RESUMO
BACKGROUND: RNA pseudouridylation is a critical post-transcriptional modification that influences gene expression and impacts various biological functions. Despite its significance, the role of mRNA pseudouridylation in cancer remains poorly understood. This study investigates the impact of pseudouridine synthase 7 (PUS7)-mediated pseudouridylation of Alpha-ketoglutarate-dependent Dioxygenase alkB Homolog 3 (ALKBH3) mRNA in gastric cancer. METHODS: Immunohistochemistry and Western blotting were used to assess PUS7 protein levels in human gastric cancer tissues. The relationship between PUS7 and gastric cancer progression was examined using 3D colony formation assays and subcutaneous xenograft models. Real-time quantitative PCR (RT-qPCR), Western blotting, and polysome profiling assays were conducted to investigate how PUS7 regulates ALKBH3. A locus-specific pseudouridine (Ψ) detection assay was used to identify Ψ sites on ALKBH3 mRNA. RESULTS: Our findings indicate a significant reduction of PUS7 in gastric cancer tissues compared to adjacent non-tumour tissues. Functional analyses reveal that PUS7 inhibits gastric cancer cell proliferation and tumour growth via its catalytic activity. Additionally, PUS7 enhances the translation efficiency of ALKBH3 mRNA by modifying the U696 site with pseudouridine, thereby attenuating tumour growth. Importantly, ALKBH3 functions as a tumour suppressor in gastric cancer, with its expression closely correlated with PUS7 levels in tumour tissues. CONCLUSIONS: PUS7-dependent pseudouridylation of ALKBH3 mRNA enhances its translation, thereby suppressing gastric cancer progression. These findings highlight the potential significance of mRNA pseudouridylation in cancer biology and suggest a therapeutic target for gastric cancer. HIGHLIGHTS: PUS7 enhances the translation efficiency of ALKBH3 through its pseudouridylation activity on ALKBH3 mRNA, thereby inhibiting gastric tumourigenesis. The expression levels of PUS7 and ALKBH3 are significantly correlated in gastric tumours, which may be potential prognostic predictors and therapeutic targets for patients with gastric cancer.
Assuntos
Homólogo AlkB 3 da Dioxigenase Dependente de alfa-Cetoglutarato , Neoplasias Gástricas , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Humanos , Homólogo AlkB 3 da Dioxigenase Dependente de alfa-Cetoglutarato/genética , Homólogo AlkB 3 da Dioxigenase Dependente de alfa-Cetoglutarato/metabolismo , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Progressão da Doença , Camundongos , Animais , Pseudouridina/metabolismo , Pseudouridina/genética , Linhagem Celular Tumoral , Camundongos Nus , Modelos Animais de Doenças , Feminino , HidroliasesRESUMO
Self-amplifying RNAs (saRNAs) are versatile vaccine platforms that take advantage of a viral RNA-dependent RNA polymerase (RdRp) to amplify the messenger RNA (mRNA) of an antigen of interest encoded within the backbone of the viral genome once inside the target cell. In recent years, more saRNA vaccines have been clinically tested with the hope of reducing the vaccination dose compared to the conventional mRNA approach. The use of N1-methyl-pseudouridine (1mΨ), which enhances RNA stability and reduces the innate immune response triggered by RNAs, is among the improvements included in the current mRNA vaccines. In the present study, we evaluated the effects of this modified nucleoside on various saRNA platforms based on different viruses. The results showed that different stages of the replication process were affected depending on the backbone virus. For TNCL, an insect virus of the Alphanodavirus genus, replication was impaired by poor recognition of viral RNA by RdRp. In contrast, the translation step was severely abrogated in coxsackievirus B3 (CVB3), a member of the Picornaviridae family. Finally, the effects of 1mΨ on Semliki forest virus (SFV), were not detrimental in in vitro studies, but no advantages were observed when immunogenicity was tested in vivo.
