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1.
Nucleic Acids Res ; 51(20): 11277-11290, 2023 11 10.
Article in English | MEDLINE | ID: mdl-37811893

ABSTRACT

Large ribosomal subunit precursors (pre-LSUs) are primarily synthesized in the nucleolus. At an undetermined step in their assembly, they are released into the nucleoplasm. Structural models of yeast pre-LSUs at various stages of assembly have been collected using cryo-EM. However, which cryo-EM model is closest to the final nucleolar intermediate of the LSU has yet to be determined. To elucidate the mechanisms of the release of pre-LSUs from the nucleolus, we assayed effects of depleting or knocking out two yeast ribosome biogenesis factors (RiBi factors), Puf6 and Nog2, and two ribosomal proteins, uL2 and eL43. These proteins function during or stabilize onto pre-LSUs between the late nucleolar stages to early nucleoplasmic stages of ribosome biogenesis. By characterizing the phenotype of these four mutants, we determined that a particle that is intermediate between the cryo-EM model State NE1 and State NE2 likely represents the final nucleolar assembly intermediate of the LSU. We conclude that the release of the RiBi factors Nip7, Nop2 and Spb1 and the subsequent stabilization of rRNA domains IV and V may be key triggers for the release of pre-LSUs from the nucleolus.


Subject(s)
Ribosomal Proteins , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae , Ribosomal Proteins/metabolism , Ribosome Subunits, Large/metabolism , Ribosome Subunits, Large, Eukaryotic/metabolism , RNA, Ribosomal/metabolism , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/metabolism
2.
Nucleic Acids Res ; 51(20): 10867-10883, 2023 11 10.
Article in English | MEDLINE | ID: mdl-35736211

ABSTRACT

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.


Subject(s)
Cell Nucleolus , Ribosomes , Saccharomyces cerevisiae , Cell Nucleolus/metabolism , Cell Nucleus/metabolism , Ribosomal Proteins/metabolism , Ribosomes/metabolism , RNA, Ribosomal/metabolism , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism
3.
Nucleic Acids Res ; 50(11): 6453-6473, 2022 06 24.
Article in English | MEDLINE | ID: mdl-35639884

ABSTRACT

During translation, nascent polypeptide chains travel from the peptidyl transferase center through the nascent polypeptide exit tunnel (NPET) to emerge from 60S subunits. The NPET includes portions of five of the six 25S/5.8S rRNA domains and ribosomal proteins uL4, uL22, and eL39. Internal loops of uL4 and uL22 form the constriction sites of the NPET and are important for both assembly and function of ribosomes. Here, we investigated the roles of eL39 in tunnel construction, 60S biogenesis, and protein synthesis. We show that eL39 is important for proper protein folding during translation. Consistent with a delay in processing of 27S and 7S pre-rRNAs, eL39 functions in pre-60S assembly during middle nucleolar stages. Our biochemical assays suggest the presence of eL39 in particles at these stages, although it is not visualized in them by cryo-electron microscopy. This indicates that eL39 takes part in assembly even when it is not fully accommodated into the body of pre-60S particles. eL39 is also important for later steps of assembly, rotation of the 5S ribonucleoprotein complex, likely through long range rRNA interactions. Finally, our data strongly suggest the presence of alternative pathways of ribosome assembly, previously observed in the biogenesis of bacterial ribosomal subunits.


Subject(s)
Ribosomal Proteins , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/metabolism , Cryoelectron Microscopy , Models, Molecular , Peptides/metabolism , Protein Folding , RNA, Ribosomal/metabolism , Ribosomal Proteins/metabolism , Ribosome Subunits, Large, Eukaryotic/metabolism , Saccharomyces cerevisiae Proteins/metabolism
4.
Curr Biol ; 31(12): 2507-2519.e4, 2021 06 21.
Article in English | MEDLINE | ID: mdl-33862007

