RESUMEN
Messenger RNA (mRNA) represents an attractive therapeutic modality for potentially a wide range of clinical indications but requires uridine chemistry modification and/or tuning of the production process to prevent activation of cellular innate immune sensors and a concomitant reduction in protein expression. To decipher the relative contributions of these factors on immune activation, here, we compared, in multiple cell and in vivo models, mRNA that encodes human erythropoietin incorporating either canonical uridine or N1-methyl-pseudouridine (1mΨ), synthesized by either a standard process shown to have double-stranded RNA (dsRNA) impurities or a modified process that yields a highly purified mRNA preparation. Our data demonstrate that the lowest stimulation of immune endpoints was with 1mΨ made by the modified process, while mRNA containing canonical uridine was immunostimulatory regardless of process. These findings confirm that uridine modification and the reduction of dsRNA impurities are both necessary and sufficient at controlling the immune-activating profile of therapeutic mRNA.
RESUMEN
Citrin deficiency is an autosomal recessive disorder caused by loss-of-function mutations in SLC25A13, encoding the liver-specific mitochondrial aspartate/glutamate transporter. It has a broad spectrum of clinical phenotypes, including life-threatening neurological complications. Conventional protein replacement therapy is not an option for these patients because of drug delivery hurdles, and current gene therapy approaches (e.g., AAV) have been hampered by immunogenicity and genotoxicity. Although dietary approaches have shown some benefits in managing citrin deficiency, the only curative treatment option for these patients is liver transplantation, which is high-risk and associated with long-term complications because of chronic immunosuppression. To develop a new class of therapy for citrin deficiency, codon-optimized mRNA encoding human citrin (hCitrin) was encapsulated in lipid nanoparticles (LNPs). We demonstrate the efficacy of hCitrin-mRNA-LNP therapy in cultured human cells and in a murine model of citrin deficiency that resembles the human condition. Of note, intravenous (i.v.) administration of the hCitrin-mRNA resulted in a significant reduction in (1) hepatic citrulline and blood ammonia levels following oral sucrose challenge and (2) sucrose aversion, hallmarks of hCitrin deficiency. In conclusion, mRNA-LNP therapy could have a significant therapeutic effect on the treatment of citrin deficiency and other mitochondrial enzymopathies with limited treatment options.
Asunto(s)
Citrulinemia/tratamiento farmacológico , Citrulinemia/metabolismo , Sistemas de Liberación de Medicamentos/métodos , Terapia Genética/métodos , Proteínas de Transporte de Membrana Mitocondrial/genética , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , ARN Mensajero/uso terapéutico , Animales , Conducta Animal/efectos de los fármacos , Modelos Animales de Enfermedad , Técnicas de Inactivación de Genes , Glucosafosfato Deshidrogenasa/genética , Células HeLa , Células Hep G2 , Humanos , Lípidos/química , Mutación con Pérdida de Función , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mitocondrias/metabolismo , Nanopartículas/química , Sistemas de Lectura Abierta/genética , ARN Mensajero/síntesis química , ARN Mensajero/química , ARN Mensajero/genética , Transfección , Resultado del TratamientoRESUMEN
Characterization of mRNA sequences is a critical aspect of mRNA drug development and regulatory filing. Herein, we developed a novel bottom-up oligonucleotide sequence mapping workflow combining multiple endonucleases that cleave mRNA at different frequencies. RNase T1, colicin E5, and mazF were applied in parallel to provide complementary sequence coverage for large mRNAs. Combined use of multiple endonucleases resulted in significantly improved sequence coverage: greater than 70% sequence coverage was achieved on mRNAs near 3000 nucleotides long. Oligonucleotide mapping simulations with large human RNA databases demonstrate that the proposed workflow can positively identify a single correct sequence from hundreds of similarly sized sequences. In addition, the workflow is sensitive and specific enough to detect minor sequence impurities such as single nucleotide polymorphisms (SNPs) with a sensitivity of less than 1%. LC-MS/MS-based oligonucleotide sequence mapping can serve as an orthogonal sequence characterization method to techniques such as Sanger sequencing or next-generation sequencing (NGS), providing high-throughput sequence identification and sensitive impurity detection.
