RESUMO
During the last ten years, developments in cryo-electron microscopy have transformed our understanding of eukaryotic ribosome assembly. As a result, the field has advanced from a list of the vast array of ribosome assembly factors toward an emerging molecular movie in which individual frames are represented by structures of stable ribosome assembly intermediates with complementary biochemical and genetic data. In this review, we discuss the mechanisms driving the assembly of yeast and human small and large ribosomal subunits. A particular emphasis is placed on the most recent findings that illustrate key concepts of ribosome assembly, such as folding of preribosomal RNA, the enforced chronology of assembly, enzyme-mediated irreversible transitions, and proofreading of preribosomal particles.
Assuntos
Microscopia Crioeletrônica , Proteínas Ribossômicas , Ribossomos , Humanos , Ribossomos/metabolismo , Ribossomos/ultraestrutura , Ribossomos/química , Ribossomos/genética , Proteínas Ribossômicas/metabolismo , Proteínas Ribossômicas/química , Proteínas Ribossômicas/genética , RNA Ribossômico/metabolismo , RNA Ribossômico/química , RNA Ribossômico/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Modelos Moleculares , Células Eucarióticas/metabolismo , Células Eucarióticas/ultraestrutura , Dobramento de RNA , Subunidades Ribossômicas Menores de Eucariotos/metabolismo , Subunidades Ribossômicas Menores de Eucariotos/química , Subunidades Ribossômicas Menores de Eucariotos/genética , Subunidades Ribossômicas Menores de Eucariotos/ultraestrutura , AnimaisRESUMO
In the past 25 years, genetic and biochemical analyses of ribosome assembly in yeast have identified most of the factors that participate in this complex pathway and have generated models for the mechanisms driving the assembly. More recently, the publication of numerous cryo-electron microscopy structures of yeast ribosome assembly intermediates has provided near-atomic resolution snapshots of ribosome precursor particles. Satisfyingly, these structural data support the genetic and biochemical models and provide additional mechanistic insight into ribosome assembly. In this Review, we discuss the mechanisms of assembly of the yeast small ribosomal subunit and large ribosomal subunit in the nucleolus, nucleus and cytoplasm. Particular emphasis is placed on concepts such as the mechanisms of RNA compaction, the functions of molecular switches and molecular mimicry, the irreversibility of assembly checkpoints and the roles of structural and functional proofreading of pre-ribosomal particles.
Assuntos
Subunidades Ribossômicas/genética , Animais , Núcleo Celular/genética , Microscopia Crioeletrônica/métodos , Citoplasma/genética , Humanos , RNA/genéticaRESUMO
The androgen receptor (AR) is a nuclear receptor that governs gene expression programs required for prostate development and male phenotype maintenance. Advanced prostate cancers display AR hyperactivation and transcriptome expansion, in part, through AR amplification and interaction with oncoprotein cofactors. Despite its biological importance, how AR domains and cofactors cooperate to bind DNA has remained elusive. Using single-particle cryo-electron microscopy, we isolated three conformations of AR bound to DNA, showing that AR forms a non-obligate dimer, with the buried dimer interface utilized by ancestral steroid receptors repurposed to facilitate cooperative DNA binding. We identify novel allosteric surfaces which are compromised in androgen insensitivity syndrome and reinforced by AR's oncoprotein cofactor, ERG, and by DNA-binding motifs. Finally, we present evidence that this plastic dimer interface may have been adopted for transactivation at the expense of DNA binding. Our work highlights how fine-tuning AR's cooperative interactions translate to consequences in development and disease.
Assuntos
Neoplasias da Próstata , Receptores Androgênicos , Microscopia Crioeletrônica , DNA/metabolismo , Dimerização , Humanos , Masculino , Neoplasias da Próstata/genética , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismo , Ativação TranscricionalRESUMO
Mitochondrial ribosomes (mitoribosomes) synthesize proteins encoded within the mitochondrial genome that are assembled into oxidative phosphorylation complexes. Thus, mitoribosome biogenesis is essential for ATP production and cellular metabolism1. Here we used cryo-electron microscopy to determine nine structures of native yeast and human mitoribosomal small subunit assembly intermediates, illuminating the mechanistic basis for how GTPases are used to control early steps of decoding centre formation, how initial rRNA folding and processing events are mediated, and how mitoribosomal proteins have active roles during assembly. Furthermore, this series of intermediates from two species with divergent mitoribosomal architecture uncovers both conserved principles and species-specific adaptations that govern the maturation of mitoribosomal small subunits in eukaryotes. By revealing the dynamic interplay between assembly factors, mitoribosomal proteins and rRNA that are required to generate functional subunits, our structural analysis provides a vignette for how molecular complexity and diversity can evolve in large ribonucleoprotein assemblies.
