Добірка наукової літератури з теми "Particule pre-60S"

Ribosome biogenesis takes place successively in the nucleolar, nucleoplasmic, and cytoplasmic compartments. Numerous nonribosomal factors transiently associate with the nascent ribosomes, but the mechanisms driving ribosome formation are mostly unknown. Here, we show that an energy-consuming enzyme, the AAA-type (ATPases associated with various cellular activities) ATPase Rix7, restructures a novel pre-60S particle at the transition from the nucleolus to nucleoplasm. Rix7 interacts genetically with Nsa1 and is targeted to the Nsa1-defined preribosomal particle. In vivo, Nsa1 cannot dissociate from pre-60S particles in rix7 mutants, causing nucleolar Nsa1 to escape to the cytoplasm, where it remains associated with aberrant 60S subunits. Altogether, our data suggest that Rix7 is required for the release of Nsa1 from a discrete preribosomal particle, thereby triggering the progression of 60S ribosome biogenesis.

Before entering translation, preribosomal particles undergo sequential late maturation steps. In the case of pre-60S particles, these steps involve the release of shuttling maturation factors and transport receptors. In this study, we report a new maturation step in the 60S biogenesis pathway in budding yeast. We show that efficient release of the nucleolar/nuclear ribosomal-like protein Mrt4 (homologous to the acidic ribosomal P-protein Rpp0) from pre-60S particles requires the highly conserved protein Yvh1, which associates only with late pre-60S particles. Cell biological and biochemical analyses reveal that Mrt4 fails to dissociate from late pre-60S particles in yvh1Δ cells, inducing a delay in nuclear pre–ribosomal RNA processing and a pre-60S export defect in yvh1Δ cells. Moreover, we have isolated gain of function alleles of Mrt4 that specifically bypass the requirement for Yvh1 and rescue all yvh1Δ-associated phenotypes. Together, our data suggest that Yvh1-mediated release of Mrt4 precedes cytoplasmic loading of Rpp0 on pre-60S particles and is an obligatory late step toward construction of translation-competent 60S subunits.

ABSTRACT Allelic forms of DRG1/AFG2 confer resistance to the drug diazaborine, an inhibitor of ribosome biogenesis in Saccharomyces cerevisiae. Our results show that the AAA-ATPase Drg1 is essential for 60S maturation and associates with 60S precursor particles in the cytoplasm. Functional inactivation of Drg1 leads to an increased cytoplasmic localization of shuttling pre-60S maturation factors like Rlp24, Arx1, and Tif6. Surprisingly, Nog1, a nuclear pre-60S factor, was also relocalized to the cytoplasm under these conditions, suggesting that it is a previously unsuspected shuttling preribosomal factor that is exported with the precursor particles and very rapidly reimported. Proteins that became cytoplasmic under drg1 mutant conditions were blocked on pre-60S particles at a step that precedes the association of Rei1, a later-acting preribosomal factor. A similar cytoplasmic accumulation of Nog1 and Rlp24 in pre-60S-bound form could be seen after overexpression of a dominant-negative Drg1 variant mutated in the D2 ATPase domain. We conclude that the ATPase activity of Drg1 is required for the release of shuttling proteins from the pre-60S particles shortly after their nuclear export. This early cytoplasmic release reaction defines a novel step in eukaryotic ribosome maturation.

We previously showed that nuclear export of the large (60S) ribosomal subunit relies on Nmd3 in a Crm1-dependent manner. Recently the general mRNA export factor, the Mtr2/Mex67 heterodimer, was shown to act as an export receptor in parallel with Crm1. These observations raise the possibility that nuclear export of the 60S subunit in Saccharomyces cerevisiae requires multiple export receptors. Here, we show that the previously characterized 60S subunit biogenesis factor, Arx1, also acts as an export receptor for the 60S subunit. We found that deletion of ARX1 was synthetic lethal with nmd3 and mtr2 mutants and was synthetic sick with several nucleoporin mutants. Deletion of ARX1 led to accumulation of pre-60S particles in the nucleus that were enriched for Nmd3, Crm1, Mex67, and Mtr2, suggesting that in the absence of Arx1, 60S export is impaired even though the subunit is loaded with export receptors. Finally, Arx1 interacted with several nucleoporins in yeast two-hybrid as well as in vitro assays. These results show that Arx1 can directly bridge the interaction between the pre-60S particle and the NPC and thus is a third export receptor for the 60S subunit in yeast.

