Eukaryotic Ribosome Assembly Research Articles

Eukaryotic Ribosome Assembly.

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.

Visualizing the nucleoplasmic maturation of human pre-60S ribosomal particles.

Eukaryotic ribosome assembly is a highly orchestrated process that involves over two hundred protein factors. After early assembly events on nascent rRNA in the nucleolus, pre-60S particles undergo continuous maturation steps in the nucleoplasm, and prepare for nuclear export. Here, we report eleven cryo-EM structures of the nuclear pre-60S particles isolated from human cells through epitope-tagged GNL2, at resolutions of 2.8-4.3 Å. These high-resolution snapshots provide fine details for several major structural remodeling events at a virtual temporal resolution. Two new human nuclear factors, L10K and C11orf98, were also identified. Comparative structural analyses reveal that many assembly factors act as successive place holders to control the timing of factor association/dissociation events. They display multi-phasic binding properties for different domains and generate complex binding inter-dependencies as a means to guide the rRNA maturation process towards its mature conformation. Overall, our data reveal that nuclear assembly of human pre-60S particles is generally hierarchical with short branch pathways, and a few factors display specific roles as rRNA chaperones by confining rRNA helices locally to facilitate their folding, such as the C-terminal domain of SDAD1.

A co-transcriptional ribosome assembly checkpoint controls nascent large ribosomal subunit maturation

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.

Caught in the act-Visualizing ribonucleases during eukaryotic ribosome assembly.

Ribosomes are essential macromolecular machines responsible for translating the genetic information encoded in mRNAs into proteins. Ribosomes are composed of ribosomal RNAs and proteins (rRNAs and RPs) and the rRNAs fulfill both catalytic and architectural functions. Excision of the mature eukaryotic rRNAs from their precursor transcript is achieved through a complex series of endoribonucleolytic cleavages and exoribonucleolytic processing steps that are precisely coordinated with other aspects of ribosome assembly. Many ribonucleases involved in pre-rRNA processing have been identified and pre-rRNA processing pathways are relatively well defined. However, momentous advances in cryo-electron microscopy have recently enabled structural snapshots of various pre-ribosomal particles from budding yeast (Saccharomyces cerevisiae) and human cells to be captured and, excitingly, these structures not only allow pre-rRNAs to be observed before and after cleavage events, but also enable ribonucleases to be visualized on their target RNAs. These structural views of pre-rRNA processing in action allow a new layer of understanding of rRNA maturation and how it is coordinated with other aspects of ribosome assembly. They illuminate mechanisms of target recognition by the diverse ribonucleases involved and reveal how the cleavage/processing activities of these enzymes are regulated. In this review, we discuss the new insights into pre-rRNA processing gained by structural analyses and the growing understanding of the mechanisms of ribonuclease regulation. This article is categorized under: Translation > Ribosome Biogenesis RNA Processing > rRNA Processing.

The small nucleolar RNA SnoR28 regulates plant growth and development by directing rRNA maturation.

Small nucleolar RNAs (snoRNAs) are noncoding RNAs (ncRNAs) that guide chemical modifications of structural RNAs, which are essential for ribosome assembly and function in eukaryotes. Although numerous snoRNAs have been identified in plants by high-throughput sequencing, the biological functions of most of these snoRNAs remain unclear. Here, we identified box C/D SnoR28.1s as important regulators of plant growth and development by screening a CRISPR/Cas9-generated ncRNA deletion mutant library in Arabidopsis thaliana. Deletion of the SnoR28.1 locus, which contains a cluster of three genes producing SnoR28.1s, resulted in defects in root and shoot growth. SnoR28.1s guide 2'-O-ribose methylation of 25S rRNA at G2396. SnoR28.1s facilitate proper and efficient pre-rRNA processing, as the SnoR28.1 deletion mutants also showed impaired ribosome assembly and function, which may account for the growth defects. SnoR28 contains a 7-bp antisense box, which is required for 2'-O-ribose methylation of 25S rRNA at G2396, and an 8-bp extra box that is complementary to a nearby rRNA methylation site and is partially responsible for methylation of G2396. Both of these motifs are required for proper and efficient pre-rRNA processing. Finally, we show that SnoR28.1s genetically interact with HIDDEN TREASURE2 and NUCLEOLIN1. Our results advance our understanding of the roles of snoRNAs in Arabidopsis.

Eukaryotic Ribosome assembly and Nucleocytoplasmic Transport.