Assuntos
Microscopia Crioeletrônica , Ribossomos Mitocondriais , Ribonucleoproteínas , Subunidades Ribossômicas Menores , Saccharomyces cerevisiae , Humanos , Proteínas Mitocondriais/química , Proteínas Mitocondriais/metabolismo , Proteínas Mitocondriais/ultraestrutura , Ribossomos Mitocondriais/química , Ribossomos Mitocondriais/metabolismo , Ribossomos Mitocondriais/ultraestrutura , Proteínas Ribossômicas/química , Proteínas Ribossômicas/metabolismo , Proteínas Ribossômicas/ultraestrutura , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , RNA Ribossômico , GTP Fosfo-Hidrolases , Ribonucleoproteínas/química , Ribonucleoproteínas/metabolismo , Ribonucleoproteínas/ultraestrutura , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/ultraestrutura , Subunidades Ribossômicas Menores/química , Subunidades Ribossômicas Menores/metabolismo , Subunidades Ribossômicas Menores/ultraestruturaRESUMO
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 de 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/ultraestruturaRESUMO
The androgen receptor (AR) is a type I nuclear hormone receptor and the primary drug target in prostate cancer due to its role as a lineage survival factor in prostate luminal epithelium. In prostate cancer, the AR cistrome is reprogrammed relative to normal prostate epithelium and particularly in cancers driven by oncogenic ETS fusion genes. The molecular basis for this change has remained elusive. Using purified proteins, we report a minimal cell-free system that demonstrates interdomain cooperativity between the ligand (LBD) and DNA binding domains (DBD) of AR, and its autoinhibition by the N terminus of AR. Furthermore, we identify ERG as a cofactor that activates AR's ability to bind DNA in both high and lower affinity contexts through direct interaction within a newly identified AR-interacting motif (AIM) in the ETS domain, independent of ERG's own DNA binding ability. Finally, we present evidence that this interaction is conserved among ETS factors whose expression is altered in prostate cancer. Our work highlights, at a biochemical level, how tumor-initiating ETS translocations result in reprogramming of the AR cistrome.
Assuntos
DNA/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias da Próstata/metabolismo , Proteínas Proto-Oncogênicas c-ets/metabolismo , Receptores Androgênicos/metabolismo , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , DNA/genética , Humanos , Masculino , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Proteínas Proto-Oncogênicas c-ets/genética , Receptores Androgênicos/química , Receptores Androgênicos/genética , Regulador Transcricional ERG/química , Regulador Transcricional ERG/genética , Regulador Transcricional ERG/metabolismo , Células Tumorais CultivadasRESUMO
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/métodosRESUMO
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 , Splicing 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 , Doenças da Imunodeficiência Primária , Receptores de Laminina/biossíntese , Proteínas Ribossômicas/biossíntese , Baço/metabolismoRESUMO
Crystallography of the cores of phosphotyrosine-activated dimers of STAT1 (132-713) and STAT3 (127-722) bound to a similar double-stranded deoxyoligonucleotide established the domain structure of the STATs and the structural basis for activation through tyrosine phosphorylation and dimerization. We reported earlier that mutants in the linker domain of STAT1 that connect the DNA-binding domain and SH2 domain can prevent transcriptional activation. Because of the pervasive importance of persistently activated STAT3 in many human cancers and the difficulty of finding useful drug candidates aimed at disrupting the pY interchange in active STAT3 dimers, we have examined effects of an array of mutants in the STAT3 linker domain. We have found several STAT3 linker domain mutants to have profound effects of inhibiting STAT3 transcriptional activation. From these results, we propose (i) there is definite functional interaction of the linker both with the DNA binding domain and with the SH2 domain, and (ii) these putative contacts provide potential new targets for small molecule-induced pSTAT3 inhibition.
