RESUMEN
Isolated methylmalonic acidemia/aciduria (MMA) due to MMUT enzyme deficiency is an ultra-rare pediatric disease with high morbidity and mortality, with no approved disease-altering therapies. Previous publications showed that systemic treatment with a codon-optimized mRNA encoding wild-type human MMUT (MMUT) is a promising strategy for treatment of MMA. We developed a second-generation drug product, mRNA-3705, comprised of an mRNA encoding the MMUT enzyme formulated in a lipid nanoparticle (LNP) with incorporation of enhancements over the previous clinical candidate mRNA-3704. Both drug products produced functional MMUT in rat livers when dosed IV, and showed long-term safety and efficacy in two mouse models of MMA. mRNA-3705 produced 2.1-3.4-fold higher levels of hepatic MMUT protein expression than the first-generation drug product mRNA-3704 when given at an identical dose level, which resulted in greater and more sustained reductions in plasma methylmalonic acid. The data presented herein provide comprehensive preclinical pharmacology to support the clinical development of mRNA-3705.
RESUMEN
The development of a protective vaccine remains a top priority for the control of the HIV/AIDS pandemic. Here, we show that a messenger RNA (mRNA) vaccine co-expressing membrane-anchored HIV-1 envelope (Env) and simian immunodeficiency virus (SIV) Gag proteins to generate virus-like particles (VLPs) induces antibodies capable of broad neutralization and reduces the risk of infection in rhesus macaques. In mice, immunization with co-formulated env and gag mRNAs was superior to env mRNA alone in inducing neutralizing antibodies. Macaques were primed with a transmitted-founder clade-B env mRNA lacking the N276 glycan, followed by multiple booster immunizations with glycan-repaired autologous and subsequently bivalent heterologous envs (clades A and C). This regimen was highly immunogenic and elicited neutralizing antibodies against the most prevalent (tier-2) HIV-1 strains accompanied by robust anti-Env CD4+ T cell responses. Vaccinated animals had a 79% per-exposure risk reduction upon repeated low-dose mucosal challenges with heterologous tier-2 simian-human immunodeficiency virus (SHIV AD8). Thus, the multiclade env-gag VLP mRNA platform represents a promising approach for the development of an HIV-1 vaccine.
Asunto(s)
Anticuerpos Neutralizantes/inmunología , Genes env , Genes gag , Anticuerpos Anti-VIH/biosíntesis , VIH-1/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio/prevención & control , Vacunas Sintéticas/inmunología , Vacunas de ARNm/inmunología , Animales , Anticuerpos Anti-VIH/inmunología , Inmunización Secundaria , Macaca mulatta , Factores de Riesgo , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Vacunas Sintéticas/administración & dosificación , Vacunas de ARNm/administración & dosificaciónRESUMEN
Nipah virus (NiV) represents a significant pandemic threat with zoonotic transmission from bats-to-humans with almost annual regional outbreaks characterized by documented human-to-human transmission and high fatality rates. Currently, no vaccine against NiV has been approved. Structure-based design and protein engineering principles were applied to stabilize the fusion (F) protein in its prefusion trimeric conformation (pre-F) to improve expression and increase immunogenicity. We covalently linked the stabilized pre-F through trimerization domains at the C-terminus to three attachment protein (G) monomers, forming a chimeric design. These studies detailed here focus on mRNA delivery of NiV immunogens in mice, assessment of mRNA immunogen-specific design elements and their effects on humoral and cellular immunogenicity. The pre-F/G chimera elicited a strong neutralizing antibody response and a superior NiV-specific Tfh and other effector T cell response compared to G alone across both the mRNA and protein platforms. These findings enabled final candidate selection of pre-F/G Fd for clinical development.
