifications on the local structural dynamics is not well characterized. Here we show that the incorporation of the modified nucleotides in tRNAfMet from Escherichia coli leads to an increase in the local conformational dynamics, ultimately resulting in the stabilization of the overall tertiary structure. Through analysis of the local dynamics by NMR spectroscopic methods we find that, although the overall thermal stability of the tRNA is higher for the modified molecule, the conformational fluctuations on the local level are increased in comparison to an unmodified tRNA. In consequence, the melting of individual base pairs in the unmodified tRNA is determined by high entropic penalties compared to the modified. Further, we find that the modifications lead to a stabilization of long-range interactions harmonizing the stability of the tRNA's secondary and tertiary structure. Our results demonstrate that the increase in chemical space through introduction of modifications enables the population of otherwise inaccessible conformational substates.
Asunto(s)
ARN de Transferencia , ARN , Emparejamiento Base , Escherichia coli/genética , Escherichia coli/metabolismo , Conformación de Ácido Nucleico , Nucleótidos , ARN/química , ARN de Transferencia/metabolismoRESUMEN
RNA methyltransferases (MTases) are ubiquitous enzymes whose hitherto low profile in medicinal chemistry, contrasts with the surging interest in RNA methylation, the arguably most important aspect of the new field of epitranscriptomics. As MTases become validated as drug targets in all major fields of biomedicine, the development of small molecule compounds as tools and inhibitors is picking up considerable momentum, in academia as well as in biotech. Here we discuss the development of small molecules for two related aspects of chemical biology. Firstly, derivates of the ubiquitous cofactor S-adenosyl-l-methionine (SAM) are being developed as bioconjugation tools for targeted transfer of functional groups and labels to increasingly visible targets. Secondly, SAM-derived compounds are being investigated for their ability to act as inhibitors of RNA MTases. Drug development is moving from derivatives of cosubstrates towards higher generation compounds that may address allosteric sites in addition to the catalytic centre. Progress in assay development and screening techniques from medicinal chemistry have led to recent breakthroughs, e.g. in addressing human enzymes targeted for their role in cancer. Spurred by the current pandemic, new inhibitors against coronaviral MTases have emerged at a spectacular rate, including a repurposed drug which is now in clinical trial.
Asunto(s)
Metiltransferasas/antagonistas & inhibidores , ARN , Desarrollo de Medicamentos , Humanos , S-Adenosilmetionina/análogos & derivadosRESUMEN
The accurate definition of an epitranscriptome is endangered by artefacts resulting from RNA degradation after cell death, a ubiquitous yet little investigated process. By tracing RNA marker modifications through tissue preparation protocols, we identified a major blind spot from daily lab routine, that has massive impact on modification analysis in small RNAs. In particular, m6,6A and Am as co-varying rRNA marker modifications, appeared in small RNA fractions following rRNA degradation in vitro and in cellulo. Analysing mouse tissue at different time points post mortem, we tracked the progress of intracellular RNA degradation after cell death, and found it reflected in RNA modification patterns. Differences were dramatic between liver, where RNA degradation commenced immediately after death, and brain, yielding essentially undamaged RNA. RNA integrity correlated with low amounts of co-varying rRNA markers. Thus validated RNA preparations featured differentially modified tRNA populations whose information content allowed a distinction even among the related brain tissues cortex, cerebellum and hippocampus. Inversely, advanced cell death correlated with high rRNA marker content, and correspondingly little with the naïve state of living tissue. Therefore, unless RNA and tissue preparations are executed with utmost care, interpretation of modification patterns in tRNA and small RNA are prone to artefacts.
Asunto(s)
Artefactos , Procesamiento Postranscripcional del ARN , Animales , Ratones , ARN/genética , ARN/metabolismo , ARN Ribosómico/genética , ARN Ribosómico/metabolismo , ARN de Transferencia/metabolismoRESUMEN
Substitution of the queuine nucleobase precursor preQ1 by an azide-containing derivative (azido-propyl-preQ1) led to incorporation of this clickable chemical entity into tRNA via transglycosylation in vitro as well as in vivo in Escherichia coli, Schizosaccharomyces pombe and human cells. The resulting semi-synthetic RNA modification, here termed Q-L1, was present in tRNAs on actively translating ribosomes, indicating functional integration into aminoacylation and recruitment to the ribosome. The azide moiety of Q-L1 facilitates analytics via click conjugation of a fluorescent dye, or of biotin for affinity purification. Combining the latter with RNAseq showed that TGT maintained its native tRNA substrate specificity in S. pombe cells. The semi-synthetic tRNA modification Q-L1 was also functional in tRNA maturation, in effectively replacing the natural queuosine in its stimulation of further modification of tRNAAsp with 5-methylcytosine at position 38 by the tRNA methyltransferase Dnmt2 in S. pombe. This is the first demonstrated in vivo integration of a synthetic moiety into an RNA modification circuit, where one RNA modification stimulates another. In summary, the scarcity of queuosinylation sites in cellular RNA, makes our synthetic q/Q system a 'minimally invasive' system for placement of a non-natural, clickable nucleobase within the total cellular RNA.