The process of eukaryotic ribosome assembly stretches across the nucleolus, the nucleoplasm and the cytoplasm, and therefore relies on efficient nucleocytoplasmic transport. In yeast, the import machinery delivers ~140,000 ribosomal proteins every minute to the nucleus for ribosome assembly. At the same time, the export machinery facilitates translocation of ~2000 pre-ribosomal particles every minute through ~200 nuclear pore complexes (NPC) into the cytoplasm. Eukaryotic ribosome assembly also requires >200 conserved assembly factors, which transiently associate with pre-ribosomal particles. Their site(s) of action on maturing pre-ribosomes are beginning to be elucidated. In this chapter, we outline protocols that enable rapid biochemical isolation of pre-ribosomal particles for single particle cryo-electron microscopy (cryo-EM) and in vitro reconstitution of nuclear transport processes. We discuss cell-biological and genetic approaches to investigate how the ribosome assembly and the nucleocytoplasmic transport machineries collaborate to produce functional ribosomes.

The Arabidopsis 2'-O-Ribose-Methylation and Pseudouridylation Landscape of rRNA in Comparison to Human and Yeast.

Eukaryotic ribosome assembly starts in the nucleolus, where the ribosomal DNA (rDNA) is transcribed into the 35S pre-ribosomal RNA (pre-rRNA). More than two-hundred ribosome biogenesis factors (RBFs) and more than two-hundred small nucleolar RNAs (snoRNA) catalyze the processing, folding and modification of the rRNA in Arabidopsis thaliana. The initial pre-ribosomal 90S complex is formed already during transcription by association of ribosomal proteins (RPs) and RBFs. In addition, small nucleolar ribonucleoprotein particles (snoRNPs) composed of snoRNAs and RBFs catalyze the two major rRNA modification types, 2′-O-ribose-methylation and pseudouridylation. Besides these two modifications, rRNAs can also undergo base methylations and acetylation. However, the latter two modifications have not yet been systematically explored in plants. The snoRNAs of these snoRNPs serve as targeting factors to direct modifications to specific rRNA regions by antisense elements. Today, hundreds of different sites of modifications in the rRNA have been described for eukaryotic ribosomes in general. While our understanding of the general process of ribosome biogenesis has advanced rapidly, the diversities appearing during plant ribosome biogenesis is beginning to emerge. Today, more than two-hundred RBFs were identified by bioinformatics or biochemical approaches, including several plant specific factors. Similarly, more than two hundred snoRNA were predicted based on RNA sequencing experiments. Here, we discuss the predicted and verified rRNA modification sites and the corresponding identified snoRNAs on the example of the model plant Arabidopsis thaliana. Our summary uncovers the plant modification sites in comparison to the human and yeast modification sites.

Genetics animates structure: leveraging genetic interactions to study the dynamics of ribosome biogenesis.

The assembly of eukaryotic ribosomes follows an assembly line-like pathway in which numerous trans-acting biogenesis factors act on discrete pre-ribosomal intermediates to progressively shape the nascent subunits into their final functional architecture. Recent advances in cryo-electron microscopy have led to high-resolution structures of many pre-ribosomal intermediates; however, these static snapshots do not capture the dynamic transitions between these intermediates. To this end, molecular genetics can be leveraged to reveal how the biogenesis factors drive these dynamic transitions. Here, we briefly review how we recently used the deletion of BUD23 (bud23∆) to understand its role in the assembly of the ribosomal small subunit. The strong growth defect of bud23∆ mutants places a selective pressure on yeast cells for the occurrence of extragenic suppressors that define a network of functional interactions among biogenesis factors. Mapping these suppressing mutations to recently published structures of pre-ribosomal complexes allowed us to contextualize these suppressing mutations and derive a detailed model in which Bud23 promotes a critical transition event to facilitate folding of the central pseudoknot of the small subunit. This mini-review highlights how genetics can be used to understand the dynamics of complex structures, such as the maturing ribosome.

Structural Insights into the Mechanism of Mitoribosomal Large Subunit Biogenesis

In contrast to the bacterial translation machinery, mitoribosomes and mitochondrial translation factors are highly divergent in terms of composition and architecture. There is increasing evidence that the biogenesis of mitoribosomes is an intricate pathway, involving many assembly factors. To better understand this process, we investigated native assembly intermediates of the mitoribosomal large subunit from the human parasite Trypanosoma brucei using cryo-electron microscopy. We identify 28 assembly factors, 6 of which are homologous to bacterial and eukaryotic ribosome assembly factors. They interact with the partially folded rRNA by specifically recognizing functionally important regions such as the peptidyltransferase center. The architectural and compositional comparison of the assembly intermediates indicates a stepwise modular assembly process, during which the rRNA folds toward its mature state. During the process, several conserved GTPases and a helicase form highly intertwined interaction networks that stabilize distinct assembly intermediates. The presented structures provide general insights into mitoribosomal maturation.

