3.2-Å-resolution structure of the 90S preribosome before A1 pre-rRNA cleavage.

The 40S small ribosomal subunit is cotranscriptionally assembled in the nucleolus as part of a large chaperone complex called the 90S preribosome or small-subunit processome. Here, we present the 3.2-Å-resolution structure of the Chaetomium thermophilum 90S preribosome, which allowed us to build atomic structures for 34 assembly factors, including the Mpp10 complex, Bms1, Utp14 and Utp18, and the complete U3 small nucleolar ribonucleoprotein. Moreover, we visualized the U3 RNA heteroduplexes with a 5' external transcribed spacer (5' ETS) and pre-18S RNA, and their stabilization by 90S factors. Overall, the structure explains how a highly intertwined network of assembly factors and pre-rRNA guide the sequential, independent folding of the individual pre-40S domains while the RNA regions forming the 40S active sites are kept immature. Finally, by identifying the unprocessed A1 cleavage site and the nearby Utp24 endonuclease, we suggest a proofreading model for regulated 5'-ETS separation and 90S-pre-40S transition.

The complete structure of the small-subunit processome.

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

Architecture of the yeast small subunit processome.

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

Exploring human 40S ribosomal proteins binding to the 18S rRNA fragment containing major 3'-terminal domain.

Association of ribosomal proteins with rRNA during assembly of ribosomal subunits is an intricate process, which is strictly regulated in vivo. As for the assembly in vitro, it was reported so far only for prokaryotic subunits. Bacterial ribosomal proteins are capable of selective binding to 16S rRNA as well as to its separate morphological domains. In this work, we explored binding of total protein of human 40S ribosomal subunit to the RNA transcript corresponding to the major 3'-domain of 18S rRNA. We showed that the resulting ribonucleoprotein particles contained almost all of the expected ribosomal proteins, whose binding sites are located in this 18S rRNA domain in the 40S subunit, together with several nonspecific proteins. The binding in solution was accompanied with aggregation of the RNA-protein complexes. Ribosomal proteins bound to the RNA transcript protected from chemical modification mostly those 18S rRNA nucleotides that are known to be involved in binding with the proteins in the 40S subunit and thereby demonstrated their ability to selectively bind to the rRNA in vitro. The possible implication of unstructured extensions of eukaryotic ribosomal proteins in their nonspecific binding with rRNA and in subsequent aggregation of the resulting complexes is discussed.

Phylogeny of choanozoa, apusozoa, and other protozoa and early eukaryote megaevolution.

The primary diversification of eukaryotes involved protozoa, especially zooflagellates-flagellate protozoa without plastids. Understanding the origins of the higher eukaryotic kingdoms (two purely heterotrophic, Animalia and Fungi, and two primarily photosynthetic, Plantae and Chromista) depends on clarifying evolutionary relationships among the phyla of the ancestral kingdom Protozoa. We therefore sequenced 18S rRNA genes from 10 strains from the protozoan phyla Choanozoa and Apusozoa. Eukaryote diversity is encompassed by three early-radiating, arguably monophyletic groups: Amoebozoa, opisthokonts, and bikonts. Our taxon-rich rRNA phylogeny for eukaryotes allowing for intersite rate variation strongly supports the opisthokont clade (animals, Choanozoa, Fungi). It agrees with the view that Choanozoa are sisters of or ancestral to animals and reveals a novel nonflagellate choanozoan lineage, Ministeriida, sister either to choanoflagellates, traditionally considered animal ancestors, or to animals. Maximum likelihood trees suggest that within animals Placozoa are derived from medusozoan Cnidaria (we therefore place Placozoa as a class within subphylum Medusozoa of the Cnidaria) and hexactinellid sponges evolved from demosponges. The bikont and amoebozoan radiations are both very ill resolved. Bikonts comprise the kingdoms Plantae and Chromista and three major protozoan groups: alveolates, excavates, and Rhizaria. Our analysis weakly suggests that Apusozoa, represented by Ancyromonas and the apusomonads ( Apusomonas and the highly diverse and much more ancient genus Amastigomonas, from which it evolved), are not closely related to other Rhizaria and may be the most divergent bikont lineages. Although Ancyromonas and apusomonads appear deeply divergent in 18S rRNA trees, the trees neither refute nor support the monophyly of Apusozoa. The bikont phylum Cercozoa weakly but consistently appears as sister to Retaria (Foraminifera; Radiolaria), together forming a hitherto largely unrecognized major protozoan assemblage (core Rhizaria) in the eukaryote tree. Both 18S rRNA sequence trees and a rare deletion show that nonciliate haplosporidian and paramyxid parasites of shellfish (together comprising the Ascetosporea) are not two separate phyla, as often thought, but part of the Cercozoa, and may be related to the plant-parasitic plasmodiophorids and phagomyxids, which were originally the only parasites included in the Cercozoa. We discuss rRNA trees in relation to other evidence concerning the basal diversification and root of the eukaryotic tree and argue that bikonts and opisthokonts, at least, are holophyletic. Amoebozoa and bikonts may be sisters-jointly called anterokonts, as they ancestrally had an anterior cilium, not a posterior one like opisthokonts; this contrasting ciliary orientation may reflect a primary divergence in feeding mode of the first eukaryotes. Anterokonts also differ from opisthokonts in sterol biosynthesis (cycloartenol versus lanosterol pathway), major exoskeletal polymers (cellulose versus chitin), and mitochondrial cristae (ancestrally tubular not flat), possibly also primary divergences.

