Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 10 de 10
Filtrar
Más filtros












Base de datos
Intervalo de año de publicación
1.
Nucleic Acids Res ; 48(11): 6210-6222, 2020 06 19.
Artículo en Inglés | MEDLINE | ID: mdl-32365182

RESUMEN

The ribotoxin α-sarcin belongs to a family of ribonucleases that cleave the sarcin/ricin loop (SRL), a critical functional rRNA element within the large ribosomal subunit (60S), thereby abolishing translation. Whether α-sarcin targets the SRL only in mature 60S subunits remains unresolved. Here, we show that, in yeast, α-sarcin can cleave SRLs within late 60S pre-ribosomes containing mature 25S rRNA but not nucleolar/nuclear 60S pre-ribosomes containing 27S pre-rRNA in vivo. Conditional expression of α-sarcin is lethal, but does not impede early pre-rRNA processing, nuclear export and the cytoplasmic maturation of 60S pre-ribosomes. Thus, SRL-cleaved containing late 60S pre-ribosomes seem to escape cytoplasmic proofreading steps. Polysome analyses revealed that SRL-cleaved 60S ribosomal subunits form 80S initiation complexes, but fail to progress to the step of translation elongation. We suggest that the functional integrity of a α-sarcin cleaved SRL might be assessed only during translation.


Asunto(s)
Endorribonucleasas/metabolismo , Proteínas Fúngicas/metabolismo , Subunidades Ribosómicas Grandes de Eucariotas/química , Subunidades Ribosómicas Grandes de Eucariotas/metabolismo , Ricina/metabolismo , Saccharomyces cerevisiae/metabolismo , Transporte Activo de Núcleo Celular , Nucléolo Celular/efectos de los fármacos , Nucléolo Celular/metabolismo , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Endorribonucleasas/farmacología , Proteínas Fúngicas/farmacología , Biosíntesis de Proteínas , ARN Ribosómico/metabolismo , Ricina/química , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/crecimiento & desarrollo
2.
Elife ; 92020 01 07.
Artículo en Inglés | MEDLINE | ID: mdl-31909713

RESUMEN

Eukaryotic ribosome precursors acquire translation competence in the cytoplasm through stepwise release of bound assembly factors, and proofreading of their functional centers. In case of the pre-60S, these steps include removal of placeholders Rlp24, Arx1 and Mrt4 that prevent premature loading of the ribosomal protein eL24, the protein-folding machinery at the polypeptide exit tunnel (PET), and the ribosomal stalk, respectively. Here, we reveal that sequential ATPase and GTPase activities license release factors Rei1 and Yvh1 to trigger Arx1 and Mrt4 removal. Drg1-ATPase activity removes Rlp24 from the GTPase Nog1 on the pre-60S; consequently, the C-terminal tail of Nog1 is extracted from the PET. These events enable Rei1 to probe PET integrity and catalyze Arx1 release. Concomitantly, Nog1 eviction from the pre-60S permits peptidyl transferase center maturation, and allows Yvh1 to mediate Mrt4 release for stalk assembly. Thus, Nog1 co-ordinates the assembly, maturation and quality control of distant functional centers during ribosome formation.


Asunto(s)
Proteínas de Unión al GTP/genética , Proteínas Nucleares/genética , Proteínas Ribosómicas/metabolismo , Ribosomas/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Proteínas de Unión al GTP/metabolismo , Proteínas Nucleares/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
3.
Nat Struct Mol Biol ; 26(2): 110-120, 2019 02.
Artículo en Inglés | MEDLINE | ID: mdl-30692646

