RESUMEN
The ribosome utilizes hydrogen bonding between mRNA codons and aminoacyl-tRNAs to ensure rapid and accurate protein production. Chemical modification of mRNA nucleobases can adjust the strength and pattern of this hydrogen bonding to alter protein synthesis. We investigate how the N1-methylpseudouridine (m1Ψ) modification, commonly incorporated into therapeutic and vaccine mRNA sequences, influences the speed and fidelity of translation. We find that m1Ψ does not substantially change the rate constants for amino acid addition by cognate tRNAs or termination by release factors. However, we also find that m1Ψ can subtly modulate the fidelity of amino acid incorporation in a codon-position and tRNA dependent manner in vitro and in human cells. Our computational modeling shows that altered energetics of mRNA:tRNA interactions largely account for the context dependence of the low levels of miscoding we observe on Ψ and m1Ψ containing codons. The outcome of translation on modified mRNA bases is thus governed by the sequence context in which they occur.
Asunto(s)
Codón , Biosíntesis de Proteínas , Seudouridina , ARN Mensajero , ARN de Transferencia , Seudouridina/metabolismo , Seudouridina/análogos & derivados , ARN Mensajero/metabolismo , ARN Mensajero/genética , Humanos , Codón/genética , ARN de Transferencia/metabolismo , ARN de Transferencia/genética , Ribosomas/metabolismo , Enlace de Hidrógeno , Células HEK293RESUMEN
Transfer RNA (tRNA) modifications are essential for the temperature adaptation of thermophilic and psychrophilic organisms as they control the rigidity and flexibility of transcripts. To further understand how specific tRNA modifications are adjusted to maintain functionality in response to temperature fluctuations, we investigated whether tRNA modifications represent an adaptation of bacteria to different growth temperatures (minimal, optimal, and maximal), focusing on closely related psychrophilic (P. halocryophilus and E. sibiricum), mesophilic (B. subtilis), and thermophilic (G. stearothermophilus) Bacillales. Utilizing an RNA sequencing approach combined with chemical pre-treatment of tRNA samples, we systematically profiled dihydrouridine (D), 4-thiouridine (s4U), 7-methyl-guanosine (m7G), and pseudouridine (Ψ) modifications at single-nucleotide resolution. Despite their close relationship, each bacterium exhibited a unique tRNA modification profile. Our findings revealed increased tRNA modifications in the thermophilic bacterium at its optimal growth temperature, particularly showing elevated levels of s4U8 and Ψ55 modifications compared to non-thermophilic bacteria, indicating a temperature-dependent regulation that may contribute to thermotolerance. Furthermore, we observed higher levels of D modifications in psychrophilic and mesophilic bacteria, indicating an adaptive strategy for cold environments by enhancing local flexibility in tRNAs. Our method demonstrated high effectiveness in identifying tRNA modifications compared to an established tool, highlighting its potential for precise tRNA profiling studies.
Asunto(s)
Procesamiento Postranscripcional del ARN , ARN de Transferencia , Temperatura , ARN de Transferencia/genética , ARN de Transferencia/metabolismo , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Seudouridina/metabolismoRESUMEN
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.
Asunto(s)
Dioxigenasa Dependiente de Alfa-Cetoglutarato, Homólogo 3 de AlkB , Neoplasias Gástricas , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patología , Humanos , Dioxigenasa Dependiente de Alfa-Cetoglutarato, Homólogo 3 de AlkB/genética , Dioxigenasa Dependiente de Alfa-Cetoglutarato, Homólogo 3 de AlkB/metabolismo , ARN Mensajero/metabolismo , ARN Mensajero/genética , Progresión de la Enfermedad , Ratones , Animales , Seudouridina/metabolismo , Seudouridina/genética , Línea Celular Tumoral , Ratones Desnudos , Modelos Animales de Enfermedad , Femenino , HidroliasasRESUMEN
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.