Asunto(s)
Antígenos Virales/genética , Liposomas/administración & dosificación , Nanopartículas/administración & dosificación , Virus Nipah/inmunología , Proteínas del Envoltorio Viral/genética , Proteínas Virales de Fusión/genética , Vacunas Virales/administración & dosificación , Vacunas de ARNm/administración & dosificación , Animales , Antígenos Virales/inmunología , Femenino , Inmunoglobulina G/sangre , Ratones , Asociación entre el Sector Público-Privado , ARN Mensajero/administración & dosificación , Linfocitos T/inmunología , Proteínas del Envoltorio Viral/inmunología , Proteínas Virales de Fusión/inmunologíaRESUMEN
Glycogen Storage Disease 1a (GSD1a) is a rare, inherited metabolic disorder caused by deficiency of glucose 6-phosphatase (G6Pase-α). G6Pase-α is critical for maintaining interprandial euglycemia. GSD1a patients exhibit life-threatening hypoglycemia and long-term liver complications including hepatocellular adenomas (HCAs) and carcinomas (HCCs). There is no treatment for GSD1a and the current standard-of-care for managing hypoglycemia (Glycosade®/modified cornstarch) fails to prevent HCA/HCC risk. Therapeutic modalities such as enzyme replacement therapy and gene therapy are not ideal options for patients due to challenges in drug-delivery, efficacy, and safety. To develop a new treatment for GSD1a capable of addressing both the life-threatening hypoglycemia and HCA/HCC risk, we encapsulated engineered mRNAs encoding human G6Pase-α in lipid nanoparticles. We demonstrate the efficacy and safety of our approach in a preclinical murine model that phenotypically resembles the human condition, thus presenting a potential therapy that could have a significant therapeutic impact on the treatment of GSD1a.
Asunto(s)
Modelos Animales de Enfermedad , Terapia Genética/métodos , Glucosa-6-Fosfatasa/genética , Enfermedad del Almacenamiento de Glucógeno/terapia , ARN Mensajero/genética , Animales , Línea Celular Tumoral , Citocinas/sangre , Citocinas/metabolismo , Glucosa-6-Fosfatasa/metabolismo , Glucógeno/metabolismo , Enfermedad del Almacenamiento de Glucógeno/genética , Enfermedad del Almacenamiento de Glucógeno/patología , Células HeLa , Humanos , Hígado/metabolismo , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Nanopartículas/administración & dosificación , Nanopartículas/química , ARN Mensajero/administración & dosificación , ARN Mensajero/química , Resultado del Tratamiento , Triglicéridos/metabolismoRESUMEN
BACKGROUND & AIMS: Progressive familial intrahepatic cholestasis type 3 (PFIC3) is a rare lethal autosomal recessive liver disorder caused by loss-of-function variations of the ABCB4 gene, encoding a phosphatidylcholine transporter (ABCB4/MDR3). Currently, no effective treatment exists for PFIC3 outside of liver transplantation. METHODS: We have produced and screened chemically and genetically modified mRNA variants encoding human ABCB4 (hABCB4 mRNA) encapsulated in lipid nanoparticles (LNPs). We examined their pharmacological effects in a cell-based model and in a new in vivo mouse model resembling human PFIC3 as a result of homozygous disruption of the Abcb4 gene in fibrosis-susceptible BALB/c.Abcb4-/- mice. RESULTS: We show that treatment with liver-targeted hABCB4 mRNA resulted in de novo expression of functional hABCB4 protein and restored phospholipid transport in cultured cells and in PFIC3 mouse livers. Importantly, repeated injections of the hABCB4 mRNA effectively rescued the severe disease phenotype in young Abcb4-/- mice, with rapid and dramatic normalisation of all clinically relevant parameters such as inflammation, ductular reaction, and liver fibrosis. Synthetic mRNA therapy also promoted favourable hepatocyte-driven liver regeneration to restore normal homeostasis, including liver weight, body weight, liver enzymes, and portal vein blood pressure. CONCLUSIONS: Our data provide strong preclinical proof-of-concept for hABCB4 mRNA therapy as a potential treatment option for patients with PFIC3. LAY SUMMARY: This report describes the development of an innovative mRNA therapy as a potential treatment for PFIC3, a devastating rare paediatric liver disease with no treatment options except liver transplantation. We show that administration of our mRNA construct completely rescues severe liver disease in a genetic model of PFIC3 in mice.
