ABSTRACT
Ribosomes are ribozymes, hence correct folding of the rRNAs during ribosome biogenesis is crucial to ensure catalytic activity. RNA helicases, which can modulate RNA-RNA and RNA/protein interactions, are proposed to participate in rRNA tridimensional folding. Here, we analyze the biochemical properties of Dbp6, a DEAD-box RNA helicase required for the conversion of the initial 90S pre-ribosomal particle into the first pre-60S particle. We demonstrate that in vitro, Dbp6 shows ATPase as well as annealing and clamping activities negatively regulated by ATP. Mutations in Dbp6 core motifs involved in ATP binding and ATP hydrolysis are lethal and impair Dbp6 ATPase activity but increase its RNA binding and RNA annealing activities. These data suggest that correct regulation of these activities is important for Dbp6 function in vivo. Using in vivo cross-linking (CRAC) experiments, we show that Dbp6 interacts with 25S rRNA sequences located in the 5' domain I and in the peptidyl transferase center (PTC), and also crosslinks to snoRNAs hybridizing to the immature PTC. We propose that the ATPase and RNA clamping/annealing activities of Dbp6 modulate interactions of snoRNAs with the immature PTC and/or contribute directly to the folding of this region.
Subject(s)
DEAD-box RNA Helicases , Ribosomes , Saccharomyces cerevisiae Proteins , Adenosine Triphosphatases/genetics , Adenosine Triphosphatases/metabolism , Adenosine Triphosphate/metabolism , DEAD-box RNA Helicases/genetics , DEAD-box RNA Helicases/metabolism , Peptidyl Transferases/genetics , Peptidyl Transferases/metabolism , Ribosomes/genetics , Ribosomes/metabolism , RNA Helicases/genetics , RNA, Ribosomal/metabolism , RNA, Small Nucleolar/metabolism , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolismABSTRACT
Eukaryotic ribosome synthesis involves more than 200 assembly factors, which promote ribosomal RNA (rRNA) processing, modification and folding, and assembly of ribosomal proteins. The formation and maturation of the earliest pre-60S particles requires structural remodeling by the Npa1 complex, but is otherwise still poorly understood. Here, we introduce Rbp95 (Ycr016w), a constituent of early pre-60S particles, as a novel ribosome assembly factor. We show that Rbp95 is both genetically and physically linked to most Npa1 complex members and to ribosomal protein Rpl3. We demonstrate that Rbp95 is an RNA-binding protein containing two independent RNA-interacting domains. In vivo, Rbp95 associates with helix H95 in the 3' region of the 25S rRNA, in close proximity to the binding sites of Npa1 and Rpl3. Additionally, Rbp95 interacts with several snoRNAs. The absence of Rbp95 results in alterations in the protein composition of early pre-60S particles. Moreover, combined mutation of Rbp95 and Npa1 complex members leads to a delay in the maturation of early pre-60S particles. We propose that Rbp95 acts together with the Npa1 complex during early pre-60S maturation, potentially by promoting pre-rRNA folding events within pre-60S particles.
Subject(s)
Nuclear Proteins/metabolism , Ribosome Subunits, Large, Eukaryotic , Saccharomyces cerevisiae Proteins/metabolism , RNA Precursors/metabolism , RNA, Ribosomal/chemistry , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Ribosomal Proteins/metabolism , Ribosome Subunits, Large, Eukaryotic/metabolism , Saccharomyces cerevisiae/geneticsABSTRACT
In eukaryotes, three of the four ribosomal RNAs (rRNAs)the 5.8S, 18S, and 25S/28S rRNAsare processed from a single pre-rRNA transcript and assembled into ribosomes. The fourth rRNA, the 5S rRNA, is transcribed by RNA polymerase III and is assembled into the 5S ribonucleoprotein particle (RNP), containing ribosomal proteins Rpl5/uL18 and Rpl11/uL5, prior to its incorporation into preribosomes. In mammals, the 5S RNP is also a central regulator of the homeostasis of the tumor suppressor p53. The nucleolar localization of the 5S RNP and its assembly into preribosomes are performed by a specialized complex composed of Rpf2 and Rrs1 in yeast or Bxdc1 and hRrs1 in humans. Here we report the structural and functional characterization of the Rpf2-Rrs1 complex alone, in complex with the 5S RNA, and within pre-60S ribosomes. We show that the Rpf2-Rrs1 complex contains a specialized 5S RNA E-loop-binding module, contacts the Rpl5 protein, and also contacts the ribosome assembly factor Rsa4 and the 25S RNA. We propose that the Rpf2-Rrs1 complex establishes a network of interactions that guide the incorporation of the 5S RNP in preribosomes in the initial conformation prior to its rotation to form the central protuberance found in the mature large ribosomal subunit.
