RESUMEN
Ribosome production is vital for every cell, and failure causes human diseases. It is driven by â¼200 assembly factors functioning along an ordered pathway from the nucleolus to the cytoplasm. Structural snapshots of biogenesis intermediates from the earliest 90S pre-ribosomes to mature 40S subunits unravel the mechanisms of small ribosome synthesis. To view this SnapShot, open or download the PDF.
Asunto(s)
Células Eucariotas , Ribosomas , Humanos , Nucléolo Celular/metabolismo , Células Eucariotas/metabolismo , Proteínas Ribosómicas/metabolismo , Subunidades Ribosómicas Pequeñas de Eucariotas/química , Subunidades Ribosómicas Pequeñas de Eucariotas/metabolismo , Ribosomas/metabolismoRESUMEN
Eukaryotic 60S ribosomal subunits are comprised of three rRNAs and â¼50 ribosomal proteins. The initial steps of their formation take place in the nucleolus, but, owing to a lack of structural information, this process is poorly understood. Using cryo-EM, we solved structures of early 60S biogenesis intermediates at 3.3 Å to 4.5 Å resolution, thereby providing insights into their sequential folding and assembly pathway. Besides revealing distinct immature rRNA conformations, we map 25 assembly factors in six different assembly states. Notably, the Nsa1-Rrp1-Rpf1-Mak16 module stabilizes the solvent side of the 60S subunit, and the Erb1-Ytm1-Nop7 complex organizes and connects through Erb1's meandering N-terminal extension, eight assembly factors, three ribosomal proteins, and three 25S rRNA domains. Our structural snapshots reveal the order of integration and compaction of the six major 60S domains within early nucleolar 60S particles developing stepwise from the solvent side around the exit tunnel to the central protuberance.
Asunto(s)
Chaetomium/química , Biogénesis de Organelos , Subunidades Ribosómicas Grandes de Eucariotas/química , Chaetomium/citología , Microscopía por Crioelectrón , Redes y Vías Metabólicas , Modelos Moleculares , Pliegue del ARN , Ribonucleoproteínas/químicaRESUMEN
The 90S pre-ribosome is an early biogenesis intermediate formed during co-transcriptional ribosome formation, composed of â¼70 assembly factors and several small nucleolar RNAs (snoRNAs) that associate with nascent pre-rRNA. We report the cryo-EM structure of the Chaetomium thermophilum 90S pre-ribosome, revealing how a network of biogenesis factors including 19 ß-propellers and large α-solenoid proteins engulfs the pre-rRNA. Within the 90S pre-ribosome, we identify the UTP-A, UTP-B, Mpp10-Imp3-Imp4, Bms1-Rcl1, and U3 snoRNP modules, which are organized around 5'-ETS and partially folded 18S rRNA. The U3 snoRNP is strategically positioned at the center of the 90S particle to perform its multiple tasks during pre-rRNA folding and processing. The architecture of the elusive 90S pre-ribosome gives unprecedented structural insight into the early steps of pre-rRNA maturation. Nascent rRNA that is co-transcriptionally folded and given a particular shape by encapsulation within a dedicated mold-like structure is reminiscent of how polypeptides use chaperone chambers for their protein folding.
Asunto(s)
Chaetomium/química , Biogénesis de Organelos , Ribosomas/química , Saccharomyces cerevisiae/química , Chaetomium/clasificación , Microscopía por Crioelectrón , Modelos Moleculares , ARN Ribosómico 18S/química , Subunidades Ribosómicas Grandes de Eucariotas/química , Subunidades Ribosómicas Pequeñas de Eucariotas/química , Ribosomas/ultraestructuraRESUMEN
Gene expression in metazoans is controlled by promoter-proximal pausing of RNA polymerase II, which can undergo productive elongation or promoter-proximal termination. Integrator-PP2A (INTAC) plays a crucial role in determining the fate of paused polymerases, but the underlying mechanisms remain unclear. Here, we establish a rapid degradation system to dissect the functions of INTAC RNA endonuclease and phosphatase modules. We find that both catalytic modules function at most if not all active promoters and enhancers, yet differentially affect polymerase fate. The endonuclease module induces promoter-proximal termination, with its disruption leading to accumulation of elongation-incompetent polymerases and downregulation of highly expressed genes, while elongation-competent polymerases accumulate at lowly expressed genes and non-coding elements, leading to their upregulation. The phosphatase module primarily prevents the release of paused polymerases and limits transcriptional activation, especially for highly paused genes. Thus, both INTAC catalytic modules have unexpectedly general yet distinct roles in dynamic transcriptional control.
