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.

Eukaryotic Ribosome Assembly.

Ribosomes, which synthesize the proteins of a cell, comprise ribosomal RNA and ribosomal proteins, which coassemble hierarchically during a process termed ribosome biogenesis. Historically, biochemical and molecular biology approaches have revealed how preribosomal particles form and mature in consecutive steps, starting in the nucleolus and terminating after nuclear export into the cytoplasm. However, only recently, due to the revolution in cryo-electron microscopy, could pseudoatomic structures of different preribosomal particles be obtained. Together with in vitro maturation assays, these findings shed light on how nascent ribosomes progress stepwise along a dynamic biogenesis pathway. Preribosomes assemble gradually, chaperoned by a myriad of assembly factors and small nucleolar RNAs, before they reach maturity and enter translation. This information will lead to a better understanding of how ribosome synthesis is linked to other cellular pathways in humans and how it can cause diseases, including cancer, if disturbed.

Transient Protein-RNA Interactions Guide Nascent Ribosomal RNA Folding.

Ribosome assembly is an efficient but complex and heterogeneous process during which ribosomal proteins assemble on the nascent rRNA during transcription. Understanding how the interplay between nascent RNA folding and protein binding determines the fate of transcripts remains a major challenge. Here, using single-molecule fluorescence microscopy, we follow assembly of the entire 3' domain of the bacterial small ribosomal subunit in real time. We find that co-transcriptional rRNA folding is complicated by the formation of long-range RNA interactions and that r-proteins self-chaperone the rRNA folding process prior to stable incorporation into a ribonucleoprotein (RNP) complex. Assembly is initiated by transient rather than stable protein binding, and the protein-RNA binding dynamics gradually decrease during assembly. This work questions the paradigm of strictly sequential and cooperative ribosome assembly and suggests that transient binding of RNA binding proteins to cellular RNAs could provide a general mechanism to shape nascent RNA folding during RNP assembly.

K29-linked free polyubiquitin chains affect ribosome biogenesis and direct ribosomal proteins to the intranuclear quality control compartment.

Ribosome assembly requires precise coordination between the production and assembly of ribosomal components. Mutations in ribosomal proteins that inhibit the assembly process or ribosome function are often associated with ribosomopathies, some of which are linked to defects in proteostasis. In this study, we examine the interplay between several yeast proteostasis enzymes, including deubiquitylases (DUBs) Ubp2 and Ubp14, and E3 ligases Ufd4 and Hul5, and we explore their roles in the regulation of the cellular levels of K29-linked unanchored polyubiquitin (polyUb) chains. Accumulating K29-linked unanchored polyUb chains associate with maturing ribosomes to disrupt their assembly, activate the ribosome assembly stress response (RASTR), and lead to the sequestration of ribosomal proteins at the intranuclear quality control compartment (INQ). These findings reveal the physiological relevance of INQ and provide insights into mechanisms of cellular toxicity associated with ribosomopathies.

Modular Assembly of the Bacterial Large Ribosomal Subunit.

The ribosome is a complex macromolecular machine and serves as an ideal system for understanding biological macromolecular assembly. Direct observation of ribosome assembly in vivo is difficult, as few intermediates have been isolated and thoroughly characterized. Herein, we deploy a genetic system to starve cells of an essential ribosomal protein, which results in the accumulation of assembly intermediates that are competent for maturation. Quantitative mass spectrometry and single-particle cryo-electron microscopy reveal 13 distinct intermediates, which were each resolved to ∼4-5 Å resolution and could be placed in an assembly pathway. We find that ribosome biogenesis is a parallel process, that blocks of structured rRNA and proteins assemble cooperatively, and that the entire process is dynamic and can be "re-routed" through different pathways as needed. This work reveals the complex landscape of ribosome assembly in vivo and provides the requisite tools to characterize additional assembly pathways for ribosomes and other macromolecular machines.

Nuclear organization.

This article discusses three reviews on the theme of nuclear organization.

Functions of ribosomal proteins in assembly of eukaryotic ribosomes in vivo.