Assuntos
RNA Viral , Replicação Viral , RNA Viral/genética , Animais , Replicon/genética , Pseudouridina/metabolismo , Vírus de RNA de Cadeia Positiva/genética , Humanos , Vírus da Floresta de Semliki/genética , RNA Polimerase Dependente de RNA/genética , RNA Polimerase Dependente de RNA/metabolismo , Estabilidade de RNA , Enterovirus Humano B/genética , Enterovirus Humano B/fisiologiaRESUMO
Pseudouridine (Ψ) is a widespread RNA modification found in various RNA species, including rRNA, tRNA, snRNA, mRNA, and long noncoding RNA (lncRNA). Understanding the function of Ψ in these RNA types requires a robust method for the detection and quantification of the Ψ level at single-nucleotide resolution. A previously used method utilizes Ψ labeling by N-cyclohexyl-N'-ß-(4-methylmorpholinium)ethylcarbodiimide (CMC). The quantification of Ψ is based on the stop ratio after reverse transcription. However, the use of CMC followed by strong alkaline treatment causes severe RNA degradation, often requiring a large amount of RNA. The removal of CMC and recovery of RNA by ethanol precipitation are also time-consuming. Here, we introduce a Bisulfite Incorporation Hindered ligation-based method (BIHIND), which can detect and quantify Ψ sites on rRNA, mRNA, and noncoding RNA. BIHIND can be coupled with quantitative PCR (BIHIND-qPCR) for quantitative detection of Ψ fraction at individual modification sites, as well as with next-generation sequencing (BIHIND-seq) for high-throughput sequencing of Ψ without requiring reverse transcription. We validated the robustness of BIHIND with the elucidation of Ψ dynamics following pseudouridine synthase depletion.
Assuntos
Pseudouridina , Sulfitos , Pseudouridina/química , Sulfitos/química , Humanos , RNA Ribossômico/química , RNA Mensageiro/genética , RNA Mensageiro/análise , RNA Longo não Codificante/genética , RNA Longo não Codificante/análiseRESUMO
Pseudouridine (Ψ), the isomer of uridine, is ubiquitously found in RNA, including tRNA, rRNA, and mRNA. Human pseudouridine synthase 3 (PUS3) catalyzes pseudouridylation of position 38/39 in tRNAs. However, the molecular mechanisms by which it recognizes its RNA targets and achieves site specificity remain elusive. Here, we determine single-particle cryo-EM structures of PUS3 in its apo form and bound to three tRNAs, showing how the symmetric PUS3 homodimer recognizes tRNAs and positions the target uridine next to its active site. Structure-guided and patient-derived mutations validate our structural findings in complementary biochemical assays. Furthermore, we deleted PUS1 and PUS3 in HEK293 cells and mapped transcriptome-wide Ψ sites by Pseudo-seq. Although PUS1-dependent sites were detectable in tRNA and mRNA, we found no evidence that human PUS3 modifies mRNAs. Our work provides the molecular basis for PUS3-mediated tRNA modification in humans and explains how its tRNA modification activity is linked to intellectual disabilities.