RESUMEN

The assembly of large multimeric complexes in the crowded cytoplasm is challenging. Here we reveal a mechanism that ensures accurate production of the yeast proteasome, involving ribosome pausing and co-translational assembly of Rpt1 and Rpt2. Interaction of nascent Rpt1 and Rpt2 then lifts ribosome pausing. We show that the N-terminal disordered domain of Rpt1 is required to ensure efficient ribosome pausing and association of nascent Rpt1 protein complexes into heavy particles, wherein the nascent protein complexes escape ribosome quality control. Immunofluorescence and in situ hybridization studies indicate that Rpt1- and Rpt2-encoding messenger RNAs co-localize in these particles that contain, and are dependent on, Not1, the scaffold of the Ccr4-Not complex. We refer to these particles as Not1-containing assemblysomes, as they are smaller than and distinct from other RNA granules such as stress granules and GW- or P-bodies. Synthesis of Rpt1 with ribosome pausing and Not1-containing assemblysome induction is conserved from yeast to human cells.


Asunto(s)
Complejo de la Endopetidasa Proteasomal/metabolismo , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Algoritmos , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Genoma Fúngico/genética , Humanos , Hibridación in Situ , Masculino , Oligonucleótidos/genética , Oligonucleótidos/metabolismo , Complejo de la Endopetidasa Proteasomal/genética , Unión Proteica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo
4.
Nat Commun ; 9(1): 3669, 2018 09 10.
Artículo en Inglés | MEDLINE | ID: mdl-30201955

RESUMEN

Disordered extensions at the termini and short internal insertions distinguish eukaryotic ribosomal proteins (r-proteins) from their anucleated archaeal counterparts. Here, we report an NMR structure of such a eukaryotic-specific segment (ESS) in the r-protein eS26 in complex with the escortin Tsr2. The structure reveals how ESS attracts Tsr2 specifically to importin:eS26 complexes entering the nucleus in order to trigger non-canonical RanGTP-independent disassembly. Tsr2 then sequesters the released eS26 and prevents rebinding to the importin, providing an alternative allosteric mechanism to terminate the process of nuclear import. Notably, a Diamond-Blackfan anemia-associated Tsr2 mutant protein is impaired in binding to ESS, unveiling a critical role for this interaction in human hematopoiesis. We propose that eS26-ESS and Tsr2 are components of a nuclear sorting system that co-evolved with the emergence of the nucleocytoplasmic barrier and transport carriers.


Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Carioferinas/metabolismo , Proteínas Ribosómicas/metabolismo , Transporte Activo de Núcleo Celular , Sitio Alostérico , Núcleo Celular/metabolismo , Dicroismo Circular , Citoplasma/metabolismo , Hematopoyesis , Humanos , Hibridación Fluorescente in Situ , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Mutación , Proteínas Nucleares/metabolismo , Fenotipo , Unión Proteica , Conformación Proteica , ARN/química , Proteínas Recombinantes/metabolismo , Ribosomas/metabolismo , Saccharomyces cerevisiae , Proteína de Unión al GTP ran/metabolismo
5.
EMBO J ; 37(7)2018 04 03.
Artículo en Inglés | MEDLINE | ID: mdl-29459436

RESUMEN

Final maturation of eukaryotic ribosomes occurs in the cytoplasm and requires the sequential removal of associated assembly factors and processing of the immature 20S pre-RNA Using cryo-electron microscopy (cryo-EM), we have determined the structure of a yeast cytoplasmic pre-40S particle in complex with Enp1, Ltv1, Rio2, Tsr1, and Pno1 assembly factors poised to initiate final maturation. The structure reveals that the pre-rRNA adopts a highly distorted conformation of its 3' major and 3' minor domains stabilized by the binding of the assembly factors. This observation is consistent with a mechanism that involves concerted release of the assembly factors orchestrated by the folding of the rRNA in the head of the pre-40S subunit during the final stages of maturation. Our results provide a structural framework for the coordination of the final maturation events that drive a pre-40S particle toward the mature form capable of engaging in translation.