Subject(s)
Models, Molecular , Nuclear Proteins/chemistry , RNA, Ribosomal, 5S/metabolism , RNA-Binding Proteins/chemistry , Saccharomyces cerevisiae Proteins/chemistry , Binding Sites , Cryoelectron Microscopy , Nuclear Proteins/metabolism , Protein Binding , Protein Structure, Quaternary , RNA, Ribosomal, 5S/chemistry , RNA-Binding Proteins/metabolism , Saccharomyces cerevisiae/chemistry , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/metabolismABSTRACT
Diamond-Blackfan anemia is a rare genetic disease characterized by erythroblastopenia and a large spectrum of developmental anomalies. The vast majority of the cases genetically described are linked to heterozygous pathogenic variants in more than 20 ribosomal protein genes. Here we report an atypical clinical case of DBA associated with a missense variant in RPL8, which encodes RPL8/uL2, a protein of the 60S large ribosomal subunit. RPL8 has been previously implicated as a candidate disease gene in one patient with DBA bearing another type of missense variant; however, evidence for pathogenicity was limited to computational tools. Using functional studies in lymphoblastoid cells as well as yeast models, we show that the RPL8 variants detected in these two patients encode functionally deficient proteins that affect ribosome production and are therefore likely pathogenic. We propose to include RPL8 in the list of DBA-associated genes.
Subject(s)
Anemia, Diamond-Blackfan , Ribosomal Proteins , Anemia, Diamond-Blackfan/genetics , Humans , Mutation , Phenotype , Ribosomal Proteins/genetics , Ribosomes/genetics , Ribosomes/metabolism , Ribosomes/pathologyABSTRACT
In vivo UV crosslinking identified numerous preribosomal RNA (pre-rRNA) binding sites for the large, highly conserved ribosome synthesis factor Rrp5. Intramolecular complementation has shown that the C-terminal domain (CTD) of Rrp5 is required for pre-rRNA cleavage at sites A0-A2 on the pathway of 18S rRNA synthesis, whereas the N-terminal domain (NTD) is required for A3 cleavage on the pathway of 5.8S/25S rRNA synthesis. The CTD was crosslinked to sequences flanking A2 and to the snoRNAs U3, U14, snR30, and snR10, which are required for cleavage at A0-A2. The NTD was crosslinked to sequences flanking A3 and to the RNA component of ribonuclease MRP, which cleaves site A3. Rrp5 could also be directly crosslinked to several large structural proteins and nucleoside triphosphatases. A key role in coordinating preribosomal assembly and processing was confirmed by chromatin spreads. Following depletion of Rrp5, cotranscriptional cleavage was lost and preribosome compaction greatly reduced.
