Добірка наукової літератури з теми "Pre-60S particle"

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.

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.

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 Eukaryotic ribosome biogenesis is an elaborate process during which ribosomal proteins assemble with the pre-rRNA while it is being processed and folded. Hundreds of assembly factors (AF) are required and transiently recruited to assist the sequential remodeling events. One of the most intricate ones is the stepwise removal of the internal transcribed spacer 2 (ITS2), between the 5.8S and 25S rRNAs, that constitutes together with five AFs the pre-60S ‘foot’. In the transition from nucleolus to nucleoplasm, Nop53 replaces Erb1 at the basis of the foot and recruits the RNA exosome for the ITS2 cleavage and foot disassembly. Here we comprehensively analyze the impact of Nop53 recruitment on the pre-60S compositional changes. We show that depletion of Nop53, different from nop53 mutants lacking the exosome-interacting motif, not only causes retention of the unprocessed foot in late pre-60S intermediates but also affects the transition from nucleolar state E particle to subsequent nuclear stages. Additionally, we reveal that Nop53 depletion causes the impairment of late maturation events such as Yvh1 recruitment. In light of recently described pre-60S cryo-EM structures, our results provide biochemical evidence for the structural role of Nop53 rearranging and stabilizing the foot interface to assist the Nog2 particle formation.

A collection of yeast strains surviving with mutant 5S RNA has been constructed. The mutant strains presented alterations of the nucleolar structure, with less granular component, and a delocalization of the 25S rRNA throughout the nucleoplasm. The 5S RNA mutations affected helix I and resulted in decreased amounts of stable 5S RNA and of the ribosomal 60S subunits. The shortage of 60S subunits was due to a specific defect in the processing of the 27SB precursor RNA that gives rise to the mature 25S and 5.8S rRNA. The processing rate of the 27SB pre-rRNA was specifically delayed, whereas the 27SA and 20S pre-rRNA were processed at a normal rate. The defect was partially corrected by increasing the amount of mutant 5S RNA. We propose that the 5S RNA is recruited by the pre-60S particle and that its recruitment is necessary for the efficient processing of the 27SB RNA precursor. Such a mechanism could ensure that all newly formed mature 60S subunits contain stoichiometric amounts of the three rRNA components.

ABSTRACT We previously identified two Trypanosoma brucei RNA binding proteins, P34 and P37, and determined that they are essential for proper ribosomal assembly in this organism. Loss of these proteins via RNA interference is lethal and causes a decrease in both 5S rRNA levels and formation of 80S ribosomes, concomitant with a decrease in total cellular protein synthesis. These data suggest that these proteins are involved at some point in the ribosomal biogenesis pathway. In the current study, we have performed subcellular fractionation in conjunction with immune capture experiments specific for 60S ribosomal proteins and accessory factors in order to determine when and where P34 and P37 are involved in the ribosomal biogenesis pathway. These studies demonstrate that P34 and P37 associate with the 60S ribosomal subunit at the stage of the nucleolar 90S particle and remain associated subsequent to nuclear export. In addition, P34 and P37 associate with conserved 60S ribosomal subunit nuclear export factors exportin 1 and Nmd3, suggesting that they are components of the 60S ribosomal subunit nuclear export complex in T. brucei. Most significantly, the pre-60S complex does not associate with exportin 1 or Nmd3 in the absence of P34 and P37. These results demonstrate that, although T. brucei 60S ribosomal subunits utilize a nuclear export complex similar to that described for other organisms, trypanosome-specific factors are essential to the process.

The coordination of RNA polymerase I transcription with pre-rRNA processing, preribosomal particle assembly, and nuclear export is a finely tuned process requiring the concerted actions of a number of accessory factors. However, the exact functions of some of these proteins and how they assemble in subcomplexes remain poorly defined. LAS1L was first described as a nucleolar protein required for maturation of the 60S preribosomal subunit. In this paper, we demonstrate that LAS1L interacts with PELP1, TEX10, and WDR18, the mammalian homologues of the budding yeast Rix1 complex, along with NOL9 and SENP3, to form a novel nucleolar complex that cofractionates with the 60S preribosomal subunit. Depletion of LAS1L-associated proteins results in a p53-dependent G1 arrest and leads to defects in processing of the pre-rRNA internal transcribed spacer 2 region. We further show that the nucleolar localization of this complex requires active RNA polymerase I transcription and the small ubiquitin-like modifier–specific protease SENP3. Taken together, our data identify a novel mammalian complex required for 60S ribosomal subunit synthesis, providing further insight into the intricate, yet poorly described, process of ribosome biogenesis in higher eukaryotes.