Asunto(s)
Microscopía por Crioelectrón , Simulación del Acoplamiento Molecular , Proteínas Ribosómicas/ultraestructura , Subunidades Ribosómicas Pequeñas de Eucariotas/ultraestructura , Proteínas de Saccharomyces cerevisiae/ultraestructura , Saccharomyces cerevisiae/ultraestructura , Citoplasma , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/ultraestructura , Conformación Proteica , Dominios Proteicos , Dominios y Motivos de Interacción de Proteínas , Proteínas Serina-Treonina Quinasas/ultraestructura , Pliegue del ARN , ARN Ribosómico/química , ARN Ribosómico/ultraestructura , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/ultraestructura , Proteínas Ribosómicas/química , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/aislamiento & purificación , Subunidades Ribosómicas Pequeñas de Eucariotas/química , Subunidades Ribosómicas Pequeñas de Eucariotas/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/aislamiento & purificación
6.
Nat Struct Mol Biol ; 24(9): 689-699, 2017 Sep 07.
Artículo en Inglés | MEDLINE | ID: mdl-28880863

RESUMEN

Eukaryotic ribosome synthesis is a complex, energy-consuming process that takes place across the nucleolus, nucleoplasm and cytoplasm and requires more than 200 conserved assembly factors. Here, we discuss mechanisms by which the ribosome assembly and nucleocytoplasmic transport machineries collaborate to produce functional ribosomes. We also highlight recent cryo-EM studies that provided unprecedented snapshots of ribosomes during assembly and quality control.


Asunto(s)
Eucariontes/metabolismo , Biogénesis de Organelos , Ribosomas/metabolismo , Transporte Biológico , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Eucariontes/citología
7.
Elife ; 52016 12 08.
Artículo en Inglés | MEDLINE | ID: mdl-27929371

RESUMEN

Spatial clustering of ribosomal proteins (r-proteins) through tertiary interactions is a striking structural feature of the eukaryotic ribosome. However, the functional importance of these intricate inter-connections, and how they are established is currently unclear. Here, we reveal that a conserved ATPase, Fap7, organizes interactions between neighboring r-proteins uS11 and eS26 prior to their delivery to the earliest ribosome precursor, the 90S. In vitro, uS11 only when bound to Fap7 becomes competent to recruit eS26 through tertiary contacts found between these r-proteins on the mature ribosome. Subsequently, Fap7 ATPase activity unloads the uS11:eS26 subcomplex onto its rRNA binding site, and therefore ensures stoichiometric integration of these r-proteins into the 90S. Fap7-depletion in vivo renders uS11 susceptible to proteolysis, and precludes eS26 incorporation into the 90S. Thus, prefabrication of a native-like r-protein subcomplex drives efficient and accurate construction of the eukaryotic ribosome.


Asunto(s)
Adenilato Quinasa/metabolismo , Sustancias Macromoleculares/metabolismo , Proteínas Nucleares/metabolismo , Nucleósido-Trifosfatasa/metabolismo , Multimerización de Proteína , Proteínas Ribosómicas/metabolismo , Ribosomas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo
8.
Elife ; 3: e03473, 2014 Aug 21.
Artículo en Inglés | MEDLINE | ID: mdl-25144938

RESUMEN

Within a single generation time a growing yeast cell imports ∼14 million ribosomal proteins (r-proteins) into the nucleus for ribosome production. After import, it is unclear how these intrinsically unstable and aggregation-prone proteins are targeted to the ribosome assembly site in the nucleolus. Here, we report the discovery of a conserved nuclear carrier Tsr2 that coordinates transfer of the r-protein eS26 to the earliest assembling pre-ribosome, the 90S. In vitro studies revealed that Tsr2 efficiently dissociates importin:eS26 complexes via an atypical RanGTP-independent mechanism that terminates the import process. Subsequently, Tsr2 binds the released eS26, shields it from proteolysis, and ensures its safe delivery to the 90S pre-ribosome. We anticipate similar carriers-termed here escortins-to securely connect the nuclear import machinery with pathways that deposit r-proteins onto developing pre-ribosomal particles.