Asunto(s)
Monoéster Fosfórico Hidrolasas , ARN Polimerasa II , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Regulación de la Expresión Génica , Activación Transcripcional , Regulación hacia Arriba , Transcripción GenéticaRESUMEN
The proper regulation of transcription is essential for maintaining genome integrity and executing other downstream cellular functions1,2. Here we identify a stable association between the genome-stability regulator sensor of single-stranded DNA (SOSS)3 and the transcription regulator Integrator-PP2A (INTAC)4-6. Through SSB1-mediated recognition of single-stranded DNA, SOSS-INTAC stimulates promoter-proximal termination of transcription and attenuates R-loops associated with paused RNA polymerase II to prevent R-loop-induced genome instability. SOSS-INTAC-dependent attenuation of R-loops is enhanced by the ability of SSB1 to form liquid-like condensates. Deletion of NABP2 (encoding SSB1) or introduction of cancer-associated mutations into its intrinsically disordered region leads to a pervasive accumulation of R-loops, highlighting a genome surveillance function of SOSS-INTAC that enables timely termination of transcription at promoters to constrain R-loop accumulation and ensure genome stability.
Asunto(s)
Inestabilidad Genómica , Regiones Promotoras Genéticas , Estructuras R-Loop , Terminación de la Transcripción Genética , Humanos , ADN de Cadena Simple/metabolismo , Inestabilidad Genómica/genética , Mutación , Estructuras R-Loop/genética , ARN Polimerasa II/metabolismo , Regiones Promotoras Genéticas/genética , Genoma Humano , Proteínas de Unión al ADN/metabolismoRESUMEN
Ribosome assembly is catalyzed by numerous trans-acting factors and coupled with irreversible pre-rRNA processing, driving the pathway toward mature ribosomal subunits. One decisive step early in this progression is removal of the 5' external transcribed spacer (5'-ETS), an RNA extension at the 18S rRNA that is integrated into the huge 90S pre-ribosome structure. Upon endo-nucleolytic cleavage at an internal site, A1, the 5'-ETS is separated from the 18S rRNA and degraded. Here we present biochemical and cryo-electron microscopy analyses that depict the RNA exosome, a major 3'-5' exoribonuclease complex, in a super-complex with the 90S pre-ribosome. The exosome is docked to the 90S through its co-factor Mtr4 helicase, a processive RNA duplex-dismantling helicase, which strategically positions the exosome at the base of 5'-ETS helices H9-H9', which are dislodged in our 90S-exosome structures. These findings suggest a direct role of the exosome in structural remodeling of the 90S pre-ribosome to drive eukaryotic ribosome synthesis.
Asunto(s)
ARN Helicasas DEAD-box/química , Endorribonucleasas/química , Exonucleasas/química , Complejo Multienzimático de Ribonucleasas del Exosoma/ultraestructura , ARN Ribosómico 18S/química , Ribosomas/ultraestructura , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Sitios de Unión , Microscopía por Crioelectrón , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , Endorribonucleasas/genética , Endorribonucleasas/metabolismo , Exonucleasas/genética , Exonucleasas/metabolismo , Complejo Multienzimático de Ribonucleasas del Exosoma/genética , Complejo Multienzimático de Ribonucleasas del Exosoma/metabolismo , Modelos Moleculares , Unión Proteica , Biosíntesis de Proteínas , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Estabilidad del ARN , ARN Ribosómico 18S/genética , ARN Ribosómico 18S/metabolismo , Ribosomas/genética , Ribosomas/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
TET proteins oxidize 5-methylcytosine (5mC) on DNA and play important roles in various biological processes. Mutations of TET2 are frequently observed in myeloid malignance. Here, we present the crystal structure of human TET2 bound to methylated DNA at 2.02 Å resolution. The structure shows that two zinc fingers bring the Cys-rich and DSBH domains together to form a compact catalytic domain. The Cys-rich domain stabilizes the DNA above the DSBH core. TET2 specifically recognizes CpG dinucleotide and shows substrate preference for 5mC in a CpG context. 5mC is inserted into the catalytic cavity with the methyl group orientated to catalytic Fe(II) for reaction. The methyl group is not involved in TET2-DNA contacts so that the catalytic cavity allows TET2 to accommodate 5mC derivatives for further oxidation. Mutations of Fe(II)/NOG-chelating, DNA-interacting, and zinc-chelating residues are frequently observed in human cancers. Our studies provide a structural basis for understanding the mechanisms of TET-mediated 5mC oxidation.
