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1.
RNA ; 25(4): 465-471, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30670483

RESUMO

The eukaryotic ribosome is assembled through a complex process involving more than 200 factors. As preribosomal RNA is transcribed, assembly factors bind the nascent pre-rRNA and guide its correct folding, modification, and cleavage. While these early events in the assembly of the small ribosomal subunit have been relatively well characterized, assembly of the large subunit precursors, or pre-60S, is less well understood. Recent structures of nucleolar intermediates of large subunit assembly have shed light on the role of many early large subunit assembly factors, but how these particles emerge is still unknown. Here, we use the expression and purification of truncated pre-rRNAs to examine the initial assembly of pre-60S particles. Using this approach, we can recapitulate the early recruitment of large subunit assembly factors mainly to the domains I, II, and VI of the assembling 25S rRNA.


Assuntos
Biogênese de Organelas , Precursores de RNA/genética , RNA Ribossômico/genética , Proteínas Ribossômicas/genética , Subunidades Ribossômicas Maiores de Eucariotos/genética , Saccharomyces cerevisiae/genética , Aptâmeros de Nucleotídeos/síntese química , Aptâmeros de Nucleotídeos/metabolismo , Clonagem Molecular , Plasmídeos/química , Plasmídeos/metabolismo , Precursores de RNA/metabolismo , RNA Ribossômico/metabolismo , Proteínas Ribossômicas/metabolismo , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , Subunidades Ribossômicas Maiores de Eucariotos/ultraestrutura , Saccharomyces cerevisiae/metabolismo , Coloração e Rotulagem/métodos
2.
Proc Natl Acad Sci U S A ; 115(34): E8007-E8016, 2018 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-30072435

RESUMO

Isolated congenital asplenia (ICA) is the only known human developmental defect exclusively affecting a lymphoid organ. In 2013, we showed that private deleterious mutations in the protein-coding region of RPSA, encoding ribosomal protein SA, caused ICA by haploinsufficiency with complete penetrance. We reported seven heterozygous protein-coding mutations in 8 of the 23 kindreds studied, including 6 of the 8 multiplex kindreds. We have since enrolled 33 new kindreds, 5 of which are multiplex. We describe here 11 new heterozygous ICA-causing RPSA protein-coding mutations, and the first two mutations in the 5'-UTR of this gene, which disrupt mRNA splicing. Overall, 40 of the 73 ICA patients (55%) and 23 of the 56 kindreds (41%) carry mutations located in translated or untranslated exons of RPSA. Eleven of the 43 kindreds affected by sporadic disease (26%) carry RPSA mutations, whereas 12 of the 13 multiplex kindreds (92%) carry RPSA mutations. We also report that 6 of 18 (33%) protein-coding mutations and the two (100%) 5'-UTR mutations display incomplete penetrance. Three mutations were identified in two independent kindreds, due to a hotspot or a founder effect. Finally, RPSA ICA-causing mutations were demonstrated to be de novo in 7 of the 23 probands. Mutations in RPSA exons can affect the translated or untranslated regions and can underlie ICA with complete or incomplete penetrance.


Assuntos
Éxons , Síndromes de Imunodeficiência/genética , Mutação , Penetrância , Biossíntese de Proteínas/genética , Processamento de RNA/genética , Receptores de Laminina/genética , Proteínas Ribossômicas/genética , Baço/anormalidades , Regiões 5' não Traduzidas , Feminino , Efeito Fundador , Heterozigoto , Humanos , Síndromes de Imunodeficiência/metabolismo , Masculino , Receptores de Laminina/biossíntese , Proteínas Ribossômicas/biossíntese , Baço/metabolismo
3.
RNA ; 24(7): 881-891, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29712726

RESUMO

The eukaryotic ribosome is made of four intricately folded ribosomal RNAs and 79 proteins. During rapid growth, yeast cells produce an incredible 2000 ribosomes every minute. Ribosome assembly involves more than 200 trans-acting factors, intervening from the transcription of the preribosomal RNA in the nucleolus to late maturation events in the cytoplasm. The biogenesis of the small ribosomal subunit, or 40S, is especially intricate, requiring more than four times the mass of the small subunit in assembly factors for its full maturation. Recent studies have provided new insights into the complex assembly of the 40S subunit. These data from cryo-electron microscopy, X-ray crystallography, and other biochemical and molecular biology methods, have elucidated the role of many factors required in small subunit maturation. Mechanisms of the regulation of ribosome assembly have also emerged from this body of work. This review aims to integrate these new results into an updated view of small subunit biogenesis and its regulation, in yeast, from transcription to the formation of the mature small subunit.


