RESUMO
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.
Assuntos
RNA Helicases DEAD-box , Ribossomos , Proteínas de Saccharomyces cerevisiae , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Peptidil Transferases/genética , Peptidil Transferases/metabolismo , Ribossomos/genética , Ribossomos/metabolismo , RNA Helicases/genética , RNA Ribossômico/metabolismo , RNA Nucleolar Pequeno/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Prp43 is a DEAH-box RNA helicase involved in both splicing and ribosome biogenesis. Its activities are directly stimulated by several co-activators that share a G-patch domain. The substrates of Prp43, its mechanism of action and the modes of interaction with and activation by G-patch proteins have been only partially characterized. We investigated how Pfa1 and PINX1, two G-patch proteins involved in ribosome biogenesis, interact with Prp43. We demonstrate that a protruding loop connecting the ß4 and ß5 strands of Prp43 OB fold is crucial for the binding of the G-patch domain of Pfa1. However, neither this loop nor the entire OB fold of Prp43 is essential for PINX1 binding. We conclude that the binding modes of Pfa1 and PINX1 G-patches to Prp43 are different. Nevertheless, stimulation of the ATPase and helicase activities of Prp43 by both full-length Pfa1 and PINX1 requires the ß4-ß5 loop. Moreover, we show that disruption of this loop completely abrogates Prp43 activity during yeast ribosome biogenesis but does not prevent its integration within pre-ribosomal particles. We propose that the ß4-ß5 loop plays a crucial role in the transmission of conformational changes induced by binding of the G-patch to Prp43 active site and substrate RNA.
Assuntos
RNA Helicases DEAD-box/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Domínio Catalítico/genética , RNA Helicases DEAD-box/química , RNA Helicases DEAD-box/genética , Escherichia coli/genética , Organismos Geneticamente Modificados , Ligação Proteica , RNA Helicases/química , RNA Helicases/genética , RNA Helicases/metabolismo , Proteínas de Ligação a RNA/genética , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
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.
Assuntos
Chaperonas Moleculares/metabolismo , Proteínas Nucleares/metabolismo , RNA Helicases/metabolismo , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , RNA Helicases DEAD-box/metabolismo , Escherichia coli , Modelos Moleculares , Peptidil Transferases/metabolismo , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Precursores de RNA/metabolismo , RNA Ribossômico/metabolismo , RNA Nucleolar Pequeno/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Recombinantes , Proteínas Ribossômicas/metabolismo , Especificidade por Substrato , Transativadores/metabolismoRESUMO
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.
Assuntos
Trifosfato de Adenosina/metabolismo , RNA Helicases DEAD-box/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Trifosfato de Adenosina/química , Substituição de Aminoácidos , Domínio Catalítico/genética , Cristalografia por Raios X , RNA Helicases DEAD-box/química , RNA Helicases DEAD-box/genética , Ativação Enzimática , Cinética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Domínios e Motivos de Interação entre Proteínas , Nucleotídeos de Pirimidina/química , Nucleotídeos de Pirimidina/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
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.
Assuntos
RNA Helicases DEAD-box/metabolismo , RNA Helicases/metabolismo , Proteínas de Ligação a RNA/fisiologia , Ribossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Adenosina Trifosfatases/metabolismo , Proteínas de Ciclo Celular , Ativação Enzimática , Células HeLa , Humanos , Estrutura Terciária de Proteína , Telomerase/antagonistas & inibidores , Proteínas Supressoras de Tumor/químicaRESUMO
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.