Asunto(s)
Cromatografía Liquida/métodos , Eritropoyetina/metabolismo , Oligonucleótidos/análisis , Polimorfismo de Nucleótido Simple , ARN Mensajero/metabolismo , Espectrometría de Masas en Tándem/métodos , alfa Catenina/metabolismo , Colicinas/metabolismo , Proteínas de Unión al ADN/metabolismo , Endorribonucleasas/metabolismo , Eritropoyetina/genética , Proteínas de Escherichia coli/metabolismo , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , ARN Mensajero/genética , Ribonucleasa T1/metabolismo , Análisis de Secuencia de ARN , Programas Informáticos , alfa Catenina/genéticaRESUMEN
Telomerase helps maintain telomeres by processive synthesis of telomere repeat DNA at their 3'-ends, using an integral telomerase RNA (TER) and telomerase reverse transcriptase (TERT). We report the cryo-electron microscopy structure of Tetrahymena telomerase at ~9 angstrom resolution. In addition to seven known holoenzyme proteins, we identify two additional proteins that form a complex (TEB) with single-stranded telomere DNA-binding protein Teb1, paralogous to heterotrimeric replication protein A (RPA). The p75-p45-p19 subcomplex is identified as another RPA-related complex, CST (CTC1-STN1-TEN1). This study reveals the paths of TER in the TERT-TER-p65 catalytic core and single-stranded DNA exit; extensive subunit interactions of the TERT essential N-terminal domain, p50, and TEB; and other subunit identities and structures, including p19 and p45C crystal structures. Our findings provide structural and mechanistic insights into telomerase holoenzyme function.
Asunto(s)
ARN/química , Telomerasa/química , Tetrahymena/enzimología , Dominio Catalítico , Microscopía por Crioelectrón , Cristalografía por Rayos X , ADN de Cadena Simple/química , Holoenzimas/química , Unión Proteica , Conformación Proteica , Subunidades de Proteína/química , Proteína de Replicación A/química , Telómero/química , Homeostasis del Telómero , Proteínas de Unión a TelómerosRESUMEN
Telomerase is the ribonucleoprotein (RNP) reverse transcriptase responsible for synthesizing the 3' ends of linear chromosomes. It plays critical roles in tumorigenesis, cellular aging, and stem cell renewal. The past two years have seen exciting progress in determining telomerase holoenzyme architecture and the structural basis of telomerase activity. Notably, the first electron microscopy structures of telomerase were reported, of the Tetrahymena thermophila telomerase holoenzyme and a human telomerase dimer. In addition to new structures of TERT and TER domains, the first structures of telomerase protein domains beyond TERT, and their complexes with TER or telomeric single-stranded DNA, were reported. Together these studies provide the first glimpse into the organization of the proteins and RNA in the telomerase RNP.
Asunto(s)
Telomerasa/química , Animales , Humanos , Modelos Moleculares , Multimerización de Proteína , Telomerasa/metabolismo , Tetrahymena thermophila/química , Tetrahymena thermophila/enzimologíaRESUMEN
The eukaryotic reverse transcriptase, telomerase, adds tandem telomeric repeats to chromosome ends to promote genome stability. The fully assembled telomerase holoenzyme contains a ribonucleoprotein (RNP) catalytic core and additional proteins that modulate the ability of the RNP catalytic core to elongate telomeres. Electron microscopy (EM) structures of Tetrahymena telomerase holoenzyme revealed a central location of the relatively uncharacterized p50 subunit. Here we have investigated the biochemical and structural basis for p50 function. We have shown that the p50-bound RNP catalytic core has a relatively low rate of tandem repeat synthesis but high processivity of repeat addition, indicative of high stability of enzyme-product interaction. The rate of tandem repeat synthesis is enhanced by p50-dependent recruitment of the holoenzyme single-stranded DNA binding subunit, Teb1. An N-terminal p50 domain is sufficient to stimulate tandem repeat synthesis and bridge the RNP catalytic core, Teb1, and the p75 subunit of the holoenzyme subcomplex p75/p19/p45. In cells, the N-terminal p50 domain assembles a complete holoenzyme that is functional for telomere maintenance, albeit at shortened telomere lengths. Also, in EM structures of holoenzymes, only the N-terminal domain of p50 is visible. Our findings provide new insights about subunit and domain interactions and functions within the Tetrahymena telomerase holoenzyme.
Asunto(s)
Holoenzimas/metabolismo , Proteínas Protozoarias/metabolismo , Tetrahymena/enzimología , Dominio Catalítico , Electroforesis en Gel de Poliacrilamida , Holoenzimas/química , Holoenzimas/ultraestructura , Imagenología Tridimensional , Microscopía Electrónica , Modelos Moleculares , Unión Proteica , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Proteínas Protozoarias/química , Proteínas Protozoarias/ultraestructura , Ribonucleoproteínas/química , Ribonucleoproteínas/metabolismo , Ribonucleoproteínas/ultraestructuraRESUMEN
Telomerase adds telomeric repeats to chromosome ends using an internal RNA template and a specialized telomerase reverse transcriptase (TERT), thereby maintaining genome integrity. Little is known about the physical relationships among protein and RNA subunits within a biologically functional holoenzyme. Here we describe the architecture of Tetrahymena thermophila telomerase holoenzyme determined by electron microscopy. Six of the seven proteins and the TERT-binding regions of telomerase RNA (TER) have been localized by affinity labelling. Fitting with high-resolution structures reveals the organization of TERT, TER and p65 in the ribonucleoprotein (RNP) catalytic core. p50 has an unanticipated role as a hub between the RNP catalytic core, p75-p19-p45 subcomplex, and the DNA-binding Teb1. A complete in vitro holoenzyme reconstitution assigns function to these interactions in processive telomeric repeat synthesis. These studies provide the first view of the extensive network of subunit associations necessary for telomerase holoenzyme assembly and physiological function.