Asunto(s)
Subfamilia B de Transportador de Casetes de Unión a ATP/deficiencia , Colestasis Intrahepática/tratamiento farmacológico , Colestasis Intrahepática/genética , Eliminación de Gen , Liposomas/química , Sistema de Administración de Fármacos con Nanopartículas/química , Nanopartículas/química , Fenotipo , ARN Mensajero/administración & dosificación , Subfamilia B de Transportador de Casetes de Unión a ATP/administración & dosificación , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Animales , Colestasis Intrahepática/metabolismo , Modelos Animales de Enfermedad , Células HEK293 , Homocigoto , Humanos , Hígado/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , ARN Mensajero/genética , Transfección , Resultado del Tratamiento , Miembro 4 de la Subfamilia B de Casete de Unión a ATPRESUMEN
Messenger RNAs (mRNAs) encode information in both their primary sequence and their higher order structure. The independent contributions of factors like codon usage and secondary structure to regulating protein expression are difficult to establish as they are often highly correlated in endogenous sequences. Here, we used 2 approaches, global inclusion of modified nucleotides and rational sequence design of exogenously delivered constructs, to understand the role of mRNA secondary structure independent from codon usage. Unexpectedly, highly expressed mRNAs contained a highly structured coding sequence (CDS). Modified nucleotides that stabilize mRNA secondary structure enabled high expression across a wide variety of primary sequences. Using a set of eGFP mRNAs with independently altered codon usage and CDS structure, we find that the structure of the CDS regulates protein expression through changes in functional mRNA half-life (i.e., mRNA being actively translated). This work highlights an underappreciated role of mRNA secondary structure in the regulation of mRNA stability.
Asunto(s)
Biosíntesis de Proteínas/fisiología , Estabilidad del ARN , ARN Mensajero/química , Semivida , Células HeLa , Humanos , Conformación de Ácido Nucleico , Proteínas/metabolismoRESUMEN
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
Despite its exceptionally low circulating concentration, apolipoprotein (apo) A-V is a potent modulator of plasma triacylglycerol levels. The secretion efficiency of nascent apoA-V was investigated in cultured cells transfected with mRNA. Following transfection of HepG2 cells with wild type apoA-V mRNA, apoA-V protein was detectable in cell lysates by 6â¯h. At 24â¯h post transfection, evidence of apoA-V secretion into media was obtained, although most apoA-V was recovered in the cell lysate fraction. By contrast, apoA-I was efficiently secreted into the culture medium. A positive correlation between culture medium fetal bovine serum content and the percentage of apoA-V recovered in conditioned media was observed. When transfected cells were cultured in serum-free media supplemented with increasing amounts of high density lipoprotein, a positive correlation with apoA-V secretion was observed. The data indicate that, following signal sequence cleavage, the bulk of nascent apoA-V remains cell associated. Transit of nascent apoA-V out of cultured cells is enhanced by the availability of extracellular lipid particle acceptors.
Asunto(s)
Apolipoproteína A-V/genética , Apolipoproteína A-V/metabolismo , Lipoproteínas HDL/metabolismo , Apolipoproteína A-V/química , Transporte Biológico Activo , Medios de Cultivo , Células HEK293 , Células Hep G2 , Humanos , Lipoproteínas HDL/sangre , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , TransfecciónRESUMEN
Fabry disease is an X-linked lysosomal storage disease caused by loss of alpha galactosidase A (α-Gal A) activity and is characterized by progressive accumulation of globotriaosylceramide and its analogs in all cells and tissues. Although enzyme replacement therapy (ERT) is considered standard of care, the long-term effects of ERT on renal and cardiac manifestations remain uncertain and thus novel therapies are desirable. We herein report preclinical studies evaluating systemic messenger RNA (mRNA) encoding human α-Gal A in wild-type (WT) mice, α-Gal A-deficient mice, and WT non-human primates (NHPs). The pharmacokinetics and distribution of h-α-Gal A mRNA encoded protein in WT mice demonstrated prolonged half-lives of α-Gal A in tissues and plasma. Single intravenous administration of h-α-Gal A mRNA to Gla-deficient mice showed dose-dependent protein activity and substrate reduction. Moreover, long duration (up to 6 weeks) of substrate reductions in tissues and plasma were observed after a single injection. Furthermore, repeat i.v. administration of h-α-Gal A mRNA showed a sustained pharmacodynamic response and efficacy in Fabry mice model. Lastly, multiple administrations to non-human primates confirmed safety and translatability. Taken together, these studies across species demonstrate preclinical proof-of-concept of systemic mRNA therapy for the treatment of Fabry disease and this approach may be useful for other lysosomal storage disorders.