Asunto(s)
Microscopía por Crioelectrón , Hidroliasas , Transferasas Intramoleculares , Seudouridina , ARN de Transferencia , Humanos , Dominio Catalítico , Células HEK293 , Hidroliasas/metabolismo , Hidroliasas/genética , Hidroliasas/química , Discapacidad Intelectual/genética , Discapacidad Intelectual/metabolismo , Discapacidad Intelectual/enzimología , Modelos Moleculares , Mutación , Unión Proteica , Seudouridina/metabolismo , Seudouridina/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN de Transferencia/metabolismo , ARN de Transferencia/genética , Especificidad por SustratoRESUMEN
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.
Asunto(s)
ARN Viral , Replicación Viral , ARN Viral/genética , Animales , Replicón/genética , Seudouridina/metabolismo , Virus ARN Monocatenarios Positivos/genética , Humanos , Virus de los Bosques Semliki/genética , ARN Polimerasa Dependiente del ARN/genética , ARN Polimerasa Dependiente del ARN/metabolismo , Estabilidad del ARN , Enterovirus Humano B/genética , Enterovirus Humano B/fisiologíaRESUMEN
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.
Asunto(s)
COVID-19 , Seudouridina , Humanos , Seudouridina/metabolismo , Seudouridina/genética , COVID-19/genética , Neoplasias/genética , Neoplasias/terapia , Neoplasias/metabolismo , Neoplasias/patología , SARS-CoV-2/genética , Procesamiento Postranscripcional del ARN/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Animales , Trastornos del Neurodesarrollo/genética , Trastornos del Neurodesarrollo/terapia , Trastornos del Neurodesarrollo/metabolismo , Trastornos del Neurodesarrollo/patología , Vacunas contra la COVID-19 , Biosíntesis de Proteínas , Empalme del ARN/genética , ARN de Transferencia/genética , ARN de Transferencia/metabolismoRESUMEN
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.
Asunto(s)
Microscopía por Crioelectrón , Modelos Moleculares , ARN Ribosómico , Ribosomas , Humanos , ARN Ribosómico/metabolismo , ARN Ribosómico/química , Ribosomas/metabolismo , Ribosomas/química , Ribosomas/ultraestructura , Conformación de Ácido Nucleico , Zinc/metabolismo , Zinc/química , Metilación , Magnesio/metabolismo , Magnesio/química , Seudouridina/metabolismo , Seudouridina/químicaRESUMEN
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.
Asunto(s)
VIH-1 , Seudouridina , ARN Mensajero , ARN Viral , Seudouridina/metabolismo , Seudouridina/genética , VIH-1/genética , Humanos , ARN Viral/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Infecciones por VIH/virología , Infecciones por VIH/genética , Procesamiento Postranscripcional del ARN , Línea CelularRESUMEN
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.
Asunto(s)
Seudouridina , ARN de Transferencia , Ribosomas , Seudouridina/metabolismo , Ribosomas/metabolismo , ARN de Transferencia/metabolismo , ARN de Transferencia/genética , Leishmania/metabolismo , Leishmania/genética , Microscopía por Crioelectrón , ARN Ribosómico/metabolismo , ARN Ribosómico/química , ARN Ribosómico/genética , Conformación de Ácido Nucleico , Modelos MolecularesRESUMEN
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.
Asunto(s)
Eritropoyesis , Células Madre Pluripotentes Inducidas , Mitocondrias , ARN de Transferencia , Animales , Ratones , Humanos , ARN de Transferencia/genética , ARN de Transferencia/metabolismo , Mitocondrias/metabolismo , Mitocondrias/patología , Células Madre Pluripotentes Inducidas/metabolismo , Seudouridina/metabolismo , Anemia Sideroblástica/genética , Anemia Sideroblástica/metabolismo , Anemia Sideroblástica/patología , ARN Mitocondrial/genética , ARN Mitocondrial/metabolismo , Hidroliasas/metabolismo , Hidroliasas/genética , Síndrome MELAS/genética , Síndrome MELAS/patología , Síndrome MELAS/metabolismo , Modelos Animales de EnfermedadRESUMEN
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.