Asunto(s)
5-Metilcitosina/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , ADN/química , ADN/metabolismo , Proteínas Proto-Oncogénicas/química , Proteínas Proto-Oncogénicas/metabolismo , Secuencia de Aminoácidos , Islas de CpG , Cristalografía por Rayos X , Metilación de ADN , Dioxigenasas , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Alineación de Secuencia , Zinc/metabolismoRESUMEN
Ribosome assembly is driven by numerous assembly factors, including the Rix1 complex and the AAA ATPase Rea1. These two assembly factors catalyze 60S maturation at two distinct states, triggering poorly understood large-scale structural transitions that we analyzed by cryo-electron microscopy. Two nuclear pre-60S intermediates were discovered that represent previously unknown states after Rea1-mediated removal of the Ytm1-Erb1 complex and reveal how the L1 stalk develops from a pre-mature nucleolar to a mature-like nucleoplasmic state. A later pre-60S intermediate shows how the central protuberance arises, assisted by the nearby Rix1-Rea1 machinery, which was solved in its pre-ribosomal context to molecular resolution. This revealed a Rix12-Ipi32 tetramer anchored to the pre-60S via Ipi1, strategically positioned to monitor this decisive remodeling. These results are consistent with a general underlying principle that temporarily stabilized immature RNA domains are successively remodeled by assembly factors, thereby ensuring failsafe assembly progression.
Asunto(s)
Subunidades Ribosómicas Grandes de Eucariotas/química , Subunidades Ribosómicas Grandes de Eucariotas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , ATPasas Asociadas con Actividades Celulares Diversas/genética , ATPasas Asociadas con Actividades Celulares Diversas/metabolismo , Nucléolo Celular/genética , Nucléolo Celular/metabolismo , Microscopía por Crioelectrón , Escherichia coli/genética , Modelos Moleculares , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Subunidades Ribosómicas Grandes de Eucariotas/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Eukaryotic ribosome biogenesis involves RNA folding and processing that depend on assembly factors and small nucleolar RNAs (snoRNAs). The 90S (SSU-processome) is the earliest pre-ribosome structurally analyzed, which was suggested to assemble stepwise along the growing pre-rRNA from 5' > 3', but this directionality may not be accurate. Here, by analyzing the structure of a series of 90S assembly intermediates from Chaetomium thermophilum, we discover a reverse order of 18S rRNA subdomain incorporation. Large parts of the 18S rRNA 3' and central domains assemble first into the 90S before the 5' domain is integrated. This final incorporation depends on a contact between a heterotrimer Enp2-Bfr2-Lcp5 recruited to the flexible 5' domain and Kre33, which reconstitutes the Kre33-Enp-Brf2-Lcp5 module on the compacted 90S. Keeping the 5' domain temporarily segregated from the 90S scaffold could provide extra time to complete the multifaceted 5' domain folding, which depends on a distinct set of snoRNAs and processing factors.