Assuntos
Subunidades Ribossômicas Menores de Eucariotos/metabolismo , Saccharomyces cerevisiae/metabolismo , Transporte Biológico , Ciclo Celular , Citoplasma/metabolismo , Biogênese de Organelas , Subunidades Ribossômicas Menores de Eucariotos/química , Saccharomyces cerevisiae/genética , Serina-Treonina Quinases TOR/metabolismo , Transcrição Genética
4.
Nature ; 556(7699): 126-129, 2018 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-29512650

RESUMO

Early co-transcriptional events during eukaryotic ribosome assembly result in the formation of precursors of the small (40S) and large (60S) ribosomal subunits. A multitude of transient assembly factors regulate and chaperone the systematic folding of pre-ribosomal RNA subdomains. However, owing to a lack of structural information, the role of these factors during early nucleolar 60S assembly is not fully understood. Here we report cryo-electron microscopy (cryo-EM) reconstructions of the nucleolar pre-60S ribosomal subunit in different conformational states at resolutions of up to 3.4 Å. These reconstructions reveal how steric hindrance and molecular mimicry are used to prevent both premature folding states and binding of later factors. This is accomplished by the concerted activity of 21 ribosome assembly factors that stabilize and remodel pre-ribosomal RNA and ribosomal proteins. Among these factors, three Brix-domain proteins and their binding partners form a ring-like structure at ribosomal RNA (rRNA) domain boundaries to support the architecture of the maturing particle. The existence of mutually exclusive conformations of these pre-60S particles suggests that the formation of the polypeptide exit tunnel is achieved through different folding pathways during subsequent stages of ribosome assembly. These structures rationalize previous genetic and biochemical data and highlight the mechanisms that drive eukaryotic ribosome assembly in a unidirectional manner.


Assuntos
Nucléolo Celular/química , Microscopia Crioeletrônica , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , Subunidades Ribossômicas Maiores de Eucariotos/ultraestrutura , Saccharomyces cerevisiae , Reagentes para Ligações Cruzadas/química , Modelos Moleculares , Mimetismo Molecular , Domínios Proteicos , Estabilidade Proteica , Dobramento de RNA , RNA Ribossômico/química , RNA Ribossômico/genética , RNA Ribossômico/metabolismo , RNA Ribossômico/ultraestrutura , Reprodutibilidade dos Testes , Proteínas Ribossômicas/química , Proteínas Ribossômicas/metabolismo , Proteínas Ribossômicas/ultraestrutura , Subunidades Ribossômicas Maiores de Eucariotos/química , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/ultraestrutura
5.
Nat Struct Mol Biol ; 24(11): 944-953, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28945246

RESUMO

The small-subunit processome represents the earliest stable precursor of the eukaryotic small ribosomal subunit. Here we present the cryo-EM structure of the Saccharomyces cerevisiae small-subunit processome at an overall resolution of 3.8 Å, which provides an essentially complete near-atomic model of this assembly. In this nucleolar superstructure, 51 ribosome-assembly factors and two RNAs encapsulate the 18S rRNA precursor and 15 ribosomal proteins in a state that precedes pre-rRNA cleavage at site A1. Extended flexible proteins are employed to connect distant sites in this particle. Molecular mimicry and steric hindrance, as well as protein- and RNA-mediated RNA remodeling, are used in a concerted fashion to prevent the premature formation of the central pseudoknot and its surrounding elements within the small ribosomal subunit.


Assuntos
Subunidades Ribossômicas Menores de Eucariotos/ultraestrutura , Saccharomyces cerevisiae/ultraestrutura , Microscopia Crioeletrônica , RNA Ribossômico 18S/ultraestrutura , Proteínas de Saccharomyces cerevisiae/ultraestrutura
6.
Science ; 355(6321)2017 01 13.
Artigo em Inglês | MEDLINE | ID: mdl-27980088

RESUMO

The small subunit (SSU) processome, a large ribonucleoprotein particle, organizes the assembly of the eukaryotic small ribosomal subunit by coordinating the folding, cleavage, and modification of nascent pre-ribosomal RNA (rRNA). Here, we present the cryo-electron microscopy structure of the yeast SSU processome at 5.1-angstrom resolution. The structure reveals how large ribosome biogenesis complexes assist the 5' external transcribed spacer and U3 small nucleolar RNA in providing an intertwined RNA-protein assembly platform for the separate maturation of 18S rRNA domains. The strategic placement of a molecular motor at the center of the particle further suggests a mechanism for mediating conformational changes within this giant particle. This study provides a structural framework for a mechanistic understanding of eukaryotic ribosome assembly in the model organism Saccharomyces cerevisiae.