ABSTRACT

In ribosomal DNA (rDNA) repeats, sequences encoding small-subunit (SSU) rRNA precede those encoding large-subunit (LSU) rRNAs. Processing the composite transcript and subunit assembly requires >100 subunit-specific nucleolar assembly factors (AFs). To investigate the functional organization of the nucleolus, we localized AFs in S. cerevisiae in which the rDNA axis was "linearized" to reduce its dimensionality, thereby revealing its coaxial organization. In this situation, rRNA synthesis and processing continue. The axis is embedded in an inner layer/phase of SSU AFs that is surrounded by an outer layer/phase of LSU AFs. When subunit production is inhibited, subsets of AFs differentially relocate between the inner and outer layers, as expected if there is a cycle of repeated relocation whereby "latent" AFs become "operative" when recruited to nascent subunits. Recognition of AF cycling and localization of segments of rRNA make it possible to infer the existence of assembly intermediates that span between the inner and outer layers and to chart the cotranscriptional assembly of each subunit. AF cycling also can explain how having more than one protein phase in the nucleolus makes possible "vectorial 2-phase partitioning" as a driving force for relocation of nascent rRNPs. Because nucleoplasmic AFs are also present in the outer layer, we propose that critical surface remodeling occurs at this site, thereby partitioning subunit precursors into the nucleoplasm for post-transcriptional maturation. Comparison to observations on higher eukaryotes shows that the coaxial paradigm is likely to be applicable for the many other organisms that have rDNA repeats.


Subject(s)
Cell Nucleolus , DNA, Ribosomal , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/genetics , Cell Nucleolus/genetics , DNA, Ribosomal/genetics , RNA, Ribosomal/genetics
5.
Nat Commun ; 11(1): 5111, 2020 10 09.
Article in English | MEDLINE | ID: mdl-33037216

ABSTRACT

The nascent polypeptide exit tunnel (NPET) is a major functional center of 60S ribosomal subunits. However, little is known about how the NPET is constructed during ribosome assembly. We utilized molecular genetics, biochemistry, and cryo-electron microscopy (cryo-EM) to investigate the functions of two NPET-associated proteins, ribosomal protein uL4 and assembly factor Nog1, in NPET assembly. Structures of mutant pre-ribosomes lacking the tunnel domain of uL4 reveal a misassembled NPET, including an aberrantly flexible ribosomal RNA helix 74, resulting in at least three different blocks in 60S assembly. Structures of pre-ribosomes lacking the C-terminal extension of Nog1 demonstrate that this extension scaffolds the tunnel domain of uL4 in the NPET to help maintain stability in the core of pre-60S subunits. Our data reveal that uL4 and Nog1 work together in the maturation of ribosomal RNA helix 74, which is required to ensure proper construction of the NPET and 60S ribosomal subunits.


Subject(s)
GTP-Binding Proteins/metabolism , Nuclear Proteins/metabolism , Ribosomal Proteins/metabolism , Ribosome Subunits, Large, Eukaryotic/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Cell Cycle Proteins/metabolism , Cryoelectron Microscopy , GTP-Binding Proteins/chemistry , GTP-Binding Proteins/genetics , Models, Molecular , Mutation , Nuclear Proteins/chemistry , Nuclear Proteins/genetics , Protein Domains , RNA Stability , RNA, Ribosomal/chemistry , RNA, Ribosomal/metabolism , Ribosomal Proteins/chemistry , Ribosomal Proteins/genetics , Ribosome Subunits, Large, Eukaryotic/chemistry , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/genetics
6.
Nat Commun ; 11(1): 3751, 2020 07 27.
Article in English | MEDLINE | ID: mdl-32719344

ABSTRACT

The protein composition and structure of assembling 60S ribosomal subunits undergo numerous changes as pre-ribosomes transition from the nucleolus to the nucleoplasm. This includes stable anchoring of the Rpf2 subcomplex containing 5S rRNA, rpL5, rpL11, Rpf2 and Rrs1, which initially docks onto the flexible domain V of rRNA at earlier stages of assembly. In this work, we tested the function of the C-terminal domain (CTD) of Rpf2 during these anchoring steps, by truncating this extension and assaying effects on middle stages of subunit maturation. The rpf2Δ255-344 mutation affects proper folding of rRNA helices H68-70 during anchoring of the Rpf2 subcomplex. In addition, several assembly factors (AFs) are absent from pre-ribosomes or in altered conformations. Consequently, major remodeling events fail to occur: rotation of the 5S RNP, maturation of the peptidyl transferase center (PTC) and the nascent polypeptide exit tunnel (NPET), and export of assembling subunits to the cytoplasm.


