Structural and functional analysis of ribosome assembly factor efg1

During synthesis of yeast ribosome, a large complex, called the 90S pre-ribosome or the small subunit processome, is assembled on the nascent precursor rRNA and mediates early processing of 18S rRNA. Utp23 contains a degenerate PIN nuclease domain followed by a long C-terminal tail and associates specifically with snR Here, we report the crystal structure of the Utp23 PIN domain at 2. The structure reveals a conserved core fold of PIN domain with degenerate active site residues, a unique CCHC Zn-finger motif, and two terminal extension elements.

Functional sites of Utp23 have been examined with conservation analysis, mutagenesis, and in vivo and in vitro assays.

structural and functional analysis of ribosome assembly factor efg1

Mutations in each of three cysteine ligands of zinc, although not the histidine ligand, were lethal or strongly inhibitory to yeast growth, indicating that the Zn-finger motif is required for Utp23 structure or function. The N-terminal helix extension harbors many highly conserved basic residues that mostly are critical for growth and in vitro RNA-binding activity of Utp Deletion of the C-terminal tail, which contains a short functionally important sequence motif, disrupted the interaction of Utp23 with snR30 and perturbed the pre-ribosomal association of Utp Our data establish a structural framework for dissecting Utp23 function in the assembly and dynamics of 90S pre-ribosomes.

Eukaryotic ribosome synthesis is a complex and dynamic process that involves transcription, modification, and processing of precursor rRNA pre-rRNAcoordinated assembly of ribosomal proteins, and export of pre-ribosomal particles from the nucleolus to the cytoplasm for review, see Tschochner and Hurt ; Henras et al.

This process starts in the nucleolus, where the small subunit 18S rRNA and the large subunit 5. Many associate with 90S particles, but their binding strength or duration can be different. Utp23 is a nucleolar protein that weakly associates with 90S pre-ribosomal particles and is required for early processing of 18S rRNA at sites A0, A1, and A2 Bleichert et al.

Typically, the PIN domain is an endoribonuclease of approximately amino acids and is involved in various aspects of RNA processing and degradation Clissold and Ponting ; Glavan et al.

However, Utp23 appears to be an inactive nuclease because its equivalent active site residues are either mutated or dispensable for function Bleichert et al. Under normal conditions, only a small fraction of snR30 cosediments with large 90S pre-ribosomes in sucrose density gradients, suggesting that the association of snR30 with 90S pre-ribosomes is weak or transitive. Utp23 has been suggested to regulate snR30 release as depletion of Utp23 led to the accumulation of snR30 in large 90S pre-ribosomes Hoareau-Aveilla et al.

In this study, we have determined the crystal structure of the Utp23 PIN domain and identified functionally important sites of Utp23 with mutagenesis and a number of functional assays.

Structural and functional analysis of Utp24, an endonuclease for processing 18S ribosomal RNA

To gain insight into the structural and functional organization of Utp23, we analyzed its sequence conservation. We searched homologs of S. Alignment of sequences showed that Utp23 contains a conserved N-terminal region residues 1—which corresponds to the predicted PIN domain, and a long C-terminal region residues — composed mainly of low complexity sequences Fig.

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Multiple sequence alignment of Utp Only sequences from Saccharomyces cerevisiae Sc and Homo sapiens Hs are displayed. The secondary structures observed in the crystal structure are indicated on the top. Triangles mark the equivalents to catalytic residues in PIN nucleases and circles denote Zn-coordinating residues. We expressed, purified, and crystallized full-length Utp23 protein and several C-terminally truncated fragments. A fragment that includes residue 1—, covering only the PIN domain, yielded best crystals.

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Since cysteine and histidine are frequent coordinating residues for metal ions such as Zn and Fe, we suspected that Utp23 may be a metal-binding protein. Indeed, characteristic radiation of Zn was detected in Utp23 crystals with X-ray fluorescence scanning at synchrotron. We therefore collected X-ray diffraction data at the peak absorption wavelength of Zn and determined the structure with single-wavelength anomalous dispersion SAD phasing.