Asunto(s)
Enfermedad de Fabry/genética , Enfermedad de Fabry/terapia , ARN Mensajero/uso terapéutico , alfa-Galactosidasa/genética , Animales , Modelos Animales de Enfermedad , Endocitosis , Terapia de Reemplazo Enzimático , Terapia Genética , Humanos , Lípidos/química , Lisosomas/metabolismo , Macaca fascicularis , Masculino , Ratones , Ratones Noqueados , ARN Mensajero/farmacocinética , Distribución Tisular , Trihexosilceramidas/metabolismoRESUMEN
Isolated methylmalonic acidemia/aciduria (MMA) is a devastating metabolic disorder with poor outcomes despite current medical treatments. Like other mitochondrial enzymopathies, enzyme replacement therapy (ERT) is not available, and although promising, AAV gene therapy can be limited by pre-existing immunity and has been associated with genotoxicity in mice. To develop a new class of therapy for MMA, we generated a pseudoU-modified codon-optimized mRNA encoding human methylmalonyl-CoA mutase (hMUT), the enzyme most frequently mutated in MMA, and encapsulated it into biodegradable lipid nanoparticles (LNPs). Intravenous (i.v.) administration of hMUT mRNA in two different mouse models of MMA resulted in a 75%-85% reduction in plasma methylmalonic acid and was associated with increased hMUT protein expression and activity in liver. Repeat dosing of hMUT mRNA reduced circulating metabolites and dramatically improved survival and weight gain. Additionally, repeat i.v. dosing did not increase markers of liver toxicity or inflammation in heterozygote MMA mice.
Asunto(s)
Errores Innatos del Metabolismo de los Aminoácidos/terapia , Terapia Genética/métodos , Metilmalonil-CoA Mutasa/genética , Nanopartículas/administración & dosificación , ARN Mensajero/genética , Administración Intravenosa , Animales , Femenino , Humanos , Lípidos/química , Hígado/metabolismo , Masculino , Metilmalonil-CoA Mutasa/metabolismo , Ratones , Nanopartículas/química , ARN Mensajero/metabolismoRESUMEN
Certain chemical modifications confer increased stability and low immunogenicity to in vitro transcribed mRNAs, thereby facilitating expression of therapeutically important proteins. Here, we demonstrate that N1-methyl-pseudouridine (N1mΨ) outperforms several other nucleoside modifications and their combinations in terms of translation capacity. Through extensive analysis of various modified transcripts in cell-free translation systems, we deconvolute the different components of the effect on protein expression independent of mRNA stability mechanisms. We show that in addition to turning off the immune/eIF2α phosphorylation-dependent inhibition of translation, the incorporated N1mΨ nucleotides dramatically alter the dynamics of the translation process by increasing ribosome pausing and density on the mRNA. Our results indicate that the increased ribosome loading of modified mRNAs renders them more permissive for initiation by favoring either ribosome recycling on the same mRNA or de novo ribosome recruitment.
Asunto(s)
Factor 2 Eucariótico de Iniciación/fisiología , Polirribosomas/metabolismo , Biosíntesis de Proteínas , Seudouridina/análogos & derivados , ARN Mensajero/genética , Animales , Línea Celular , Sistema Libre de Células , Activación Enzimática , Fibroblastos , Células HEK293 , Células HeLa , Humanos , Ratones , Fosforilación , Procesamiento Proteico-Postraduccional , Seudouridina/metabolismo , ARN/metabolismo , Estabilidad del ARN , ARN Mensajero/química , Transfección , eIF-2 Quinasa/metabolismoRESUMEN
mRNA degradation represents a critical regulated step in gene expression. Although the major pathways in turnover have been identified, accounting for disparate half-lives has been elusive. We show that codon optimality is one feature that contributes greatly to mRNA stability. Genome-wide RNA decay analysis revealed that stable mRNAs are enriched in codons designated optimal, whereas unstable mRNAs contain predominately non-optimal codons. Substitution of optimal codons with synonymous, non-optimal codons results in dramatic mRNA destabilization, whereas the converse substitution significantly increases stability. Further, we demonstrate that codon optimality impacts ribosome translocation, connecting the processes of translation elongation and decay through codon optimality. Finally, we show that optimal codon content accounts for the similar stabilities observed in mRNAs encoding proteins with coordinated physiological function. This work demonstrates that codon optimization exists as a mechanism to finely tune levels of mRNAs and, ultimately, proteins.