Subject(s)
Ribonucleoproteins/metabolism , Ribosome Subunits, Large/metabolism , Rotation , Saccharomyces cerevisiae/metabolism , Active Transport, Cell Nucleus , Cell Nucleus/metabolism , Models, Molecular , Mutant Proteins/chemistry , Mutant Proteins/genetics , Mutation/genetics , Protein Domains , Protein Folding , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/metabolism , RNA-Binding Proteins/ultrastructure , Ribosome Subunits, Large/chemistry , Saccharomyces cerevisiae/growth & development , Saccharomyces cerevisiae/ultrastructure , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae Proteins/ultrastructure
7.
Nat Rev Mol Cell Biol ; 20(2): 116-131, 2019 02.
Article in English | MEDLINE | ID: mdl-30467428

ABSTRACT

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.


Subject(s)
Ribosome Subunits/genetics , Animals , Cell Nucleus/genetics , Cryoelectron Microscopy/methods , Cytoplasm/genetics , Humans , RNA/genetics
8.
J Cell Biol ; 217(7): 2503-2518, 2018 07 02.
Article in English | MEDLINE | ID: mdl-29691304

ABSTRACT

Ribosome biogenesis involves numerous preribosomal RNA (pre-rRNA) processing events to remove internal and external transcribed spacer sequences, ultimately yielding three mature rRNAs. Removal of the internal transcribed spacer 2 spacer RNA is the final step in large subunit pre-rRNA processing and begins with endonucleolytic cleavage at the C2 site of 27SB pre-rRNA. C2 cleavage requires the hierarchical recruitment of 11 ribosomal proteins and 14 ribosome assembly factors. However, the function of these proteins in C2 cleavage remained unclear. In this study, we have performed a detailed analysis of the effects of depleting proteins required for C2 cleavage and interpreted these results using cryo-electron microscopy structures of assembling 60S subunits. This work revealed that these proteins are required for remodeling of several neighborhoods, including two major functional centers of the 60S subunit, suggesting that these remodeling events form a checkpoint leading to C2 cleavage. Interestingly, when C2 cleavage is directly blocked by depleting or inactivating the C2 endonuclease, assembly progresses through all other subsequent steps.


Subject(s)
RNA Precursors/ultrastructure , RNA, Ribosomal/ultrastructure , Ribosomal Proteins/ultrastructure , Ribosomes/ultrastructure , Cryoelectron Microscopy , Crystallography, X-Ray , DNA, Ribosomal Spacer/genetics , DNA, Ribosomal Spacer/ultrastructure , RNA Precursors/chemistry , RNA Precursors/genetics , RNA, Ribosomal/chemistry , RNA, Ribosomal/genetics , Ribosomal Proteins/biosynthesis , Ribosomal Proteins/genetics , Ribosomes/chemistry , Ribosomes/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/ultrastructure
9.
RNA Biol ; 14(10): 1306-1313, 2017 10 03.
Article in English | MEDLINE | ID: mdl-28267408

ABSTRACT

Ribosomes are responsible for translating the genome, in the form of mRNA, into the proteome in all organisms. Biogenesis of ribosomes in eukaryotes is a complex process involving numerous remodeling events driven in part by the concerted actions of hundreds of protein assembly factors. A major challenge in studying eukaryotic ribosome assembly has, until recently, been a lack of structural data to facilitate understanding of the conformational and compositional changes the pre-ribosome undergoes during its construction. Cryo-electron microscopy (cryo-EM) has begun filling these gaps; recent advances in cryo-EM have enabled the determination of several high resolution pre-ribosome structures. This review focuses mainly on lessons learned from the study of pre-60S particles purified from yeast using the assembly factor Nog2 as bait. These Nog2 particles provide insight into many aspects of nuclear stages of 60S subunit assembly, including construction of major 60S subunit functional centers and processing of the ITS2 spacer RNA.