Localization in situ of specific RNA by electron microscopy.

To better understand the metabolism of RNA in nuclei, the analysis of precise nuclear distribution of specific RNA would be essential. For this purpose, nonradioactive electron microscopic (EM) in situ hybridization may be the most appropriate technique while the details required for the technique have not been fully established. In the present study, we attempted to localize 28S and 18S rRNAs in the nuclei of mouse Sertoli cells by EM in situ hybridization as a model system. After various preliminary experiments we chose the pre-embedding method; fresh-frozen sections of mouse testis were fixed with a mixture of 4% paraformaldehyde and 0.1% glutaraldehyde, digested with 10 microg/ml of proteinase K and hybridized with thymine-thymine (T-T) dimerized oligodeoxynucleotides (oligo-DNA) complementary to a part of 28S and 18S rRNAs. Then, the T-T dimers were detected enzyme-immunohistochemically with horseradish peroxidase (HRP) labeled anti-T-T dimer. After osmification of HRP products, the sections were embedded in Epon resin, cut into 100 nm ultra-thin sections and observed under a transmission electron microscope. As a result, we successfully localized both 28S and 18S rRNAs in the dense fibrillar and granular components of the nucleolus, showing the usefulness of nonradioactive EM in situ hybridization in the nuclear localization of specific RNA.

Physical mapping, expression patterns and interphase organisation of rDNA loci in Portuguese endemic Silene cintrana and Silene rothmaleri.

Double target in situ hybridization to root tip metaphase and interphase cells of Silene cintrana and Silene rothmaleri was used to allocate the position of 18S-5.8S-25S and 5S rRNA genes. In both species, the 18S-5.8S-25S rDNA probe labelled four sites located on the short arms of two submetacentric chromosomes. Only one locus for 5S rDNA was mapped adjacent to 18S-5.8S-25S genes in a subterminal position on the centromere side: in S. rothmaleri the 5S rDNA locus was adjacent to the small 18S-5.8S-25S locus while in S. cintrana it was near the large one. The NOR activity analysed by Ag-staining in metaphase cells revealed proportionality between in situ labelling dimensions and Ag-NORs. In both species all rDNA loci were potentially active, although in S. rothmaleri a tendency for the expression of only one locus was observed. Interphase organisation analysis of rDNA showed some differences between both species that were correlated with NOR activity.

Correlation of the expansion segments in mammalian rRNA with the fine structure of the 80 S ribosome; a cryoelectron microscopic reconstruction of the rabbit reticulocyte ribosome at 21 A resolution.

Samples of 80 S ribosomes from rabbit reticulocytes were subjected to electron cryomicroscopy combined with angular reconstitution. A three-dimensional reconstruction at 21 A resolution was obtained, which was compared with the corresponding (previously published) reconstruction of Escherichia coli 70 S ribosomes carrying tRNAs at the A and P sites. In the region of the intersubunit cavity, the principal features observed in the 70 S ribosome (such as the L1 protuberance, the central protuberance and A site finger in the large subunit) could all be clearly identified in the 80 S particle. On the other hand, significant additional features were observed in the 80 S ribosomes on the solvent sides and lower regions of both subunits. In the case of the small (40 S) subunit, the most prominent additions are two extensions at the base of the particle. By comparing the secondary structure of the rabbit 18 S rRNA with our model for the three-dimensional arrangement of E. coli 16 S rRNA, these two extensions could be correlated with the rabbit expansion segments (each totalling ca 170 bases) in the regions of helix 21, and of helices 8, 9 and 44, respectively. A similar comparison of the secondary structures of mammalian 28 S rRNA and E. coli 23 S rRNA, combined with preliminary modelling studies on the 23 S rRNA within the 50 S subunit, enabled the additional features in the 60 S subunit to be sub-divided into five groups. The first (corresponding to a total of ca 335 extra bases in helices 45, 98 and 101) is located on the solvent side of the 60 S subunit, close to the L7/L12 area. The second (820 bases in helices 25 and 38) is centrally placed on the solvent side of the subunit, whereas the third group (totaling 225 bases in helices 18/19, 27/29, 52 and 54) lies towards the L1 side of the subunit. The fourth feature (80 bases in helices 78 and 79) lies within or close to the L1 protuberance itself, and the fifth (560 bases in helix 63) is located underneath the L1 protuberance on the interface side of the 60 S subunit.