Assuntos
Microscopia Crioeletrônica , Hidroliases , Transferases Intramoleculares , Pseudouridina , RNA de Transferência , Humanos , Domínio Catalítico , Células HEK293 , Hidroliases/metabolismo , Hidroliases/genética , Hidroliases/química , Deficiência Intelectual/genética , Deficiência Intelectual/metabolismo , Deficiência Intelectual/enzimologia , Modelos Moleculares , Mutação , Ligação Proteica , Pseudouridina/metabolismo , Pseudouridina/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Transferência/metabolismo , RNA de Transferência/genética , Especificidade por SubstratoRESUMO
The rodent-borne Andes virus (ANDV) causes a severe disease in humans. We developed an ANDV mRNA vaccine based on the M segment of the viral genome, either with regular uridine (U-mRNA) or N1-methylpseudouridine (m1Ψ-mRNA). Female mice immunized by m1Ψ-mRNA developed slightly greater germinal center (GC) responses than U-mRNA-immunized mice. Single cell RNA and BCR sequencing of the GC B cells revealed similar levels of activation, except an additional cluster of cells exhibiting interferon response in animals vaccinated with U-mRNA but not m1Ψ-mRNA. Similar immunoglobulin class-switching and somatic hypermutations were observed in response to the vaccines. Female Syrian hamsters were immunized via a prime-boost regimen with two doses of each vaccine. The titers of glycoprotein-binding antibodies were greater for U-mRNA construct than for m1Ψ-mRNA construct; however, the titers of ANDV-neutralizing antibodies were similar. Vaccinated animals were challenged with a lethal dose of ANDV, along with a naïve control group. All control animals and two animals vaccinated with a lower dose of m1Ψ-mRNA succumbed to infection whereas other vaccinated animals survived without evidence of virus replication. The data demonstrate the development of a protective vaccine against ANDV and the lack of a substantial effect of m1Ψ modification on immunogenicity and protection in rodents.
Assuntos
Mesocricetus , Uridina , Vacinas Virais , Animais , Feminino , Camundongos , Vacinas Virais/imunologia , Vacinas Virais/administração & dosagem , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Mensageiro/imunologia , Anticorpos Antivirais/imunologia , Orthohantavírus/imunologia , Orthohantavírus/genética , Anticorpos Neutralizantes/imunologia , Centro Germinativo/imunologia , Pseudouridina/imunologia , Cricetinae , Vacinas de mRNA , Febre Hemorrágica Americana/prevenção & controle , Febre Hemorrágica Americana/imunologia , Febre Hemorrágica Americana/virologia , RNA Viral/genética , RNA Viral/imunologia , Linfócitos B/imunologia , Humanos , Desenvolvimento de VacinasRESUMO
Pseudouridine (Ψ), the most abundant RNA modification, plays a role in pre-mRNA splicing, RNA stability, protein translation efficiency, and cellular responses to environmental stress. Dysregulation of pseudouridylation is linked to human diseases. This review explores recent insights into the role of RNA pseudouridylation alterations in human disorders and the therapeutic potential of Ψ. We discuss the impact of the reduction of Ψ levels in ribosomal, messenger, and transfer RNA in RNA processing, protein translation, and consequently its role in neurodevelopmental diseases and cancer. Furthermore, we review the success of N1-methyl-Ψ messenger RNA vaccines against COVID-19 and the development of RNA-guided pseudouridylation enzymes for treating genetic diseases caused by premature stop codons.
Assuntos
COVID-19 , Pseudouridina , Humanos , Pseudouridina/metabolismo , Pseudouridina/genética , COVID-19/genética , Neoplasias/genética , Neoplasias/terapia , Neoplasias/metabolismo , Neoplasias/patologia , SARS-CoV-2/genética , Processamento Pós-Transcricional do RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Animais , Transtornos do Neurodesenvolvimento/genética , Transtornos do Neurodesenvolvimento/terapia , Transtornos do Neurodesenvolvimento/metabolismo , Transtornos do Neurodesenvolvimento/patologia , Vacinas contra COVID-19 , Biossíntese de Proteínas , Splicing de RNA/genética , RNA de Transferência/genética , RNA de Transferência/metabolismoRESUMO
The ribosomal RNA of the human protein synthesis machinery comprises numerous chemical modifications that are introduced during ribosome biogenesis. Here we present the 1.9 Å resolution cryo electron microscopy structure of the 80S human ribosome resolving numerous new ribosomal RNA modifications and functionally important ions such as Zn2+, K+ and Mg2+, including their associated individual water molecules. The 2'-O-methylation, pseudo-uridine and base modifications were confirmed by mass spectrometry, resulting in a complete investigation of the >230 sites, many of which could not be addressed previously. They choreograph key interactions within the RNA and at the interface with proteins, including at the ribosomal subunit interfaces of the fully assembled 80S ribosome. Uridine isomerization turns out to be a key mechanism for U-A base pair stabilization in RNA in general. The structural environment of chemical modifications and ions is primordial for the RNA architecture of the mature human ribosome, hence providing a structural framework to address their role in healthy states and in human diseases.