Subject(s)
Fungal Proteins/genetics , RNA Precursors/genetics , RNA Processing, Post-Transcriptional , RNA, Fungal/genetics , RNA, Ribosomal/genetics , Ribosomes/genetics , Base Sequence , Binding Sites , Endoribonucleases/genetics , Endoribonucleases/metabolism , Fungal Proteins/metabolism , Molecular Sequence Data , Nucleoside-Triphosphatase/genetics , Nucleoside-Triphosphatase/metabolism , RNA Precursors/metabolism , RNA, Ribosomal/metabolism , Yeasts/genetics , Yeasts/metabolismABSTRACT
The early steps of the production of the large ribosomal subunit are probably the least understood stages of eukaryotic ribosome biogenesis. The first specific precursor to the yeast large ribosomal subunit, the first pre-60S particle, contains 30 assembly factors (AFs), including 8 RNA helicases. These helicases, presumed to drive conformational rearrangements, usually lack substrate specificity in vitro. The mechanisms by which they are targeted to their correct substrate within pre-ribosomal particles and their precise molecular roles remain largely unknown. We demonstrate that the Dbp6p helicase, essential for the normal accumulation of the first pre-60S pre-ribosomal particle in S. cerevisiae, associates with a complex of four AFs, namely Npa1p, Npa2p, Nop8p and Rsa3p, prior to their incorporation into the 90S pre-ribosomal particles. By tandem affinity purifications using yeast extracts depleted of one component of the complex, we show that Npa1p forms the backbone of the complex. We provide evidence that Npa1p and Npa2p directly bind Dbp6p and we demonstrate that Npa1p is essential for the insertion of the Dbp6p helicase within 90S pre-ribosomal particles. In addition, by an in vivo cross-linking analysis (CRAC), we map Npa1p rRNA binding sites on 25S rRNA adjacent to the root helices of the first and last secondary structure domains of 25S rRNA. This finding supports the notion that Npa1p and Dbp6p function in the formation and/or clustering of root helices of large subunit rRNAs which creates the core of the large ribosomal subunit RNA structure. Npa1p also crosslinks to snoRNAs involved in decoding center and peptidyl transferase center modifications and in the immediate vicinity of the binding sites of these snoRNAs on 25S rRNA. Our data suggest that the Dbp6p helicase and the Npa1p complex play key roles in the compaction of the central core of 25S rRNA and the control of snoRNA-pre-rRNA interactions.
Subject(s)
Molecular Chaperones/metabolism , Nuclear Proteins/metabolism , RNA Helicases/metabolism , Ribosome Subunits, Large, Eukaryotic/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , DEAD-box RNA Helicases/metabolism , Escherichia coli , Models, Molecular , Peptidyl Transferases/metabolism , Protein Interaction Domains and Motifs , Protein Structure, Secondary , RNA Precursors/metabolism , RNA, Ribosomal/metabolism , RNA, Small Nucleolar/metabolism , RNA-Binding Proteins/metabolism , Recombinant Proteins , Ribosomal Proteins/metabolism , Substrate Specificity , Trans-Activators/metabolismABSTRACT
Assembly of eukaryotic ribosomal subunits is a very complex and sequential process that starts in the nucleolus and finishes in the cytoplasm with the formation of functional ribosomes. Over the past few years, characterization of the many molecular events underlying eukaryotic ribosome biogenesis has been drastically improved by the "resolution revolution" of cryo-electron microscopy (cryo-EM). However, if very early maturation events have been well characterized for both yeast ribosomal subunits, little is known regarding the final maturation steps occurring to the small (40S) ribosomal subunit. To try to bridge this gap, we have used proteomics together with cryo-EM and single particle analysis to characterize yeast pre-40S particles containing the ribosome biogenesis factor Tsr1. Our analyses lead us to refine the timing of the early pre-40S particle maturation steps. Furthermore, we suggest that after an early and structurally stable stage, the beak and platform domains of pre-40S particles enter a "vibrating" or "wriggling" stage, that might be involved in the final maturation of 18S rRNA as well as the fitting of late ribosomal proteins into their mature position.
Subject(s)
Proteomics/methods , Ribosomes/metabolism , Ribosomes/ultrastructure , Computational Biology , Cryoelectron Microscopy/methods , RNA, Ribosomal, 18S/metabolism , Ribosome Subunits, Small/metabolism , Ribosome Subunits, Small/ultrastructure , Tandem Mass SpectrometryABSTRACT
The DEAH box helicase Prp43 is a bifunctional enzyme from the DEAH/RHA helicase family required both for the maturation of ribosomes and for lariat intron release during splicing. It interacts with G-patch domain containing proteins which activate the enzymatic activity of Prp43 in vitro by an unknown mechanism. In this work, we show that the activation by G-patch domains is linked to the unique nucleotide binding mode of this helicase family. The base of the ATP molecule is stacked between two residues, R159 of the RecA1 domain (R-motif) and F357 of the RecA2 domain (F-motif). Using Prp43 F357A mutants or pyrimidine nucleotides, we show that the lack of stacking of the nucleotide base to the F-motif decouples the NTPase and helicase activities of Prp43. In contrast the R159A mutant (R-motif) showed reduced ATPase and helicase activities. We show that the Prp43 R-motif mutant induces the same phenotype as the absence of the G-patch protein Gno1, strongly suggesting that the processing defects observed in the absence of Gno1 result from a failure to activate the Prp43 helicase. Overall we propose that the stacking between the R- and F-motifs and the nucleotide base is important for the activity and regulation of this helicase family.