Abstract The synthesis of new ribosomes is a fundamental conserved process in all cells. Ribosomes are pre-assembled in the nucleus and subsequently exported to the cytoplasm where they acquire functionality through a series of final maturation steps that include formation of the catalytic center, recruitment of the last remaining ribosomal proteins and the removal of inhibitory assembly factors. Surprisingly, a number of key factors (SBDS, DNAJC21, RPL10 (uL16)) involved in late cytoplasmic maturation of the large (60S) ribosomal subunit are mutated in both inherited and sporadic forms of leukemia. In particular, biallelic mutations in the SBDS gene cause Shwachman-Diamond syndrome (SDS), a recessive bone marrow failure disorder with significant predisposition to acute myeloid leukemia. By using the latest advances in single-particle cryo-electron microscopy to elucidate the function of the SBDS protein, we have uncovered an elegant mechanism that couples final maturation of the 60S subunit to a quality control assessment of the structural integrity of the active sites of the ribosome. Further molecular dissection of this pathway may inform novel therapeutic strategies for SDS and leukemia more generally. References: 1. Weis F, Giudice E, Churcher M,et al. Mechanism of eIF6 release from the nascent 60S ribosomal subunit. Nat Struct Mol Biol, (2015) Nov;22(11):914-9. 2. Wong CC, Traynor D, Basse N, et al. Defective ribosome assembly in Shwachman-Diamond syndrome. Plenary Paper, Blood. 2011 Oct 20;118(16):4305-12. 3. Finch AJ, Hilcenko C, Basse N, et al. Uncoupling of GTP hydrolysis from eIF6 release on the ribosome causes Shwachman-Diamond syndrome. Genes Dev (2011) 25: 917-929. 4. Menne TM, Goyenechea B, Sánchez-Puig N, et al. The Shwachman-Bodian-Diamond syndrome protein mediates translational activation of ribosomes in yeast. Nature Genetics (2007) 39: 486-95. Disclosures No relevant conflicts of interest to declare.

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.

For a safe operation of gas pipelines, the prevention of propagating brittle facture is one of the most important requirements. To evaluate the transition temperature of a propagating fracture, the Drop Weight Tear (DWT) Test was developed in the 60s. Fracture surfaces of DWT specimens have been shown to correspond well to the fracture surface of a pipe exposed to a propagating fracture at a certain temperature. Historically, there have always been observations of the fracture initiating in a ductile manner in the DWT test. Nevertheless, the most widely used test standard rules out such behavior, known as inverse or abnormal fracture. As an option to prevent ductile initiation, an alternative notch is proposed. While this might have served in the earlier days, high toughness steels of today are known to provide a high resistance against crack initiation and are therefore prone to inverse fracture, even when making use of the suggested alternative notch. Other, non-standard notch types have been investigated and discussed in literature, amongst these the static pre-crack and brittle weld notch. Observations of the DWT test, especially comparing material showing non-inverse and inverse behaviour, show delayed crack initiation resulting in large deflection when the specimens are inverse. This high degree of pre-deformation of the material will have an adverse influence on the material performance by the time the crack propagates into it. This implies that the appearance of inverse fracture is a test effect in the laboratory test, and not an inherent material property, leading to the question if such DWT test results still correspond to the behavior of pipes. If the correlation is shown to be valid, the brittle initiation requirement as such becomes questionable. This study summarises investigations of different notch types in DWT tests. West Jefferson tests that have been conducted to verify the correlation to shear area fraction in DWT tests. The investigation revealed that ductile initiation could not be reliably suppressed. While neither Chevron nor static pre-crack specimen lead to any reduction of the occurrence of inverse fracture, test series of brittle weld specimens did have a higher number of valid specimens. Interestingly, the results of valid, non-inverse specimens and invalid, inverse specimens showed no shift in transitional behavior. Correspondingly, both valid and invalid specimens showed a good representation of the pipe behaviour in the upper transition region.