Assuntos
Microscopia Crioeletrônica , Modelos Moleculares , RNA Ribossômico , Ribossomos , Humanos , RNA Ribossômico/metabolismo , RNA Ribossômico/química , Ribossomos/metabolismo , Ribossomos/química , Ribossomos/ultraestrutura , Conformação de Ácido Nucleico , Zinco/metabolismo , Zinco/química , Metilação , Magnésio/metabolismo , Magnésio/química , Pseudouridina/metabolismo , Pseudouridina/químicaRESUMO
According to the CDC, both Pfizer and Moderna COVID-19 vaccines contain nucleoside-modified messenger RNA (mRNA) encoding the viral spike glycoprotein of severe acute respiratory syndrome caused by corona virus (SARS-CoV-2), administered via intramuscular injections. Despite their worldwide use, very little is known about how nucleoside modifications in mRNA sequences affect their breakdown, transcription and protein synthesis. It was hoped that resident and circulating immune cells attracted to the injection site make copies of the spike protein while the injected mRNA degrades within a few days. It was also originally estimated that recombinant spike proteins generated by mRNA vaccines would persist in the body for a few weeks. In reality, clinical studies now report that modified SARS-CoV-2 mRNA routinely persist up to a month from injection and can be detected in cardiac and skeletal muscle at sites of inflammation and fibrosis, while the recombinant spike protein may persist a little over half a year in blood. Vaccination with 1-methylΨ (pseudouridine enriched) mRNA can elicit cellular immunity to peptide antigens produced by +1 ribosomal frameshifting in major histocompatibility complex-diverse people. The translation of 1-methylΨ mRNA using liquid chromatography tandem mass spectrometry identified nine peptides derived from the mRNA +1 frame. These products impact on off-target host T cell immunity that include increased production of new B cell antigens with far reaching clinical consequences. As an example, a highly significant increase in heart muscle 18-flourodeoxyglucose uptake was detected in vaccinated patients up to half a year (180 days). This review article focuses on medical biochemistry, proteomics and deutenomics principles that explain the persisting spike phenomenon in circulation with organ-related functional damage even in asymptomatic individuals. Proline and hydroxyproline residues emerge as prominent deuterium (heavy hydrogen) binding sites in structural proteins with robust isotopic stability that resists not only enzymatic breakdown, but virtually all (non)-enzymatic cleavage mechanisms known in chemistry.
Assuntos
Vacinas contra COVID-19 , COVID-19 , RNA Mensageiro , Glicoproteína da Espícula de Coronavírus , Humanos , COVID-19/prevenção & controle , COVID-19/imunologia , Vacinas contra COVID-19/imunologia , Vacinas de mRNA/imunologia , Pseudouridina , Proteínas Recombinantes/administração & dosagem , RNA Viral , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/genética , Vacinação , Vacinas Sintéticas/imunologia , Vacinas Sintéticas/administração & dosagemRESUMO
The identification of RNA modifications at single nucleotide resolution has become an emerging area of interest within biology and specifically among virologists seeking to ascertain how this untapped area of RNA regulation may be altered or hijacked upon viral infection. Herein, we describe a straightforward biochemical approach modified from two original published Ψ mapping protocols, BID-seq and PRAISE, to specifically identify pseudouridine modifications on mRNA transcripts from an HIV-1 infected T cell line. This protocol could readily be adapted for other viral infected cell types and additionally for populations of purified virions from infected cells.
