SnapShot: Eukaryotic ribosome biogenesis II.

Ribosome production is essential for cell growth. Approximately 200 assembly factors drive this complicated pathway that starts in the nucleolus and ends in the cytoplasm. A large number of structural snapshots of the pre-60S pathway have revealed the principles behind large subunit synthesis. To view this SnapShot, open or download the PDF.

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.

Structural insights into coordinating 5S RNP rotation with ITS2 pre-RNA processing during ribosome formation.

The rixosome defined in Schizosaccharomyces pombe and humans performs diverse roles in pre-ribosomal RNA processing and gene silencing. Here, we isolate and describe the conserved rixosome from Chaetomium thermophilum, which consists of two sub-modules, the sphere-like Rix1-Ipi3-Ipi1 and the butterfly-like Las1-Grc3 complex, connected by a flexible linker. The Rix1 complex of the rixosome utilizes Sda1 as landing platform on nucleoplasmic pre-60S particles to wedge between the 5S rRNA tip and L1-stalk, thereby facilitating the 180° rotation of the immature 5S RNP towards its mature conformation. Upon rixosome positioning, the other sub-module with Las1 endonuclease and Grc3 polynucleotide-kinase can reach a strategic position at the pre-60S foot to cleave and 5' phosphorylate the nearby ITS2 pre-rRNA. Finally, inward movement of the L1 stalk permits the flexible Nop53 N-terminus with its AIM motif to become positioned at the base of the L1-stalk to facilitate Mtr4 helicase-exosome participation for completing ITS2 removal. Thus, the rixosome structure elucidates the coordination of two central ribosome biogenesis events, but its role in gene silencing may adapt similar strategies.

Identification and characterization of sugar-regulated promoters in Chaetomium thermophilum.

The thermophilic fungus Chaetomium thermophilum has been used extensively for biochemical and high-resolution structural studies of protein complexes. However, subsequent functional analyses of these assemblies have been hindered owing to the lack of genetic tools compatible with this thermophile, which are typically suited to other mesophilic eukaryotic model organisms, in particular the yeast Saccharomyces cerevisiae. Hence, we aimed to find genes from C. thermophilum that are expressed under the control of different sugars and examine their associated 5' untranslated regions as promoters responsible for sugar-regulated gene expression. To identify sugar-regulated promoters in C. thermophilum, we performed comparative xylose- versus glucose-dependent gene expression studies, which uncovered a number of enzymes with induced expression in the presence of xylose but repressed expression in glucose-supplemented media. Subsequently, we cloned the promoters of the two most stringently regulated genes, the xylosidase-like gene (XYL) and xylitol dehydrogenase (XDH), obtained from this genome-wide analysis in front of a thermostable yellow fluorescent protein (YFP) reporter. With this, we demonstrated xylose-dependent YFP expression by both Western blotting and live-cell imaging fluorescence microscopy. Prompted by these results, we expressed the C. thermophilum orthologue of a well-characterized dominant-negative ribosome assembly factor mutant, under the control of the XDH promoter, which allowed us to induce a nuclear export defect on the pre-60S subunit when C. thermophilum cells were grown in xylose- but not glucose-containing medium. Altogether, our study identified xylose-regulatable promoters in C. thermophilum, which might facilitate functional studies of genes of interest in this thermophilic eukaryotic model organism.

Structure of nascent 5S RNPs at the crossroad between ribosome assembly and MDM2-p53 pathways.

The 5S ribonucleoprotein (RNP) is assembled from its three components (5S rRNA, Rpl5/uL18 and Rpl11/uL5) before being incorporated into the pre-60S subunit. However, when ribosome synthesis is disturbed, a free 5S RNP can enter the MDM2-p53 pathway to regulate cell cycle and apoptotic signaling. Here we reconstitute and determine the cryo-electron microscopy structure of the conserved hexameric 5S RNP with fungal or human factors. This reveals how the nascent 5S rRNA associates with the initial nuclear import complex Syo1-uL18-uL5 and, upon further recruitment of the nucleolar factors Rpf2 and Rrs1, develops into the 5S RNP precursor that can assemble into the pre-ribosome. In addition, we elucidate the structure of another 5S RNP intermediate, carrying the human ubiquitin ligase Mdm2, which unravels how this enzyme can be sequestered from its target substrate p53. Our data provide molecular insight into how the 5S RNP can mediate between ribosome biogenesis and cell proliferation.

Mechanism of 5S RNP recruitment and helicase-surveilled rRNA maturation during pre-60S biogenesis.