Subject(s)
Adenosine Triphosphate/metabolism , DEAD-box RNA Helicases/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Adenosine Triphosphate/chemistry , Amino Acid Substitution , Catalytic Domain/genetics , Crystallography, X-Ray , DEAD-box RNA Helicases/chemistry , DEAD-box RNA Helicases/genetics , Enzyme Activation , Kinetics , Models, Molecular , Mutagenesis, Site-Directed , Protein Interaction Domains and Motifs , Pyrimidine Nucleotides/chemistry , Pyrimidine Nucleotides/metabolism , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/geneticsABSTRACT
During biogenesis of the 40S and 60S ribosomal subunits, the pre-40S particles are exported to the cytoplasm prior to final cleavage of the 20S pre-rRNA to mature 18S rRNA. Amongst the factors involved in this maturation step, Fap7 is unusual, as it both interacts with ribosomal protein Rps14 and harbors adenylate kinase activity, a function not usually associated with ribonucleoprotein assembly. Human hFap7 also regulates Cajal body assembly and cell cycle progression via the p53-MDM2 pathway. This work presents the functional and structural characterization of the Fap7-Rps14 complex. We report that Fap7 association blocks the RNA binding surface of Rps14 and, conversely, Rps14 binding inhibits adenylate kinase activity of Fap7. In addition, the affinity of Fap7 for Rps14 is higher with bound ADP, whereas ATP hydrolysis dissociates the complex. These results suggest that Fap7 chaperones Rps14 assembly into pre-40S particles via RNA mimicry in an ATP-dependent manner. Incorporation of Rps14 by Fap7 leads to a structural rearrangement of the platform domain necessary for the pre-rRNA to acquire a cleavage competent conformation.
Subject(s)
Adenylate Kinase/genetics , Gene Expression Regulation, Fungal , Nuclear Proteins/genetics , Nucleoside-Triphosphatase/genetics , Ribosomal Proteins/genetics , Ribosome Subunits, Small, Eukaryotic/genetics , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae/genetics , Adenosine Diphosphate/chemistry , Adenosine Diphosphate/metabolism , Adenosine Triphosphate/chemistry , Adenosine Triphosphate/metabolism , Adenylate Kinase/chemistry , Adenylate Kinase/metabolism , Amino Acid Sequence , Escherichia coli/genetics , Escherichia coli/metabolism , Humans , Models, Molecular , Molecular Mimicry , Molecular Sequence Data , Nuclear Proteins/chemistry , Nuclear Proteins/metabolism , Nucleoside-Triphosphatase/chemistry , Nucleoside-Triphosphatase/metabolism , Pyrococcus abyssi/genetics , Pyrococcus abyssi/metabolism , RNA, Ribosomal, 18S/chemistry , RNA, Ribosomal, 18S/genetics , RNA, Ribosomal, 18S/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Ribosomal Proteins/chemistry , Ribosomal Proteins/metabolism , Ribosome Subunits, Large, Eukaryotic/genetics , Ribosome Subunits, Large, Eukaryotic/metabolism , Ribosome Subunits, Small, Eukaryotic/metabolism , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/metabolism , Sequence AlignmentABSTRACT
Ribosomal protein L3 is an evolutionarily conserved protein that participates in the assembly of early pre-60S particles. We report that the rpl3[W255C] allele, which affects the affinity and function of translation elongation factors, impairs cytoplasmic maturation of 20S pre-rRNA. This was not seen for other mutations in or depletion of L3 or other 60S ribosomal proteins. Surprisingly, pre-40S particles containing 20S pre-rRNA form translation-competent 80S ribosomes, and translation inhibition partially suppresses 20S pre-rRNA accumulation. The GTP-dependent translation initiation factor Fun12 (yeast eIF5B) shows similar in vivo binding to ribosomal particles from wild-type and rpl3[W255C] cells. However, the GTPase activity of eIF5B failed to stimulate processing of 20S pre-rRNA when assayed with ribosomal particles purified from rpl3[W255C] cells. We conclude that L3 plays an important role in the function of eIF5B in stimulating 3' end processing of 18S rRNA in the context of 80S ribosomes that have not yet engaged in translation. These findings indicate that the correct conformation of the GTPase activation region is assessed in a quality control step during maturation of cytoplasmic pre-ribosomal particles.
