Ribosome Assembly Intermediates 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.

The RNA helicase Dbp10 coordinates assembly factor association with PTC maturation during ribosome biogenesis.

During ribosome biogenesis a plethora of assembly factors and essential enzymes drive the unidirectional maturation of nascent pre-ribosomal subunits. The DEAD-box RNA helicase Dbp10 is suggested to restructure pre-ribosomal rRNA of the evolving peptidyl-transferase center (PTC) on nucleolar ribosomal 60S assembly intermediates. Here, we show that point mutations within conserved catalytic helicase-core motifs of Dbp10 yield a dominant-lethal growth phenotype. Such dbp10 mutants, which stably associate with pre-60S intermediates, impair pre-60S biogenesis at a nucleolar stage prior to the release of assembly factor Rrp14 and stable integration of late nucleolar factors such as Noc3. Furthermore, the binding of the GTPase Nug1 to particles isolated directly via mutant Dbp10 bait proteins is specifically inhibited. The N-terminal domain of Nug1 interacts with Dbp10 and the methyltransferase Spb1, whose pre-60S incorporation is also reduced in absence of functional Dbp10 resulting in decreased methylation of 25S rRNA nucleotide G2922. Our data suggest that Dbp10's helicase activity generates the necessary framework for assembly factor docking thereby permitting PTC rRNA methylation and the progression of pre-60S maturation.

RNA Post-transcriptional Modifications of an Early-Stage Large-Subunit Ribosomal Intermediate.

Protein production by ribosomes is fundamental to life, and proper assembly of the ribosome is required for protein production. The RNA, which is post-transcriptionally modified, provides the platform for ribosome assembly. Thus, a complete understanding of ribosome assembly requires the determination of the RNA post-transcriptional modifications in all of the ribosome assembly intermediates and on each pathway. There are 26 RNA post-transcriptional modifications in 23S RNA of the mature Escherichia coli (E. coli) large ribosomal subunit. The levels of these modifications have been investigated extensively only for a small number of large subunit intermediates and under a limited number of cellular and environmental conditions. In this study, we determined the level of incorporations of 2-methyl adenosine, 3-methyl pseudouridine, 5-hydroxycytosine, and seven pseudouridines in an early-stage E. coli large-subunit assembly intermediate with a sedimentation coefficient of 27S. The 27S intermediate is one of three large subunit intermediates accumulated in E. coli cells lacking the DEAD-box RNA helicase DbpA and expressing the helicase inactive R331A DbpA construct. The majority of the investigated modifications are incorporated into the 27S large subunit intermediate to similar levels to those in the mature 50S large subunit, indicating that these early modifications or the enzymes that incorporate them play important roles in the initial events of large subunit ribosome assembly.

Purification and Characterization of Authentic 30S Ribosomal Precursors Induced by Heat Shock.

Ribosome biogenesis is a complex and multistep process that depends on various assembly factors. To understand this process and identify the ribosome assembly intermediates, most studies have set out to delete or deplete these assembly factors. Instead, we took advantage of the impact of heat stress (45 °C) on the late stages of the biogenesis of the 30S ribosomal subunit to explore authentic precursors. Under these conditions, reduced levels of the DnaK chaperone proteins devoted to ribosome assembly lead to the transient accumulation of 21S ribosomal particles, which are 30S precursors. We constructed strains with different affinity tags on one early and one late 30S ribosomal protein and purified the 21S particles that form under heat shock. A combination of relative quantification using mass spectrometry-based proteomics and cryo-electron microscopy (cryo-EM) was then used to determine their protein contents and structures.

Attachment of the RNA degradosome to the bacterial inner cytoplasmic membrane prevents wasteful degradation of rRNA in ribosome assembly intermediates