Assuntos
RNA Helicases DEAD-box/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimologia , Trifosfato de Adenosina/metabolismo , Cristalografia por Raios X , RNA Helicases DEAD-box/metabolismo , Modelos Moleculares , Conformação Proteica , Estrutura Terciária de Proteína , RNA Fúngico/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
RNA helicases perform essential housekeeping and regulatory functions in all domains of life by binding and unwinding RNA molecules. The Ski2-like proteins are primordial helicases that play an active role in eukaryotic RNA homeostasis pathways, with multiple homologs having specialized functions. The significance of the expansion and diversity of Ski2-like proteins in Archaea, the third domain of life, has not yet been established. Here, by studying the phylogenetic diversity of Ski2-like helicases among archaeal genomes and the enzymatic activities of those in Thermococcales, we provide further evidence of the function of this protein family in archaeal metabolism of nucleic acids. We show that, in the course of evolution, ASH-Ski2 and Hel308-Ski2, the two main groups of Ski2-like proteins, have diverged in their biological functions. Whereas Hel308 has been shown to mainly act on DNA, we show that ASH-Ski2, previously described to be associated with the 5'-3' aRNase J exonuclease, acts on RNA by supporting an efficient annealing activity, but also an RNA unwinding with a 3'-5' polarity. To gain insights into the function of Ski2, we also analyse the transcriptome of Thermococcus barophilus ΔASH-Ski2 mutant strain and provide evidence of the importance of ASH-Ski2 in cellular metabolism pathways related to translation.
RESUMO
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.
Assuntos
Adenosina Trifosfatases/química , RNA Helicases DEAD-box/fisiologia , Regulação Fúngica da Expressão Gênica , Fosfopiruvato Hidratase/fisiologia , RNA Helicases/química , Ribossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , RNA Helicases DEAD-box/metabolismo , Proteínas de Ligação ao GTP/química , Modelos Biológicos , Ligação Proteica , Estrutura Terciária de Proteína , Precursores de RNA/química , RNA Ribossômico/química , RNA Ribossômico 18S/química , Ribossomos/química , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/fisiologiaRESUMO
Helicase proteins are known to use the energy of ATP to unwind nucleic acids and to remodel protein-nucleic acid complexes. They are involved in almost every aspect of DNA and RNA metabolisms and participate in numerous repair mechanisms that maintain cellular integrity. The archaeal Lhr-type proteins are SF2 helicases that are mostly uncharacterized. They have been proposed to be DNA helicases that act in DNA recombination and repair processes in Sulfolobales and Methanothermobacter. In Thermococcales, a protein annotated as an Lhr2 protein was found in the network of proteins involved in RNA metabolism. To investigate this, we performed in-depth phylogenomic analyses to report the classification and taxonomic distribution of Lhr-type proteins in Archaea, and to better understand their relationship with bacterial Lhr. Furthermore, with the goal of envisioning the role(s) of aLhr2 in Thermococcales cells, we deciphered the enzymatic activities of aLhr2 from Thermococcus barophilus (Tbar). We showed that Tbar-aLhr2 is a DNA/RNA helicase with a significant annealing activity that is involved in processes dependent on DNA and RNA transactions.
Assuntos
DNA Helicases/genética , RNA Helicases/genética , Thermococcales/enzimologia , Adenosina Trifosfatases/genética , Proteínas Arqueais/química , DNA/química , DNA Helicases/isolamento & purificação , DNA Helicases/metabolismo , Filogenia , RNA/química , RNA Helicases/isolamento & purificação , RNA Helicases/metabolismo , Homologia de Sequência de Aminoácidos , Thermococcales/genética , Thermococcales/metabolismoRESUMO
Synthesis of eukaryotic ribosomes involves the assembly and maturation of precursor particles (pre-ribosomal particles) containing ribosomal RNA (rRNA) precursors, ribosomal proteins (RPs) and a plethora of assembly factors (AFs). Formation of the earliest precursors of the 60S ribosomal subunit (pre-60S r-particle) is among the least understood stages of ribosome biogenesis. It involves the Npa1 complex, a protein module suggested to play a key role in the early structuring of the pre-rRNA. Npa1 displays genetic interactions with the DExD-box protein Dbp7 and interacts physically with the snR190 box C/D snoRNA. We show here that snR190 functions as a snoRNA chaperone, which likely cooperates with the Npa1 complex to initiate compaction of the pre-rRNA in early pre-60S r-particles. We further show that Dbp7 regulates the dynamic base-pairing between snR190 and the pre-rRNA within the earliest pre-60S r-particles, thereby participating in structuring the peptidyl transferase center (PTC) of the large ribosomal subunit.