Subject(s)
GTP Phosphohydrolases/metabolism , Ribosome Subunits, Large, Eukaryotic/chemistry , Ribosome Subunits, Large, Eukaryotic/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Cryoelectron Microscopy , DNA, Ribosomal Spacer , Models, Molecular , Protein Biosynthesis , Protein Conformation , Protein Multimerization , Saccharomyces cerevisiae/chemistry , Saccharomyces cerevisiae Proteins/chemistry
10.
Nucleic Acids Res ; 45(8): 4853-4865, 2017 05 05.
Article in English | MEDLINE | ID: mdl-28115637

ABSTRACT

A major gap in our understanding of ribosome assembly is knowing the precise function of each of the ∼200 assembly factors. The steps in subunit assembly in which these factors participate have been examined for the most part by depleting each protein from cells. Depletion of the assembly factor Erb1 prevents stable assembly of seven other interdependent assembly factors with pre-60S subunits, resulting in turnover of early preribosomes, before the ITS1 spacer can be removed from 27SA3 pre-rRNA. To investigate more specific functions of Erb1, we constructed eight internal deletions of 40-60 amino acid residues each, spanning the amino-terminal half of Erb1. The erb1Δ161-200 and erb1Δ201-245 deletion mutations block a later step than depletion of Erb1, namely cleavage of the C2 site that initiates removal of the ITS2 spacer. Two other remodeling events fail to occur in these erb1 mutants: association of twelve different assembly factors with domain V of 25S rRNA, including the neighborhood surrounding the peptidyl transferase center, and stable association of ribosomal proteins with rRNA surrounding the polypeptide exit tunnel. This suggests that successful initiation of construction of these functional centers is a checkpoint for committing to spacer removal.


Subject(s)
DNA, Ribosomal Spacer/genetics , RNA Precursors/genetics , Ribosomal Proteins/genetics , Ribosomes/genetics , Saccharomyces cerevisiae Proteins/genetics , Cell Nucleus/genetics , Ribosome Subunits, Large, Eukaryotic/genetics , Saccharomyces cerevisiae/genetics
11.
Nat Struct Mol Biol ; 24(3): 214-220, 2017 03.
Article in English | MEDLINE | ID: mdl-28112732

ABSTRACT

A key step in ribosome biogenesis is the nuclear export of pre-ribosomal particles. Nmd3, a highly conserved protein in eukaryotes, is a specific adaptor required for the export of pre-60S particles. Here we used cryo-electron microscopy (cryo-EM) to characterize Saccharomyces cerevisiae pre-60S particles purified with epitope-tagged Nmd3. Our structural analysis indicates that these particles belong to a specific late stage of cytoplasmic pre-60S maturation in which ribosomal proteins uL16, uL10, uL11, eL40 and eL41 are deficient, but ribosome assembly factors Nmd3, Lsg1, Tif6 and Reh1 are present. Nmd3 and Lsg1 are located near the peptidyl-transferase center (PTC). In particular, Nmd3 recognizes the PTC in its near-mature conformation. In contrast, Reh1 is anchored to the exit of the polypeptide tunnel, with its C terminus inserted into the tunnel. These findings pinpoint a structural checkpoint role for Nmd3 in PTC assembly, and provide information about functional and mechanistic roles of these assembly factors in the maturation of the 60S ribosomal subunit.


Subject(s)
Cytoplasm/metabolism , Ribosome Subunits, Large, Eukaryotic/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Cytoplasm/ultrastructure , Models, Molecular , Peptides/chemistry , Peptides/metabolism , Peptidyl Transferases/metabolism , Protein Binding , Protein Domains , RNA, Ribosomal/chemistry , RNA, Ribosomal/metabolism , Ribosomal Proteins/chemistry , Ribosomal Proteins/metabolism , Ribosome Subunits, Large, Eukaryotic/chemistry , Ribosome Subunits, Large, Eukaryotic/ultrastructure , Saccharomyces cerevisiae/ultrastructure , Saccharomyces cerevisiae Proteins/chemistry
12.
Biochem J ; 474(2): 195-214, 2017 01 15.
Article in English | MEDLINE | ID: mdl-28062837

ABSTRACT

Ribosome biogenesis requires the intertwined processes of folding, modification, and processing of ribosomal RNA, together with binding of ribosomal proteins. In eukaryotic cells, ribosome assembly begins in the nucleolus, continues in the nucleoplasm, and is not completed until after nascent particles are exported to the cytoplasm. The efficiency and fidelity of ribosome biogenesis are facilitated by >200 assembly factors and ∼76 different small nucleolar RNAs. The pathway is driven forward by numerous remodeling events to rearrange the ribonucleoprotein architecture of pre-ribosomes. Here, we describe principles of ribosome assembly that have emerged from recent studies of biogenesis of the large ribosomal subunit in the yeast Saccharomyces cerevisiae We describe tools that have empowered investigations of ribosome biogenesis, and then summarize recent discoveries about each of the consecutive steps of subunit assembly.


