Sebastian Klinge

Crystal Structure of the Eukaryotic 60S Ribosomal Subunit in Complex with Initiation Factor 6

The 3.5 angstrom–resolution structure provides insights into the architecture of the eukaryotic ribosome and its regulation. Protein synthesis in all organisms is catalyzed by ribosomes. In comparison to their prokaryotic counterparts, eukaryotic ribosomes are considerably larger and are subject to more complex regulation. The large ribosomal subunit (60S) catalyzes peptide bond formation and contains the nascent polypeptide exit tunnel. We present the structure of the 60S ribosomal subunit from Tetrahymena thermophila in complex with eukaryotic initiation factor 6 (eIF6), cocrystallized with the antibiotic cycloheximide (a eukaryotic-specific inhibitor of protein synthesis), at a resolution of 3.5 angstroms. The structure illustrates the complex functional architecture of the eukaryotic 60S subunit, which comprises an intricate network of interactions between eukaryotic-specific ribosomal protein features and RNA expansion segments. It reveals the roles of eukaryotic ribosomal protein elements in the stabilization of the active site and the extent of eukaryotic-specific differences in other functional regions of the subunit. Furthermore, it elucidates the molecular basis of the interaction with eIF6 and provides a structural framework for further studies of ribosome-associated diseases and the role of the 60S subunit in the initiation of protein synthesis.

Atomic structures of the eukaryotic ribosome

Eukaryotic ribosomes are significantly larger and more complex than their prokaryotic counterparts. This parallels the increased complexity of the associated cellular machinery responsible for translation initiation, ribosome assembly, and the regulation of protein synthesis in eukaryotic cells. The recently determined crystal structures of the small (40S) and large (60S) ribosomal subunits and the 80S ribosome now provide an atomic description of this essential molecular machine and reveal its eukaryote-specific features. In this review, we discuss the common structural principles underlying the evolution of both ribosomal subunits. The recently obtained structural information provides a framework for further genetic, biochemical and structural studies of eukaryotic ribosomes. At the same time, it facilitates a direct comparison between prokaryotic and eukaryotic ribosomal features.

Stage-specific assembly events of the 6-MDa small-subunit processome initiate eukaryotic ribosome biogenesis

Eukaryotic ribosome biogenesis involves a plethora of ribosome-assembly factors, and their temporal order of association with preribosomal RNA is largely unknown. By using Saccharomyces cerevisiae as a model organism, we developed a system that recapitulates and arrests ribosome assembly at early stages, thus providing in vivo snapshots of nascent preribosomal particles. Here we report the stage-specific order in which 70 ribosome-assembly factors associate with preribosomal RNA domains, thereby forming the 6-MDa small-subunit processome.