The ribosome assembly factor Nsa2 is part of the Rea1-Rsa4-Nsa2 interconnected relay on nuclear pre-60S particles that is essential for 60S ribosome biogenesis. Cryo-EM structures depict Nsa2 docked via its C-terminal β-barrel domain to nuclear pre-60S particles, whereas the extended N-terminus, consisting of three α-helical segments, meanders between various 25S rRNA helices with the extreme N-terminus in close vicinity to the Nog1 GTPase center. Here, we tested whether this unappreciated proximity between Nsa2 and Nog1 is of functional importance. Our findings demonstrate that a conservative mutation, Nsa2 Q3N, abolished cell growth and impaired 60S biogenesis. Subsequent genetic and biochemical analyses verified that the Nsa2 N-terminus is required to target Nsa2 to early pre-60S particles. However, overexpression of the Nsa2 N-terminus abolished cytoplasmic recycling of the Nog1 GTPase, and both Nog1 and the Nsa2-N (1-58) construct, but not the respective Nsa2-N (1-58) Q3N mutant, were found arrested on late cytoplasmic pre-60S particles. These findings point to specific roles of the different Nsa2 domains for 60S ribosome biogenesis.

Many analyses have examined subnucleolar structures in eukaryotic cells, but the relationship between morphological structures, pre-rRNA processing, and ribosomal particle assembly has remained unclear. Using a visual assay for export of the 60S ribosomal subunit, we isolated a ts-lethal mutation, rix9-1, which causes nucleolar accumulation of an Rpl25p-eGFP reporter construct. The mutation results in a single amino acid substitution (F176S) in Rlp7p, an essential nucleolar protein related to ribosomal protein Rpl7p. The rix9-1 (rlp7-1) mutation blocks the late pre-RNA cleavage at site C2 in ITS2, which separates the precursors to the 5.8S and 25S rRNAs. Consistent with this, synthesis of the mature 5.8S and 25S rRNAs was blocked in the rlp7-1 strain at nonpermissive temperature, whereas 18S rRNA synthesis continued. Moreover, pre-rRNA containing ITS2 accumulates in the nucleolus of rix9-1 cells as revealed by in situ hybridization. Finally, tagged Rlp7p was shown to associate with a pre-60S particle, and fluorescence microscopy and immuno-EM localized Rlp7p to a subregion of the nucleolus, which could be the granular component (GC). All together, these data suggest that pre-rRNA cleavage at site C2 specifically requires Rlp7p and occurs within pre-60S particles located in the GC region of the nucleolus.

ABSTRACT Ribosome biogenesis in eukaryotes depends on the coordinated action of ribosomal and nonribosomal proteins that guide the assembly of preribosomal particles. These intermediate particles follow a maturation pathway in which important changes in their protein composition occur. The mechanisms involved in the coordinated assembly of the ribosomal particles are poorly understood. We show here that the association of preribosomal factors with pre-60S complexes depends on the presence of earlier factors, a phenomenon essential for ribosome biogenesis. The analysis of the composition of purified preribosomal complexes blocked in maturation at specific steps allowed us to propose a model of sequential protein association with, and dissociation from, early pre-60S complexes for several preribosomal factors such as Mak11, Ssf1, Rlp24, Nog1, and Nog2. The presence of either Ssf1 or Nog2 in complexes that contain the 27SB pre-rRNA defines novel, distinct pre-60S particles that contain the same pre-rRNA intermediates and that differ only by the presence or absence of specific proteins. Physical and functional interactions between Rlp24 and Nog1 revealed that the assembly steps are, at least in part, mediated by direct protein-protein interactions.

Formation of eukaryotic ribosomes is driven by energy-consuming enzymes. The AAA-ATPase Drg1 is essential for the release of several shuttling proteins from cytoplasmic pre-60S particles and the loading of late joining proteins. However, its exact role in ribosome biogenesis has been unknown. Here we show that the shuttling protein Rlp24 recruited Drg1 to pre-60S particles and stimulated its ATPase activity. ATP hydrolysis in the second AAA domain of Drg1 was required to release shuttling proteins. In vitro, Drg1 specifically and exclusively extracted Rlp24 from purified pre-60S particles. Rlp24 release required ATP and was promoted by the interaction of Drg1 with the nucleoporin Nup116. Subsequent ATP hydrolysis in the first AAA domain dissociated Drg1 from Rlp24, liberating both proteins for consecutive cycles of activity. Our results show that release of Rlp24 by Drg1 defines a key event in large subunit formation that is a prerequisite for progression of cytoplasmic pre-60S maturation.