Structural snapshots of human pre-60S ribosomal particles before and after nuclear export

Ribosome biogenesis is an elaborate and energetically expensive program that involve two hundred protein factors in eukaryotes. Nuclear export of pre-ribosomal particles is one central step which also serves as an internal structural checkpoint to ensure the proper completion of nuclear assembly events. Here we present four structures of human pre-60S particles isolated through a nuclear export factor NMD3, representing assembly stages immediately before and after nuclear export. These structures reveal locations of a dozen of human factors, including an uncharacterized factor TMA16 localized between the 5S RNA and the P0 stalk. Comparison of these structures shows a progressive maturation for the functional regions, such as peptidyl transferase centre and peptide exit tunnel, and illustrate a sequence of factor-assisted rRNA maturation events. These data facilitate our understanding of the global conservation of ribosome assembly in eukaryotes and species-specific features of human assembly factors.

SnoRNPs: Functions in Ribosome Biogenesis.

Ribosomes are perhaps the most critical macromolecular machine as they are tasked with carrying out protein synthesis in cells. They are incredibly complex structures composed of protein components and heavily chemically modified RNAs. The task of assembling mature ribosomes from their component parts consumes a massive amount of energy and requires greater than 200 assembly factors. Among the most critical of these are small nucleolar ribonucleoproteins (snoRNPs). These are small RNAs complexed with diverse sets of proteins. As suggested by their name, they localize to the nucleolus, the site of ribosome biogenesis. There, they facilitate multiple roles in ribosomes biogenesis, such as pseudouridylation and 2′-O-methylation of ribosomal (r)RNA, guiding pre-rRNA processing, and acting as molecular chaperones. Here, we reviewed their activity in promoting the assembly of ribosomes in eukaryotes with regards to chemical modification and pre-rRNA processing.

Co-Assembly of 40S and 60S Ribosomal Proteins in Early Steps of Eukaryotic Ribosome Assembly.

In eukaryotes three of the four ribosomal RNA (rRNA) molecules are transcribed as a long precursor that is processed into mature rRNAs concurrently with the assembly of ribosomal subunits. However, the relative timing of association of ribosomal proteins with the ribosomal precursor particles and the cleavage of the precursor rRNA into the subunit-specific moieties is not known. To address this question, we searched for ribosomal precursors containing components from both subunits. Particles containing specific ribosomal proteins were targeted by inducing synthesis of epitope-tagged ribosomal proteins followed by pull-down with antibodies targeting the tagged protein. By identifying other ribosomal proteins and internal rRNA transcribed spacers (ITS1 and ITS2) in the immuno-purified ribosomal particles, we showed that eS7/S7 and uL4/L4 bind to nascent ribosomes prior to the separation of 40S and 60S specific segments, while uS4/S9, uL22, and eL13/L13 are bound after, or simultaneously with, the separation. Thus, the incorporation of ribosomal proteins from the two subunits begins as a co-assembly with a single rRNA molecule, but is finished as an assembly onto separate precursors for the two subunits.

Cryo-EM structure of an early precursor of large ribosomal subunit reveals a half-assembled intermediate

Assembly of eukaryotic ribosome is a complicated and dynamic process that involves a series of intermediates. It is unknown how the highly intertwined structure of 60S large ribosomal subunits is established. Here, we report the structure of an early nucleolar pre-60S ribosome determined by cryo-electron microscopy at 3.7 Å resolution, revealing a half-assembled subunit. Domains I, II and VI of 25S/5.8S rRNA pack tightly into a native-like substructure, but domains III, IV and V are not assembled. The structure contains 12 assembly factors and 19 ribosomal proteins, many of which are required for early processing of large subunit rRNA. The Brx1-Ebp2 complex would interfere with the assembly of domains IV and V. Rpf1, Mak16, Nsa1 and Rrp1 form a cluster that consolidates the joining of domains I and II. Our structure reveals a key intermediate on the path to establishing the global architecture of 60S subunits.

Modular assembly of the nucleolar pre-60S ribosomal subunit.

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.

Eukaryotic ribosome assembly, transport and quality control.

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.

The cryo-EM structure of YjeQ bound to the 30S subunit suggests a fidelity checkpoint function for this protein in ribosome assembly

Recent work suggests that bacterial YjeQ (RsgA) participates in the late stages of assembly of the 30S subunit and aids the assembly of the decoding center but also binds the mature 30S subunit with high affinity. To determine the function and mechanisms of YjeQ in the context of the mature subunit, we determined the cryo-EM structure of the fully assembled 30S subunit in complex with YjeQ at 5.8-Å resolution. We found that binding of YjeQ stabilizes helix 44 into a conformation similar to that adopted by the subunit during proofreading. This finding indicates that, along with acting as an assembly factor, YjeQ has a role as a checkpoint protein, consisting of testing the proofreading ability of the 30S subunit. The structure also informs the mechanism by which YjeQ implements the release from the 30S subunit of a second assembly factor, called RbfA. Finally, it reveals how the 30S subunit stimulates YjeQ GTPase activity and leads to release of the protein. Checkpoint functions have been described for eukaryotic ribosome assembly factors; however, this work describes an example of a bacterial assembly factor that tests a specific translation mechanism of the 30S subunit.