The proteome of cells is synthesized by ribosomes, complex ribonucleoproteins that in eukaryotes contain 79-80 proteins and four ribosomal RNAs (rRNAs) more than 5,400 nucleotides long. How these molecules assemble together and how their assembly is regulated in concert with the growth and proliferation of cells remain important unanswered questions. Here, we review recently emerging principles to understand how eukaryotic ribosomal proteins drive ribosome assembly in vivo. Most ribosomal proteins assemble with rRNA cotranscriptionally; their association with nascent particles is strengthened as assembly proceeds. Each subunit is assembled hierarchically by sequential stabilization of their subdomains. The active sites of both subunits are constructed last, perhaps to prevent premature engagement of immature ribosomes with active subunits. Late-assembly intermediates undergo quality-control checks for proper function. Mutations in ribosomal proteins that affect mostly late steps lead to ribosomopathies, diseases that include a spectrum of cell type-specific disorders that often transition from hypoproliferative to hyperproliferative growth.

Snapshots of native pre-50S ribosomes reveal a biogenesis factor network and evolutionary specialization.

Ribosome biogenesis is a fundamental multi-step cellular process that culminates in the formation of ribosomal subunits, whose production and modification are regulated by numerous biogenesis factors. In this study, we analyze physiologic prokaryotic ribosome biogenesis by isolating bona fide pre-50S subunits from an Escherichia coli strain with the biogenesis factor ObgE, affinity tagged at its native gene locus. Our integrative structural approach reveals a network of interacting biogenesis factors consisting of YjgA, RluD, RsfS, and ObgE on the immature pre-50S subunit. In addition, our study provides mechanistic insight into how the GTPase ObgE, in concert with other biogenesis factors, facilitates the maturation of the 50S functional core and reveals both conserved and divergent evolutionary features of ribosome biogenesis between prokaryotes and eukaryotes.

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.

Construction of the Central Protuberance and L1 Stalk during 60S Subunit Biogenesis.

Ribosome assembly is driven by numerous assembly factors, including the Rix1 complex and the AAA ATPase Rea1. These two assembly factors catalyze 60S maturation at two distinct states, triggering poorly understood large-scale structural transitions that we analyzed by cryo-electron microscopy. Two nuclear pre-60S intermediates were discovered that represent previously unknown states after Rea1-mediated removal of the Ytm1-Erb1 complex and reveal how the L1 stalk develops from a pre-mature nucleolar to a mature-like nucleoplasmic state. A later pre-60S intermediate shows how the central protuberance arises, assisted by the nearby Rix1-Rea1 machinery, which was solved in its pre-ribosomal context to molecular resolution. This revealed a Rix12-Ipi32 tetramer anchored to the pre-60S via Ipi1, strategically positioned to monitor this decisive remodeling. These results are consistent with a general underlying principle that temporarily stabilized immature RNA domains are successively remodeled by assembly factors, thereby ensuring failsafe assembly progression.

Making ends meet: a universal driver of large ribosomal subunit biogenesis.

A common aspect of ribosome assembly, conserved across all domains of life, is the establishment of connections between the 5' and 3' ends of the large subunit (LSU) ribosomal RNA (rRNA) to initiate rRNA domain compaction and subunit assembly. We discuss the diverse mechanisms employed in different organisms to accomplish this important event.

Compartmentalization of RNA Degradosomes in Bacteria Controls Accessibility to Substrates and Ensures Concerted Degradation of mRNA to Nucleotides.

RNA degradosomes are multienzyme complexes composed of ribonucleases, RNA helicases, and metabolic enzymes. RNase E-based degradosomes are widespread in Proteobacteria. The Escherichia coli RNA degradosome is sequestered from transcription in the nucleoid and translation in the cytoplasm by localization to the inner cytoplasmic membrane, where it forms short-lived clusters that are proposed to be sites of mRNA degradation. In Caulobacter crescentus, RNA degradosomes localize to ribonucleoprotein condensates in the interior of the cell [bacterial ribonucleoprotein-bodies (BR-bodies)], which have been proposed to drive the concerted degradation of mRNA to nucleotides. The turnover of mRNA in growing cells is important for maintaining pools of nucleotides for transcription and DNA replication.Membrane attachment of the E. coli RNA degradosome is necessary to avoid wasteful degradation of intermediates in ribosome assembly. Sequestering RNA degradosomes to C. crescentus BR-bodies, which exclude structured RNA, could have a similar role in protecting intermediates in ribosome assembly from degradation.

Thermophile 90S Pre-ribosome Structures Reveal the Reverse Order of Co-transcriptional 18S rRNA Subdomain Integration.