Ribosome biogenesis proceeds along a multifaceted pathway from the nucleolus to the cytoplasm that is extensively coupled to several quality control mechanisms. However, the mode by which 5S ribosomal RNA is incorporated into the developing pre-60S ribosome, which in humans links ribosome biogenesis to cell proliferation by surveillance by factors such as p53-MDM2, is poorly understood. Here, we report nine nucleolar pre-60S cryo-EM structures from Chaetomium thermophilum, one of which clarifies the mechanism of 5S RNP incorporation into the early pre-60S. Successive assembly states then represent how helicases Dbp10 and Spb4, and the Pumilio domain factor Puf6 act in series to surveil the gradual folding of the nearby 25S rRNA domain IV. Finally, the methyltransferase Spb1 methylates a universally conserved guanine nucleotide in the A-loop of the peptidyl transferase center, thereby licensing further maturation. Our findings provide insight into the hierarchical action of helicases in safeguarding rRNA tertiary structure folding and coupling to surveillance mechanisms that culminate in local RNA modification.

SnapShot: Eukaryotic ribosome biogenesis I.

Ribosome production is vital for every cell, and failure causes human diseases. It is driven by ∼200 assembly factors functioning along an ordered pathway from the nucleolus to the cytoplasm. Structural snapshots of biogenesis intermediates from the earliest 90S pre-ribosomes to mature 40S subunits unravel the mechanisms of small ribosome synthesis. To view this SnapShot, open or download the PDF.

Distinct domains in Ndc1 mediate its interaction with the Nup84 complex and the nuclear membrane.

Nuclear pore complexes (NPCs) are embedded in the nuclear envelope and built from ∼30 different nucleoporins (Nups) in multiple copies, few are integral membrane proteins. One of these transmembrane nucleoporins, Ndc1, is thought to function in NPC assembly at the fused inner and outer nuclear membranes. Here, we show a direct interaction of Ndc1's transmembrane domain with Nup120 and Nup133, members of the pore membrane coating Y-complex. We identify an amphipathic helix in Ndc1's C-terminal domain binding highly curved liposomes. Upon overexpression, this amphipathic motif is toxic and dramatically alters the intracellular membrane organization in yeast. Ndc1's amphipathic motif functionally interacts with related motifs in the C-terminus of the nucleoporins Nup53 and Nup59, important for pore membrane binding and interconnecting NPC modules. The essential function of Ndc1 can be suppressed by deleting the amphipathic helix from Nup53. Our data indicate that nuclear membrane and presumably NPC biogenesis depends on a balanced ratio between amphipathic motifs in diverse nucleoporins.

Structural inventory of cotranslational protein folding by the eukaryotic RAC complex.

The challenge of nascent chain folding at the ribosome is met by the conserved ribosome-associated complex (RAC), which forms a chaperone triad with the Hsp70 protein Ssb in fungi, and consists of the non-canonical Hsp70 Ssz1 and the J domain protein Zuotin (Zuo1). Here we determine cryo-EM structures of Chaetomium thermophilum RAC bound to 80S ribosomes. RAC adopts two distinct conformations accommodating continuous ribosomal rotation by a flexible lever arm. It is held together by a tight interaction between the Ssz1 substrate-binding domain and the Zuo1 N terminus, and additional contacts between the Ssz1 nucleotide-binding domain and Zuo1 J- and Zuo1 homology domains, which form a rigid unit. The Zuo1 HPD motif conserved in J-proteins is masked in a non-canonical interaction by the Ssz1 nucleotide-binding domain, and allows the positioning of Ssb for activation by Zuo1. Overall, we provide the basis for understanding how RAC cooperates with Ssb in a dynamic nascent chain interaction and protein folding.

Concurrent remodelling of nucleolar 60S subunit precursors by the Rea1 ATPase and Spb4 RNA helicase.

Biogenesis intermediates of nucleolar ribosomal 60S precursor particles undergo a number of structural maturation steps before they transit to the nucleoplasm and are finally exported into the cytoplasm. The AAA+-ATPase Rea1 participates in the nucleolar exit by releasing the Ytm1-Erb1 heterodimer from the evolving pre-60S particle. Here, we show that the DEAD-box RNA helicase Spb4 with its interacting partner Rrp17 is further integrated into this maturation event. Spb4 binds to a specific class of late nucleolar pre-60S intermediates, whose cryo-EM structure revealed how its helicase activity facilitates melting and restructuring of 25S rRNA helices H62 and H63/H63a prior to Ytm1-Erb1 release. In vitro maturation of such Spb4-enriched pre-60S particles, incubated with purified Rea1 and its associated pentameric Rix1-complex in the presence of ATP, combined with cryo-EM analysis depicted the details of the Rea1-dependent large-scale pre-ribosomal remodeling. Our structural insights unveil how the Rea1 ATPase and Spb4 helicase remodel late nucleolar pre-60S particles by rRNA restructuring and dismantling of a network of several ribosomal assembly factors.