RNA processing and degradation shape the transcriptome by generating stable molecules that are necessary for translation (rRNA and tRNA) and by facilitating the turnover of mRNA, which is necessary for the posttranscriptional control of gene expression. In bacteria and the plant chloroplast, RNA degradosomes are multienzyme complexes that process and degrade RNA. In many bacterial species, the endoribonuclease RNase E is the central component of the RNA degradosome. RNase E-based RNA degradosomes are inner membrane proteins in a large family of gram-negative bacteria (β- and γ-Proteobacteria). Until now, the reason for membrane localization was not understood. Here, we show that a mutant strain of Escherichia coli, in which the RNA degradosome is localized to the interior of the cell, has high levels of 20S and 40S particles that are defective intermediates in ribosome assembly. These particles have aberrant protein composition and contain rRNA precursors that have been cleaved by RNase E. After RNase E cleavage, rRNA fragments are degraded to nucleotides by exoribonucleases. In vitro, rRNA in intact ribosomes is resistant to RNase E cleavage, whereas protein-free rRNA is readily degraded. We conclude that RNA degradosomes in the nucleoid of the mutant strain interfere with cotranscriptional ribosome assembly. We propose that membrane-attached RNA degradosomes in wild-type cells control the quality of ribosome assembly after intermediates are released from the nucleoid. That is, the compact structure of mature ribosomes protects rRNA against cleavage by RNase E. Turnover of a proportion of intermediates in ribosome assembly explains slow growth of the mutant strain. Competition between mRNA and rRNA degradation could be the cause of slower mRNA degradation in the mutant strain. We conclude that attachment of the RNA degradosome to the bacterial inner cytoplasmic membrane prevents wasteful degradation of rRNA precursors, thus explaining the reason for conservation of membrane-attached RNA degradosomes throughout the β- and γ-Proteobacteria.

Additional principles that govern the release of pre-ribosomes from the nucleolus into the nucleoplasm in yeast.

During eukaryotic ribosome biogenesis, pre-ribosomes travel from the nucleolus, where assembly is initiated, to the nucleoplasm and then are exported to the cytoplasm, where assembly concludes. Although nuclear export of pre-ribosomes has been extensively investigated, the release of pre-ribosomes from the nucleolus is an understudied phenomenon. Initial data indicate that unfolded rRNA interacts in trans with nucleolar components and that, when rRNA folds due to ribosomal protein (RP) binding, the number of trans interactions drops below the threshold necessary for nucleolar retention. To validate and expand on this idea, we performed a bioinformatic analysis of the protein components of the Saccharomyces cerevisiae ribosome assembly pathway. We found that ribosome biogenesis factors (RiBi factors) contain significantly more predicted trans interacting regions than RPs. We also analyzed cryo-EM structures of ribosome assembly intermediates to determine how nucleolar pre-ribosomes differ from post-nucleolar pre-ribosomes, specifically the capacity of RPs, RiBi factors, and rRNA components to interact in trans. We observed a significant decrease in the theoretical trans-interacting capability of pre-ribosomes between nucleolar and post-nucleolar stages of assembly due to the release of RiBi factors from particles and the folding of rRNA. Here, we provide a mechanism for the release of pre-ribosomes from the nucleolus.

Artificial intelligence-assisted cryoEM structure of Bfr2-Lcp5 complex observed in the yeast small subunit processome

Eukaryotic ribosome is maturated through an elaborate process that includes modification, processing and folding of pre-ribosomal RNA (pre-rRNAs) by a series of ribosome assembly intermediates. More than 70 factors participate in the dynamic assembly and disassembly of the small subunit processome (90S) inside nucleolus, leading to the early maturation of small subunit. The 5’ domain of the 18S rRNA is the last to be incorporated into the stable 90S prior to the cleavage of pre-rRNA at the A1 site. This step is facilitated by the Kre33-Enp2-Bfr2-Lcp5 protein module with the participation of the DEAD-box protein Dbp4. Though structures of Kre33 and Enp2 have been modeled in previously observed 90S structures, that of Bfr2-Lcp5 complex remains unavailable. Here, we report an AlphaFold-assisted structure determination of the Bfr2-Lcp5 complex captured in a 3.99 Å − 7.24 Å cryoEM structure of 90S isolated from yeast cells depleted of Pih1, a chaperone protein of the 90S core assembly. The structure model is consistent with the protein-protein interaction results and the secondary structures of recombinant Bfr2 and Bfr2-Lcp5 complex obtained by Circular Dichroism. The Bfr2-Lcp5 complex interaction mimics that of exosome factors Rrp6-Rrp47 and acts to regulate 90S transitions.

Quantitative mining of compositional heterogeneity in cryo-EM datasets of ribosome assembly intermediates

Single-particle cryoelectron microscopy (cryo-EM) offers a unique opportunity to characterize macromolecular structural heterogeneity by virtue of its ability to place distinct particle populations into different groups through computational classification. However, there is a dearth of tools for surveying the heterogeneity landscape, quantitatively analyzing heterogeneous particle populations after classification, deciding how many unique classes are represented by the data, and accurately cross-comparing reconstructions. Here, we develop a workflow that contains discovery and analysis modules to quantitatively mine cryo-EM data for sets of structures with maximal diversity. This workflow was applied to a dataset of E.coli 50S ribosome assembly intermediates, which are characterized by significant structural heterogeneity. We identified more detailed branchpoints in the assembly process and characterized the interactions of an assembly factor with immature intermediates. While the tools described here were developed for ribosome assembly, they should be broadly applicable to the analysis of other heterogeneous cryo-EM datasets.