Assuntos
HIV-1 , Pseudouridina , RNA Mensageiro , RNA Viral , Pseudouridina/metabolismo , Pseudouridina/genética , HIV-1/genética , Humanos , RNA Viral/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Infecções por HIV/virologia , Infecções por HIV/genética , Processamento Pós-Transcricional do RNA , Linhagem CelularRESUMO
Nucleotide modifications are occurrent in all types of RNA and play an important role in RNA structure formation and stability. Modified bases not only possess the ability to shift the RNA structure ensemble towards desired functional confirmations. By changes in the base pairing partner preference, they may even enlarge or reduce the conformational space, i.e., the number and types of structures the RNA molecule can adopt. However, most methods to predict RNA secondary structure do not provide the means to include the effect of modifications on the result. With the help of a heavily modified transfer RNA (tRNA) molecule, this chapter demonstrates how to include the effect of different base modifications into secondary structure prediction using the ViennaRNA Package. The constructive approach demonstrated here allows for the calculation of minimum free energy structure and suboptimal structures at different levels of modified base support. In particular we, show how to incorporate the isomerization of uridine to pseudouridine ( Ψ ) and the reduction of uridine to dihydrouridine (D).
Assuntos
Conformação de Ácido Nucleico , RNA , RNA/química , RNA de Transferência/química , RNA de Transferência/metabolismo , Nucleotídeos/química , Pareamento de Bases , Biologia Computacional/métodos , Termodinâmica , Software , Uridina/química , Modelos Moleculares , Pseudouridina/químicaRESUMO
Nanopore direct RNA sequencing provided a promising solution for unraveling the landscapes of modifications on single RNA molecules. Here, we proposed NanoMUD, a computational framework for predicting the RNA pseudouridine modification (Ψ) and its methylated analog N1-methylpseudouridine (m1Ψ), which have critical application in mRNA vaccination, at single-base and single-molecule resolution from direct RNA sequencing data. Electric signal features were fed into a bidirectional LSTM neural network to achieve improved accuracy and predictive capabilities. Motif-specific models (NNUNN, N = A, C, U or G) were trained based on features extracted from designed dataset and achieved superior performance on molecule-level modification prediction (Ψ models: min AUC = 0.86, max AUC = 0.99; m1Ψ models: min AUC = 0.87, max AUC = 0.99). We then aggregated read-level predictions for site stoichiometry estimation. Given the observed sequence-dependent bias in model performance, we trained regression models based on the distribution of modification probabilities for sites with known stoichiometry. The distribution-based site stoichiometry estimation method allows unbiased comparison between different contexts. To demonstrate the feasibility of our work, three case studies on both in vitro and in vivo transcribed RNAs were presented. NanoMUD will make a powerful tool to facilitate the research on modified therapeutic IVT RNAs and provides useful insight to the landscape and stoichiometry of pseudouridine and N1-pseudouridine on in vivo transcribed RNA species.
Assuntos
Pseudouridina , Análise de Sequência de RNA , Pseudouridina/química , Análise de Sequência de RNA/métodos , RNA/química , Nanoporos , Redes Neurais de Computação , Sequenciamento por Nanoporos/métodosRESUMO
Leishmania is the causative agent of cutaneous and visceral diseases affecting millions of individuals worldwide. Pseudouridine (Ψ), the most abundant modification on rRNA, changes during the parasite life cycle. Alterations in the level of a specific Ψ in helix 69 (H69) affected ribosome function. To decipher the molecular mechanism of this phenotype, we determine the structure of ribosomes lacking the single Ψ and its parental strain at â¼2.4-3 Å resolution using cryo-EM. Our findings demonstrate the significance of a single Ψ on H69 to its structure and the importance for its interactions with helix 44 and specific tRNAs. Our study suggests that rRNA modification affects translation of mRNAs carrying codon bias due to selective accommodation of tRNAs by the ribosome. Based on the high-resolution structures, we propose a mechanism explaining how the ribosome selects specific tRNAs.