The nucleoplasmic phase of pre-40S formation prior to nuclear export.

Biogenesis of the small ribosomal subunit in eukaryotes starts in the nucleolus with the formation of a 90S precursor and ends in the cytoplasm. Here, we elucidate the enigmatic structural transitions of assembly intermediates from human and yeast cells during the nucleoplasmic maturation phase. After dissociation of all 90S factors, the 40S body adopts a close-to-mature conformation, whereas the 3' major domain, later forming the 40S head, remains entirely immature. A first coordination is facilitated by the assembly factors TSR1 and BUD23-TRMT112, followed by re-positioning of RRP12 that is already recruited early to the 90S for further head rearrangements. Eventually, the uS2 cluster, CK1 (Hrr25 in yeast) and the export factor SLX9 associate with the pre-40S to provide export competence. These exemplary findings reveal the evolutionary conserved mechanism of how yeast and humans assemble the 40S ribosomal subunit, but reveal also a few minor differences.

Cms1 coordinates stepwise local 90S pre-ribosome assembly with timely snR83 release.

Ribosome synthesis begins in the nucleolus with 90S pre-ribosome construction, but little is known about how the many different snoRNAs that modify the pre-rRNA are timely guided to their target sites. Here, we report a role for Cms1 in such a process. Initially, we discovered CMS1 as a null suppressor of a nop14 mutant impaired in Rrp12-Enp1 factor recruitment to the 90S. Further investigations detected Cms1 at the 18S rRNA 3' major domain of an early 90S that carried H/ACA snR83, which is known to guide pseudouridylation at two target sites within the same subdomain. Cms1 co-precipitates with many 90S factors, but Rrp12-Enp1 encircling the 3' major domain in the mature 90S is decreased. We suggest that Cms1 associates with the 3' major domain during early 90S biogenesis to restrict premature Rrp12-Enp1 binding but allows snR83 to timely perform its modification role before the next 90S assembly steps coupled with Cms1 release take place.

In vitro structural maturation of an early stage pre-40S particle coupled with U3 snoRNA release and central pseudoknot formation.

The transition of the 90S to the pre-40S pre-ribosome is a decisive step in eukaryotic small subunit biogenesis leading to a first pre-40S intermediate (state Dis-C or primordial pre-40S), where the U3 snoRNA keeps the nascent 18S rRNA locally immature. We in vitro reconstitute the ATP-dependent U3 release from this particle, catalyzed by the helicase Dhr1, and follow this process by cryo-EM revealing two successive pre-40S intermediates, Dis-D and Dis-E. The latter has lost not only U3 but all residual 90S factors including the GTPase Bms1. In vitro remodeling likewise induced the formation of the central pseudoknot, a universally conserved tertiary RNA structure that comprises the core of the small subunit decoding center. Thus, we could structurally reveal a key tertiary RNA folding step that is essential to form the active 40S subunit.

The C-terminal tail of ribosomal protein Rps15 is engaged in cytoplasmic pre-40S maturation.

The small ribosomal subunit protein Rps15/uS19 is involved in early nucleolar ribosome biogenesis and subsequent nuclear export of pre-40S particles to the cytoplasm. In addition, the C-terminal tail of Rps15 was suggested to play a role in mature ribosomes, namely during translation elongation. Here, we show that Rps15 not only functions in nucleolar ribosome assembly but also in cytoplasmic pre-40S maturation, which is indicated by a strong genetic interaction between Rps15 and the 40S assembly factor Ltv1. Specifically, mutations either in the globular or C-terminal domain of Rps15 when combined with the non-essential ltv1 null allele are lethal or display a strong growth defect. However, not only rps15 ltv1 double mutants but also single rps15 C-terminal deletion mutants exhibit an accumulation of the 20S pre-rRNA in the cytoplasm, indicative of a cytoplasmic pre-40S maturation defect. Since in pre-40S particles, the C-terminal tail of Rps15 is positioned between assembly factors Rio2 and Tsr1, we further tested whether Tsr1 is genetically linked to Rps15, which indeed could be demonstrated. Thus, the integrity of the Rps15 C-terminal tail plays an important role during late pre-40S maturation, perhaps in a quality control step to ensure that only 40S ribosomal subunits with functional Rps15 C-terminal tail can efficiently enter translation. As mutations in the C-terminal tail of human RPS15 have been observed in connection with chronic lymphocytic leukaemia, it is possible that apart from defects in translation, an impaired late pre-40S maturation step in the cytoplasm could also be a reason for this disease.

Emergence of the primordial pre-60S from the 90S pre-ribosome.