Subject(s)
Organelle Biogenesis , RNA, Ribosomal/chemistry , RNA, Small Nucleolar/chemistry , Ribosomal Proteins/chemistry , Ribosome Subunits, Large, Eukaryotic/metabolism , Saccharomyces cerevisiae/metabolism , Active Transport, Cell Nucleus , Binding Sites , Cell Nucleolus/genetics , Cell Nucleolus/metabolism , Cell Nucleolus/ultrastructure , Cytoplasm/genetics , Cytoplasm/metabolism , Cytoplasm/ultrastructure , Models, Molecular , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , RNA Precursors/chemistry , RNA Precursors/genetics , RNA Precursors/metabolism , RNA, Ribosomal/genetics , RNA, Ribosomal/metabolism , RNA, Small Nucleolar/genetics , RNA, Small Nucleolar/metabolism , Ribosomal Proteins/genetics , Ribosomal Proteins/metabolism , Ribosome Subunits, Large, Eukaryotic/genetics , Ribosome Subunits, Large, Eukaryotic/ultrastructure , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/ultrastructure
13.
Protein Sci ; 26(1): 103-112, 2017 01.
Article in English | MEDLINE | ID: mdl-27643814

ABSTRACT

The assembly of ribosomal subunits starts in the nucleus, initiated by co-transcriptional folding of nascent ribosomal RNA (rRNA) transcripts and binding of ribosomal proteins and assembly factors. The internal transcribed spacer 2 (ITS2) is a precursor sequence to be processed from the intermediate 27S rRNA in the nucleoplasm; its removal is required for nuclear export of pre-60S particles. The proper processing of the ITS2 depends on multiple associated assembly factors and RNases. However, none of the structures of the known ITS2-binding factors is available. Here, we describe the modeling of the ITS2 subcomplex, including five assembly factors Cic1, Nop7, Nop15, Nop53, and Rlp7, using a combination of cryo-electron microscopy and cross-linking of proteins coupled with mass spectrometry approaches. The resulting atomic models provide structural insights into their function in ribosome assembly, and establish a framework for further dissection of their molecular roles in ITS2 processing.


Subject(s)
Models, Molecular , RNA, Ribosomal/ultrastructure , Ribosome Subunits, Large, Eukaryotic/ultrastructure , Saccharomyces cerevisiae/ultrastructure , Carrier Proteins/chemistry , Carrier Proteins/metabolism , Cryoelectron Microscopy , Mass Spectrometry , Nuclear Proteins/chemistry , Nuclear Proteins/metabolism , RNA, Ribosomal/chemistry , RNA, Ribosomal/metabolism , Ribosomal Proteins/chemistry , Ribosomal Proteins/metabolism , Ribosome Subunits, Large, Eukaryotic/chemistry , Ribosome Subunits, Large, Eukaryotic/metabolism , Saccharomyces cerevisiae/chemistry , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/metabolism
14.
Proc Natl Acad Sci U S A ; 113(43): 12174-12179, 2016 10 25.
Article in English | MEDLINE | ID: mdl-27791004

ABSTRACT

Ribosomes of trypanosomatids, a family of protozoan parasites causing debilitating human diseases, possess multiply fragmented rRNAs that together are analogous to 28S rRNA, unusually large rRNA expansion segments, and r-protein variations compared with other eukaryotic ribosomes. To investigate the architecture of the trypanosomatid ribosomes, we determined the 2.5-Å structure of the Trypanosoma cruzi ribosome large subunit by single-particle cryo-EM. Examination of this structure and comparative analysis of the yeast ribosomal assembly pathway allowed us to develop a stepwise assembly model for the eight pieces of the large subunit rRNAs and a number of ancillary "glue" proteins. This model can be applied to the characterization of Trypanosoma brucei and Leishmania spp. ribosomes as well. Together with other details, our atomic-level structure may provide a foundation for structure-based design of antitrypanosome drugs.