The structure was refined at 2. The crystal belongs to space group P3 2 21 and contains two Utp23 molecules per asymmetric unit.

structural and functional analysis of ribosome assembly factor efg1

Other than that, the two molecules have little contact. Gel filtration chromatography showed that Utp23 1— is predominantly monomeric in solution Supplemental Fig. S1suggesting that the observed dimer in crystal is not the physiologically relevant conformation. Domain-swapped dimers have been observed for other crystallized proteins, for example, the FHA domain of Chfr mitotic checkpoint protein Stavridi et al. High protein concentration used in crystallization could induce formation of a small fraction of thermodynamically equivalent domain-swapped dimer, which sometimes crystallizes readily.

Structure of Utp23 PIN domain.Ribosome biogenesis in eukaryotes is a complicated process that involves association and dissociation of numerous assembly factors and snoRNAs. The yeast small ribosomal subunit is first assembled into 90S pre-ribosomes in an ordered and dynamic mann The yeast small ribosomal subunit is first assembled into 90S pre-ribosomes in an ordered and dynamic manner.

Efg1 is a protein with no recognizable domain that is associated with early 90S particles. Here, we determine the crystal structure of Efg1 from Chaetomium thermophilum at 3. Efg1 is not located in recently determined cryo-EM densities of 90S likely due to its low abundance in mature 90S.

Genetic analysis in Saccharomyces cerevisiae shows that the functional core of Efg1 contains two helical hairpins composed of highly conserved residues. Efg1 is initially recruited by the 5' domain of 18S rRNA. Our study shows that Efg1 is required for early assembly and reorganization of the 5' domain of 18S rRNA.

Warning You are using a web browser that we do not support. Our website will not work properly. Please update to a newer version or download a new web browser, such as Chrome or Firefox. Asymmetric Unit. Macromolecule Content Total Structure Weight: This is version 1. Structural and functional analysis of ribosome assembly factor Efg1. Shu, S.

Hide Full Abstract. Reference Sequence. Small Molecules. View more in-depth experimental data. Chaetomium thermophilum var. Protein Feature View Expand.Ribosome biogenesis in eukaryotes is a complicated process that involves association and dissociation of numerous assembly factors and snoRNAs.

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The yeast small ribosomal subunit is first assembled into 90S pre-ribosomes in an ordered and dynamic manner. Efg1 is a protein with no recognizable domain that is associated with early 90S particles. Here, we determine the crystal structure of Efg1 from Chaetomium thermophilum at 3.

Efg1 is not located in recently determined cryo-EM densities of 90S likely due to its low abundance in mature 90S. Genetic analysis in Saccharomyces cerevisiae shows that the functional core of Efg1 contains two helical hairpins composed of highly conserved residues. Ribosomes are essential and conserved nanomachines in all organisms and are responsible for proteins synthesis.

Consisting of four ribosomal RNAs rRNAs and 79 ribosomal proteins, the ribosome in Saccharomyces cerevisiae is assembled in a highly complicated and dynamic process 1—3. A series of pre-ribosomal particles of the small 40S and large 60S subunits are formed, coupled with pre-rRNA processing, structural reorganization and translocation from the nucleolus through the nucleoplasm to the cytoplasm.

Early assembly of both small and large subunits occurs in a stepwise manner during the transcription of pre-rRNA 4—7. Following dramatic structural reorganization, a preS ribosome is released, exported to the cytoplasm and matures into 40S subunits.

Efg1 is a protein component of 90S pre-ribosomes 45 and contains an uncharacterized domain DUF or Efg1 domain. Efg1 deletion is synthetic lethal with mutants of Emg1, another 90S AF and leads to increased sensitivity to paromomycin It is unclear whether and where Efg1 is located in the cryo-EM density of 90S in the absence of the Efg1 structure. In this study, we determine the crystal structure of Efg1 from Chaetomium thermophilum ctEfg1 and analyze the function of Efg1 in pre-rRNA processing and 90S assembly using the model organism S.

The ctEfg1 gene was amplified from C. The construct was confirmed by DNA sequencing. Selenomethione SeMet -substituted ctEfg1 was expressed in M9 medium as described previously After centrifugal clarification, the supernatant was filtered through 0.