Assuntos
Mutação de Sentido Incorreto , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Fator de Transcrição STAT3/metabolismo , Ativação Transcricional , Substituição de Aminoácidos , Linhagem Celular Tumoral , Humanos , Proteínas de Neoplasias/genética , Neoplasias/genética , Neoplasias/patologia , Fosforilação , Multimerização Proteica , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/metabolismo , Fator de Transcrição STAT3/genéticaRESUMO
Eukaryotic ribosomes are significantly larger and more complex than their prokaryotic counterparts. This parallels the increased complexity of the associated cellular machinery responsible for translation initiation, ribosome assembly, and the regulation of protein synthesis in eukaryotic cells. The recently determined crystal structures of the small (40S) and large (60S) ribosomal subunits and the 80S ribosome now provide an atomic description of this essential molecular machine and reveal its eukaryote-specific features. In this review, we discuss the common structural principles underlying the evolution of both ribosomal subunits. The recently obtained structural information provides a framework for further genetic, biochemical and structural studies of eukaryotic ribosomes. At the same time, it facilitates a direct comparison between prokaryotic and eukaryotic ribosomal features.
Assuntos
Biossíntese de Proteínas , RNA Ribossômico/química , Proteínas Ribossômicas/química , Ribossomos/química , Cristalografia por Raios X , Células Eucarióticas/metabolismo , Modelos Moleculares , RNA Ribossômico/metabolismo , Ribonucleoproteínas/química , Ribonucleoproteínas/metabolismo , Proteínas Ribossômicas/metabolismo , Ribossomos/metabolismoRESUMO
Eukaryotic DNA replication requires the coordinated activity of the multi-subunit DNA polymerases: Pol alpha, Pol delta and Pol epsilon. The conserved catalytic and regulatory B subunits associate in a constitutive heterodimer that represents the functional core of all three replicative polymerases. Here, we combine X-ray crystallography and electron microscopy (EM) to describe subunit interaction and 3D architecture of heterodimeric yeast Pol alpha. The crystal structure of the C-terminal domain (CTD) of the catalytic subunit bound to the B subunit illustrates a conserved mechanism of accessory factor recruitment by replicative polymerases. The EM reconstructions of Pol alpha reveal a bilobal shape with separate catalytic and regulatory modules. Docking of the B-CTD complex in the EM reconstruction shows that the B subunit is tethered to the polymerase domain through a structured but flexible linker. Our combined findings provide a structural template for the common functional architecture of the three major replicative DNA polymerases.
Assuntos
DNA Polimerase I/química , DNA Polimerase I/metabolismo , Saccharomyces cerevisiae/enzimologia , Sequência de Aminoácidos , Animais , Cristalografia por Raios X , DNA Polimerase I/genética , Escherichia coli/genética , Humanos , Microscopia Eletrônica , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Estrutura Terciária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Alinhamento de SequênciaRESUMO
The Pol α/primase complex or primosome is the primase/polymerase complex that initiates nucleic acid synthesis during eukaryotic replication. Within the primosome, the primase synthesizes short RNA primers that undergo limited extension by Pol α. The resulting RNA-DNA primers are utilized by Pol δ and Pol ε for processive elongation on the lagging and leading strands, respectively. Despite its importance, the mechanism of RNA-DNA primer synthesis remains poorly understood. Here, we describe a structural model of the yeast primosome based on electron microscopy and functional studies. The 3D architecture of the primosome reveals an asymmetric, dumbbell-shaped particle. The catalytic centers of primase and Pol α reside in separate lobes of high relative mobility. The flexible tethering of the primosome lobes increases the efficiency of primer transfer between primase and Pol α. The physical organization of the primosome suggests that a concerted mechanism of primer hand-off between primase and Pol α would involve coordinated movements of the primosome lobes. The first three-dimensional map of the eukaryotic primosome at 25 Å resolution provides an essential structural template for understanding initiation of eukaryotic replication.