Asunto(s)
Chaetomium/metabolismo , Proteínas Fúngicas/metabolismo , Conformación de Ácido Nucleico , ARN de Hongos/metabolismo , ARN Ribosómico 18S/metabolismo , Ribosomas/metabolismo , Chaetomium/genética , Proteínas Fúngicas/genética , ARN de Hongos/genética , ARN Ribosómico 18S/genética , Ribosomas/genéticaRESUMEN
In eukaryotes, most secretory and membrane proteins are targeted by an N-terminal signal sequence to the endoplasmic reticulum, where the trimeric Sec61 complex serves as protein-conducting channel (PCC). In the post-translational mode, fully synthesized proteins are recognized by a specialized channel additionally containing the Sec62, Sec63, Sec71, and Sec72 subunits. Recent structures of this Sec complex in the idle state revealed the overall architecture in a pre-opened state. Here, we present a cryo-EM structure of the yeast Sec complex bound to a substrate, and a crystal structure of the Sec62 cytosolic domain. The signal sequence is inserted into the lateral gate of Sec61α similar to previous structures, yet, with the gate adopting an even more open conformation. The signal sequence is flanked by two Sec62 transmembrane helices, the cytoplasmic N-terminal domain of Sec62 is more rigidly positioned, and the plug domain is relocated. We crystallized the Sec62 domain and mapped its interaction with the C-terminus of Sec63. Together, we obtained a near-complete and integrated model of the active Sec complex.
Asunto(s)
Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/metabolismo , Proteínas de Transporte de Membrana/química , Proteínas de Transporte de Membrana/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Sitios de Unión , Microscopía por Crioelectrón , Cristalografía por Rayos X , Retículo Endoplásmico/metabolismo , Modelos Moleculares , Unión Proteica , Conformación Proteica , Dominios Proteicos , Procesamiento Proteico-Postraduccional , Saccharomyces cerevisiae/químicaRESUMEN
In eukaryotic translation, termination and ribosome recycling phases are linked to subsequent initiation of a new round of translation by persistence of several factors at ribosomal sub-complexes. These comprise/include the large eIF3 complex, eIF3j (Hcr1 in yeast) and the ATP-binding cassette protein ABCE1 (Rli1 in yeast). The ATPase is mainly active as a recycling factor, but it can remain bound to the dissociated 40S subunit until formation of the next 43S pre-initiation complexes. However, its functional role and native architectural context remains largely enigmatic. Here, we present an architectural inventory of native yeast and human ABCE1-containing pre-initiation complexes by cryo-EM. We found that ABCE1 was mostly associated with early 43S, but also with later 48S phases of initiation. It adopted a novel hybrid conformation of its nucleotide-binding domains, while interacting with the N-terminus of eIF3j. Further, eIF3j occupied the mRNA entry channel via its ultimate C-terminus providing a structural explanation for its antagonistic role with respect to mRNA binding. Overall, the native human samples provide a near-complete molecular picture of the architecture and sophisticated interaction network of the 43S-bound eIF3 complex and the eIF2 ternary complex containing the initiator tRNA.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Subunidades Ribosómicas Pequeñas de Eucariotas/metabolismo , Línea Celular , Proteínas de Unión al ADN/metabolismo , Factor 2 Eucariótico de Iniciación/metabolismo , Células HEK293 , Humanos , Unión Proteica/fisiología , Biosíntesis de Proteínas/fisiología , ARN Mensajero/metabolismo , ARN de Transferencia/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Ribosome biogenesis proceeds along a multifaceted pathway from the nucleolus to the cytoplasm that is extensively coupled to several quality control mechanisms. However, the mode by which 5S ribosomal RNA is incorporated into the developing pre-60S ribosome, which in humans links ribosome biogenesis to cell proliferation by surveillance by factors such as p53-MDM2, is poorly understood. Here, we report nine nucleolar pre-60S cryo-EM structures from Chaetomium thermophilum, one of which clarifies the mechanism of 5S RNP incorporation into the early pre-60S. Successive assembly states then represent how helicases Dbp10 and Spb4, and the Pumilio domain factor Puf6 act in series to surveil the gradual folding of the nearby 25S rRNA domain IV. Finally, the methyltransferase Spb1 methylates a universally conserved guanine nucleotide in the A-loop of the peptidyl transferase center, thereby licensing further maturation. Our findings provide insight into the hierarchical action of helicases in safeguarding rRNA tertiary structure folding and coupling to surveillance mechanisms that culminate in local RNA modification.