Assuntos
Pseudouridina , RNA de Transferência , Ribossomos , Pseudouridina/metabolismo , Ribossomos/metabolismo , RNA de Transferência/metabolismo , RNA de Transferência/genética , Leishmania/metabolismo , Leishmania/genética , Microscopia Crioeletrônica , RNA Ribossômico/metabolismo , RNA Ribossômico/química , RNA Ribossômico/genética , Conformação de Ácido Nucleico , Modelos MolecularesRESUMO
Over 150 types of chemical modifications have been identified in RNA to date, with pseudouridine (Ψ) being one of the most prevalent modifications in RNA. Ψ plays vital roles in various biological processes, and precise, base-resolution detection methods are fundamental for deep analysis of its distribution and function. In this study, we introduced a novel base-resolution Ψ detection method named pseU-TRACE. pseU-TRACE relied on the fact that RNA containing Ψ underwent a base deletion after treatment of bisulfite (BS) during reverse transcription, which enabled efficient ligation of two probes complementary to the cDNA sequence on either side of the Ψ site and successful amplification in subsequent real-time quantitative PCR (qPCR), thereby achieving selective and accurate Ψ detection. Our method accurately and sensitively detected several known Ψ sites in 28S, 18S, 5.8S, and even mRNA. Moreover, pseU-TRACE could be employed to measure the Ψ fraction in RNA and explore the Ψ metabolism of different pseudouridine synthases (PUSs), providing valuable insights into the function of Ψ. Overall, pseU-TRACE represents a reliable, time-efficient and sensitive Ψ detection method.
Assuntos
Pseudouridina , Reação em Cadeia da Polimerase em Tempo Real , Sulfitos , Humanos , Pseudouridina/química , Pseudouridina/genética , Pseudouridina/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/métodos , RNA/química , RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Sulfitos/químicaRESUMO
Pseudouridine is an RNA modification that is widely distributed in both prokaryotes and eukaryotes, and plays a critical role in numerous biological activities. Despite its importance, the precise identification of pseudouridine sites through experimental approaches poses significant challenges, requiring substantial time and resources.Therefore, there is a growing need for computational techniques that can reliably and quickly identify pseudouridine sites from vast amounts of RNA sequencing data. In this study, we propose fuzzy kernel evidence Random Forest (FKeERF) to identify pseudouridine sites. This method is called PseU-FKeERF, which demonstrates high accuracy in identifying pseudouridine sites from RNA sequencing data. The PseU-FKeERF model selected four RNA feature coding schemes with relatively good performance for feature combination, and then input them into the newly proposed FKeERF method for category prediction. FKeERF not only uses fuzzy logic to expand the original feature space, but also combines kernel methods that are easy to interpret in general for category prediction. Both cross-validation tests and independent tests on benchmark datasets have shown that PseU-FKeERF has better predictive performance than several state-of-the-art methods. This new method not only improves the accuracy of pseudouridine site identification, but also provides a certain reference for disease control and related drug development in the future.
Assuntos
Pseudouridina , Algoritmo Florestas Aleatórias , Pseudouridina/genética , RNA/genética , Sequência de BasesRESUMO
Due to the health emergency created by SARS-CoV-2, the virus that causes the COVID-19 disease, the rapid implementation of a new vaccine technology was necessary. mRNA vaccines, being one of the cutting-edge new technologies, attracted significant interest and offered a lot of hope. The potential of these vaccines in preventing admission to hospitals and serious illness in people with comorbidities has recently been called into question due to the vaccines' rapidly waning immunity. Mounting evidence indicates that these vaccines, like many others, do not generate sterilizing immunity, leaving people vulnerable to recurrent infections. Additionally, it has been discovered that the mRNA vaccines inhibit essential immunological pathways, thus impairing early interferon signaling. Within the framework of COVID-19 vaccination, this inhibition ensures an appropriate spike protein synthesis and a reduced immune activation. Evidence is provided that adding 100 % of N1-methyl-pseudouridine (m1Ψ) to the mRNA vaccine in a melanoma model stimulated cancer growth and metastasis, while non-modified mRNA vaccines induced opposite results, thus suggesting that COVID-19 mRNA vaccines could aid cancer development. Based on this compelling evidence, we suggest that future clinical trials for cancers or infectious diseases should not use mRNA vaccines with a 100 % m1Ψ modification, but rather ones with the lower percentage of m1Ψ modification to avoid immune suppression.