Asunto(s)
Codón , ARN de Hongos/genética , ARN de Hongos/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Saccharomyces cerevisiae/metabolismo , Biosíntesis de Proteínas , Estabilidad del ARN , ARN de Hongos/química , ARN Mensajero/químicaRESUMEN
TbRGG2 is an essential kinetoplastid RNA editing accessory factor that acts specifically on pan-edited RNAs. To understand the mechanism of TbRGG2 action, we undertook an in-depth analysis of edited RNA populations in TbRGG2 knockdown cells and an in vitro examination of the biochemical activities of the protein. We demonstrate that TbRGG2 down-regulation more severely impacts editing at the 5' ends of pan-edited RNAs than at their 3' ends. The initiation of editing is reduced to some extent in TbRGG2 knockdown cells. In addition, TbRGG2 plays a post-initiation role as editing becomes stalled in TbRGG2-depleted cells, resulting in an overall decrease in the 3' to 5' progression of editing. Detailed analyses of edited RNAs from wild-type and TbRGG2-depleted cells reveal that TbRGG2 facilitates progression of editing past intrinsic pause sites that often correspond to the 3' ends of cognate guide RNAs (gRNAs). In addition, noncanonically edited junction regions are either absent or significantly shortened in TbRGG2-depleted cells, consistent with impaired gRNA transitions. Sequence analysis further suggests that TbRGG2 facilitates complete utilization of certain gRNAs. In vitro RNA annealing and in vivo RNA unwinding assays demonstrate that TbRGG2 can modulate RNA-RNA interactions. Collectively, these data are consistent with a model in which TbRGG2 facilitates initiation and 3' to 5' progression of editing through its ability to affect gRNA utilization, both during the transition between specific gRNAs and during usage of certain gRNAs.
Asunto(s)
Cinetocoros/metabolismo , Plastidios/metabolismo , Proteínas Protozoarias/metabolismo , Edición de ARN , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismo , Secuencia de Bases , Datos de Secuencia Molecular , ARN Protozoario/genéticaRESUMEN
Mitochondrial RNA metabolism in Trypanosoma brucei is a complex process involving both extensive RNA editing and control of RNA stability. MRP1/2 and RBP16 are two factors that have been implicated in regulating the editing and stability of specific mRNAs. These two factors exhibit similar nonspecific RNA binding and RNA-annealing activities, suggesting that some of their actions may have been previously masked by functional redundancy. Here, we examine the functional interaction of MRP1/2 and RBP16 by separate and simultaneous RNA interference and by overexpressing RBP16 in an MRP1/2-depleted background. Simultaneous depletion of these factors resulted in synthetic lethality in procyclic trypanosomes. Analysis of mitochondrial RNAs in procyclic cells revealed distinct functions for MRP1/2 and RBP16 toward edited apocytochrome b mRNA, redundant functions in stabilization of edited ATPase subunit 6 and cytochrome oxidase subunit 3 mRNAs, and concentration-dependent positive and negative functions for RBP16 toward edited RPS12 mRNAs. While simultaneous MRP1/2-RBP16 depletion had no effect on the growth of bloodstream form cells, massive adverse effects on the levels of almost all mitochondrial RNAs were observed. These studies greatly expand our knowledge regarding the functions of MRP1/2 and RBP16 and suggest that both RNA-specific and life cycle stage-specific factors impact MRP1/2 and RBP16 functions.
Asunto(s)
Proteínas Mitocondriales/metabolismo , Proteínas Protozoarias/metabolismo , Proteínas de Unión al ARN/metabolismo , ARN/metabolismo , Trypanosoma brucei brucei/metabolismo , Animales , Citocromos b/genética , Proteínas Mitocondriales/genética , NADH Deshidrogenasa/genética , NADH Deshidrogenasa/metabolismo , Proteínas Protozoarias/genética , Edición de ARN , ARN Guía de Kinetoplastida/metabolismo , ARN Mensajero/metabolismo , ARN Mitocondrial , Proteínas de Unión al ARN/genética , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismo , Trypanosoma brucei brucei/genéticaRESUMEN
Arginine methylation is a widespread post-translational modification of proteins catalyzed by a family of protein arginine methyltransferases (PRMTs). The ancient protozoan parasite, Trypanosoma brucei, possesses five putative PRMTs, a relatively large number for a single-celled eukaryote. Trypanosomatids lack gene regulation at the level of transcription, instead relying on post-transcriptional control mechanisms that act at the levels of RNA turnover, translation, and editing, all processes that likely involve multiple RNA-binding proteins, which are common targets of arginine methylation. Here, we report the characterization of a trypanosome PRMT, TbPRMT7, which is homologous to human PRMT7. Interestingly, trypanosomatids are the only single-celled eukaryotes known to harbor a PRMT7 homologue. TbPRMT7 differs dramatically from all known metazoan PRMT7 homologues in lacking the second AdoMet binding-like domain that is required for activity of the human enzyme. Nevertheless, bacterially expressed TbPRMT7 exhibits robust methyltransferase activity toward multiple targets in vitro. High resolution ion exchange chromatography analysis of methylated substrates reveals that TbPRMT7 is a type III PRMT, catalyzing the formation of only monomethylarginine, thereby representing the only exclusively type III PRMT identified to date. TbPRMT7 is expressed in both mammalian and insect stage T. brucei and is apparently dispensable for growth in both life cycle stages. The enzyme is cytoplasmically localized and is a component of several higher order complexes in vivo. Together, our studies indicate that TbPRMT7 is a Type III PRMT, and its robust activity and presence in numerous complexes suggest it plays multiple roles during the complex T. brucei life cycle.