Asunto(s)
ARN Ribosómico , Proteínas de Saccharomyces cerevisiae , Humanos , ARN Ribosómico/genética , ARN Ribosómico/metabolismo , Ribosomas/genética , ARN Ribosómico 5S/genética , ARN Ribosómico 5S/metabolismo , ADN Helicasas/metabolismo , Unión Proteica , Proteínas Ribosómicas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
The rixosome defined in Schizosaccharomyces pombe and humans performs diverse roles in pre-ribosomal RNA processing and gene silencing. Here, we isolate and describe the conserved rixosome from Chaetomium thermophilum, which consists of two sub-modules, the sphere-like Rix1-Ipi3-Ipi1 and the butterfly-like Las1-Grc3 complex, connected by a flexible linker. The Rix1 complex of the rixosome utilizes Sda1 as landing platform on nucleoplasmic pre-60S particles to wedge between the 5S rRNA tip and L1-stalk, thereby facilitating the 180° rotation of the immature 5S RNP towards its mature conformation. Upon rixosome positioning, the other sub-module with Las1 endonuclease and Grc3 polynucleotide-kinase can reach a strategic position at the pre-60S foot to cleave and 5' phosphorylate the nearby ITS2 pre-rRNA. Finally, inward movement of the L1 stalk permits the flexible Nop53 N-terminus with its AIM motif to become positioned at the base of the L1-stalk to facilitate Mtr4 helicase-exosome participation for completing ITS2 removal. Thus, the rixosome structure elucidates the coordination of two central ribosome biogenesis events, but its role in gene silencing may adapt similar strategies.
Asunto(s)
Proteínas de Saccharomyces cerevisiae , Schizosaccharomyces , Humanos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas Nucleares/metabolismo , Rotación , ARN Ribosómico/metabolismo , Ribosomas/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Procesamiento Postranscripcional del ARN , Proteínas Ribosómicas/genéticaRESUMEN
Change history: In this Letter, the bottom blot in Fig. 2g (for 'IB: Myc') was missing. This has been corrected online.
RESUMEN
Ribosome-associated quality control (RQC) provides a rescue pathway for eukaryotic cells to process faulty proteins after translational stalling of cytoplasmic ribosomes1-6. After dissociation of ribosomes, the stalled tRNA-bound peptide remains associated with the 60S subunit and extended by Rqc2 by addition of C-terminal alanyl and threonyl residues (CAT tails)7-9, whereas Vms1 catalyses cleavage and release of the peptidyl-tRNA before or after addition of CAT tails10-12. In doing so, Vms1 counteracts CAT-tailing of nuclear-encoded mitochondrial proteins that otherwise drive aggregation and compromise mitochondrial and cellular homeostasis13. Here we present structural and functional insights into the interaction of Saccharomyces cerevisiae Vms1 with 60S subunits in pre- and post-peptidyl-tRNA cleavage states. Vms1 binds to 60S subunits with its Vms1-like release factor 1 (VLRF1), zinc finger and ankyrin domains. VLRF1 overlaps with the Rqc2 A-tRNA position and interacts with the ribosomal A-site, projecting its catalytic GSQ motif towards the CCA end of the tRNA, its Y285 residue dislodging the tRNA A73 for nucleolytic cleavage. Moreover, in the pre-state, we found the ABCF-type ATPase Arb1 in the ribosomal E-site, which stabilizes the delocalized A73 of the peptidyl-tRNA and stimulates Vms1-dependent tRNA cleavage. Our structural analysis provides mechanistic insights into the interplay of the RQC factors Vms1, Rqc2 and Arb1 and their role in the protection of mitochondria from the aggregation of toxic proteins.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/metabolismo , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/metabolismo , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Homeostasis , Proteínas Mitocondriales/metabolismo , Ribosomas/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/ultraestructura , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/ultraestructura , Secuencia de Aminoácidos , Proteínas Portadoras/ultraestructura , Microscopía por Crioelectrón , Modelos Moleculares , Proteoma/metabolismo , Proteínas de Unión al ARN/antagonistas & inhibidores , Proteínas de Unión al ARN/metabolismo , Subunidades Ribosómicas Grandes de Eucariotas/química , Subunidades Ribosómicas Grandes de Eucariotas/genética , Subunidades Ribosómicas Grandes de Eucariotas/metabolismo , Ribosomas/química , Ribosomas/genética , Proteínas de Saccharomyces cerevisiae/antagonistas & inhibidores , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/ultraestructuraRESUMEN
Inhibitory codon pairs and poly(A) tracts within the translated mRNA cause ribosome stalling and reduce protein output. The molecular mechanisms that drive these stalling events, however, are still unknown. Here, we use a combination of in vitro biochemistry, ribosome profiling, and cryo-EM to define molecular mechanisms that lead to these ribosome stalls. First, we use an in vitro reconstituted yeast translation system to demonstrate that inhibitory codon pairs slow elongation rates which are partially rescued by increased tRNA concentration or by an artificial tRNA not dependent on wobble base-pairing. Ribosome profiling data extend these observations by revealing that paused ribosomes with empty A sites are enriched on these sequences. Cryo-EM structures of stalled ribosomes provide a structural explanation for the observed effects by showing decoding-incompatible conformations of mRNA in the A sites of all studied stall- and collision-inducing sequences. Interestingly, in the case of poly(A) tracts, the inhibitory conformation of the mRNA in the A site involves a nucleotide stacking array. Together, these data demonstrate a novel mRNA-induced mechanisms of translational stalling in eukaryotic ribosomes.
