RESUMEN
The 3' exonucleolytic processing of stable RNAs is conserved throughout biology. Yeast strains lacking the exoribonuclease Rex1 are defective in the 3' processing of stable RNAs, including 5S rRNA and tRNA. The equivalent RNA processing steps in Escherichia coli are carried out by RNase T. Rex1 is larger than RNase T, the catalytic DEDD domain being embedded within uncharacterized amino- and carboxy-terminal regions. Here we report that both amino- and carboxy-terminal regions of Rex1 are essential for its function, as shown by genetic analyses and 5S rRNA profiling. Full-length Rex1, but not mutants lacking amino- or carboxy-terminal regions, accurately processed a 3' extended 5S rRNA substrate. Crosslinking analyses showed that both amino- and carboxy-terminal regions of Rex1 directly contact RNA in vivo. Sequence homology searches identified YFE9 in Schizosaccharomyces pombe and SDN5 in Arabidopsis thaliana as closely related proteins to Rex1. In addition to the DEDD domain, these proteins share a domain, referred to as the RYS (Rex1, YFE9 and SDN5) domain, that includes elements of both the amino- and caroxy-terminal flanking regions. We also characterize a nuclear localization signal in the amino-terminal region of Rex1. These studies reveal a novel dual domain structure at the core of Rex1-related ribonucleases, wherein the catalytic DEDD domain and the RYS domain are aligned such that they both contact the bound substrate. The domain organization of Rex1 is distinct from that of other previously characterized DEDD family nucleases and expands the known repertoire of structures for this fundamental family of RNA processing enzymes.
Asunto(s)
Exorribonucleasas , Saccharomyces cerevisiae , Endorribonucleasas/metabolismo , Exorribonucleasas/genética , Exorribonucleasas/metabolismo , Procesamiento Postranscripcional del ARN , ARN de Transferencia/química , Saccharomyces cerevisiae/metabolismoRESUMEN
Debaryomyces hansenii is a yeast with considerable biotechnological potential as an osmotolerant, stress-tolerant oleaginous microbe. However, targeted genome modification tools are limited and require a strain with auxotrophic markers. Gene targeting by homologous recombination has been reported to be inefficient, but here we describe a set of reagents and a method that allows gene targeting at high efficiency in wild-type isolates. It uses a simple polymerase chain reaction (PCR)-based amplification that extends a completely heterologous selectable marker with 50 bp flanks identical to the target site in the genome. Transformants integrate the PCR product through homologous recombination at high frequency (>75%). We illustrate the potential of this method by disrupting genes at high efficiency and by expressing a heterologous protein from a safe chromosomal harbour site. These methods should stimulate and facilitate further analysis of D. hansenii strains and open the way to engineer strains for biotechnology.
Asunto(s)
Debaryomyces , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Reacción en Cadena de la Polimerasa , Marcación de Gen , BiotecnologíaRESUMEN
The exosome is a conserved multi-subunit ribonuclease complex that functions in 3' end processing, turnover and surveillance of nuclear and cytoplasmic RNAs. In the yeast nucleus, the 10-subunit core complex of the exosome (Exo-10) physically and functionally interacts with the Rrp6 exoribonuclease and its associated cofactor Rrp47, the helicase Mtr4 and Mpp6. Here, we show that binding of Mtr4 to Exo-10 in vitro is dependent upon both Rrp6 and Rrp47, whereas Mpp6 binds directly and independently of other cofactors. Crystallographic analyses reveal that the N-terminal domains of Rrp6 and Rrp47 form a highly intertwined structural unit. Rrp6 and Rrp47 synergize to create a composite and conserved surface groove that binds the N-terminus of Mtr4. Mutation of conserved residues within Rrp6 and Mtr4 at the structural interface disrupts their interaction and inhibits growth of strains expressing a C-terminal GFP fusion of Mtr4. These studies provide detailed structural insight into the interaction between the Rrp6-Rrp47 complex and Mtr4, revealing an important link between Mtr4 and the core exosome.