Subject(s)
Ribosomal Proteins/genetics , Ribosome Subunits, Large, Eukaryotic/genetics , Ribosome Subunits, Small, Eukaryotic/genetics , Saccharomyces cerevisiae/genetics , Alleles , Cytoplasm/genetics , Cytoplasm/metabolism , Eukaryotic Initiation Factors/genetics , Mutation , Proteasome Endopeptidase Complex/genetics , Proteasome Endopeptidase Complex/metabolism , Protein Binding , RNA Precursors/genetics , RNA, Ribosomal, 18S/genetics , Ribosomal Protein L3 , Ribosomal Proteins/metabolism , Ribosome Subunits, Large, Eukaryotic/metabolism , Ribosome Subunits, Small, Eukaryotic/metabolismABSTRACT
During the last step in 40S ribosome subunit biogenesis, the PIN-domain endonuclease Nob1 cleaves the 20S pre-rRNA at site D, to form the mature 18S rRNAs. Here we report that cleavage occurs in particles that have largely been stripped of previously characterized pre-40S components, but retain the endonuclease Nob1, its binding partner Pno1 (Dim2) and the atypical ATPase Rio1. Within the Rio1-associated pre-40S particles, in vitro pre-rRNA cleavage was strongly stimulated by ATP and required nucleotide binding by Rio1. In vivo binding sites for Rio1, Pno1 and Nob1 were mapped by UV cross-linking in actively growing cells. Nob1 and Pno1 bind overlapping regions within the internal transcribed spacer 1, and both bind directly over cleavage site D. Binding sites for Rio1 were within the core of the 18S rRNA, overlapping tRNA interaction sites and distinct from the related kinase Rio2. Site D cleavage occurs within pre-40S-60S complexes and Rio1-associated particles efficiently assemble into these complexes, whereas Pno1 appeared to be depleted relative to Nob1. We speculate that Rio1-mediated dissociation of Pno1 from cleavage site D is the trigger for final 18S rRNA maturation.
Subject(s)
Adenosine Triphosphate/metabolism , Protein Serine-Threonine Kinases/metabolism , Ribosome Subunits, Small, Eukaryotic/enzymology , Saccharomyces cerevisiae Proteins/metabolism , Binding Sites , Models, Molecular , Nuclear Proteins/metabolism , RNA Cleavage , RNA Precursors/metabolism , RNA, Ribosomal/metabolism , Ribosomal Proteins/chemistry , Ribosomal Proteins/metabolism , Ribosome Subunits, Small, Eukaryotic/chemistry , Ribosome Subunits, Small, Eukaryotic/metabolismABSTRACT
We provide evidence that a central player in ribosome synthesis, the ribonucleic acid helicase Prp43p, can be activated by yeast Gno1p and its human ortholog, the telomerase inhibitor PINX1. Gno1p and PINX1 expressed in yeast interact with Prp43p and the integrity of their G-patch domain is required for this interaction. Moreover, PINX1 interacts with human PRP43 (DHX15) in HeLa cells. PINX1 directly binds to yeast Prp43p and stimulates its adenosine triphosphatase activity, while alterations of the G patch abolish formation of the PINX1/Prp43p complex and the stimulation of Prp43p. In yeast, lack of Gno1p leads to a decrease in the levels of pre-40S and intermediate pre-60S pre-ribosomal particles, defects that can be corrected by PINX1 expression. We show that Gno1p associates with 90S and early pre-60S pre-ribosomal particles and is released from intermediate pre-60S particles. G-patch alterations in Gno1p or PINX1 that inhibit their interactions with Prp43p completely abolish their function in yeast ribosome biogenesis. Altogether, our results suggest that activation of Prp43p by Gno1p/PINX1 within early pre-ribosomal particles is crucial for their subsequent maturation.