Resolving Late-Stage Intermediates of Eukaryotic Ribosome Assembly

The assembly of eukaryotic ribosomes requires about 200 assembly factors promoting RNA modification, folding, cleavage, and ribosomal protein association. In this issue of Structure, Johnson etal. (2017) report structures of several late-stage intermediates of pre-40S ribosomal subunit assembly. This work provides detailed testable insights into assembly factor function.

Nop9 binds the central pseudoknot region of 18S rRNA.

The assembly of eukaryotic ribosomes requires numerous factors that transiently associate with evolving pre-ribosomal particles. The Pumilio repeat-containing protein Nop9 briefly associates with the 90S pre-ribosome during its co-transcriptional assembly. Here, we show that Nop9 specifically binds an 11-nucleotide sequence of 18S rRNA that forms the 3΄ side of the central pseudoknot and helix 28 in the mature subunit. Crystal structures of Nop9 in the free and RNA-bound states reveal a new type of Pumilio repeat protein with a distinct structure, target sequence and RNA-binding mode. Nop9 contains 10 Pumilio repeats arranged into a U-shaped scaffold. The target RNA is recognized by two stretches of repeats in a bipartite manner, and three central bases are unrecognized as a result of the degeneracy of repeats 6 and 7. Our data suggest that Nop9 regulates the folding of 18S rRNA at early assembly stages of 90S.

Potent, Reversible, and Specific Chemical Inhibitors of Eukaryotic Ribosome Biogenesis

SummaryAll cellular proteins are synthesized by ribosomes, whose biogenesis in eukaryotes is a complex multi-step process completed within minutes. Several chemical inhibitors of ribosome function are available and used as tools or drugs. By contrast, we lack potent validated chemical probes to analyze the dynamics of eukaryotic ribosome assembly. Here, we combine chemical and genetic approaches to discover ribozinoindoles (or Rbins), potent and reversible triazinoindole-based inhibitors of eukaryotic ribosome biogenesis. Analyses of Rbin sensitivity and resistance conferring mutations in fission yeast, along with biochemical assays with recombinant proteins, provide evidence that Rbins’ physiological target is Midasin, an essential ∼540-kDa AAA+ (ATPases associated with diverse cellular activities) protein. Using Rbins to acutely inhibit or activate Midasin function, in parallel experiments with inhibitor-sensitive or inhibitor-resistant cells, we uncover Midasin’s role in assembling Nsa1 particles, nucleolar precursors of the 60S subunit. Together, our findings demonstrate that Rbins are powerful probes for eukaryotic ribosome assembly.

The N-terminal extension of yeast ribosomal protein L8 is involved in two major remodeling events during late nuclear stages of 60S ribosomal subunit assembly.

Assaying effects on pre-rRNA processing and ribosome assembly upon depleting individual ribosomal proteins (r-proteins) provided an initial paradigm for assembly of eukaryotic ribosomes in vivo—that each structural domain of ribosomal subunits assembles in a hierarchical fashion. However, two features suggest that a more complex pathway may exist: (i) Some r-proteins contain extensions that reach long distances across ribosomes to interact with multiple rRNA domains as well as with other r-proteins. (ii) Individual r-proteins may assemble in a stepwise fashion. For example, the globular domain of an r-protein might assemble separately from its extensions. Thus, these extensions might play roles in assembly that could not be revealed by depleting the entire protein. Here, we show that deleting or mutating extensions of r-proteins L7 (uL30) and L35 (uL29) from yeast reveal important roles in early and middle steps during 60S ribosomal subunit biogenesis. Detailed analysis of the N-terminal terminal extension of L8 (eL8) showed that it is necessary for late nuclear stages of 60S subunit assembly involving two major remodeling events: removal of the ITS2 spacer; and reorganization of the central protuberance (CP) containing 5S rRNA and r-proteins L5 (uL18) and L11 (uL5). Mutations in the L8 extension block processing of 7S pre-rRNA, prevent release of assembly factors Rpf2 and Rrs1 from pre-ribosomes, which is required for rotation of the CP, and block association of Sda1, the Rix1 complex, and the Rea1 ATPase involved in late steps of remodeling.