Synthesis of ribosomes begins in the nucleolus with formation of the 90S pre-ribosome, during which the pre-40S and pre-60S pathways diverge by pre-rRNA cleavage. However, it remains unclear how, after this uncoupling, the earliest pre-60S subunit continues to develop. Here, we reveal a large-subunit intermediate at the beginning of its construction when still linked to the 90S, the precursor to the 40S subunit. This primordial pre-60S is characterized by the SPOUT domain methyltransferase Upa1-Upa2, large α-solenoid scaffolds, Mak5, one of several RNA helicases, and two small nucleolar RNA (snoRNAs), C/D box snR190 and H/ACA box snR37. The emerging pre-60S does not efficiently disconnect from the 90S pre-ribosome in a dominant mak5 helicase mutant, allowing a 70-nm 90S-pre-60S bipartite particle to be visualized by electron microscopy. Our study provides insight into the assembly pathway when the still-connected nascent 40S and 60S subunits are beginning to separate.

Transformation of Chaetomium thermophilum and Affinity Purification of Native Thermostable Protein Complexes.

Chaetomium thermophilum, a eukaryotic thermophile, is an aspiring organism holding great potential for various biochemical and biotechnological applications. Prerequisite for genetic manipulation is a reliable transformation system for target genes combined with selection markers operating at the high growth temperatures of the fungus. Here, we present a detailed protocol for Chaetomium thermophilum protoplast transformation to allow stable chromosomal integration of constructs into its genome, rendering this eukaryotic thermophile a valuable resource for affinity purification of native thermostable protein complexes, like nuclear pore subcomplexes.

A Homologous Recombination System to Generate Epitope-Tagged Target Genes in Chaetomium thermophilum: A Genetic Approach to Investigate Native Thermostable Proteins.

Chaetomium thermophilum is an attractive eukaryotic model organism which, due to its unusually high temperature tolerance (optimal growth at 50-52 °C), has a thermostable proteome that can be exploited for biochemical, structural and biotechnological applications. Site directed gene manipulation for the expression of labeled target genes is a desirable approach to study the structure and function of thermostable proteins and their organization in complexes, which has not been established for this thermophile yet. Here, we describe the development of a homologous recombination system to epitope-tag chromosomal genes of interest in Chaetomium thermophilum with the goal to exploit the derived thermostable fusion proteins for tandem-affinity purification. This genetic approach was facilitated by the engineering of suitable strains, in which factors of the non-homologous end-joining pathway were deleted, thereby improving the efficiency of homologous integration at specific gene loci. Following this strategy, we could demonstrate that gene tagging via homologous recombination improved the yield of purified bait proteins and co-precipitated factors, paving the way for related studies in fundamental research and industrial applications.

Global Transcriptome Characterization and Assembly of the Thermophilic Ascomycete Chaetomium thermophilum.

A correct genome annotation is fundamental for research in the field of molecular and structural biology. The annotation of the reference genome of Chaetomium thermophilum has been reported previously, but it is essentially limited to open reading frames (ORFs) of protein coding genes and contains only a few noncoding transcripts. In this study, we identified and annotated full-length transcripts of C. thermophilum by deep RNA sequencing. We annotated 7044 coding genes and 4567 noncoding genes. Astonishingly, 23% of the coding genes are alternatively spliced. We identified 679 novel coding genes as well as 2878 novel noncoding genes and corrected the structural organization of more than 50% of the previously annotated genes. Furthermore, we substantially extended the Gene Ontology (GO) and Enzyme Commission (EC) lists, which provide comprehensive search tools for potential industrial applications and basic research. The identified novel transcripts and improved annotation will help to understand the gene regulatory landscape in C. thermophilum. The analysis pipeline developed here can be used to build transcriptome assemblies and identify coding and noncoding RNAs of other species.

Structure of the Maturing 90S Pre-ribosome in Association with the RNA Exosome.

Ribosome assembly is catalyzed by numerous trans-acting factors and coupled with irreversible pre-rRNA processing, driving the pathway toward mature ribosomal subunits. One decisive step early in this progression is removal of the 5' external transcribed spacer (5'-ETS), an RNA extension at the 18S rRNA that is integrated into the huge 90S pre-ribosome structure. Upon endo-nucleolytic cleavage at an internal site, A1, the 5'-ETS is separated from the 18S rRNA and degraded. Here we present biochemical and cryo-electron microscopy analyses that depict the RNA exosome, a major 3'-5' exoribonuclease complex, in a super-complex with the 90S pre-ribosome. The exosome is docked to the 90S through its co-factor Mtr4 helicase, a processive RNA duplex-dismantling helicase, which strategically positions the exosome at the base of 5'-ETS helices H9-H9', which are dislodged in our 90S-exosome structures. These findings suggest a direct role of the exosome in structural remodeling of the 90S pre-ribosome to drive eukaryotic ribosome synthesis.