Subject(s)
DEAD-box RNA Helicases/metabolism , RNA Helicases/metabolism , RNA-Binding Proteins/physiology , Ribosomes/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Tumor Suppressor Proteins/metabolism , Adenosine Triphosphatases/metabolism , Cell Cycle Proteins , Enzyme Activation , HeLa Cells , Humans , Protein Structure, Tertiary , Telomerase/antagonists & inhibitors , Tumor Suppressor Proteins/chemistryABSTRACT
The DEAH/RNA helicase A (RHA) helicase family comprises proteins involved in splicing, ribosome biogenesis and transcription regulation. We report the structure of yeast Prp43p, a DEAH/RHA helicase remarkable in that it functions in both splicing and ribosome biogenesis. Prp43p displays a novel structural architecture with an unforeseen homology with the Ski2-like Hel308 DNA helicase. Together with the presence of a beta-hairpin in the second RecA-like domain, Prp43p contains all the structural elements of a processive helicase. Moreover, our structure reveals that the C-terminal domain contains an oligonucleotide/oligosaccharide-binding (OB)-fold placed at the entrance of the putative nucleic acid cavity. Deletion or mutations of this domain decrease the affinity of Prp43p for RNA and severely reduce Prp43p ATPase activity in the presence of RNA. We also show that this domain constitutes the binding site for the G-patch-containing domain of Pfa1p. We propose that the C-terminal domain, specific to DEAH/RHA helicases, is a central player in the regulation of helicase activity by binding both RNA and G-patch domain proteins.
Subject(s)
DEAD-box RNA Helicases/chemistry , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae/enzymology , Adenosine Triphosphate/metabolism , Crystallography, X-Ray , DEAD-box RNA Helicases/metabolism , Models, Molecular , Protein Conformation , Protein Structure, Tertiary , RNA, Fungal/metabolism , Saccharomyces cerevisiae Proteins/metabolismABSTRACT
Prp43p is a RNA helicase required for pre-mRNA splicing and for the synthesis of large and small ribosomal subunits. The molecular functions and modes of regulation of Prp43p during ribosome biogenesis remain unknown. We demonstrate that the G-patch protein Pfa1p, a component of pre-40S pre-ribosomal particles, directly interacts with Prp43p. We also show that lack of Gno1p, another G-patch protein associated with Prp43p, specifically reduces Pfa1p accumulation, whereas it increases the levels of the pre-40S pre-ribosomal particle component Ltv1p. Moreover, cells lacking Pfa1p and depleted for Ltv1p show strong 20S pre-rRNA accumulation in the cytoplasm and reduced levels of 18S rRNA. Finally, we demonstrate that Pfa1p stimulates the ATPase and helicase activities of Prp43p. Truncated Pfa1p variants unable to fully stimulate the activity of Prp43p fail to complement the 20S pre-rRNA processing defect of Deltapfa1 cells depleted for Ltv1p. Our results strongly suggest that stimulation of ATPase/helicase activities of Prp43p by Pfa1p is required for efficient 20S pre-rRNA-to-18S rRNA conversion.
Subject(s)
Adenosine Triphosphatases/chemistry , DEAD-box RNA Helicases/physiology , Gene Expression Regulation, Fungal , Phosphopyruvate Hydratase/physiology , RNA Helicases/chemistry , Ribosomes/metabolism , Saccharomyces cerevisiae Proteins/metabolism , DEAD-box RNA Helicases/metabolism , GTP-Binding Proteins/chemistry , Models, Biological , Protein Binding , Protein Structure, Tertiary , RNA Precursors/chemistry , RNA, Ribosomal/chemistry , RNA, Ribosomal, 18S/chemistry , Ribosomes/chemistry , Saccharomyces cerevisiae , Saccharomyces cerevisiae Proteins/physiologyABSTRACT
In Saccharomyces cerevisiae, ribosome biogenesis requires, in addition to rRNA and ribosomal proteins, a myriad of small nucleolar RNAs (snoRNAs) and over two hundred protein trans-acting factors. There are protein trans-acting factors predicted to participate in ribosome biogenesis that have not been so far characterized. Here, we report the functional analysis of the Nucleolar protein 6 (Nop6) in ribosome biogenesis. Our results show that Nop6 is needed for optimal 40S ribosomal subunit biogenesis. Deletion of NOP6 leads to an appropriate 20% reduction in 18S rRNA levels and therefore in 40S ribosomal subunits. This is due to mild inhibition of pre-rRNA processing at cleavage site A 2. Tandem affinity purification followed by mass spectrometry and northern blot analyses indicate that Nop6 is a component of 90S pre-ribosomal particles. rDNA chromatin immunoprecipitation experiments and analysis of the intracellular localisation of Nop6-eGFP after in vivo shut down of pre-rRNA transcription strongly suggest that Nop6 binds to the pre-rRNA early during transcription. Genetic data suggest that Nop6 and the snoRNA snR57 both interact similarly with the protein trans-acting factor Nep1. It has been proposed that snR57 and Nep1 participate in a pre-rRNA conformational switch that allows the proper assembly of 40S ribosomal protein S19. Our results strongly suggest that the role Nop6 might have in this conformational switch is independent of snR57.