Architecture of the yeast small subunit processome

A machine for building ribosomes The ribosome is a very large protein and RNA complex responsible for the difficult process of synthesizing proteins. Construction of the ribosome itself involves several molecular machines and an army of helper proteins and RNAs. Chaker-Margot et al. determined the structure of one of those machines, the yeast small subunit processome. The structure reveals how the processome helps in the maturation of individual domains of the ribosome and suggests that the mechanism involves a molecular motor to drive conformational changes. Science, this issue p. 10.1126/science.aal1880 The structure of one of the molecular machines that helps build the ribosome reveals how it partitions ribosomal RNA domains. INTRODUCTION The eukaryotic ribosome is assembled through an intricate process that involves in excess of 250 nonribosomal proteins and small nucleolar RNAs (snoRNAs). Ribosomal RNA (rRNA) that later gives rise to the small ribosomal subunit is initially transcribed as a long precursor, the 35S pre-rRNA, which also contains a 5′ external transcribed spacer (5′ ETS). Ribosome biogenesis factors assemble cotranscriptionally on nascent pre-rRNA and coordinate the folding and cleavage of the 35S pre-rRNA, forming large terminal knobs, which can be observed in Miller spreads. The identity of these large terminal structures, referred to as small and large subunit processomes, remained elusive for decades. Recent studies revealed that the small subunit (SSU) processome is a large ribonucleoprotein particle composed of approximately 70 nonribosomal proteins, pre-rRNA, and the U3 snoRNA. It organizes the assembly of the eukaryotic small ribosomal subunit at the early stages by coordinating the folding and modification of nascent pre-rRNA. In addition, the SSU processome facilitates the cleavage of the precursor RNA at distinct sites in the 5′ ETS (A0 and A1) and the internal transcribed spacer 1 (ITS1; A2) to give rise to the mature SSU. RATIONALE For decades, a lack of structural information has precluded a mechanistic understanding of early ribosome biogenesis. Mean- while, important genetic and biochemical insights into eukaryotic ribosome assembly have been obtained by using the model organism Saccharomyces cerevisiae. To bridge this gap of knowledge, we sought to determine the structure of the SSU processome from S. cerevisiae in order to elucidate the architecture of the functional core of this particle. RESULTS Here, we present the cryo–electron microscopy (cryo-EM) structure of the yeast SSU processome at 5.1-Å resolution. We describe the organization of the 5′ ETS and its role, together with the U3 snoRNA, in providing a structural blueprint for the entire particle. This very 5′ end of the pre-rRNA folds into several helices that coordinate the recruitment of large ribosome biogenesis complexes, such as UtpA and UtpB, to the SSU processome. UtpA, UtpB, and the U3 snoRNA, which pairs at two sites with the base 5′ ETS, bridge the A0-cleaved 5′ ETS with the 18S precursor RNA. In conjunction with many other essential ribosome biogenesis factors, this structure forms an intertwined RNA-protein assembly platform for the 18S rRNA. This platform guarantees the spatial segregation of 18S rRNA domains, facilitating the recruitment of enzymes and other assembly factors required for their maturation. Several large helical repeat proteins mediate long-range interactions between distant domains of the SSU processome. We discovered structural similarities between the subcomplexes UtpA and UtpB, such as a conserved helical tetramerization domain, that suggest that these complexes share a common evolutionary origin. The strategic placement of the 135-kDa guanosine triphosphatase Bms1 at the center of the particle near long architectural helices suggests a mechanism for mediating conformational changes within this giant particle. These motions could facilitate subsequent cleavage reactions and rearrangements required in the maturation of the small ribosomal subunit. CONCLUSION The architecture of the yeast SSU processome allows us to rationalize a wealth of biochemical and genetic data available for the model organism S. cerevisiae. Most notably, it sheds light on the central role of the 5′ ETS and the U3 snoRNA in initiating and organizing SSU processome assembly. In addition, the structural information allows us to contextualize biochemical data on SSU processome assembly as a function of transcription. Proteins that bind within the 5′ ETS are located at the base of the SSU processome structure, whereas factors associated with 18S rRNA domains form the core of the particle. Last, proteins recruited only after 18S rRNA completion form the outer tier of the structure. This study provides an improved structural framework for a mechanistic understanding of eukaryotic ribosome assembly in S. cerevisiae. Targeted functional studies will now be possible to further elucidate the individual roles of many ribosome assembly factors in the model organism for ribosome biogenesis. Architecture of the S. cerevisiae SSU processome. Front view of the segmented cryo-EM density map of the complete particle, with color-coded protein and RNA elements (left), and an outline of a 10-Å low-pass filtered map, with only RNA elements shown in full color (right). 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.

Structural insights into eukaryotic ribosomes and the initiation of translation.

The initiation of protein biosynthesis entails the ordered assembly of elongation-competent ribosomes, with an initiator tRNA basepaired to an appropriate mRNA start codon. In eukaryotes, this process is more complex than in prokaryotes and involves numerous protein factors that mediate tRNA delivery, mRNA binding, start codon selection and subunit joining. The recent 40S:eIF1, 80S and eIF2:tRNA:GDPNP ternary complex structures provide an initial structural framework toward a molecular understanding of the eukaryotic translation initiation process. Updated homology models of larger initiation complexes provide first insights into the likely arrangements of these higher-order complexes, but also reveal the limits of our current understanding of the eukaryotic translation initiation process.

UtpA and UtpB chaperone nascent pre-ribosomal RNA and U3 snoRNA to initiate eukaryotic ribosome assembly

Early eukaryotic ribosome biogenesis involves large multi-protein complexes, which co-transcriptionally associate with pre-ribosomal RNA to form the small subunit processome. The precise mechanisms by which two of the largest multi-protein complexes—UtpA and UtpB—interact with nascent pre-ribosomal RNA are poorly understood. Here, we combined biochemical and structural biology approaches with ensembles of RNA–protein cross-linking data to elucidate the essential functions of both complexes. We show that UtpA contains a large composite RNA-binding site and captures the 5′ end of pre-ribosomal RNA. UtpB forms an extended structure that binds early pre-ribosomal intermediates in close proximity to architectural sites such as an RNA duplex formed by the 5′ ETS and U3 snoRNA as well as the 3′ boundary of the 18S rRNA. Both complexes therefore act as vital RNA chaperones to initiate eukaryotic ribosome assembly.