Subject(s)
Ribosome Subunits, Large, Eukaryotic/ultrastructure , Ribosomes/ultrastructure , Trypanosoma cruzi/chemistry , Chagas Disease/drug therapy , Chagas Disease/parasitology , Cryoelectron Microscopy , Crystallography, X-Ray , Humans , RNA, Ribosomal/genetics , RNA, Ribosomal/ultrastructure , Ribosomal Proteins/chemistry , Ribosomal Proteins/genetics , Ribosome Subunits, Large, Eukaryotic/chemistry , Ribosome Subunits, Large, Eukaryotic/genetics , Ribosomes/genetics , Trypanosoma cruzi/genetics , Trypanosoma cruzi/ultrastructure
15.
RNA ; 22(9): 1386-99, 2016 09.
Article in English | MEDLINE | ID: mdl-27390266

ABSTRACT

Assaying effects on pre-rRNA processing and ribosome assembly upon depleting individual ribosomal proteins (r-proteins) provided an initial paradigm for assembly of eukaryotic ribosomes in vivo-that each structural domain of ribosomal subunits assembles in a hierarchical fashion. However, two features suggest that a more complex pathway may exist: (i) Some r-proteins contain extensions that reach long distances across ribosomes to interact with multiple rRNA domains as well as with other r-proteins. (ii) Individual r-proteins may assemble in a stepwise fashion. For example, the globular domain of an r-protein might assemble separately from its extensions. Thus, these extensions might play roles in assembly that could not be revealed by depleting the entire protein. Here, we show that deleting or mutating extensions of r-proteins L7 (uL30) and L35 (uL29) from yeast reveal important roles in early and middle steps during 60S ribosomal subunit biogenesis. Detailed analysis of the N-terminal terminal extension of L8 (eL8) showed that it is necessary for late nuclear stages of 60S subunit assembly involving two major remodeling events: removal of the ITS2 spacer; and reorganization of the central protuberance (CP) containing 5S rRNA and r-proteins L5 (uL18) and L11 (uL5). Mutations in the L8 extension block processing of 7S pre-rRNA, prevent release of assembly factors Rpf2 and Rrs1 from pre-ribosomes, which is required for rotation of the CP, and block association of Sda1, the Rix1 complex, and the Rea1 ATPase involved in late steps of remodeling.


Subject(s)
Ribosomal Proteins/metabolism , Ribosome Subunits, Large, Eukaryotic/metabolism , Saccharomyces cerevisiae/metabolism , Amino Acid Motifs , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Protein Binding , RNA, Ribosomal/metabolism , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Ribosomal Proteins/chemistry , Ribosomal Proteins/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism
16.
RNA ; 22(8): 1153-62, 2016 08.
Article in English | MEDLINE | ID: mdl-27317789

ABSTRACT

The secondary structure of ribosomal RNA (rRNA) is largely conserved across all kingdoms of life. However, eukaryotes have evolved extra blocks of rRNA sequences, relative to those of prokaryotes, called expansion segments (ES). A thorough characterization of the potential roles of ES remains to be done, possibly because of limitations in the availability of robust systems to study rRNA mutants. We sought to systematically investigate the potential functions, if any, of the ES in 25S rRNA of Saccharomyces cerevisiae by deletion mutagenesis. We deleted 14 of the 16 different eukaryote-specific ES in yeast 25S rRNA individually and assayed their phenotypes. Our results show that all but two of the ES tested are necessary for optimal growth and are required for production of 25S rRNA, suggesting that ES play roles in ribosome biogenesis. Further, we classified expansion segments into groups that participate in early nucleolar, middle, and late nucleoplasmic steps of ribosome biogenesis, by assaying their pre-rRNA processing phenotypes. This study is the first of its kind to systematically identify the functions of eukaryote-specific expansion segments by showing that they play roles in specific steps of ribosome biogenesis. The catalog of phenotypes we identified, combined with previous investigations of the roles ribosomal proteins in large subunit biogenesis, leads us to infer that assembling ribosomes are composed of distinct RNA and protein structural neighborhood clusters that participate in specific steps of ribosome biogenesis.