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The column was washed with buffer A containing 25 mM imidazole and eluted with a linear gradient of 25— mM imidazole in buffer A. All buffers used in the following purification procedures included 1 mM dithiothreitol DTT. The statistics for data processing and structural refinement are summarized in Table 1. The crystal belongs to space group P6 2 and contains one molecule per asymmetric unit. The final model contains residues 47— and — of ctEfg1. Yeast experiments were performed according to standard protocols.

Plasmids were constructed by the In-Fusion approach and deletion mutations were introduced by the QuikChange method. All plasmids were confirmed by DNA sequencing. Single clones of the transformants of similar size were cultured, 5-fold serially diluted and spotted onto plates containing SC medium with galactose or glucose. Immunoprecipitation was conducted as described below. Ribosome profile assays were performed as previously described The cells were lysed using steel balls and clarified by centrifugation.

The supernatant was incubated with rabbit IgG-coated magnetic Beaver beads Beaverbio for 30 min. The beads were washed five times with 1 ml of lysis buffer and submitted to mass spectrometric analysis or northern blot analysis. Mass spectrometric analysis was conducted as described 6. RNA extraction and northern blotting were carried out as described The reactions were incubated at room temperature for 30 min and resolved in a native polyacrylamide gel run in Tris-glycine buffer pH 8.

We initially attempted to crystallize Efg1 from S. We then shifted to ctEfg1 from the thermophilic fungus C.Toggle navigation. Advanced search. Refine results. Options Page Size. RSS feed. Scholarly journals. Full text online. Exclude newspapers.

Library catalogue. Expand beyond library holdings. Publication Date. Full Text Role of the yeast Rrp1 protein in the dynamics of pre-ribosome maturation. The Saccharomyces cerevisiae gene RRP1 encodes an essential, evolutionarily conserved protein necessary for biogenesis of 60S ribosomal subunits.

Processing of Digital rights. Loading Rights. Full Text Disruption of ribosome assembly in yeast blocks cotranscriptional pre-rRNA processing and affects the global hierarchy of ribosome biogenesis. In higher eukaryotes, pre-rRNA processing occurs almost exclusively post-transcriptionally. This is not the case in rapidly dividing yeast, as the majority of Full Text Molecular architecture of the 90S small subunit pre-ribosome.

Eukaryotic small ribosomal subunits are first assembled into 90S pre-ribosomes.

Functions of mRNA, rRNA, and tRNA

The complete 90S is a gigantic complex with a molecular mass of approximatelyWrote the paper: KY. A multitude of proteins and small nucleolar RNAs transiently associate with eukaryotic ribosomal RNAs to direct their modification and processing and the assembly of ribosomal proteins. Utp22 and Rrp7, two interacting proteins with no recognizable domain, are components of the 90S preribosome or the small subunit processome that conducts early processing of 18S rRNA.

Here, we determine the cocrystal structure of Utp22 and Rrp7 complex at 1. Rrp7 binds extensively to Utp22 using a deviant RNA recognition motif and an extended linker. Functional sites on the two proteins were identified by structure-based mutagenesis in yeast. We show that Rrp7 contains a flexible RNA-binding C-terminal tail that is essential for association with preribosomes. Depletion of snR30 prevents the stable assembly of Rrp7 into preribosomes. Our results provide insight into the evolutionary origin and functional context of Utp22 and Rrp7.

Ribosomes are large RNA—protein complexes that manufacture proteins in all living organisms. Synthesis of large and small ribosomal subunits is a fundamental and enormous task that requires activities of approximately assembly factors in eukaryotic cells.

These factors transiently associate with the ribosome, forming a series of pre-ribosomal particles. We currently have a poor understanding of the structure and assembly of ribosome precursors.

Ar dmr barrel

Utp22 and Rrp7 are two interacting proteins present in early precursors of the small ribosomal subunit. In this study, we determined the structure of the Utp22 and Rrp7 complex by X-ray crystallography and NMR and dissected their functional domains by mutagenesis. The structure of Utp22 reveals an unexpected structural similarity to the tRNA CCA-adding enzyme, providing insight into the evolutionary origin of Utp Utp22 apparently lacks any enzymatic activity and functions instead as a structural building block.