Mitochondrial RNA quality control in trypanosomes.

Unicellular parasites Trypanosoma brucei spp. cause African human and animal trypanosomiasis, a spectrum of diseases that jeopardize public health and afflict the economy in sub-Saharan Africa. These hemoflagellates are distinguished by a single mitochondrion, which contains a kinetoplast nucleoid composed of DNA and histone-like proteins. Kinetoplast DNA (kDNA) represents a densely packed network of interlinked relaxed circular molecules: a few ~23-kb maxicircles encoding ribosomal RNAs (rRNAs) and proteins, and approximately 5,000 1-kb minicircles bearing guide RNA (gRNA) genes. The transcription start site defines the mRNA's 5' terminus while the primary RNA is remodeled into a monocistronic messenger by 3'-5' exonucleolytic trimming, 5' and 3' end modifications, and, in most cases, by internal U-insertion/deletion editing. Ribosomal and guide RNA precursors are also trimmed, and the processed molecules are uridylated. For 35 years, mRNA editing has attracted a major effort, but more recently the essential pre- and postediting processing and turnover events have been discovered and the key effectors have been identified. Among these, pentatricopeptide repeat (PPR) RNA binding proteins emerged as conduits coupling modifications of mRNA termini with internal sequence changes introduced by editing. Among 39 annotated PPRs, 20 belong to ribosomal subunits or assembly intermediates, four function as polyadenylation factors, a single factor directs 5' mRNA modification, and one protein is found in F1-ATPase. Nuclear and mitochondrial RNases P consist of a single PPR polypeptide, PRORP1 and PROP2, respectively. Here, we review PPR-mediated mitochondrial processes and discuss their potential roles in mRNA maturation, quality control, translational activation, and decay. This article is categorized under: RNA Processing > Capping and 5' End Modifications RNA Processing > 3' End Processing RNA Processing > RNA Editing and Modification.

Quantitative Analysis of Late-Stage Ribosome Assembly with Cryo-EM

Despite the “resolution revolution” in cryo-electron microscopy (cryo-EM), compositionally and conformationally dynamic systems still remain a challenge for traditional cryo-EM workflows. Recently, we developed a novel cryo-EM workflow to harvest all of the information content possible with current computational tools from compositionally and conformationally dynamic ribosome assembly intermediates. This workflow includes modifications of conventional classification strategies--adding steps for iterative sub-classification, a statistically significant difference analysis derived from the signal-to-noise ration from cryo-EM maps, a quantitative strategy for deciding on the number of classes, and a catalog of features to facilitate interpretation of the massive amounts of data gathered from cryo-EM data collection. We developed this strategy to interrogate the process of ribosome assembly. The ribosome is a large, cellular machine responsible for protein biosynthesis. While much is known about how ribosomes translate mRNA into proteins, comparatively little is known about how this machine assembles in the first place. Furthermore, what is known about ribosome assembly focuses on protein binding networks and excludes information about rRNA folding. Recently, the Williamson lab has developed a novel genetic approach to put the r-protein bL17 under the control of a titratable promoter that controls the level of r-protein available to the host (bL17-lim). As a consequence, ribosome assembly intermediates build up and can be analyzed by cryo-EM. Here, we present our latest work applying our workflow to new r-protein limitation strains to delineate the effects that were specific to bL17 depletion from a more general ribosome assembly pathway. We use this data to build a map of interdependence between r-protein binding and rRNA folding downstream of bL17 and build on our knowledge of these interactions to determine a potential mechanism for ribosome assembly in vivo.

SrmB Rescues Trapped Ribosome Assembly Intermediates

RNA helicases play various roles in ribosome biogenesis depending on the ribosome assembly pathway and stress state of the cell. However, it is unclear how most RNA helicases interact with ribosome assembly intermediates or participate in other cell processes to regulate ribosome assembly. SrmB is a DEAD-box helicase that acts early in the ribosome assembly process, although very little is known about its mechanism of action. Here, we use a combined quantitative mass spectrometry/cryo-electron microscopy approach to detail the protein inventory, rRNA modification state, and structures of 40S ribosomal intermediates that form upon SrmB deletion. We show that the binding site of SrmB is unperturbed by SrmB deletion, but the peptidyl transferase center, the uL7/12 stalk, and 30S contact sites all show severe assembly defects. Taking into account existing data on SrmB function and the experiments presented here, we propose several mechanisms by which SrmB could guide assembling particles from kinetic traps to competent subunits during the 50S ribosome assembly process.