Assuntos
DNA Polimerase I/química , DNA Polimerase I/ultraestrutura , DNA Primase/química , DNA Primase/ultraestrutura , Sequência de Aminoácidos , DNA Polimerase I/metabolismo , DNA Primase/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Subunidades Proteicas/química , RNA/química , Saccharomyces cerevisiae/enzimologiaRESUMO
During the early stages of human large ribosomal subunit (60S) biogenesis, an ensemble of assembly factors establishes and fine-tunes the essential RNA functional centers of pre-60S particles by an unknown mechanism. Here, we report a series of cryo-electron microscopy structures of human nucleolar and nuclear pre-60S assembly intermediates at resolutions of 2.5 to 3.2 angstroms. These structures show how protein interaction hubs tether assembly factor complexes to nucleolar particles and how guanosine triphosphatases and adenosine triphosphatase couple irreversible nucleotide hydrolysis steps to the installation of functional centers. Nuclear stages highlight how a conserved RNA-processing complex, the rixosome, couples large-scale RNA conformational changes with pre-ribosomal RNA processing by the RNA degradation machinery. Our ensemble of human pre-60S particles provides a rich foundation with which to elucidate the molecular principles of ribosome formation.
Assuntos
RNA Ribossômico , Subunidades Ribossômicas Maiores de Eucariotos , Humanos , Núcleo Celular/química , Núcleo Celular/metabolismo , Microscopia Crioeletrônica , Proteínas Ribossômicas/química , Proteínas Ribossômicas/metabolismo , Subunidades Ribossômicas Maiores de Eucariotos/química , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , RNA Ribossômico/química , RNA Ribossômico/metabolismo , Saccharomyces cerevisiae , Conformação ProteicaRESUMO
During early stages of human large ribosomal subunit (60 S ) biogenesis, an ensemble of assembly factors establishes and fine-tunes the essential RNA functional centers of pre-60 S particles by an unknown mechanism. Here, we report a series of cryo-electron microscopy structures of human nucleolar and nuclear pre-60 S assembly intermediates at resolutions of 2.5-3.2 Ã . These structures show how protein interaction hubs tether assembly factor complexes to nucleolar particles and how GTPases and ATPases couple irreversible nucleotide hydrolysis steps to the installation of functional centers. Nuclear stages highlight how a conserved RNA processing complex, the rixosome, couples large-scale RNA conformational changes to pre-rRNA processing by the RNA degradation machinery. Our ensemble of human pre-60 S particles provides a rich foundation to elucidate the molecular principles of ribosome formation. One-Sentence Summary: High-resolution cryo-EM structures of human pre-60S particles reveal new principles of eukaryotic ribosome assembly.
RESUMO
One of the challenges faced by current CRISPR/Cas9 editing strategies is the difficulty in rapidly selecting clonal populations of biallelically edited cells. Here we present Surface engiNeered fluorEscence Assisted Kit with Protein Epitope Enhanced Capture (SNEAK PEEC), a platform that combines human genome editing with cell-surface display, which enables the direct identification of biallelically edited clones with minimal screening.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Humanos , Sistemas CRISPR-Cas/genética , Membrana Celular , Células Clonais , EpitoposRESUMO
During transcription of eukaryotic ribosomal DNA in the nucleolus, assembly checkpoints exist that guarantee the formation of stable precursors of small and large ribosomal subunits. While the formation of an early large subunit assembly checkpoint precedes the separation of small and large subunit maturation, its mechanism of action and function remain unknown. Here, we report the cryo-electron microscopy structure of the yeast co-transcriptional large ribosomal subunit assembly intermediate that serves as a checkpoint. The structure provides the mechanistic basis for how quality-control pathways are established through co-transcriptional ribosome assembly factors, that structurally interrogate, remodel and, together with ribosomal proteins, cooperatively stabilize correctly folded pre-ribosomal RNA. Our findings thus provide a molecular explanation for quality control during eukaryotic ribosome assembly in the nucleolus.