La synthèse de sous-unités ribosomiques eucaryotes implique l'assemblage et la maturation de particules précurseurs complexes (particules pré-ribosomiques) contenant des précurseurs de l'ARN ribosomique (ARNr), des protéines ribosomiques (RP ou r-protéines) et une pléthore de facteurs d'assemblage et de maturation (AMF). La première partie de ma thèse à porté sur l'hétérodimère BXDC1-RRS1. BXDC1 et RRS1 sont les homologues humains des facteurs d'assemblage et de maturation de la levure Rpf2 et Rrs1, respectivement. Chez S. cerevisiae, Rpf2 et Rrs1 sont impliqués dans le recrutement de la 5S RNP dans les particules pré-60S. De plus, des études récentes menées dans mon équipe d'accueil à Toulouse ont identifié l'hétérodimère Rpf2-Rrs1 comme un nouveau complexe nucléolaire impliqué dans la régulation de la transcription de Pol I dans les cellules de levure. Dans les cellules humaines, BXDC1 et RRS1 sont également impliqués dans l'incorporation de la 5S RNP dans les pré-ribosomes et jouent en outre un rôle central dans la coordination entre la synthèse des ribosomes et la progression du cycle cellulaire. Ce projet visait à déterminer si, à l'instar de son homologue de levure, le complexe BXDC1 / RRS1 est également impliqué dans la régulation de la transcription de Pol I dans les cellules humaines. Nous avons utilisé des tests d'immunoprécipitation de la chromatine (ChIP) pour déterminer si BXDC1 interagit avec l'ADNr. Nous avons également testé si l'absence de BXDC1 affecte l'association de l'ARN Pol I avec l'ADNr. Malheureusement, nous n'avons pas réussi à démontrer une interaction entre BXDC1 et ADNr et en outre, l'interaction Pol I avec l'ADNr n'a pas été affectée lors de la depletion de BXDC1 médiée par l'ARNi. Ces données obtenues ne nous ont pas encouragés à approfondir cette partie de ma thèse. La deuxième partie de ma thèse a consisté à etudier la fonction du snoARN a boîte C/D snR190 et son interaction avec la DEAD-box ATPase Dbp7 chez la levure. SnR190 a longtemps été prédit d'agir comme un guide de méthylation snoRNA ciblant un nucléotide du centre peptidyl transférase (PTC) de l'ARNr 25S, bien que la méthylation cible n'ait jamais été détectée. Ce snoARN interagit préférentiellement avec un module protéique composé de cinq facteurs appelés "complexe Npa1", suggéré de jouer un rôle clé dans la compaction de l'ARNr 25S au sein des particules pré-60S précoces. Nous montrons que snR190 est nécessaire pour une prolifération optimale des levures et une maturation efficace des particules pré-60S précoces. Nous proposons que snR190 fonctionne comme un nouveau snoARN chaperon, qui coopère avec le complexe Npa1 pour favoriser la compaction du pré-ARNr dans les premières particules pré-60S, grâce à deux éléments antisens conservés de manière évolutive. Notre étude a en outre révélé un nouveau lien génétique entre snR190 et l'hélicase ARN Dbp7, qui présente des interactions génétiques avec tous les membres du complexe Npa1. Nous montrons en outre que l'absence de Dbp7 conduit à une rétention aberrante dans les particules pré-60S de snR190 et plusieurs snoARNs guides de modification ciblant la région PTC de l'ARNr 25S. De plus, le knock-out de snR190 dans une souche dépourvue de Dbp7 atténue partiellement son défaut de croissance et restaure dans une certaine mesure la maturation précoce des particules pré-60S. Nous proposons que l'hélicase ARN Dbp7 régule l'appariement de bases dynamique entre snR190 et le pré-ARNr au sein des premières particules pré-60S, participant ainsi à la structuration de la région PTC de la grande sous-unité ribosomique
Synthesis of eukaryotic ribosomal subunits involves assembly and maturation of complex precursor particles (pre-ribosomal particles) containing ribosomal RNA (rRNA) precursors, ribosomal proteins (RPs or r-proteins) and a plethora of assembly and maturation factors (AMFs). The first part of my thesis focused on the BXDC1-RRS1 heterodimer. BXDC1 and RRS1 are the human homologues of the yeast assembly and maturation factors Rpf2 and Rrs1, respectively. In S. cerevisiae, Rpf2 and Rrs1 are involved in the recruitment of the 5S RNP into pre-60S particles. Moreover, recent studies performed in my host team in Toulouse identified the Rpf2-Rrs1 heterodimer as a novel nucleolar complex involved in the regulation of Pol I transcription in yeast cells. In human cells, BXDC1 and RRS1 are also implicated in the incorporation of the 5S RNP into pre-ribosomes and further plays a central role in the coordination between ribosome synthesis and the cell cycle progression. This project aimed to determine whether, similarly to its yeast counterpart, the BXDC1/RRS1 complex is also involved in the regulation of Pol I transcription in human cells. We used Chromatin Immunoprecipitation (ChIP) assays to determine whether BXDC1 interacts with rDNA. We also tested if the absence of BXDC1 affects RNA Pol I association with rDNA. Unfortunately, we failed to demonstrate any interaction between BXDC1 and rDNA and furthermore, Pol I interaction with rDNA was not affected upon RNAi-mediated depletion of BXDC1. These obtained data did not encourage us to further explore this part of my thesis. The second part of my thesis consisted in deciphering the function of the box C/D snoRNA snR190 and its interplay with the DEAD-box ATPase Dbp7 in yeast. snR190 has long been predicted to act as a methylation guide snoRNA targeting a nucleotide of the peptidyl transferase center (PTC) of the 25S rRNA, although the target methylation has never been detected. This snoRNA interacts preferentially with a protein module composed of five factors called the "Npa1 complex", suggested to play a key role in the compaction of the 25S rRNA within the earliest pre-60S particles. We show that snR190 is required for optimal yeast proliferation and efficient maturation of early pre-60S particles. We propose that snR190 functions as a novel snoRNA chaperone, which cooperates with the Npa1 complex to promote the compaction of the pre-rRNA in the first pre-60S particles, through two evolutionarily conserved antisense elements. Our study further revealed a novel genetic link between snR190 and the Dbp7 RNA helicase, which displays genetic interactions with all members of the Npa1 complex. We further show that the absence of Dbp7 leads to an aberrant retention within pre-60S particles of snR190 and several modification guide snoRNAs targeting the PTC region of the 25S rRNA. In addition, knockout of snR190 in a strain lacking Dbp7 partially alleviates its growth defect and restores early pre-60S particle maturation to some extent. We propose that the Dbp7 RNA helicase regulates the dynamic base-pairing between snR190 and the pre-rRNA within the earliest pre-60S particles, thereby participating in the structuring of the PTC region of the large ribosomal subunit