Rrp7 associates extensively with Utp22 and appears to be anchored to pre-ribosomes via a flexible RNA-binding tail. We used RNA—protein crosslinking to identify the binding site and neighboring factor of Rrp7 on pre-ribosomes.

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Our study provides a detailed insight into the structure of small ribosomal subunit precursors. In addition to their greater structural complexity, eukaryotic ribosomes are assembled through a substantially more complex process that involves approximately trans -acting protein factors and 75 small nucleolar RNAs snoRNAs in the yeast Saccharomyces cerevisiae.

In contrast, only several tens of ribosome synthesis factors have been found in bacteria [3]. These conserved eukaryotic factors are involved in modification and processing of pre-rRNAs, coordination of rRNA folding, and the assembly of ribosomal proteins and exportation of preribosomal particles from the nucleolus to the cytoplasm see recent reviews [4] — [7]. The 35S pre-rRNA associates cotranscriptionally with nearly 50 nonribosomal proteins, U3 snoRNA, and a subset of SSU r-proteins into the enormous 90S preribosome or small subunit processome, which can be visualized as a terminal ball on nascent rRNAs by electron microscopy [4][8][9].

Within the 90S preribosome, the 35S pre-rRNA is sequentially cleaved at sites A0, A1, and A2, and these early cleavages can occur during or after transcription [10]. The preS particle is exported to the cytoplasm and associates with LSU to complete its maturation [13][14]. U3, U14, and snR30 are essential in yeast and thought to be universally conserved in eukaryotes, whereas snR10 is nonessential and appears to be yeast-specific.

The former three are known to function by binding to complementary sites on pre-rRNA, but the mode of action remains unknown for snR10 [16][17]. Most ribosome synthesis factors probably have been identified in yeast through genetic study and biochemical purification of various preribosomal particles. Their association with rRNA processing steps and preribosome types has been generally assigned.

The current major challenge is to understand the molecular function of individual factors and the structure and assembly of preribosomal particles. Several complexes and factors were shown to assemble into the 90S preribosome in a hierarchical order [23][24].Here we determine the crystal structure of Utp24 from Schizosaccharomyces pombe at 2.

Utp24 structurally resembles the ribosome assembly factor Utp23 and both contain a Zn-finger motif. Functional analysis in Saccharomyces cerevisiae shows that depletion of Utp24 disturbs the assembly of 90S and abolishes cleavage at sites A0, A1 and A2. The 90S assembled with inactivated Utp24 is arrested at a post-A0-cleavage state and contains enriched nuclear exosome for degradation of 5' ETS.

Despite of high sequence conservation, Utp24 from other organisms is unable to form an active 90S in S. Our study provides biochemical and structural insight into the role of Utp24 in 90S assembly and activity.

This is an open access article distributed under the terms of the Creative Commons Attribution Licensewhich permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: All coordinates and structural factors files are available from the Protein Data Bank database accession number 5YZ4.

The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. Assembly of yeast ribosome requires processing and modification of rRNAs and association of 79 ribosomal proteins [ 12 ]. The four spacers need to be removed by a series of endo- and exo-nucleolytic activities in the context of various pre-ribosomal particles [ 3 ].

Early processing of 18S rRNA occurs in the nucleolus within the 90S pre-ribosome or the small subunit processome [ 45 ], which is the early assembly intermediate of small ribosomal subunits. The cryo-EM structures of 90S have been recently determined, revealing that the nascent 40S ribosome is assembled into several isolated subdomains and stabilized by numerous AFs [ 10 — 14 ].

The 23S pre-rRNA is also considered as a non-functional intermediate that strongly accumulates under stress conditions [ 16 ].

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It typically functions as an endonuclease in degradation and processing of various RNAs. Utp24 is the endonuclease proposed to cleave the A1 and A2 sites. Depletion of Utp24, aka Fcf1 Faf1 copurifying factor 1inhibits cleavage at sites A0, A1 and A2 in yeast [ 2426 ], whereas mutations of the active site of Utp24 specifically block cleavage at sites A1 and A2, but not at site A0 [ 24 ]. This suggests that Utp24 plays both a structural and catalytic role in 90S.