Eukaryotic ribosome biogenesis involves RNA folding and processing that depend on assembly factors and small nucleolar RNAs (snoRNAs). The 90S (SSU-processome) is the earliest pre-ribosome structurally analyzed, which was suggested to assemble stepwise along the growing pre-rRNA from 5' > 3', but this directionality may not be accurate. Here, by analyzing the structure of a series of 90S assembly intermediates from Chaetomium thermophilum, we discover a reverse order of 18S rRNA subdomain incorporation. Large parts of the 18S rRNA 3' and central domains assemble first into the 90S before the 5' domain is integrated. This final incorporation depends on a contact between a heterotrimer Enp2-Bfr2-Lcp5 recruited to the flexible 5' domain and Kre33, which reconstitutes the Kre33-Enp-Brf2-Lcp5 module on the compacted 90S. Keeping the 5' domain temporarily segregated from the 90S scaffold could provide extra time to complete the multifaceted 5' domain folding, which depends on a distinct set of snoRNAs and processing factors.

Structural Visualization of the Formation and Activation of the 50S Ribosomal Subunit during In Vitro Reconstitution.

The assembly of ribosomal subunits is an essential prerequisite for protein biosynthesis in all domains of life. Although biochemical and biophysical approaches have advanced our understanding of ribosome assembly, our mechanistic comprehension of this process is still limited. Here, we perform an in vitro reconstitution of the Escherichia coli 50S ribosomal subunit. Late reconstitution products were subjected to high-resolution cryo-electron microscopy and multiparticle refinement analysis to reconstruct five distinct precursors of the 50S subunit with 4.3-3.8 Å resolution. These assembly intermediates define a progressive maturation pathway culminating in a late assembly particle, whose structure is more than 96% identical to a mature 50S subunit. Our structures monitor the formation and stabilization of structural elements in a nascent particle in unprecedented detail and identify the maturation of the rRNA-based peptidyl transferase center as the final critical step along the 50S assembly pathway.

A roadmap for rRNA folding and assembly during transcription.

Ribonucleoprotein (RNP) assembly typically begins during transcription when folding of the newly synthesized RNA is coupled with the recruitment of RNA-binding proteins (RBPs). Upon binding, the proteins induce structural rearrangements in the RNA that are crucial for the next steps of assembly. Focusing primarily on bacterial ribosome assembly, we discuss recent work showing that early RNA-protein interactions are more dynamic than previously supposed, and remain so, until sufficient proteins are recruited to each transcript to consolidate an entire domain of the RNP. We also review studies showing that stable assembly of an RNP competes against modification and processing of the RNA. Finally, we discuss how transcription sets the timeline for competing and cooperative RNA-RBP interactions that determine the fate of the nascent RNA. How this dance is coordinated is the focus of this review.

The ATPase Fap7 Tests the Ability to Carry Out Translocation-like Conformational Changes and Releases Dim1 during 40S Ribosome Maturation.

Late in their maturation, nascent small (40S) ribosomal subunits bind 60S subunits to produce 80S-like ribosomes. Because of the analogy of this translation-like cycle to actual translation, and because 80S-like ribosomes do not produce any protein, it has been suggested that this represents a quality control mechanism for subunit functionality. Here we use genetic and biochemical experiments to show that the essential ATPase Fap7 promotes formation of the rotated state, a key intermediate in translocation, thereby releasing the essential assembly factor Dim1 from pre-40S subunits. Bypassing this quality control step produces defects in reading frame maintenance. These results show how progress in the maturation cascade is linked to a test for a key functionality of 40S ribosomes: their ability to translocate the mRNA⋅tRNA pair. Furthermore, our data demonstrate for the first time that the translation-like cycle is a quality control mechanism that ensures the fidelity of the cellular ribosome pool.

Mitoribosomal small subunit maturation involves formation of initiation-like complexes.

Mitochondrial ribosomes (mitoribosomes) play a central role in synthesizing mitochondrial inner membrane proteins responsible for oxidative phosphorylation. Although mitoribosomes from different organisms exhibit considerable structural variations, recent insights into mitoribosome assembly suggest that mitoribosome maturation follows common principles and involves a number of conserved assembly factors. To investigate the steps involved in the assembly of the mitoribosomal small subunit (mt-SSU) we determined the cryoelectron microscopy structures of middle and late assembly intermediates of the Trypanosoma brucei mitochondrial small subunit (mt-SSU) at 3.6- and 3.7-Å resolution, respectively. We identified five additional assembly factors that together with the mitochondrial initiation factor 2 (mt-IF-2) specifically interact with functionally important regions of the rRNA, including the decoding center, thereby preventing premature mRNA or large subunit binding. Structural comparison of assembly intermediates with mature mt-SSU combined with RNAi experiments suggests a noncanonical role of mt-IF-2 and a stepwise assembly process, where modular exchange of ribosomal proteins and assembly factors together with mt-IF-2 ensure proper 9S rRNA folding and protein maturation during the final steps of assembly.