Asunto(s)
COVID-19 , Neoplasias , Seudouridina , SARS-CoV-2 , Humanos , COVID-19/inmunología , COVID-19/prevención & control , SARS-CoV-2/inmunología , Neoplasias/inmunología , Seudouridina/metabolismo , Vacunas contra la COVID-19/inmunología , Animales , Vacunas de ARNm , Pandemias , Neumonía Viral/inmunología , Neumonía Viral/virología , Neumonía Viral/prevención & control , Betacoronavirus/inmunología , Infecciones por Coronavirus/prevención & control , Infecciones por Coronavirus/inmunología , Infecciones por Coronavirus/virologíaRESUMEN
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.
Asunto(s)
Proteínas de Escherichia coli , Escherichia coli , Escherichia coli/metabolismo , Seudouridina/metabolismo , Glucosa/metabolismo , Uracilo/metabolismo , Reactores Biológicos , Fermentación , Ingeniería Metabólica , Proteínas de Transporte de Membrana/metabolismo , Proteínas de Escherichia coli/metabolismoRESUMEN
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.
Asunto(s)
Escherichia coli , Seudouridina , Seudouridina/genética , Seudouridina/química , Seudouridina/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Uridina/genética , Uridina/química , Uridina/metabolismo , Catálisis , Hidrolasas/metabolismoRESUMEN
Pseudouridine, one of the most abundant RNA modifications, is synthesized by stand-alone or RNA-guided pseudouridine synthases. Here, we comprehensively mapped pseudouridines in rRNAs, tRNAs and small RNAs in the archaeon Sulfolobus islandicus and identified Cbf5-associated H/ACA RNAs. Through genetic deletion and in vitro modification assays, we determined the responsible enzymes for these modifications. The pseudouridylation machinery in S. islandicus consists of the stand-alone enzymes aPus7 and aPus10, and six H/ACA RNA-guided enzymes that account for all identified pseudouridines. These H/ACA RNAs guide the modification of all eleven sites in rRNAs, two sites in tRNAs, and two sites in CRISPR RNAs. One H/ACA RNA shows exceptional versatility by targeting eight different sites. aPus7 and aPus10 are responsible for modifying positions 13, 54 and 55 in tRNAs. We identified four atypical H/ACA RNAs that lack the lower stem and the ACA motif and confirmed their function both in vivo and in vitro. Intriguingly, atypical H/ACA RNAs can be modified by Cbf5 in a guide-independent manner. Our data provide the first global view of pseudouridylation in archaea and reveal unexpected structures, substrates, and activities of archaeal H/ACA RNPs.
Asunto(s)
Seudouridina , ARN de Archaea , ARN de Transferencia , Sulfolobus , Seudouridina/metabolismo , Sulfolobus/genética , Sulfolobus/metabolismo , ARN de Transferencia/metabolismo , ARN de Transferencia/genética , ARN de Archaea/genética , ARN de Archaea/metabolismo , ARN de Archaea/química , ARN Ribosómico/metabolismo , ARN Ribosómico/genética , Proteínas Arqueales/metabolismo , Proteínas Arqueales/genética , Procesamiento Postranscripcional del ARN , ARN Guía de Sistemas CRISPR-Cas/genética , ARN Guía de Sistemas CRISPR-Cas/metabolismo , Transferasas Intramoleculares/genética , Transferasas Intramoleculares/metabolismoRESUMEN
RNA-guided pseudouridylation, a widespread post-transcriptional RNA modification, has recently gained recognition for its role in cellular processes such as pre-mRNA splicing and the modulation of premature termination codon (PTC) readthrough. This review provides insights into its mechanisms, functions, and potential therapeutic applications. It examines the mechanisms governing RNA-guided pseudouridylation, emphasizing the roles of guide RNAs and pseudouridine synthases in catalyzing uridine-to-pseudouridine conversion. A key focus is the impact of RNA-guided pseudouridylation of U2 small nuclear RNA on pre-mRNA splicing, encompassing its influence on branch site recognition and spliceosome assembly. Additionally, the review discusses the emerging role of RNA-guided pseudouridylation in regulating PTC readthrough, impacting translation termination and genetic disorders. Finally, it explores the therapeutic potential of pseudouridine modifications, offering insights into potential treatments for genetic diseases and cancer and the development of mRNA vaccine.