Asunto(s)
Nucleósido Q , Schizosaccharomyces , 5-Metilcitosina/metabolismo , Azidas , Biotina/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Colorantes Fluorescentes/metabolismo , Humanos , Nucleósido Q/química , ARN de Transferencia/metabolismo , ARN de Transferencia de Aspártico/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , ARNt Metiltransferasas/metabolismoRESUMEN
Actinobacterial genus Streptomyces (streptomycetes) represents one of the largest cultivable group of bacteria famous for their ability to produce valuable specialized (secondary) metabolites. Regulation of secondary metabolic pathways inextricably couples the latter to essential cellular processes that determine levels of amino acids, carbohydrates, phosphate, etc. Post-transcriptional tRNA modifications remain one of the least studied aspects of streptomycete physiology, albeit a few of them were recently shown to impact antibiotic production. In this study, we describe the diversity of post-transcriptional tRNA modifications in model strain Streptomyces albus (albidoflavus) J1074 by combining mass spectrometry and genomic data. Our results show that J1074 can produce more chemically distinct tRNA modifications than previously thought. An in silico approach identified orthologs for enzymes governing most of the identified tRNA modifications. Yet, genetic control of certain modifications remained elusive, suggesting early divergence of tRNA modification pathways in Streptomyces from the better studied model bacteria, such as Escherichia coli and Bacillus subtilis. As a first point in case, our data point to the presence of a non-canonical MiaE enzyme performing hydroxylation of prenylated adenosines. A further finding concerns the methylthiotransferase MiaB, which requires previous modification of adenosines by MiaA to i6A for thiomethylation to ms2i6A. We show here that the J1074 ortholog, when overexpressed, yields ms2A in a ΔmiaA background. Our results set the working ground for and justify a more detailed studies of biological significance of tRNA modification pathways in streptomycetes. IMPORTANCE Post-transcriptional tRNA modifications (PTTMs) play an important role in maturation and functionality of tRNAs. Little is known about tRNA modifications in the antibiotic-producing actinobacterial genus Streptomyces, even though peculiar tRNA-based regulatory mechanisms operate in this taxon. We provide a first detailed description of the chemical diversity of PTTMs in the model species, S. albidoflavus J1074, and identify most plausible genes for these PTTMs. Some of the PTTMs are described for the first time for Streptomyces. Production of certain PTTMs in J1074 appears to depend on enzymes that show no sequence similarity to known PTTM enzymes from model species. Our findings are of relevance for interrogation of genetic basis of PTTMs in pathogenic actinobacteria, such as M. tuberculosis.
Asunto(s)
Streptomyces , Streptomyces/genética , Streptomyces/metabolismo , Genómica , Espectrometría de Masas , ARN de Transferencia/genética , ARN de Transferencia/metabolismo , Antibacterianos/metabolismoRESUMEN
Newly growing evidence highlights the essential role that epitranscriptomic marks play in the development of many cancers; however, little is known about the role and implications of altered epitranscriptome deposition in prostate cancer. Here, we show that the transfer RNA N7-methylguanosine (m7G) transferase METTL1 is highly expressed in primary and advanced prostate tumours. Mechanistically, we find that METTL1 depletion causes the loss of m7G tRNA methylation and promotes the biogenesis of a novel class of small non-coding RNAs derived from 5'tRNA fragments. 5'tRNA-derived small RNAs steer translation control to favour the synthesis of key regulators of tumour growth suppression, interferon pathway, and immune effectors. Knockdown of Mettl1 in prostate cancer preclinical models increases intratumoural infiltration of pro-inflammatory immune cells and enhances responses to immunotherapy. Collectively, our findings reveal a therapeutically actionable role of METTL1-directed m7G tRNA methylation in cancer cell translation control and tumour biology.