Nop53 e uma protena nucleolar, conservada evolutivamente e essencial na levedura Saccharomyces cerevisiae para a biogênese da subunidade maior do ribossomo, 60S. O principal fenotipo causado pela repressão da expressão de Nop53 e o acumulo do intermedi ario de processamento de pre-Rrna, 7S, que tambem e substrato do complexo exossomo na formação do rRNA maduro 5:8S. Nop53 interage diretamente com a subunidade do exossomo Rrp6 e com a subunidade Mtr4 do co-ativador do exossomo TRAMP. O objetivo principal deste trabalho foi o de analisar como a interação entre Nop53 e o exossomo pode modular a atividade deste ultimo. Para isso, foram utilizados metodos bioqumicos, geneticos e de biologia molecular. Os resultados mostrados aqui demonstram que a depleção de Nop53 faz com que mais protenas ribossomais, principalmente da subunidade maior, sejam co-imunoprecipitadas com o core do exossomo, sugerindo que Nop53 possa ter um papel na liberação do exossomo da subunidade pre-60S depois da formação do rRNA maduro 5:8S. Esta hipotese foi conrmada atraves da separação de complexos por centrifugação em gradiente de glicerol, que mostrou a presenca de subunidades do exossomo em complexos maiores na ausência de Nop53, provavelmente correspondendo a partculas pre-ribossomais. Co-imunoprecipitação de RNA com o exossomo na ausência de Nop53 tambem conrmou uma maior associação deste complexo com o pre-rRNA 7S. Como tambem mostrado aqui, alem de interagir com Rrp6, Nop53 interage com subunidades do core do exossomo e a superexpressão de uma destas subunidades, Rrp43, complementa parcialmente a ausência de Nop53 na celula. Estes resultados levaram a conclusão de que Nop53 pode recrutar o exossomo para a partcula ribossomal pre-60S para a maturação do pre-rRNA 7S a 5:8S, e atue tambem na liberação do exossomo, possivelmente atraves de sua interação com a helicase Mtr4.
Abstract Nop53 is a nucleolar, conserved and essential protein in the yeast Saccharomyces cerevisiae, involved in the biogenesis of the large ribosomal subunit 60S. The main phenotype of the depletion of Nop53 in yeast cells is the accumulation of the prerRNA processing intermediate 7S, which is also the substrate of the exosome complex for the formation of the mature rRNA 5:8S. Nop53 directly interacts with the exosome subunit Rrp6, and with the subunit Mtr4 of the TRAMP complex, an exosome co-activator. The main objective of this work was the analysis of the interaction between Nop53 and the exosome and the identication of the mechanism through which Nop53 regulates the exosome activity. The results shown here demonstrate that the depletion of Nop53 leads to a more stable association of the exosome with the pre-60S ribosome particle, as determined by co-immunoprecipitation of proteins with one of the exosome core subunits, and by fractionation of complexes through glycerol gradients. These results suggested that Nop53 could play a role in the release of the exosome after the formation of the mature rRNA 5:8S. This hypothesis was conrmed through the co-immunoprecipitation of pre-rRNA 7S with the exosome in the absence of Nop53. In addition to the interaction with the exosome subunit Rrp6, as shown here, Nop53 also interacts with core subunits of the complex. Interestingly, overexpression of one of these subunits, Rrp43, partially complements the depletion of Nop53. These results led to the conclusion that Nop53 may recruit the exosome to the pre-60S particle for the maturation of the pre-rRNA 7S to the mature 5:8S, but Nop53 may also be involved in the release of the exosome, possibly through its interaction with the helicase Mtr4.