Asunto(s)
Telomerasa/química , Telomerasa/ultraestructura , Tetrahymena thermophila/enzimología , Dominio Catalítico , Holoenzimas/química , Holoenzimas/genética , Holoenzimas/ultraestructura , Microscopía Electrónica , Modelos Moleculares , Conformación de Ácido Nucleico , Docilidad , Estructura Terciaria de Proteína , Subunidades de Proteína/análisis , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Proteínas Protozoarias/química , Proteínas Protozoarias/metabolismo , Proteínas Protozoarias/ultraestructura , ARN/química , ARN/metabolismo , ARN/ultraestructura , Ribonucleoproteínas/química , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Ribonucleoproteínas/ultraestructura , Telomerasa/genética , Telomerasa/metabolismo , Tetrahymena thermophila/química , Tetrahymena thermophila/genética , Tetrahymena thermophila/ultraestructuraRESUMEN
The pseudouridine synthase TruB handles 5-fluorouridine in RNA as a substrate, converting it into two isomeric hydrated products. Unexpectedly, the two products differ not in the hydrated pyrimidine ring but in the pentose ring, which is epimerized to arabinose in the minor product. This inversion of stereochemistry at C2' suggests that pseudouridine generation may proceed by a mechanism involving a glycal intermediate or that the previously proposed mechanism involving an acylal intermediate operates but with an added reaction manifold for 5-fluorouridine versus uridine. The arabino product strongly disfavors a mechanism involving a Michael addition to the pyrimidine ring.
Asunto(s)
Transferasas Intramoleculares/metabolismo , Uridina/análogos & derivados , Transferasas Intramoleculares/química , Estructura Molecular , Teoría Cuántica , ARN/química , ARN/metabolismo , Estereoisomerismo , Uridina/biosíntesis , Uridina/químicaRESUMEN
RNA containing 5-fluorouridine (F(5)U) had previously been used to examine the mechanism of the pseudouridine synthase TruA, formerly known as pseudouridine synthase I [Gu et al. (1999) Proc. Natl. Acad. Sci. U.S.A. 96, 14270-14275]. From that work, it was reasonably concluded that the pseudouridine synthases proceed via a mechanism involving a Michael addition by an active site aspartic acid residue to the pyrimidine ring of uridine or F(5)U. Those conclusions rested on the assumption that the hydrate of F(5)U was obtained after digestion of the product RNA and that hydration resulted from hydrolysis of the ester intermediate between the aspartic acid residue and F(5)U. As reported here, (18)O labeling definitively demonstrates that ester hydrolysis does not give rise to the observed hydrated product and that digestion generates not the expected mononucleoside product but rather a dinucleotide between a hydrated isomer of F(5)U and the following nucleoside in RNA. The discovery that digestion products are dinucleotides accounts for the previously puzzling differences in the isolated products obtained following the action of the pseudouridine synthases TruB and RluA on F(5)U in RNA.
Asunto(s)
Transferasas Intramoleculares/química , Sondas Moleculares/química , Uridina/análogos & derivados , Ácido Aspártico/química , Proteínas de Escherichia coli/química , Ésteres/química , Hidrólisis , Nucleósidos/química , ARN/química , Uridina/químicaRESUMEN
As part of the investigation of the pseudouridine synthases, 5-fluorouridine in RNA was employed as a mechanistic probe. The hydrated, rearranged product of 5-fluorouridine was isolated as part of a dinucleotide and found to undergo unusual fragmentation during mass spectrometry, with the facile loss of HNCO from the product pyrimidine ring favored over phosphodiester bond rupture. Although the loss of HNCO from uridine and pseudouridine is well established, the pericyclic process leading to their fragmentation cannot operate with the saturated pyrimidine ring in the product of 5-fluorouridine. Based on the MS(n) results and calculations reported here, a new mechanism relying on the peculiar disposition of the functional groups of the product pyrimidine ring is proposed to account for the unusually facile fragmentation.