Ribosome assembly coming into focus.

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.

MTORC1 Controls the Physical Properties of the Nucleus

The mechanisms that determine the physical properties of the nucleus remain largely unknown. We recently developed Genetically Encoded Multimeric (GEM) nanoparticles to study the physical properties of the cell. GEMs are homomultimeric proteins that self-assemble into bright, stable fluorescent particles of defined size and shape. GEMs are extremely useful as tracer particles to probe the biophysical properties of cells. We added a nuclear localization signal (NLS), to create nuc-GEMs that enabled rapid microrheology experiments within the nucleus. We leveraged this technology for a genetic screen and found that inhibition of mTORC1 dramatically altered the motion of nuc-GEMs, doubling their effective diffusion coefficient. Epistasis experiments revealed that this biophysical effect was due to decreased ribosome production. These results suggest that ribosomal assembly intermediates, which are estimated to occupy five-times more nuclear volume than chromosomes, dominate the physical properties of the nucleus. Thus, mTORC1, a central regulator of metabolism controls the biophysical properties of the nucleus by tuning macromolecular crowding. Using nuc-GEMs in S. cerevisiae, we have discovered a mechanism for tuning the properties of the nucleus that should be universal to all eukaryotes.

High-throughput RNA structure probing reveals critical folding events during early 60S ribosome assembly in yeast

While the protein composition of various yeast 60S ribosomal subunit assembly intermediates has been studied in detail, little is known about ribosomal RNA (rRNA) structural rearrangements that take place during early 60S assembly steps. Using a high-throughput RNA structure probing method, we provide nucleotide resolution insights into rRNA structural rearrangements during nucleolar 60S assembly. Our results suggest that many rRNA-folding steps, such as folding of 5.8S rRNA, occur at a very specific stage of assembly, and propose that downstream nuclear assembly events can only continue once 5.8S folding has been completed. Our maps of nucleotide flexibility enable making predictions about the establishment of protein–rRNA interactions, providing intriguing insights into the temporal order of protein–rRNA as well as long-range inter-domain rRNA interactions. These data argue that many distant domains in the rRNA can assemble simultaneously during early 60S assembly and underscore the enormous complexity of 60S synthesis.

Probing the structure of ribosome assembly intermediates in vivo using DMS and hydroxyl radical footprinting

The assembly of the Escherichia coli ribosome has been widely studied and characterized in vitro. Despite this, ribosome biogenesis in living cells is only partly understood because assembly is coupled with transcription, modification and processing of the pre-ribosomal RNA. We present a method for footprinting and isolating pre-rRNA as it is synthesized in E. coli cells. Pre-rRNA synthesis is synchronized by starvation, followed by nutrient upshift. RNA synthesized during outgrowth is metabolically labeled to facilitate isolation of recent transcripts. Combining this technique with two in vivo RNA probing methods, hydroxyl radical and DMS footprinting, allows the structure of nascent RNA to be probed over time. Together, these can be used to determine changes in the structures of ribosome assembly intermediates as they fold in vivo.

Functional Profiling of Ribosomal RNA Modifications using Quantitative Mass Spectrometry

Ribosomal RNA (rRNA) modifications, including ribose, base methylations and pseudouridines, are important players in the ribosome biogenesis process and translation. Here we introduce mass spectrometry (MS) combined with stable isotope labeling to quantitatively monitor rRNA modifications in bacteria and yeast. By looking at the ribosome assembly intermediates isolated from E. coli cells, this approach enabled us to characterize the timing of individual modification events along the ribosome biogenesis pathway. Additionally, using mutant strains lacking the specific assembly factors RimP and SrmB we observe functional crosstalk between modifications and other assembly steps. More recently, we extended quantitative MS application for studies of rRNA from S. Cerevisiae, where we successfully identified and quantified 80% of over 100 modified nucleosides present in both 18S and 28S RNA. Gaining new mechanistic details, we hope to globally map rRNA modifications on to the existing framework for ribosome assembly, and to explain how modification steps may assist in maintaining the speed and accuracy of the fundamental to life ribosome biogenesis process.