Subject(s)
RNA-Binding Proteins/metabolism , Ribosome Subunits, Small, Eukaryotic/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Cell Nucleolus/genetics , Cell Nucleolus/metabolism , Gene Deletion , Mutation , Phenotype , Protein Binding , RNA Processing, Post-Transcriptional , RNA, Small Nucleolar/metabolism , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/genetics , Ribosomal Proteins/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/growth & development , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/geneticsABSTRACT
Preventing premature interaction of pre-ribosomes with the translation apparatus is essential for translational accuracy. Hence, the final maturation step releasing functional 40S ribosomal subunits, namely processing of the 18S ribosomal RNA 3' end, is safeguarded by the protein DIM2, which both interacts with the endoribonuclease NOB1 and masks the rRNA cleavage site. To elucidate the control mechanism that unlocks NOB1 activity, we performed cryo-electron microscopy analysis of late human pre-40S particles purified using a catalytically inactive form of the ATPase RIO1. These structures, together with in vivo and in vitro functional analyses, support a model in which ATP-loaded RIO1 cooperates with ribosomal protein RPS26/eS26 to displace DIM2 from the 18S rRNA 3' end, thereby triggering final cleavage by NOB1; release of ADP then leads to RIO1 dissociation from the 40S subunit. This dual key lock mechanism requiring RIO1 and RPS26 guarantees the precise timing of pre-40S particle conversion into translation-competent ribosomal subunits.
Subject(s)
Protein Serine-Threonine Kinases/metabolism , Ribosomal Proteins/metabolism , Ribosome Subunits, Small, Eukaryotic/metabolism , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Humans , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Protein Serine-Threonine Kinases/genetics , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Ribosomal Proteins/genetics , Ribosome Subunits, Small, Eukaryotic/geneticsABSTRACT
We report the characterization of the yeast Npa2p (Urb2p) protein, which is essential for 60S ribosomal subunit biogenesis. We identified this protein in a synthetic lethal screening with the rsa3 null allele. Rsa3p is a genetic partner of the putative RNA helicase Dbp6p. Mutation or depletion of Npa2p leads to a net deficit in 60S subunits and a decrease in the levels all 27S pre-rRNAs and mature 25S and 5.8S rRNAs. This is likely due to instability of early pre-60S particles. Consistent with a role of Npa2p in 60S subunit biogenesis, green fluorescent protein-tagged Npa2p localizes predominantly to the nucleolus and TAP-tagged Npa2p sediments with large complexes in sucrose gradients and is associated mainly with 27SA(2) pre-rRNA-containing preribosomal particles. In addition, we reveal a genetic synthetic interaction between Npa2p, several factors required for early steps of 60S subunit biogenesis (Dbp6p, Dbp7p, Dbp9p, Npa1p, Nop8p, and Rsa3p), and the 60S protein Rpl3p. Furthermore, coimmunoprecipitation and gel filtration analyses demonstrated that at least Npa2p, Dbp6p, Npa1p, Nop8p, and Rsa3p are present together in a subcomplex of low molecular mass whose integrity is independent of RNA. Our results support the idea that these five factors work in concert during the early steps of 60S subunit biogenesis.