Assuntos
Subunidades Ribossômicas Menores de Eucariotos/química , Subunidades Ribossômicas Menores de Eucariotos/ultraestrutura , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/ultraestrutura , Microscopia Crioeletrônica , Conformação de Ácido Nucleico , Conformação Proteica em Folha beta , RNA Fúngico/química , RNA Fúngico/ultraestrutura , RNA Ribossômico/química , RNA Ribossômico/ultraestrutura , RNA Ribossômico 18S/química , RNA Ribossômico 18S/ultraestrutura , RNA Nucleolar Pequeno/química , RNA Nucleolar Pequeno/ultraestrutura , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/ultraestrutura
7.
Nat Commun ; 7: 12090, 2016 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-27354316

RESUMO

Early eukaryotic ribosome biogenesis involves large multi-protein complexes, which co-transcriptionally associate with pre-ribosomal RNA to form the small subunit processome. The precise mechanisms by which two of the largest multi-protein complexes-UtpA and UtpB-interact with nascent pre-ribosomal RNA are poorly understood. Here, we combined biochemical and structural biology approaches with ensembles of RNA-protein cross-linking data to elucidate the essential functions of both complexes. We show that UtpA contains a large composite RNA-binding site and captures the 5' end of pre-ribosomal RNA. UtpB forms an extended structure that binds early pre-ribosomal intermediates in close proximity to architectural sites such as an RNA duplex formed by the 5' ETS and U3 snoRNA as well as the 3' boundary of the 18S rRNA. Both complexes therefore act as vital RNA chaperones to initiate eukaryotic ribosome assembly.


Assuntos
Chaperonas Moleculares/fisiologia , RNA Fúngico/metabolismo , RNA Nucleolar Pequeno/metabolismo , Ribossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Regulação Fúngica da Expressão Gênica , Precursores de RNA/genética , Processamento Pós-Transcricional do RNA , RNA Ribossômico 18S , RNA Nucleolar Pequeno/genética , RNA Nucleolar Pequeno/fisiologia , Proteínas Ribossômicas/metabolismo , Proteínas de Saccharomyces cerevisiae/genética
8.
Nat Struct Mol Biol ; 22(11): 920-3, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26479197

RESUMO

Eukaryotic ribosome biogenesis involves a plethora of ribosome-assembly factors, and their temporal order of association with preribosomal RNA is largely unknown. By using Saccharomyces cerevisiae as a model organism, we developed a system that recapitulates and arrests ribosome assembly at early stages, thus providing in vivo snapshots of nascent preribosomal particles. Here we report the stage-specific order in which 70 ribosome-assembly factors associate with preribosomal RNA domains, thereby forming the 6-MDa small-subunit processome.


Assuntos
Biogênese de Organelas , RNA Ribossômico/metabolismo , Proteínas Ribossômicas/metabolismo , Saccharomyces cerevisiae/fisiologia , Substâncias Macromoleculares/metabolismo , Modelos Biológicos , Saccharomyces cerevisiae/metabolismo
9.
Structure ; 23(1): 126-138, 2015 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-25497731

RESUMO

PML and several other proteins localizing in PML-nuclear bodies (PML-NB) contain phosphoSIMs (SUMO-interacting motifs), and phosphorylation of this motif plays a key role in their interaction with SUMO family proteins. We examined the role that phosphorylation plays in the binding of the phosphoSIMs of PML and Daxx to SUMO1 at the atomic level. The crystal structures of SUMO1 bound to unphosphorylated and tetraphosphorylated PML-SIM peptides indicate that three phosphoserines directly contact specific positively charged residues of SUMO1. Surprisingly, the crystal structure of SUMO1 bound to a diphosphorylated Daxx-SIM peptide indicate that the hydrophobic residues of the phosphoSIM bind in a manner similar to that seen with PML, but important differences are observed when comparing the phosphorylated residues. Together, the results provide an atomic level description of how specific acetylation patterns within different SUMO family proteins can work together with phosphorylation of phosphoSIM's regions of target proteins to regulate binding specificity.


Assuntos
Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Domínios e Motivos de Interação entre Proteínas , Proteína SUMO-1/química , Proteína SUMO-1/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sequência de Aminoácidos , Cristalografia por Raios X , Células HEK293 , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Fosforilação , Proteína da Leucemia Promielocítica , Ligação Proteica , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos
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