Asunto(s)
ARN Helicasas DEAD-box/metabolismo , Proteínas de Unión al ADN/metabolismo , Complejo Multienzimático de Ribonucleasas del Exosoma/metabolismo , Modelos Moleculares , Complejos Multiproteicos/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Western Blotting , Calorimetría , Cromatografía en Gel , Cristalización , ARN Helicasas DEAD-box/química , Proteínas de Unión al ADN/química , Electroforesis en Gel de Poliacrilamida , Escherichia coli , Complejo Multienzimático de Ribonucleasas del Exosoma/química , Polarización de Fluorescencia , Complejos Multiproteicos/química , Proteínas Nucleares/química , Sondas de Oligonucleótidos , Conformación Proteica , Proteínas de Unión al ARN/química , Colorantes de Rosanilina , Proteínas de Saccharomyces cerevisiae/químicaRESUMEN
The eukaryotic exosome exoribonuclease Rrp6 forms a complex with Rrp47 that functions in nuclear RNA quality control mechanisms, the degradation of cryptic unstable transcripts (CUTs), and in the 3' end maturation of stable RNAs. Stable expression of Rrp47 is dependent upon its interaction with the N-terminal domain of Rrp6 (Rrp6NT). To address the function of Rrp47 independently of Rrp6, we developed a DECOID (decreased expression of complexes by overexpression of interacting domains) strategy to resolve the Rrp6/Rrp47 complex in vivo and employed mpp6Δ and rex1Δ mutants that are synthetic lethal with loss-of-function rrp47 mutants. Strikingly, Rrp47 was able to function in mpp6Δ and rex1Δ mutants when separated from the catalytic and exosome-binding domains of Rrp6, whereas a truncated Rrp47 protein lacking its C-terminal region caused a block in cell growth. Northern analyses of the conditional mutants revealed a specific block in the 3' maturation of box C/D snoRNAs in the rex1 rrp47 mutant and widespread inhibition of Rrp6-mediated RNA surveillance processes in the mpp6 rrp47 mutant. In contrast, growth analyses and RNA northern blot hybridization analyses showed no effect on the rrp47Δ mutant upon overexpression of the Rrp6NT domain. These findings demonstrate that Rrp47 and Rrp6 have resolvable functions in Rrp6-mediated RNA surveillance and processing pathways. In addition, this study reveals a redundant requirement for Rrp6 or Rex1 in snoRNA maturation and demonstrates the effective use of the DECOID strategy for the resolution and functional analysis of protein complexes.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Complejo Multienzimático de Ribonucleasas del Exosoma/metabolismo , Complejo Multienzimático de Ribonucleasas del Exosoma/fisiología , Proteínas Nucleares/fisiología , Proteínas de Unión al ARN/fisiología , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiología , Saccharomyces cerevisiae/enzimología , Unión Competitiva , Dominio Catalítico , Proteínas de Unión al ADN/química , Exorribonucleasas/genética , Exorribonucleasas/metabolismo , Complejo Multienzimático de Ribonucleasas del Exosoma/química , Complejo Multienzimático de Ribonucleasas del Exosoma/genética , Expresión Génica , Proteínas Nucleares/química , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Procesamiento Postranscripcional del ARN , Estabilidad del ARN , ARN de Hongos/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Eliminación de SecuenciaRESUMEN
Rrp6 is a key catalytic subunit of the nuclear RNA exosome that plays a pivotal role in the processing, degradation, and quality control of a wide range of cellular RNAs. Here we report our findings on the assembly of the complex involving Rrp6 and its associated protein Rrp47, which is required for many Rrp6-mediated RNA processes. Recombinant Rrp47 is expressed as a non-globular homodimer. Analysis of the purified recombinant Rrp6·Rrp47 complex revealed a heterodimer, suggesting that Rrp47 undergoes a structural reconfiguration upon interaction with Rrp6. Studies using GFP fusion proteins show that Rrp6 and Rrp47 are localized to the yeast cell nucleus independently of one another. Consistent with this data, Rrp6, but not Rrp47, is found associated with the nuclear import adaptor protein Srp1. We show that the interaction with Rrp6 is critical for Rrp47 stability in vivo; in the absence of Rrp6, newly synthesized Rrp47 is rapidly degraded in a proteasome-dependent manner. These data resolve independent nuclear import routes for Rrp6 and Rrp47, reveal a structural reorganization of Rrp47 upon its interaction with Rrp6, and demonstrate a proteasome-dependent mechanism that efficiently suppresses the expression of Rrp47 in the absence of Rrp6.