Asunto(s)
Proteína-Arginina N-Metiltransferasas/fisiología , Secuencia de Aminoácidos , Animales , Cromatografía por Intercambio Iónico/métodos , Clonación Molecular , Citoplasma/metabolismo , Dimerización , Humanos , Cinética , Metiltransferasas/química , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Proteína-Arginina N-Metiltransferasas/metabolismo , Interferencia de ARN , Homología de Secuencia de Aminoácido , Factores de Tiempo , Trypanosoma brucei bruceiRESUMEN
In the mitochondria of kinetoplastid protozoa, including Trypanosoma brucei, RNA editing inserts and/or deletes uridines from pre-mRNAs to produce mature, translatable mRNAs. RNA editing is carried out by several related multiprotein complexes known as editosomes, which contain all of the enzymatic components required for catalysis of editing. In addition, noneditosome accessory factors necessary for editing of specific RNAs have also been described. Here, we report the in vitro and in vivo characterization of the mitochondrial TbRGG2 protein (originally termed TbRGGm) and demonstrate that it acts as an editing accessory factor. TbRGG2 is an RNA-binding protein with a preference for poly(U). TbRGG2 protein levels are up-regulated 10-fold in procyclic form T. brucei compared with bloodstream forms. Nevertheless, the protein is essential for growth in both life cycle stages. TbRGG2 associates with RNase-sensitive and RNase-insensitive mitochondrial complexes, and a small fraction of the protein co-immunoprecipitates with editosomes. RNA interference-mediated depletion of TbRGG2 in both procyclic and bloodstream form T. brucei leads to a dramatic decrease in pan-edited RNAs and in some cases a corresponding increase in the pre-edited RNA. TbRGG2 down-regulation also results in moderate stabilization of never-edited and minimally edited RNAs. Thus, our data are consistent with a model in which TbRGG2 is multifunctional, strongly facilitating the editing of pan-edited RNAs and modestly destabilizing minimally edited and never-edited RNAs. This is the first example of an RNA editing accessory factor that functions in the mammalian infective T. brucei life cycle stage.
Asunto(s)
Proteínas Protozoarias/química , Edición de ARN , Proteínas de Unión al ARN/fisiología , ARN/genética , Trypanosoma brucei brucei/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Clonación Molecular , ADN de Cinetoplasto/química , Inmunoprecipitación , Mitocondrias/metabolismo , Modelos Biológicos , Datos de Secuencia Molecular , Interferencia de ARN , Proteínas de Unión al ARN/metabolismoRESUMEN
Human APOBEC3G (hA3G) is a host factor that defends against HIV-1 as well as other exogenous retroviruses and endogenous retroelements. To this end, hA3G is restricted to the cytoplasm of T lymphocytes where it interacts with viral RNA and proteins to assemble with viral particles causing a post-entry block during reverse transcription. hA3G also exhibits a mechanism to inhibit the reverse transcription of retroelements by RNA binding and sequestration into mRNA processing centers in the cytoplasm. We have determined that the molecular basis for this specialized property of hA3G is a novel cytoplasmic retention signal (CRS) that is necessary and sufficient to restrict wild-type hA3G and chimeric constructs to the cytoplasm. The CRS resides within amino acids 113-128 and is embedded within a basic flanking sequence and does not require RNA binding to retain hA3G in the cytoplasm. Paralogs of hA3G that have nuclear or cytoplasmic distributions differ from hA3G within the region encompassing the CRS motif with respect to charge and amino acid composition. We propose that the CRS enables hA3G to interact with cytoplasmic factors, and thereby enables hA3G to serve in host cell defense by restricting an antiviral sentinel to the cytoplasm. The CRS lies in a region involved in both Gag and Vif interactions; therefore, identification of this motif has important implications for the design of therapeutics that target HIV-1 while maintaining antiviral and cellular functions.