Fluorescence-based monitoring of ribosome assembly landscapes

BackgroundRibosomes and functional complexes of them have been analyzed at the atomic level. Far less is known about the dynamic assembly and degradation events that define the half-life of ribosomes and guarantee their quality control.ResultsWe developed a system that allows visualization of intact ribosomal subunits and assembly intermediates (i.e. assembly landscapes) by convenient fluorescence-based analysis. To this end, we labeled the early assembly ribosomal proteins L1 and S15 with the fluorescent proteins mAzami green and mCherry, respectively, using chromosomal gene insertion. The reporter strain harbors fluorescently labeled ribosomal subunits that operate wild type-like, as shown by biochemical and growth assays. Using genetic and chemical perturbations by depleting genes encoding the ribosomal proteins L3 and S17, respectively, or using ribosome-targeting antibiotics, we provoked ribosomal subunit assembly defects. These defects were readily identified by fluorometric analysis after sucrose density centrifugation in unprecedented resolution.ConclusionThis strategy is useful to monitor and characterize subunit specific assembly defects caused by ribosome-targeting drugs that are currently used and to characterize new molecules that affect ribosome assembly and thereby constitute new classes of antibacterial agents.Electronic supplementary materialThe online version of this article (doi:10.1186/s12867-015-0031-y) contains supplementary material, which is available to authorized users.

Using Hydroxyl Radical Footprinting to Observe Ribosome Assembly Intermediates in vivo

The assembly of the E. coli ribosome small subunit has been widely studied and characterized in vitro. Despite this, ribosome biogenesis in living cells remains poorly understood. This is a very complex process in which an rRNA is transcribed, folded, cleaved, and modified, while also binding with 20 different proteins. Very little is known about how the tertiary structure of the ribosomal RNA changes during assembly. There are a number of structure-probing methods that can be used to study rRNA in vivo, but virtually all of them lack the time resolution necessary to study a process like ribosome synthesis, which is completed within a few minutes.Hydroxyl radical footprinting can be used to probe in vivo rRNA structure. The hydroxyl radicals which probe the rRNA can be produced in milliseconds using synchrotron X-rays. With this technique it is possible to examine ribosome assembly with meaningful time resolution. The hydroxyl radicals cleave the RNA backbone in solvent accessible regions, giving cleavage patterns that reflect regions of flexibility and rigidity within an RNA.For the purpose of examining ribosome assembly, it is nascent ribosomes that are of interest, not pre-existing ribosomes that are already assembled. Therefore, the nascent ribosomes must be isolated from the background of pre-existing ribosomes. It has been shown that cells can take up labeled nucleosides that have been added to their growth media and incorporate them into nascent RNA transcripts. These can then be isolated using affinity methods. Once the nascent, assembling rRNA has been isolated, it can be analyzed by primer extension. The reverse transcriptase terminates at the cleavage sites. The cDNA fragments are then able to be analyzed by either slab gel, capillary electrophoresis, or high-throughput sequencing methods.

A combined quantitative mass spectrometry and electron microscopy analysis of ribosomal 30S subunit assembly in E. coli.

Ribosome assembly is a complex process involving the folding and processing of ribosomal RNAs (rRNAs), concomitant binding of ribosomal proteins (r-proteins), and participation of numerous accessory cofactors. Here, we use a quantitative mass spectrometry/electron microscopy hybrid approach to determine the r-protein composition and conformation of 30S ribosome assembly intermediates in Escherichia coli. The relative timing of assembly of the 3' domain and the formation of the central pseudoknot (PK) structure depends on the presence of the assembly factor RimP. The central PK is unstable in the absence of RimP, resulting in the accumulation of intermediates in which the 3'-domain is unanchored and the 5'-domain is depleted for r-proteins S5 and S12 that contact the central PK. Our results reveal the importance of the cofactor RimP in central PK formation, and introduce a broadly applicable method for characterizing macromolecular assembly in cells.

Dynamics of the Ribosome Assembly Line in Living Cells

The process of ribosome assembly is a major metabolic program that accounts for a third of the energy budget of a rapidly growing bacterium. using Stable Isotope Pulse Labeling and Quantitative Mass Spectrometry, we are investigating the dynamics of ribosome biogenesis in living cells. A distribution of ribosome assembly intermediates is observed that provides direct information on the order of ribosomal protein binding to the nascent ribosomal RNA transcript. Proteomics reveals the participation of many assembly cofactors at specific stages of assembly. using specific deletion strains and growth under perturbed conditions, we are developing a systems level approach to understanding the critical assembly dynamics required to ensure the capacity for protein synthesis.