Asunto(s)
Núcleo Celular , Proteínas de Unión al ADN , Complejo Multienzimático de Ribonucleasas del Exosoma , Regulación Fúngica de la Expresión Génica/fisiología , Proteínas Nucleares , Multimerización de Proteína/fisiología , Proteínas de Unión al ARN , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Transporte Activo de Núcleo Celular/fisiología , Núcleo Celular/química , Núcleo Celular/genética , Núcleo Celular/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Complejo Multienzimático de Ribonucleasas del Exosoma/química , Complejo Multienzimático de Ribonucleasas del Exosoma/genética , Complejo Multienzimático de Ribonucleasas del Exosoma/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Complejo de la Endopetidasa Proteasomal/química , Complejo de la Endopetidasa Proteasomal/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteolisis , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
The exosome ribonuclease complex functions in both the limited trimming of the 3'-ends of nuclear substrates during RNA processing events and the complete destruction of nuclear and cytoplasmic RNAs. The two RNases of the eukaryotic exosome, Rrp44 (rRNA-processing protein 44) and Rrp6, are bound at either end of a catalytically inert cylindrical core. RNA substrates are threaded through the internal channel of the core to Rrp44 by RNA helicase components of the nuclear TRAMP complex (Trf4-Air2-Mtr4 polyadenylation complex) or the cytoplasmic Ski (superkiller) complex. Recent studies reveal that Rrp44 can also associate directly with substrates via channel-independent routes. Although the substrates of the exosome are known, it is not clear whether specific substrates are restricted to one or other pathway. Data currently available support the model that processed substrates are targeted directly to the catalytic subunits, whereas at least some substrates that are directed towards discard pathways must be threaded through the exosome core.
Asunto(s)
Complejo Multienzimático de Ribonucleasas del Exosoma/metabolismo , Exosomas/metabolismo , Animales , Humanos , Ribonucleasas/metabolismoRESUMEN
Debaryomyces hansenii is considered an unconventional yeast with a strong biotechnological potential, which can produce and store high amounts of lipids. However, relatively little is known about its lipid metabolism, and genetic tools for this yeast have been limited. The aim of this study was to explore the fatty acid ß-oxidation pathway in D. hansenii. To this end, we employed recently developed methods to generate multiple gene deletions and tag open reading frames with GFP in their chromosomal context in this yeast. We found that, similar as in other yeasts, the ß-oxidation of fatty acids in D. hansenii was restricted to peroxisomes. We report a series of experiments in D. hansenii and the well-studied yeast Saccharomyces cerevisiae that show that the homeostasis of NAD+ in D. hansenii peroxisomes is dependent upon the peroxisomal membrane protein Pmp47 and two peroxisomal dehydrogenases, Mdh3 and Gpd1, which both export reducing equivalents produced during ß-oxidation to the cytosol. Pmp47 is the first identified NAD+ carrier in yeast peroxisomes.
RESUMEN
Cells lacking the exosome-associated protein Rrp47 show similar defects in stable RNA processing to those observed in the absence of the catalytic subunit Rrp6, but the precise mechanism(s) by which Rrp47 functions together with Rrp6 remains unclear. Deletion complementation analyses defined an N-terminal region of Rrp47, largely coincident with the bioinformatically defined Sas10/C1D domain, which was sufficient for protein function in vivo. In vitro protein interaction studies demonstrated that this domain of Rrp47 binds the PMC2NT domain of Rrp6. Expression of the N-terminal domain of Rrp47 in yeast complemented most RNA-processing defects associated with the rrp47Δ mutant but failed to complement the defect observed in 3'-end maturation of box C/D small nucleolar RNAs. Consistent with these results, protein capture assays revealed an interaction between the C-terminal region of Rrp47 and the small nucleolar ribonucleoproteins Nop56 and Nop58. Filter binding assays demonstrated that deletion of the lysine-rich sequence at the C terminus of Rrp47 blocked RNA binding in vitro. Furthermore, a protein mutated both at the C terminus and within the N-terminal domain showed a synergistic defect in RNA binding without impacting on its ability to interact with Rrp6. These studies provide evidence for a role of Rrp47 in registering a small nucleolar ribonucleoprotein particle assembly, functionally characterize the Sas10/C1D domain of Rrp47, and show that both the C terminus of Rrp47 and the N-terminal domain contribute to its RNA-binding activity.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas Nucleares/metabolismo , Procesamiento Postranscripcional del ARN/fisiología , ARN Nucleolar Pequeño/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Unión al ADN/genética , Exorribonucleasas/genética , Exorribonucleasas/metabolismo , Complejo Multienzimático de Ribonucleasas del Exosoma , Prueba de Complementación Genética , Mutación , Proteínas Nucleares/genética , Unión Proteica , Estructura Terciaria de Proteína , ARN Nucleolar Pequeño/genética , Proteínas de Unión al ARN/genética , Ribonucleoproteínas Nucleolares Pequeñas/genética , Ribonucleoproteínas Nucleolares Pequeñas/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
We describe the events surrounding the identification of the exosome complex and the subsequent early development of the field. Like many scientific discoveries, the initial identification and characterization of the exosome was based on a combination of skill, good fortune-and the availability of cutting edge technology.