Asunto(s)
Proteínas Portadoras/metabolismo , Núcleo Celular/metabolismo , Proteínas Ribosómicas/metabolismo , Ribosomas/metabolismo , Saccharomyces cerevisiae/metabolismo , Transporte Activo de Núcleo Celular , Secuencia de Aminoácidos , Proteínas Portadoras/química , Proteínas Portadoras/genética , Núcleo Celular/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Activadoras de GTPasa/química , Proteínas Activadoras de GTPasa/genética , Proteínas Activadoras de GTPasa/metabolismo , Carioferinas/química , Carioferinas/genética , Carioferinas/metabolismo , Datos de Secuencia Molecular , Unión Proteica , Estabilidad Proteica , Proteolisis , ARN Ribosómico 18S/química , ARN Ribosómico 18S/genética , ARN Ribosómico 18S/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Ribosómicas/química , Proteínas Ribosómicas/genética , Ribosomas/química , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , beta Carioferinas/química , beta Carioferinas/genética , beta Carioferinas/metabolismo
9.
Chromosoma ; 123(4): 327-44, 2014 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-24817020

RESUMEN

The ribosome is responsible for the final step of decoding genetic information into proteins. Therefore, correct assembly of ribosomes is a fundamental task for all living cells. In eukaryotes, the construction of the ribosome which begins in the nucleolus requires coordinated efforts of >350 specialized factors that associate with pre-ribosomal particles at distinct stages to perform specific assembly steps. On their way through the nucleus, diverse energy-consuming enzymes are thought to release assembly factors from maturing pre-ribosomal particles after accomplishing their task(s). Subsequently, recruitment of export factors prepares pre-ribosomal particles for transport through nuclear pore complexes. Pre-ribosomes are exported into the cytoplasm in a functionally inactive state, where they undergo final maturation before initiating translation. Accumulating evidence indicates a tight coupling between nuclear export, cytoplasmic maturation, and final proofreading of the ribosome. In this review, we summarize our current understanding of nuclear export of pre-ribosomal subunits and cytoplasmic maturation steps that render pre-ribosomal subunits translation-competent.


Asunto(s)
Núcleo Celular/metabolismo , Ribosomas/metabolismo , Saccharomycetales/metabolismo , Transporte Activo de Núcleo Celular , Modelos Moleculares , Subunidades Ribosómicas/metabolismo
10.
Cell Microbiol ; 12(6): 765-80, 2010 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-20070309

RESUMEN

Infection of Dictyostelium discoideum with Legionella pneumophila resulted in a large number of differentially regulated genes among them three core autophagy genes, ATG8, ATG9 and ATG16. Macroautophagy contributes to many physiological and pathological processes and might also constitute an important mechanism in cell-autonomous immunity. For further studies we selected the highly conserved ATG9. In colocalization studies with GFP-tagged ATG9 and different organelle marker proteins we neither observed colocalization with mitochondria, the ER nor lysosomes. However, there was partial colocalization with the Golgi apparatus and many ATG9-GFP-containing vesicles localized along microtubules and accumulated around the microtubule organizing centre. ATG9-deficient cells had pleiotropic defects. In addition to growth defects they displayed severe developmental defects, consistent with the known role of autophagy in Dictyostelium development. Unexpectedly, the ATG9 mutant also had a strong phagocytosis defect that was particularly apparent when infecting the cells with L. pneumophila. However, those Legionellae that entered the host could multiply better in mutant than in wild-type cells, because of a less efficient clearance in the early and a more efficient replication in the late phase of infection. We conclude that ATG9 and hence macroautophagy has a protective role during pathogen infection.


Asunto(s)
Dictyostelium/genética , Legionella pneumophila/crecimiento & desarrollo , Fagocitosis , Proteínas Protozoarias/genética , Dictyostelium/crecimiento & desarrollo , Dictyostelium/inmunología , Dictyostelium/microbiología , Técnicas de Inactivación de Genes , Aparato de Golgi/química , Microtúbulos/química , Proteínas Protozoarias/análisis , Proteínas Protozoarias/fisiología
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA
...