Subject(s)
Nuclear Proteins/metabolism , Ribosomes/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Alleles , Cell Nucleolus/metabolism , DEAD-box RNA Helicases , Molecular Weight , Multiprotein Complexes/metabolism , Mutant Proteins/isolation & purification , Mutant Proteins/metabolism , Mutation/genetics , Nuclear Proteins/deficiency , Phenotype , Protein Binding , Protein Transport , RNA Nucleotidyltransferases/metabolism , RNA Precursors/genetics , RNA Precursors/metabolism , RNA Processing, Post-Transcriptional , RNA, Ribosomal/metabolism , RNA-Binding Proteins/metabolism , Ribosomal Proteins/metabolism , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/growth & development , Saccharomyces cerevisiae Proteins/isolation & purification , Trans-Activators/metabolismABSTRACT
The synthesis of ribosomal subunits in eukaryotes requires the interplay of numerous maturation and assembly factors (AFs) that intervene in the insertion of ribosomal proteins within pre-ribosomal particles, the ribosomal subunit precursors, as well as in pre-ribosomal RNA (rRNA) processing and folding. Here, we review the intricate nuclear and cytoplasmic maturation steps of pre-40S particles, the precursors to the small ribosomal subunits, in both yeast and human cells, with particular emphasis on the timing and mechanisms of AF association with and dissociation from pre-40S particles and the roles of these AFs in the maturation process. We highlight the particularly complex pre-rRNA processing pathway in human cells, compared to yeast, to generate the mature 18S rRNA. We discuss the information gained from the recently published cryo-electron microscopy atomic models of yeast and human pre-40S particles, as well as the checkpoint/quality control systems that seem to operate to probe functional sites within yeast cytoplasmic pre-40S particles. This article is categorized under: RNA Processing > rRNA Processing Translation > Ribosome Biogenesis.
Subject(s)
RNA Precursors , RNA, Ribosomal , Animals , Humans , Ribosomal Proteins/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/metabolismABSTRACT
Prp43p is a putative helicase of the DEAH family which is required for the release of the lariat intron from the spliceosome. Prp43p could also play a role in ribosome synthesis, since it accumulates in the nucleolus. Consistent with this hypothesis, we find that depletion of Prp43p leads to accumulation of 35S pre-rRNA and strongly reduces levels of all downstream pre-rRNA processing intermediates. As a result, the steady-state levels of mature rRNAs are greatly diminished following Prp43p depletion. We present data arguing that such effects are unlikely to be solely due to splicing defects. Moreover, we demonstrate by a combination of a comprehensive two-hybrid screen, tandem-affinity purification followed by mass spectrometry, and Northern analyses that Prp43p is associated with 90S, pre-60S, and pre-40S ribosomal particles. Prp43p seems preferentially associated with Pfa1p, a novel specific component of pre-40S ribosomal particles. In addition, Prp43p interacts with components of the RNA polymerase I (Pol I) transcription machinery and with mature 18S and 25S rRNAs. Hence, Prp43p might be delivered to nascent 90S ribosomal particles during pre-rRNA transcription and remain associated with preribosomal particles until their final maturation steps in the cytoplasm. Our data also suggest that the ATPase activity of Prp43p is required for early steps of pre-rRNA processing and normal accumulation of mature rRNAs.
Subject(s)
Adenosine Triphosphatases/genetics , RNA Helicases/genetics , RNA Precursors/genetics , RNA, Fungal/genetics , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae/genetics , Adenosine Triphosphatases/metabolism , DEAD-box RNA Helicases , Mass Spectrometry , Protein Splicing , RNA Helicases/metabolism , RNA Polymerase I/genetics , RNA Polymerase I/metabolism , RNA Precursors/metabolism , RNA Processing, Post-Transcriptional , RNA, Fungal/metabolism , RNA, Ribosomal/genetics , RNA, Ribosomal/metabolism , Ribosomal Proteins/genetics , Ribosomal Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Two-Hybrid System TechniquesABSTRACT
Ribosomal RNAs, the most abundant cellular RNA species, have evolved as the structural scaffold and the catalytic center of protein synthesis in every living organism. In eukaryotes, they are produced from a long primary transcript through an intricate sequence of processing steps that include RNA cleavage and folding and nucleotide modification. The mechanisms underlying this process in human cells have long been investigated, but technological advances have accelerated their study in the past decade. In addition, the association of congenital diseases to defects in ribosome synthesis has highlighted the central place of ribosomal RNA maturation in cell physiology regulation and broadened the interest in these mechanisms. Here, we give an overview of the current knowledge of pre-ribosomal RNA processing in human cells in light of recent progress and discuss how dysfunction of this pathway may contribute to the physiopathology of congenital diseases.