Cooperative role of AtRsmD and AtRimM proteins in modification and maturation of 16S rRNA in plastids.

Chloroplast pre-ribosomal RNA (rRNA) undergoes maturation, which is critical for ribosome assembly. While the central and auxiliary factors in rRNA maturation have been elucidated in bacteria, their mode of action remains largely unexplored in chloroplasts. We now reveal chloroplast-specific factors involved in 16S rRNA maturation, Arabidopsis thaliana orthologs of bacterial RsmD methyltransferase (AtRsmD) and ribosome maturation factor RimM (AtRimM). A forward genetic screen aimed to find suppressors of the Arabidopsis yellow variegated 2 (var2) mutant defective in photosystem II quality control found a causal nonsense mutation in AtRsmD. The substantially impaired 16S rRNA maturation and translation due to the mutation rescued the leaf variegation phenotype by lowering the levels of chloroplast-encoded proteins, including photosystem II core proteins, in var2. The subsequent co-immunoprecipitation coupled with mass spectrometry analyses and bimolecular fluorescence complementation assay found that AtRsmD interacts with AtRimM. Consistent with their interaction, loss of AtRimM also considerably impairs 16S rRNA maturation with decelerated m2 G915 modification in 16S rRNA catalyzed by AtRsmD. The atrimM mutation also rescued var2 mutant phenotypes, corroborating the functional interplay between AtRsmD and AtRimM towards modification and maturation of 16S rRNA and chloroplast proteostasis. The maturation and post-transcriptional modifications of rRNA are critical to assembling ribosomes responsible for protein translation. Here, we revealed that the cooperative regulation of 16S rRNA m2 G915 modifications by AtRsmD methyltransferase and ribosome assembly factor AtRimM contributes to 16S rRNA maturation, ribosome assembly, and proteostasis in chloroplasts.

The modifying enzyme Tsr3 establishes the hierarchy of Rio kinase binding in 40S ribosome assembly.

Ribosome assembly is an intricate process, which in eukaryotes is promoted by a large machinery comprised of over 200 assembly factors (AFs) that enable the modification, folding, and processing of the ribosomal RNA (rRNA) and the binding of the 79 ribosomal proteins. While some early assembly steps occur via parallel pathways, the process overall is highly hierarchical, which allows for the integration of maturation steps with quality control processes that ensure only fully and correctly assembled subunits are released into the translating pool. How exactly this hierarchy is established, in particular given that there are many instances of RNA substrate "handover" from one highly related AF to another, remains to be determined. Here we have investigated the role of Tsr3, which installs a universally conserved modification in the P-site of the small ribosomal subunit late in assembly. Our data demonstrate that Tsr3 separates the binding of the Rio kinases, Rio2 and Rio1, with whom it shares a binding site. By binding after Rio2 dissociation, Tsr3 prevents rebinding of Rio2, promoting forward assembly. After rRNA modification is complete, Tsr3 dissociates, thereby allowing for recruitment of Rio1 into its functional site. Inactive Tsr3 blocks Rio1 function, which can be rescued using mutants that bypass the requirement for Rio1 activity. Finally, yeast strains lacking Tsr3 randomize the binding of the two kinases, leading to the release of immature ribosomes into the translating pool. These data demonstrate a role for Tsr3 and its modification activity in establishing a hierarchy for the function of the Rio kinases.

Orchestrating ribosomal RNA folding during ribosome assembly.

Construction of the eukaryotic ribosome is a complex process in which a nascent ribosomal RNA (rRNA) emerging from RNA Polymerase I hierarchically folds into a native three-dimensional structure. Modular assembly of individual RNA domains through interactions with ribosomal proteins and a myriad of assembly factors permit efficient disentanglement of the error-prone RNA folding process. Following these dynamic events, long-range tertiary interactions are orchestrated to compact rRNA. A combination of genetic, biochemical, and structural studies is now providing clues into how a nascent rRNA is transformed into a functional ribosome with high precision. With this essay, we aim to draw attention to the poorly understood process of establishing correct RNA tertiary contacts during ribosome formation.