Assuntos
COVID-19 , Neoplasias , Pseudouridina , SARS-CoV-2 , Humanos , COVID-19/imunologia , COVID-19/prevenção & controle , SARS-CoV-2/imunologia , Neoplasias/imunologia , Pseudouridina/metabolismo , Vacinas contra COVID-19/imunologia , Animais , Vacinas de mRNA , Pandemias , Pneumonia Viral/imunologia , Pneumonia Viral/virologia , Pneumonia Viral/prevenção & controle , Betacoronavirus/imunologia , Infecções por Coronavirus/prevenção & controle , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologiaRESUMO
Here we report the efficient synthetic access to 13C/15N-labelled pseudouridine phosphoramidites, which were incorporated into a binary H/ACA box guide RNA/product complex comprising 77 nucleotides (nts) in total and into a 75â nt E. coli tRNAGly. The stable isotope (SI) labelled pseudouridines were produced via a highly efficient chemo-enzymatic synthesis. 13C/15N labelled uracils were produced via chemical synthesis and enzymatically converted to pseudouridine 5'-monophosphate (ΨMP) by using YeiN, a Ψ-5'-monophosphate C-glycosidase. Removal of the 5'-phosphate group yielded the desired pseudouridine nucleoside (Ψ), which was transformed into a phosphoramidite building suitable for RNA solid phase synthesis. A Ψ -building block carrying both a 13C and a 15N label was incorporated into a product RNA and the complex formation with a 63â nt H/ACA box RNA could be observed via NMR. Furthermore, the SI labelled pseudouridine building block was used to determine imino proton bulk water exchange rates of a 75â nt E. coli tRNAGly CCmnm5U, identifying the TΨC-loop 5-methyluridine as a modifier of the exchange rates. The efficient synthetic access to SI-labelled Ψ building blocks will allow the solution and solid-state NMR spectroscopic studies of Ψ containing RNAs and will facilitate the mass spectrometric analysis of Ψ-modified nucleic acids.
Assuntos
Escherichia coli , Marcação por Isótopo , Isótopos de Nitrogênio , Compostos Organofosforados , Pseudouridina , Pseudouridina/química , Compostos Organofosforados/química , Isótopos de Nitrogênio/química , Marcação por Isótopo/métodos , RNA/química , Isótopos de Carbono/química , Espectroscopia de Ressonância Magnética/métodosRESUMO
ABSTRACT: Pseudouridine is the most prevalent RNA modification, and its aberrant function is implicated in various human diseases. However, the specific impact of pseudouridylation on hematopoiesis remains poorly understood. Here, we investigated the role of transfer RNA (tRNA) pseudouridylation in erythropoiesis and its association with mitochondrial myopathy, lactic acidosis, and sideroblastic anemia syndrome (MLASA) pathogenesis. By using patient-specific induced pluripotent stem cells (iPSCs) carrying a genetic pseudouridine synthase 1 (PUS1) mutation and a corresponding mutant mouse model, we demonstrated impaired erythropoiesis in MLASA-iPSCs and anemia in the MLASA mouse model. Both MLASA-iPSCs and mouse erythroblasts exhibited compromised mitochondrial function and impaired protein synthesis. Mechanistically, we revealed that PUS1 deficiency resulted in reduced mitochondrial tRNA levels because of pseudouridylation loss, leading to aberrant mitochondrial translation. Screening of mitochondrial supplements aimed at enhancing respiration or heme synthesis showed limited effect in promoting erythroid differentiation. Interestingly, the mammalian target of rapamycin (mTOR) inhibitor rapamycin facilitated erythroid differentiation in MLASA-iPSCs by suppressing mTOR signaling and protein synthesis, and consistent results were observed in the MLASA mouse model. Importantly, rapamycin treatment partially ameliorated anemia phenotypes in a patient with MLASA. Our findings provide novel insights into the crucial role of mitochondrial tRNA pseudouridylation in governing erythropoiesis and present potential therapeutic strategies for patients with anemia facing challenges related to protein translation.