Subject(s)
RNA, Fungal/genetics , RNA, Ribosomal/metabolism , Ribosomes/metabolism , Saccharomyces cerevisiae/genetics
17.
Nature ; 534(7605): 133-7, 2016 06 02.
Article in English | MEDLINE | ID: mdl-27251291

ABSTRACT

Ribosome biogenesis is a highly complex process in eukaryotes, involving temporally and spatially regulated ribosomal protein (r-protein) binding and ribosomal RNA remodelling events in the nucleolus, nucleoplasm and cytoplasm. Hundreds of assembly factors, organized into sequential functional groups, facilitate and guide the maturation process into productive assembly branches in and across different cellular compartments. However, the precise mechanisms by which these assembly factors function are largely unknown. Here we use cryo-electron microscopy to characterize the structures of yeast nucleoplasmic pre-60S particles affinity-purified using the epitope-tagged assembly factor Nog2. Our data pinpoint the locations and determine the structures of over 20 assembly factors, which are enriched in two areas: an arc region extending from the central protuberance to the polypeptide tunnel exit, and the domain including the internal transcribed spacer 2 (ITS2) that separates 5.8S and 25S ribosomal RNAs. In particular, two regulatory GTPases, Nog2 and Nog1, act as hub proteins to interact with multiple, distant assembly factors and functional ribosomal RNA elements, manifesting their critical roles in structural remodelling checkpoints and nuclear export. Moreover, our snapshots of compositionally and structurally different pre-60S intermediates provide essential mechanistic details for three major remodelling events before nuclear export: rotation of the 5S ribonucleoprotein, construction of the active centre and ITS2 removal. The rich structural information in our structures provides a framework to dissect molecular roles of diverse assembly factors in eukaryotic ribosome assembly.


Subject(s)
Cryoelectron Microscopy , Ribosomal Proteins/metabolism , Ribosomal Proteins/ultrastructure , Ribosome Subunits, Large, Eukaryotic/chemistry , Ribosome Subunits, Large, Eukaryotic/ultrastructure , Saccharomyces cerevisiae/chemistry , Saccharomyces cerevisiae/ultrastructure , Active Transport, Cell Nucleus , Base Sequence , Catalytic Domain , Cell Nucleus/chemistry , Cell Nucleus/metabolism , Cell Nucleus/ultrastructure , Cytoplasm/metabolism , DNA, Ribosomal Spacer/chemistry , DNA, Ribosomal Spacer/genetics , DNA, Ribosomal Spacer/metabolism , DNA, Ribosomal Spacer/ultrastructure , GTP Phosphohydrolases/chemistry , GTP Phosphohydrolases/metabolism , GTP Phosphohydrolases/ultrastructure , GTP-Binding Proteins/chemistry , GTP-Binding Proteins/metabolism , GTP-Binding Proteins/ultrastructure , Models, Molecular , Molecular Sequence Data , Nuclear Proteins/chemistry , Nuclear Proteins/metabolism , Nuclear Proteins/ultrastructure , Protein Binding , RNA, Fungal/genetics , RNA, Fungal/metabolism , RNA, Fungal/ultrastructure , RNA, Ribosomal/genetics , RNA, Ribosomal/metabolism , RNA, Ribosomal/ultrastructure , Ribonucleoproteins/chemistry , Ribonucleoproteins/metabolism , Ribonucleoproteins/ultrastructure , Ribosomal Proteins/chemistry , Ribosomal Proteins/isolation & purification , Ribosome Subunits, Large, Eukaryotic/metabolism , Rotation , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/isolation & purification , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae Proteins/ultrastructure
18.
RNA ; 22(6): 852-66, 2016 06.
Article in English | MEDLINE | ID: mdl-27036125