Assuntos
RNA Helicases DEAD-box/metabolismo , Chaperonas Moleculares/metabolismo , RNA Nucleolar Pequeno/metabolismo , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Pareamento de Bases , RNA Helicases DEAD-box/genética , Chaperonas Moleculares/genética , Mutação , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Biogênese de Organelas , Dobramento de RNA , Precursores de RNA/química , Precursores de RNA/genética , Precursores de RNA/metabolismo , RNA Ribossômico/química , RNA Ribossômico/genética , RNA Ribossômico/metabolismo , RNA Nucleolar Pequeno/genética , Subunidades Ribossômicas Maiores de Eucariotos/química , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
During ribosome biogenesis, the RNA precursor to mature rRNAs undergoes numerous post-transcriptional chemical modifications of bases, including conversions of uridines to pseudouridines. In archaea and eukaryotes, these conversions are performed by box H/ACA small ribonucleoprotein particles (box H/ACA RNPs), which contain a small guide RNA responsible for the selection of substrate uridines and four proteins, including the pseudouridine synthase, Cbf5p. So far, no in vitro reconstitution of eukaryotic box H/ACA RNPs from purified components has been achieved, principally due to difficulties in purifying recombinant eukaryotic Cbf5p. In this study, we present the purification of a truncated derivative of yeast Cbf5p (Cbf5(Delta)p) that retains the highly conserved TRUB and PUA domains. We have used band retardation assays to show that Cbf5(Delta)p on its own binds to box H/ACA small nucleolar (sno)RNAs. We demonstrate that the conserved H and ACA boxes enhance the affinity of the protein for the snoRNA. Furthermore, like its archaeal homologs, Cbf5(Delta)p can bind to a single stem-loop-box ACA RNA. Finally, we report the first enzymatic footprinting analysis of a Cbf5-RNA complex. Our results are compatible with the view that two molecules of Cbf5p interact with a binding platform constituted by the 5' end of the RNA, the single-stranded hinge domain containing the conserved H box, and the 3' end of the molecule, including the conserved ACA box.
Assuntos
Hidroliases/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , RNA Fúngico/metabolismo , RNA Nucleolar Pequeno/metabolismo , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação/genética , Sequência Conservada , Hidroliases/química , Hidroliases/genética , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/genética , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Ligação Proteica , Pegadas de Proteínas , Estrutura Terciária de Proteína , RNA Fúngico/química , RNA Fúngico/genética , RNA Nucleolar Pequeno/química , RNA Nucleolar Pequeno/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ribonucleases , Ribonucleoproteínas Nucleares Pequenas/química , Ribonucleoproteínas Nucleares Pequenas/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Homologia de Sequência de AminoácidosRESUMO
Box C/D and box H/ACA small ribonucleoprotein particles (sRNPs) are found from archaea to humans, and some of these play key roles during the biogenesis of ribosomes or components of the splicing apparatus. The protein composition of the core of both types of particles is well established and the assembly pathway of box C/D sRNPs has been extensively investigated both in archaeal and eukaryotic systems. In contrast, knowledge concerning the mode of assembly and final structure of box H/ACA sRNPs is much more limited. In the present study, we have investigated the protein/protein interactions taking place between the four protein components of yeast box H/ACA small nucleolar RNPs (snoRNPs), Cbf5p, Gar1p, Nhp2p, and Nop10p. We provide evidence that Cbf5p, Gar1p, and Nop10p can form a complex devoid of Nhp2p and small nucleolar RNA (snoRNA) components of the particles and that Cbf5p and Nop10p can directly bind to each other. We also show that the absence of any component necessary for assembly of box H/ACA snoRNPs inhibits accumulation of Cbf5p, Gar1p, or Nop10p, whereas Nhp2p levels are little affected.