In humans, inactivation of UTP24 similarly inhibits cleavage at the equivalent 1 and 2a sites [ 2728 ]. Structurally, Utp24 crosslinks with the 5' end of 18S [ 27 ] and is positioned near the A1 site in the cryo-EM structures of 90S [ 10 — 14 ].

These data strongly support that Utp24 is the endonuclease for site A1.Proteins Rpf2 and Rrs1 are required for 60S ribosomal subunit maturation. Here we present the crystal structure of the Aspergillus nidulans An Rpf2-Rrs1 core complex. The core complex contains the tightly interlocked N-terminal domains of Rpf2 and Rrs1.

structural and functional analysis of ribosome assembly factor efg1

The Rpf2 N-terminal domain includes a Brix domain characterized by similar N- and C-terminal architecture. The conserved proline-rich linker connecting the N- and C-terminal domains of Rrs1 wrap around the side of Rpf2 and anchor the C-terminal domain of Rrs1 to a specific site on Rpf2.

Further analysis of Rpf2-Rrs1 mutants demonstrated that Saccharomyces cerevisiae Rpf2 R corresponds to R of An Rpf2 plays a significant role in this binding. Based on these studies and previous reports, we have proposed a model for ribosomal component recruitment to the 90S ribosome precursor. These steps determine the splitting of the 90S precursor into two independent complexes, the preS and preS ribosomal particles.

Subsequently, ribosomal subunits exit the nucleolus through the nucleoplasm to the cytoplasm, where they are further assembled into 80S ribosomes for translation 4. This step is thought to require major conformational pre-rRNP rearrangements 67.

Rpf2 and Rrs1 are two such factors. Rpf2 is an Imp4 superfamily protein. Although proteins in this family play distinct roles at different stages of ribosome biogenesis, all have a similar domain architecture consisting of a central globular Brix domain and optional highly charged N- and C-terminal segments 9.

Brix domain function was characterized in a study of Imp4. A structural analysis of the Imp4-like protein Mil revealed the characteristic architecture of the Brix domain, which features a similar arrangement of structural elements in its N- and C-terminal halves Although the Brix domain serves as a scaffold for interactions with several binding partners, these interactions are not well understood.

Rrs1 was isolated as a factor related to a secretory defect that caused the transcriptional repression of both rRNA and ribosomal protein genes This protein was known to localize to the nucleolus and nuclear periphery. In the nucleolus, Rrs1 acts as a ribosome assembly factor. At the nuclear periphery, it directly interacts with the membrane-spanning SUN domain protein Mps3 and silent information factor Sir4, which are involved in telomere clustering and silencing Genetic depletion of each of the four proteins inhibited the recruitment of the other three proteins and 5S rRNA to the 90S precursor Yeast two-hybrid assays and GST pull-down experiments revealed direct interactions between these proteins 17 — Among these interactions, the strong interaction between Rpf2 and Rrs1 suggested that these proteins function as a heterodimer.

We conducted a structural and functional analysis of the Rpf2-Rrs1 complex. Here we describe the crystal structure of the Rpf2-Rrs1 core complex, which contains the N-terminal domains of both Rpf2 and Rrs1. The complex structure revealed that the N-terminal domains of both Rpf2 and Rrs1 interact tightly at three regions.

Structural and functional analysis of the Rpf2-Rrs1 complex in ribosome biogenesis

Interestingly, the highly conserved proline-rich loop of Rrs1 wraps around Rpf2 and facilitates separation of the N- and C-terminal domains of Rrs1. Preparation of the Aspergillus nidulans An Rpf2-Rrs1 complex has been described previously The size exclusion chromatography showed that the stoichiometry of An Rpf2-Rrs1 complex is in solution.

Details of the templates and primers used in this study are described in the Supplementary Tables S1 and S2. All vectors were confirmed by plasmid DNA sequencing. The expressed Sc Rpf2-Rrs1 full-length complex was purified using a previously described method The reaction mixture was subsequently isopropanol-precipitated, purified by denaturing urea-polyacrylamide gel electrophoresis and extracted with an Elutrap Electroelution system Whatman plc, Maidstone, UK.

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