Plusieurs études ont permis la caractérisation de la structure et de la fonction du ribosome. En ce qui attrait à la biogénèse du ribosome, nombreux aspects restent à être découverts et compris de façon plus dynamique. En effet, cette biogénèse englobe une variété de voies de modifications et d’assemblages requises pour la maturation des ARNr et pour leurs liaisons avec les protéines ribosomales. De ce fait, les protéines Noc ont été caractérisées comme des facteurs d’assemblages et ont permis la découverte d’une des premières indications sur l’ordre spatio-temporel de la maturation du ribosome. Ainsi, en utilisant la levure comme modèle, notre objectif est d’étudier d’avantage l’échange des complexes composés des protéines Noc ainsi que leur localisation intranucléaire. Ainsi, la nature des interactions de Noc2p avec Noc1p et Noc3p et l’influence de l’arrêt du transport intranucléaire ont été étudiés en utilisant des promoteurs inductibles, la microscopie à fluorescence, des immunobuvardages, qRT-PCR et des purifications par affinité.
Several studies have been performed to characterize the ribosome as far as to understand its structure and its function. However, major aspects of ribosome biogenesis remain elusive or gave only a static picture of the process. In fact, ribosome biogenesis involves dynamic processing and assembly pathways that are required for rRNA modification and folding, in addition to rRNA binding with some ribosomal proteins. One set of assembly factors, the Noc proteins, allowed one of the first indications about the spatio-temporal ordering of ribosome maturation. By using yeast as model, our objective is to provide a dynamic picture of the Noc proteins complexes exchange and nuclear localization by determining the nature of Noc2p interactions with Noc1p and Noc3p and by studying the influence of reversibly arrested intranuclear transport on these proteins and on Rix7p, an AAA-ATPase. In order to achieve these aims, inducible promoter, fluorescent microscopy, western blot, qRT-PCR and affinity purification analyses were used.