Asunto(s)
Biosíntesis de Proteínas , ARN Mensajero/genética , Ribosomas/metabolismo , Saccharomyces cerevisiae/metabolismo , Codón , Microscopía por Crioelectrón , Modelos Moleculares , Conformación de Ácido Nucleico , Poli A/metabolismo , ARN Mensajero/química , ARN Mensajero/metabolismo , ARN de Transferencia/metabolismo , Saccharomyces cerevisiae/genéticaRESUMEN
BACKGROUND: The aim of this study was to investigate the factors affecting plasma valproic acid (VPA) concentration in pediatric patients with epilepsy and the clinical significance of CYP2C9 gene polymorphisms in personalized dosing using therapeutic drug monitoring and pharmacogenetic testing. METHODS: The medical records of children with epilepsy who underwent therapeutic drug monitoring at our institution between July 2022 and July 2023 and met the inclusion criteria were reviewed. Statistical analysis was performed to determine whether age, sex, blood ammonia, liver function, kidney function, and other characteristics affected the concentration-to-dose ratio of VPA (CDRV) in these patients. To investigate the effect of CYP2C9 polymorphisms on CDRV, DNA samples were collected from patients and the CYP2C9 genotypes were identified using real-time quantitative PCR. RESULTS: The mean age of 208 pediatric patients with epilepsy was 5.50 ± 3.50 years. Among these patients, 182 had the CYP2C9 *1/*1 genotype, with a mean CDRV (mcg.kg/mL.mg) of 2.64 ± 1.46, 24 had the CYP2C9 *1/*3 genotype, with a mean CDRV of 3.28 ± 1.74, and 2 had the CYP2C9 *3/*3 genotype, with a mean CDRV of 6.46 ± 3.33. There were statistical differences among these 3 genotypes ( P < 0.05). The CDRV in these patients were significantly influenced by age, aspartate aminotransferase, total bilirubin, direct bilirubin, globulin, albumin/globulin ratio, prealbumin, creatinine, and CYP2C9 polymorphisms. In addition, multivariate linear regression analysis identified total bilirubin, direct bilirubin, and CYP2C9 polymorphisms as independent risk factors for high CDRV. CONCLUSIONS: Liver problems and mutations in the CYP2C9 gene increase VPA levels. This underscores the importance of considering these factors when prescribing VPA to children with epilepsy, thereby enhancing the safety and efficacy of the therapy.