ABSTRACT

In higher eukaryotes, pre-rRNA processing occurs almost exclusively post-transcriptionally. This is not the case in rapidly dividing yeast, as the majority of nascent pre-rRNAs are processed cotranscriptionally, with cleavage at the A2 site first releasing a pre-40S ribosomal subunit followed by release of a pre-60S ribosomal subunit upon transcription termination. Ribosome assembly is driven in part by hierarchical association of assembly factors and r-proteins. Groups of proteins are thought to associate with pre-ribosomes cotranscriptionally during early assembly steps, whereas others associate later, after transcription is completed. Here we describe a previously uncharacterized phenotype observed upon disruption of ribosome assembly, in which normally late-binding proteins associate earlier, with pre-ribosomes containing 35S pre-rRNA. As previously observed by many other groups, we show that disruption of 60S subunit biogenesis results in increased amounts of 35S pre-rRNA, suggesting that a greater fraction of pre-rRNAs are processed post-transcriptionally. Surprisingly, we found that early pre-ribosomes containing 35S pre-rRNA also contain proteins previously thought to only associate with pre-ribosomes after early pre-rRNA processing steps have separated maturation of the two subunits. We believe the shift to post-transcriptional processing is ultimately due to decreased cellular division upon disruption of ribosome assembly. When cells are grown under stress or to high density, a greater fraction of pre-rRNAs are processed post-transcriptionally and follow an alternative processing pathway. Together, these results affirm the principle that ribosome assembly occurs through different, parallel assembly pathways and suggest that there is a kinetic foot-race between the formation of protein binding sites and pre-rRNA processing events.


Subject(s)
RNA Precursors/metabolism , RNA Processing, Post-Transcriptional , RNA, Fungal/metabolism , Ribosomes/metabolism , Yeasts/metabolism , Yeasts/genetics
19.
Biochemistry ; 55(12): 1749-57, 2016 Mar 29.
Article in English | MEDLINE | ID: mdl-26950608

ABSTRACT

Potential guanine (G) quadruplex-forming sequences (QFSs) found throughout the genomes and transcriptomes of organisms have emerged as biologically relevant structures. These G-quadruplexes represent novel opportunities for gene regulation at the DNA and RNA levels. Recently, the definition of functional QFSs has been expanding to include a variety of unconventional motifs, including relatively long loop sequences (i.e., >7 nucleotides) separating adjacent G-tracts. We have identified a QFS within the 25S rDNA gene from Saccharomyces cerevisae that features a long loop separating the two 3'-most G-tracts. An oligonucleotide based on this sequence, QFS3, folds into a stable G-quadruplex in vitro. We have studied the interaction between QFS3 and several loop mutants with a small, homologous (G-rich) peptide nucleic acid (PNA) oligomer that is designed to form a DNA/PNA heteroquadruplex. The PNA successfully invades the DNA quadruplex target to form a stable heteroquadruplex, but with surprisingly high PNA:DNA ratios based on surface plasmon resonance and mass spectrometric results. A model for high stoichiometry PNA-DNA heteroquadruplexes is proposed, and the implications for quadruplex targeting by G-rich PNA are discussed.


Subject(s)
DNA/chemistry , DNA/metabolism , G-Quadruplexes , Peptide Nucleic Acids/chemistry , Peptide Nucleic Acids/metabolism , Nucleic Acid Hybridization/physiology , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/metabolism
20.
Annu Rev Biochem ; 84: 93-129, 2015.
Article in English | MEDLINE | ID: mdl-25706898

ABSTRACT

The proteome of cells is synthesized by ribosomes, complex ribonucleoproteins that in eukaryotes contain 79-80 proteins and four ribosomal RNAs (rRNAs) more than 5,400 nucleotides long. How these molecules assemble together and how their assembly is regulated in concert with the growth and proliferation of cells remain important unanswered questions. Here, we review recently emerging principles to understand how eukaryotic ribosomal proteins drive ribosome assembly in vivo. Most ribosomal proteins assemble with rRNA cotranscriptionally; their association with nascent particles is strengthened as assembly proceeds. Each subunit is assembled hierarchically by sequential stabilization of their subdomains. The active sites of both subunits are constructed last, perhaps to prevent premature engagement of immature ribosomes with active subunits. Late-assembly intermediates undergo quality-control checks for proper function. Mutations in ribosomal proteins that affect mostly late steps lead to ribosomopathies, diseases that include a spectrum of cell type-specific disorders that often transition from hypoproliferative to hyperproliferative growth.


Subject(s)
Eukaryotic Cells/metabolism , Ribosomal Proteins/metabolism , Ribosomes/chemistry , Ribosomes/metabolism , Animals , Eukaryotic Cells/chemistry , Humans , Models, Molecular , Ribosomal Proteins/chemistry
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