Assuntos
RNA Ribossômico , Proteínas de Saccharomyces cerevisiae , Microscopia Crioeletrônica , RNA Ribossômico/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas Ribossômicas/metabolismo , Subunidades Ribossômicas Maiores/metabolismo , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , Subunidades Ribossômicas Menores de Eucariotos/metabolismoRESUMO
Primases synthesize the RNA primers that are necessary for replication of the parental DNA strands. Here we report that the heterodimeric archaeal/eukaryotic primase is an iron-sulfur (Fe-S) protein. Binding of the Fe-S cluster is mediated by an evolutionarily conserved domain at the C terminus of the large subunit. We further show that the Fe-S domain is essential to the unique ability of the eukaryotic primase to start DNA replication.
Assuntos
DNA Primase/metabolismo , Proteínas Ferro-Enxofre/metabolismo , RNA , DNA Primase/química , Espectroscopia de Ressonância de Spin Eletrônica , Ligação ProteicaRESUMO
The biogenesis of the eukaryotic ribosome is a tightly regulated and energetically demanding process involving more than 200 ribosome assembly factors. These factors work in concert to ensure accurate assembly and maturation of both ribosomal subunits. Cryo-electron microscopy (cryo-EM) structures of numerous eukaryotic ribosome assembly intermediates have provided a wealth of structural insights highlighting the molecular interplay of a cast of assembly factors. In this review, we focus on recently determined structures of maturing small subunit (SSU) processomes, giant precursors of the small ribosomal subunit. Based on these structures and complementary biochemical and genetic studies, we discuss an emerging mechanism involving exosome-mediated SSU processome maturation and disassembly.
Assuntos
Subunidades Ribossômicas Menores , Proteínas de Saccharomyces cerevisiae , Microscopia Crioeletrônica , Células Eucarióticas , Proteínas Ribossômicas/química , Subunidades Ribossômicas Menores/química , RibossomosRESUMO
The human small subunit processome mediates early maturation of the small ribosomal subunit by coupling RNA folding to subsequent RNA cleavage and processing steps. We report the high-resolution cryoelectron microscopy structures of maturing human small subunit (SSU) processomes at resolutions of 2.7 to 3.9 angstroms. These structures reveal the molecular mechanisms that enable crucial progressions during SSU processome maturation. RNA folding states within these particles are communicated to and coordinated with key enzymes that drive irreversible steps such as targeted exosome-mediated RNA degradation, protein-guided site-specific endonucleolytic RNA cleavage, and tightly controlled RNA unwinding. These conserved mechanisms highlight the SSU processome's impressive structural plasticity, which endows this 4.5-megadalton nucleolar assembly with the distinctive ability to mature the small ribosomal subunit from within.
Assuntos
Nucléolo Celular/ultraestrutura , Dobramento de RNA , Estabilidade de RNA , RNA Nucleolar Pequeno/química , Nucléolo Celular/metabolismo , Microscopia Crioeletrônica , RNA Helicases DEAD-box/química , Humanos , Clivagem do RNA , Fatores de Processamento de RNA/químicaRESUMO
Microsporidia are eukaryotic parasites that infect essentially all animal species, including many of agricultural importance1-3, and are significant opportunistic parasites of humans4. They are characterized by having a specialized infection apparatus, an obligate intracellular lifestyle5, rudimentary mitochondria and the smallest known eukaryotic genomes5-7. Extreme genome compaction led to minimal gene sizes affecting even conserved ancient complexes such as the ribosome8-10. In the present study, the cryo-electron microscopy structure of the ribosome from the microsporidium Vairimorpha necatrix is presented, which illustrates how genome compaction has resulted in the smallest known eukaryotic cytoplasmic ribosome. Selection pressure led to the loss of two ribosomal proteins and removal of essentially all eukaryote-specific ribosomal RNA (rRNA) expansion segments, reducing the rRNA to a functionally conserved core. The structure highlights how one microsporidia-specific and several repurposed existing ribosomal proteins compensate for the extensive rRNA reduction. The microsporidian ribosome is kept in an inactive state by two previously uncharacterized dormancy factors that specifically target the functionally important E-site, P-site and polypeptide exit tunnel. The present study illustrates the distinct effects of evolutionary pressure on RNA and protein-coding genes, provides a mechanism for ribosome inhibition and can serve as a structural basis for the development of inhibitors against microsporidian parasites.