Native 3D structure of eukaryotic 80s ribosome: morphological homology with E. coli 70S ribosome.

A three-dimensional reconstruction of the eukaryotic 80S monosome from a frozen-hydrated electron microscopic preparation reveals the native structure of this macromolecular complex. The new structure, at 38A resolution, shows a marked resemblance to the structure determined for the E. coli 70S ribosome (Frank, J., A. Verschoor, Y. Li, J. Zhu, R.K. Lata, M. Radermacher, P. Penczek, R. Grassucci, R.K. Agrawal, and Srivastava. 1996b. In press; Frank, J., J. Zhu, P. Penczek, Y. Li, S. Srivastava ., A. Verschoor, M. Radermacher, R. Grassucci, R.K. Lata, and R. Agrawal. 1995. Nature (Lond.).376:441-444.) limited to a comparable resolution, but with a number of eukaryotic elaborations superimposed. Although considerably greater size and intricacy of the features is seen in the morphology of the large subunit (60S vs 50S), the most striking differences are in the small subunit morphology (40S vs 30S): the extended beak and crest features of the head, the back lobes, and the feet. However, the structure underlying these extra features appears to be remarkably similar in form to the 30S portion of the 70S structure. The intersubunit space also appears to be strongly conserved, as might be expected from the degree of functional conservation of the ribosome among kingdoms (Eukarya, Eubacteria, and Archaea). The internal organization of the 80S structure appears as an armature or core of high-density material for each subunit, with the two cores linked by a single bridge between the platform region of the 40S subunit and the region below the presumed peptidyltransferase center of the 60S subunit. This may be equated with a close contact of the 18S and 28S rRNAs in the translational domain centered on the upper subunit:subunit interface.

Structures of small subunit ribosomal RNAs in situ from Escherichia coli and Thermomyces lanuginosus.

Small ribosomal subunits from the prokaryote Escherichia coli and the eukaryote Thermomyces lanuginosus were imaged electron spectroscopically, and single particle analysis used to yield three-dimensional reconstructions of the net phosphorus distribution representing the nucleic acid (RNA) backbone. This direct approach showed both ribosomal RNAs to have a three domain structure and other characteristic morphological features. The eukaryotic small ribosomal subunit had a prominent bill present in the head domain, while the prokaryotic subunit had a small vestigial bill. Both ribosomal subunits contained a thick 'collar' central domain which correlates to the site of the evolutionarily conserved ribosomal RNA core, and the location of the majority of ribosomal RNA bases that have been implicated in translation. The reconstruction of the prokaryotic subunit had a prominent protrusion extending from the collar, forming a channel approximately 1.5 nm wide and potentially representing a 'bridge' to the large subunit in the intact monosome. The basal domain of the prokaryotic ribosomal subunit was protein free. In this region of the eukaryotic subunit, there were two basal lobes composed of ribosomal RNA, consistent with previous hypotheses that this is a site for the 'non-conserved core' ribosomal RNA.

Ultrastructural rRNA localization in plant cell nucleoli. RNA/RNA in situ hybridization, autoradiography and cytochemistry.

The distribution of ribosomal transcripts in the plant nucleolus has been studied by non-isotopic in situ hybridization in ultrathin Lowicryl K4M sections and by high-resolution autoradiography after labelling with tritiated uridine. In parallel, cytochemical techniques were applied to localize RNA on different plant nucleolar components of Allium cepa L. root meristematic cells and Capsicum annuum L. pollen grains. For RNA/RNA in situ hybridization, several biotinylated single-stranded ribosomal RNA probes were used for mapping different fragments of the 18 S and the 25 S rRNA gene transcribed regions. Ribosomal RNAs (from pre-rRNAs to mature 18 and 25 S RNAs) were found in the nucleolus, in the dense fibrillar (DFC) and granular components (GC). Hybridization signal was found at the periphery of some fibrillar centres (FCs) with probes recognizing both 18 and 25 S rRNA sequences. A quantitative study was performed to analyze the significance of this labelling. Incorporation of tritiated uridine into roots was carried out and, later, after a long time-exposure, autoradiography revealed the presence of newly synthesized RNA mainly in the DFC and at the periphery of the FCs. The presence of RNA in these areas was also confirmed by the cytochemical techniques used in this study. Taken together, these data favour the hypothesis that transcription can begin at the periphery of the FCs, although we cannot exclude the possibility that the DFC plays a role in this process.