Asunto(s)
Seudouridina , Precursores del ARN , Seudouridina/genética , Seudouridina/metabolismo , Precursores del ARN/metabolismo , ARN Guía de Sistemas CRISPR-Cas , ARN/metabolismo , Procesamiento Postranscripcional del ARN , Biosíntesis de ProteínasRESUMEN
Plasma nucleosides-pseudouridine (PU) and N2N2-dimethyl guanosine (DMG) predict the progression of type 2 diabetic kidney disease (DKD) to end-stage renal disease, but the mechanisms underlying this relationship are not well understood. We used a well-characterized model of type 2 diabetes (db/db mice) and control nondiabetic mice (db/m mice) to characterize the production and excretion of PU and DMG levels using liquid chromatography-mass spectrometry. The fractional excretion of PU and DMG was decreased in db/db mice compared with control mice at 24 wk before any changes to renal function. We then examined the dynamic changes in nucleoside metabolism using in vivo metabolic flux analysis with the injection of labeled nucleoside precursors. Metabolic flux analysis revealed significant decreases in the ratio of urine-to-plasma labeling of PU and DMG in db/db mice compared with db/m mice, indicating significant tubular dysfunction in diabetic kidney disease. We observed that the gene and protein expression of the renal tubular transporters involved with nucleoside transport in diabetic kidneys in mice and humans was reduced. In conclusion, this study strongly suggests that tubular handling of nucleosides is altered in early DKD, in part explaining the association of PU and DMG with human DKD progression observed in previous studies.NEW & NOTEWORTHY Tubular dysfunction explains the association between the nucleosides pseudouridine and N2N2-dimethyl guanosine and diabetic kidney disease.
Asunto(s)
Diabetes Mellitus Tipo 2 , Nefropatías Diabéticas , Humanos , Ratones , Animales , Nefropatías Diabéticas/metabolismo , Seudouridina/metabolismo , Diabetes Mellitus Tipo 2/complicaciones , Diabetes Mellitus Tipo 2/metabolismo , Nucleósidos/metabolismo , Eliminación Renal , Riñón/metabolismo , Guanosina/metabolismoRESUMEN
In vitro-transcribed (IVT) mRNAs are modalities that can combat human disease, exemplified by their use as vaccines for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). IVT mRNAs are transfected into target cells, where they are translated into recombinant protein, and the biological activity or immunogenicity of the encoded protein exerts an intended therapeutic effect1,2. Modified ribonucleotides are commonly incorporated into therapeutic IVT mRNAs to decrease their innate immunogenicity3-5, but their effects on mRNA translation fidelity have not been fully explored. Here we demonstrate that incorporation of N1-methylpseudouridine into mRNA results in +1 ribosomal frameshifting in vitro and that cellular immunity in mice and humans to +1 frameshifted products from BNT162b2 vaccine mRNA translation occurs after vaccination. The +1 ribosome frameshifting observed is probably a consequence of N1-methylpseudouridine-induced ribosome stalling during IVT mRNA translation, with frameshifting occurring at ribosome slippery sequences. However, we demonstrate that synonymous targeting of such slippery sequences provides an effective strategy to reduce the production of frameshifted products. Overall, these data increase our understanding of how modified ribonucleotides affect the fidelity of mRNA translation, and although there are no adverse outcomes reported from mistranslation of mRNA-based SARS-CoV-2 vaccines in humans, these data highlight potential off-target effects for future mRNA-based therapeutics and demonstrate the requirement for sequence optimization.