Asunto(s)
Anticonvulsivantes , Citocromo P-450 CYP2C9 , Monitoreo de Drogas , Epilepsia , Genotipo , Ácido Valproico , Humanos , Citocromo P-450 CYP2C9/genética , Epilepsia/tratamiento farmacológico , Epilepsia/genética , Epilepsia/sangre , Ácido Valproico/uso terapéutico , Ácido Valproico/sangre , Femenino , Niño , Masculino , Preescolar , Anticonvulsivantes/uso terapéutico , Anticonvulsivantes/sangre , Anticonvulsivantes/farmacocinética , Monitoreo de Drogas/métodos , Adolescente , Medicina de Precisión/métodos , Lactante , Estudios Retrospectivos , Polimorfismo Genético/genética , Relevancia ClínicaRESUMEN
The formation of eukaryotic ribosomal subunits extends from the nucleolus to the cytoplasm and entails hundreds of assembly factors. Despite differences in the pathways of ribosome formation, high-resolution structural information has been available only from fungi. Here we present cryo-electron microscopy structures of late-stage human 40S assembly intermediates, representing one state reconstituted in vitro and five native states that range from nuclear to late cytoplasmic. The earliest particles reveal the position of the biogenesis factor RRP12 and distinct immature rRNA conformations that accompany the formation of the 40S subunit head. Molecular models of the late-acting assembly factors TSR1, RIOK1, RIOK2, ENP1, LTV1, PNO1 and NOB1 provide mechanistic details that underlie their contribution to a sequential 40S subunit assembly. The NOB1 architecture displays an inactive nuclease conformation that requires rearrangement of the PNO1-bound 3' rRNA, thereby coordinating the final rRNA folding steps with site 3 cleavage.
Asunto(s)
Microscopía por Crioelectrón , Subunidades Ribosómicas Pequeñas de Eucariotas/metabolismo , Subunidades Ribosómicas Pequeñas de Eucariotas/ultraestructura , Secuencia de Bases , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Humanos , Modelos Moleculares , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Proteínas Nucleares/ultraestructura , Conformación de Ácido Nucleico , Dominios Proteicos , Pliegue del ARN , ARN Ribosómico/química , ARN Ribosómico/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/ultraestructura , Proteínas Ribosómicas/metabolismo , Proteínas Ribosómicas/ultraestructura , Subunidades Ribosómicas Pequeñas de Eucariotas/químicaRESUMEN
Huntingtin (HTT) is a large (348 kDa) protein that is essential for embryonic development and is involved in diverse cellular activities such as vesicular transport, endocytosis, autophagy and the regulation of transcription. Although an integrative understanding of the biological functions of HTT is lacking, the large number of identified HTT interactors suggests that it serves as a protein-protein interaction hub. Furthermore, Huntington's disease is caused by a mutation in the HTT gene, resulting in a pathogenic expansion of a polyglutamine repeat at the amino terminus of HTT. However, only limited structural information regarding HTT is currently available. Here we use cryo-electron microscopy to determine the structure of full-length human HTT in a complex with HTT-associated protein 40 (HAP40; encoded by three F8A genes in humans) to an overall resolution of 4 Å. HTT is largely α-helical and consists of three major domains. The amino- and carboxy-terminal domains contain multiple HEAT (huntingtin, elongation factor 3, protein phosphatase 2A and lipid kinase TOR) repeats arranged in a solenoid fashion. These domains are connected by a smaller bridge domain containing different types of tandem repeats. HAP40 is also largely α-helical and has a tetratricopeptide repeat-like organization. HAP40 binds in a cleft and contacts the three HTT domains by hydrophobic and electrostatic interactions, thereby stabilizing the conformation of HTT. These data rationalize previous biochemical results and pave the way for improved understanding of the diverse cellular functions of HTT.
Asunto(s)
Proteína Huntingtina/ultraestructura , Microscopía por Crioelectrón , Humanos , Proteína Huntingtina/química , Proteína Huntingtina/metabolismo , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Proteínas Nucleares/ultraestructura , Unión Proteica , Dominios Proteicos , Estructura Secundaria de Proteína , Electricidad EstáticaRESUMEN
The transition of the 90S to the pre-40S pre-ribosome is a decisive step in eukaryotic small subunit biogenesis leading to a first pre-40S intermediate (state Dis-C or primordial pre-40S), where the U3 snoRNA keeps the nascent 18S rRNA locally immature. We in vitro reconstitute the ATP-dependent U3 release from this particle, catalyzed by the helicase Dhr1, and follow this process by cryo-EM revealing two successive pre-40S intermediates, Dis-D and Dis-E. The latter has lost not only U3 but all residual 90S factors including the GTPase Bms1. In vitro remodeling likewise induced the formation of the central pseudoknot, a universally conserved tertiary RNA structure that comprises the core of the small subunit decoding center. Thus, we could structurally reveal a key tertiary RNA folding step that is essential to form the active 40S subunit.