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.

Mutations in the linker domain affect phospho-STAT3 function and suggest targets for interrupting STAT3 activity

Significance Examination of mutants in linker domain of STAT3 suggests contacts with both the DNA binding and SH2 domains that may cause structural changes and affect pSTAT3-dependent transcription, opening the possibility of new targets for drug inhibition of pSTAT3. Crystallography of the cores of phosphotyrosine-activated dimers of STAT1 (132–713) and STAT3 (127–722) bound to a similar double-stranded deoxyoligonucleotide established the domain structure of the STATs and the structural basis for activation through tyrosine phosphorylation and dimerization. We reported earlier that mutants in the linker domain of STAT1 that connect the DNA-binding domain and SH2 domain can prevent transcriptional activation. Because of the pervasive importance of persistently activated STAT3 in many human cancers and the difficulty of finding useful drug candidates aimed at disrupting the pY interchange in active STAT3 dimers, we have examined effects of an array of mutants in the STAT3 linker domain. We have found several STAT3 linker domain mutants to have profound effects of inhibiting STAT3 transcriptional activation. From these results, we propose (i) there is definite functional interaction of the linker both with the DNA binding domain and with the SH2 domain, and (ii) these putative contacts provide potential new targets for small molecule-induced pSTAT3 inhibition.

Incomplete penetrance for isolated congenital asplenia in humans with mutations in translated and untranslated RPSA exons

Significance Isolated congenital asplenia (ICA) is characterized by the absence of a spleen at birth without any other developmental defect. ICA predisposes individuals to severe bacterial infections early in childhood. In 2013, we showed that very rare deleterious mutations in the protein-coding region of RPSA, which codes for a protein in the ribosome, caused ICA in 8 of 23 kindreds. We have since enrolled 33 more kindreds and identified 11 new ICA-causing RPSA protein-coding mutations, as well as the first two ICA-causing mutations in the 5′-UTR of this gene. A few individuals carrying one of the new RPSA mutations had a spleen, indicating that mutations in RPSA can cause ICA with incomplete penetrance. Isolated congenital asplenia (ICA) is the only known human developmental defect exclusively affecting a lymphoid organ. In 2013, we showed that private deleterious mutations in the protein-coding region of RPSA, encoding ribosomal protein SA, caused ICA by haploinsufficiency with complete penetrance. We reported seven heterozygous protein-coding mutations in 8 of the 23 kindreds studied, including 6 of the 8 multiplex kindreds. We have since enrolled 33 new kindreds, 5 of which are multiplex. We describe here 11 new heterozygous ICA-causing RPSA protein-coding mutations, and the first two mutations in the 5′-UTR of this gene, which disrupt mRNA splicing. Overall, 40 of the 73 ICA patients (55%) and 23 of the 56 kindreds (41%) carry mutations located in translated or untranslated exons of RPSA. Eleven of the 43 kindreds affected by sporadic disease (26%) carry RPSA mutations, whereas 12 of the 13 multiplex kindreds (92%) carry RPSA mutations. We also report that 6 of 18 (33%) protein-coding mutations and the two (100%) 5′-UTR mutations display incomplete penetrance. Three mutations were identified in two independent kindreds, due to a hotspot or a founder effect. Finally, RPSA ICA-causing mutations were demonstrated to be de novo in 7 of the 23 probands. Mutations in RPSA exons can affect the translated or untranslated regions and can underlie ICA with complete or incomplete penetrance.

Assembly and structure of the SSU processome — a nucleolar precursor of the small ribosomal subunit

The small subunit processome is the first precursor of the small eukaryotic ribosomal subunit. During its assembly in the nucleolus, many ribosome biogenesis factors, an RNA chaperone, and ribosomal proteins associate with the nascent pre-rRNA. Biochemical studies have elucidated the rRNA-subdomain dependent recruitment of these factors during SSU processome assembly and have been complemented by structural studies of the assembled particle. Ribosome biogenesis factors encapsulate and guide subdomains of pre-ribosomal RNA in distinct compartments. This prevents uncoordinated maturation and enables processing of regions not accessible in the mature subunit. By sequentially reducing conformational freedom, flexible proteins facilitate the incorporation of dynamic subcomplexes into a globular particle. Large rearrangements within the SSU processome are required for compaction into the mature small ribosomal subunit.