Asunto(s)
Sistema de Lectura Ribosómico , Seudouridina , ARN Mensajero , Animales , Humanos , Ratones , Vacuna BNT162/efectos adversos , Vacuna BNT162/genética , Vacuna BNT162/inmunología , Sistema de Lectura Ribosómico/genética , ARN Mensajero/química , ARN Mensajero/genética , ARN Mensajero/metabolismo , Seudouridina/análogos & derivados , Seudouridina/metabolismo , Ribosomas/metabolismo , Biosíntesis de ProteínasRESUMEN
Pseudouridine is a noncanonical C-nucleoside containing a C-C glycosidic linkage between uracil and ribose. In the two-step degradation of pseudouridine, pseudouridine 5'-monophosphate glycosylase (PUMY) is responsible for the second step and catalyses the cleavage of the C-C glycosidic bond in pseudouridine 5'-monophosphate (ΨMP) into uridine and ribose 5'-phosphate, which are recycled via other metabolic pathways. Structural features of Escherichia coli PUMY have been reported, but the details of the substrate specificity of ΨMP were unknown. Here, we present three crystal structures of Arabidopsis thaliana PUMY in different ligation states and a kinetic analysis of ΨMP degradation. The results indicate that Thr149 and Asn308, which are conserved in the PUMY family, are structural determinants for recognizing the nucleobase of ΨMP. The distinct binding modes of ΨMP and ribose 5'-phosphate also suggest that the nucleobase, rather than the phosphate group, of ΨMP dictates the substrate-binding mode. An open-to-close transition of the active site is essential for catalysis, which is mediated by two α-helices, α11 and α12, near the active site. Mutational analysis validates the proposed roles of the active site residues in catalysis. Our structural and functional analyses provide further insight into the enzymatic features of PUMY towards ΨMP.
Asunto(s)
Arabidopsis , Seudouridina , Seudouridina/metabolismo , Cinética , Ribosa/metabolismo , Escherichia coli/metabolismo , Nucleósidos/metabolismo , Fosfatos , Catálisis , Especificidad por Sustrato , Cristalografía por Rayos XRESUMEN
The uridine modifications pseudouridine (Ψ), dihydrouridine, and 5-methyluridine are present in eukaryotic mRNAs. Many uridine-modifying enzymes are associated with human disease, underscoring the importance of uncovering the functions of uridine modifications in mRNAs. These modified uridines have chemical properties distinct from those of canonical uridines, which impact RNA structure and RNA-protein interactions. Ψ, the most abundant of these uridine modifications, is present across (pre-)mRNAs. Recent work has shown that many Ψs are present at intermediate to high stoichiometries that are likely conducive to function and at locations that are poised to influence pre-/mRNA processing. Technological innovations and mechanistic investigations are unveiling the functions of uridine modifications in pre-mRNA splicing, translation, and mRNA stability, which are discussed in this review.
Asunto(s)
Seudouridina , ARN , Humanos , Seudouridina/genética , Seudouridina/metabolismo , ARN Mensajero/metabolismo , ARN/metabolismo , Uridina/química , Uridina/metabolismo , Procesamiento Postranscripcional del ARN , Precursores del ARN/genéticaRESUMEN
Trypanosomes are protozoan parasites that cycle between insect and mammalian hosts and are the causative agent of sleeping sickness. Here, we describe the changes of pseudouridine (Ψ) modification on rRNA in the two life stages of the parasite using four different genome-wide approaches. CRISPR-Cas9 knock-outs of all four snoRNAs guiding Ψ on helix 69 (H69) of the large rRNA subunit were lethal. A single knock-out of a snoRNA guiding Ψ530 on H69 altered the composition of the 80S monosome. These changes specifically affected the translation of only a subset of proteins. This study correlates a single site Ψ modification with changes in ribosomal protein stoichiometry, supported by a high-resolution cryo-EM structure. We propose that alteration in rRNA modifications could generate ribosomes preferentially translating state-beneficial proteins.