Assuntos
Eritropoese , Células-Tronco Pluripotentes Induzidas , Mitocôndrias , RNA de Transferência , Animais , Camundongos , Humanos , RNA de Transferência/genética , RNA de Transferência/metabolismo , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Pseudouridina/metabolismo , Anemia Sideroblástica/genética , Anemia Sideroblástica/metabolismo , Anemia Sideroblástica/patologia , RNA Mitocondrial/genética , RNA Mitocondrial/metabolismo , Hidroliases/metabolismo , Hidroliases/genética , Síndrome MELAS/genética , Síndrome MELAS/patologia , Síndrome MELAS/metabolismo , Modelos Animais de DoençasRESUMO
Pseudouridine is the most abundant modified nucleoside found in non-coding RNA and is widely used in biological and pharmaceutical fields. However, current methods for pseudouridine production suffer from drawbacks such as complex procedures, low efficiency and high costs. This study presents a novel enzymatic cascade reaction route in Escherichia coli, enabling the whole-cell catalytic synthesis of pseudouridine from uridine. Initially, a metabolic pathway was established through plasmid-mediated overexpression of endogenous pseudouridine-5-phosphase glycosidase, ribokinase, and ribonucleoside hydrolase, resulting in the accumulation of pseudouridine. Subsequently, highly active endogenous ribonucleoside hydrolase was screened to enhance uridine hydrolysis and provide more precursors for pseudouridine synthesis. Furthermore, modifications were made to the substrates and products transport pathways to increase the pseudouridine yield while avoiding the accumulation of by-product uridine. The resulting recombinant strain Ψ-7 catalyzed the conversion of 30 g/L uridine into 27.24 g/L pseudouridine in 24 h, achieving a conversion rate of 90.8% and a production efficiency of 1.135 g/(L·h). These values represent the highest reported yield and production efficiency achieved by enzymatic catalysis methods to date.
Assuntos
Escherichia coli , Pseudouridina , Pseudouridina/genética , Pseudouridina/química , Pseudouridina/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Uridina/genética , Uridina/química , Uridina/metabolismo , Catálise , Hidrolases/metabolismoRESUMO
We combined the CRISPR-Cas13a system with CMC chemical labeling, developing an approach that enables precise identification of pseudouridine (Ψ) sites at specific loci within ribosomal RNA (rRNA), messenger RNA (mRNA) and small nuclear RNAs (snRNA). This method, with good efficiency and simplicity, detects Ψ sites through fluorescence measurement, providing a straightforward and fast validation for targeted Ψ sites of interest.
Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Pseudouridina , Pseudouridina/genética , RNA Nuclear Pequeno/genética , RNA Ribossômico , RNA Mensageiro/genéticaRESUMO
In this study, we proposed a biological approach to efficiently produce pseudouridine (Ψ) from glucose and uracil in vivo using engineered Escherichia coli. By screening host strains and core enzymes, E. coli MG1655 overexpressing Ψ monophosphate (ΨMP) glycosidase and ΨMP phosphatase was obtained, which displayed the highest Ψ concentration. Then, optimization of the RBS sequences, enhancement of ribose 5-phosphate supply in the cells, and overexpression of the membrane transport protein UraA were investigated. Finally, fed-batch fermentation of Ψ in a 5 L fermentor can reach 27.5 g/L with a yield of 89.2 mol % toward uracil and 25.6 mol % toward glucose within 48 h, both of which are the highest to date. In addition, the Ψ product with a high purity of 99.8% can be purified from the fermentation broth after crystallization. This work provides an efficient and environmentally friendly protocol for allowing for the possibility of Ψ bioproduction on an industrial scale.