Asunto(s)
Exorribonucleasas , Exosomas , HumanosRESUMEN
This chapter reviews the present state of knowledge on the activity of enzymes that function with the RNA exosome in the nucleus. In this compartment, the exosome interacts physically and functionally with the exoribonuclease Rrp6 and several cofactors, most prominently Rrp47 and the TRAMP complex. These interactions decide the fate of RNA precursors from transcription through the formation of mature ribonucleoprotein particles (RNPs) and the export of the RNPs to the cytoplasm. The nuclear exosome catalyzes the formation of the mature 3' ends of many of these RNAs, but in other cases degrades the RNAs to mononucleotides. Cofactors such as Mpp6, TRAMP and the Nrd1/Nab3 complex play important roles in determining the outcome of the interaction of RNPs with the nuclear exosome. The details that govern the specificity of these decisions remain a rich source for future investigation.
Asunto(s)
Exosomas , Proteínas de Saccharomyces cerevisiae , Complejo Multienzimático de Ribonucleasas del Exosoma , Exosomas/metabolismo , Humanos , Proteínas Nucleares/metabolismo , Proteínas de Unión al ARN/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
The Sas10/C1D domain is found in a small group of eukaryotic proteins that have functions in RNA processing events, translational control and DNA repair mechanisms. The domain is predicted to be alpha-helical in nature and comprises approx. 80 amino acid residues. Whereas the Sas10/C1D domain has yet to be functionally characterized, available results suggest that this domain forms a binding surface for specific interactions with other proteins and can concomitantly interact with RNA or DNA. This property of the Sas10/C1D domain may facilitate this family of proteins to dock other proteins on to nucleic acid substrates.
Asunto(s)
Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/fisiología , Proteínas Nucleares/química , Proteínas Nucleares/fisiología , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/fisiología , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/fisiología , Secuencia de Aminoácidos , Animales , ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Complejo Multienzimático de Ribonucleasas del Exosoma , Humanos , Modelos Biológicos , Datos de Secuencia Molecular , Proteínas Nucleares/metabolismo , Unión Proteica/fisiología , Dominios y Motivos de Interacción de Proteínas/fisiología , ARN/metabolismo , Proteínas de Unión al ARN/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiología , Proteínas de Saccharomyces cerevisiae/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
We describe the events surrounding the identification of the exosome complex and the subsequent early development of the field. Like many scientific discoveries, the initial identification and characterization ofthe exosome was based on a combination of skill, good fortune--and the availability of cutting edge technology.
Asunto(s)
Exorribonucleasas , Exosomas , Precursores del ARN/metabolismo , ARN Ribosómico/metabolismo , Humanos , ARN de Hongos/metabolismoRESUMEN
This chapter reviews the present state of knowledge on the activity of enzymes that function with the RNA exosome in the nucleus. In this compartment, the exosome interacts physically and functionally with the exoribonuclease Rrp6 and several cofactors, most prominently Rrp47 and the TRAMP complex. These interactions decide the fate of RNA precursors from transcription through the formation of mature ribonucleoprotein particles (RNPs) and the export of the RNPs to the cytoplasm. The nuclear exosome catalyzes the formation of the mature 3' ends of many of these RNAs, but in other cases degrades the RNAs to mononucleotides. Cofactors such as Mpp6, TRAMP and the Nrd1/Nab3 complex play important roles in determining the outcome of the interaction of RNPs with the nuclear exosome. The details that govern the specificity of these decisions remain a rich source for future investigation.
Asunto(s)
Coenzimas/metabolismo , Exorribonucleasas/química , Exorribonucleasas/metabolismo , Exosomas/química , Exosomas/metabolismo , Proteínas Nucleares/metabolismo , Coenzimas/química , Complejo Multienzimático de Ribonucleasas del Exosoma , Humanos , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Modelos Moleculares , Proteínas Nucleares/química , Conformación Proteica , ARN/metabolismo , Precursores del ARN/metabolismo , ARN no Traducido/metabolismoRESUMEN
The exosome complex is a key component of the cellular RNA surveillance machinery and is required for normal 3' end processing of many stable RNAs. Exosome activity requires additional factors such as the Ski or TRAMP complexes to activate the complex or facilitate substrate binding. Rrp47p promotes the catalytic activity of the exosome component Rrp6p, but its precise function is unknown. Here we show that recombinant Rrp47p is expressed as an apparently hexameric complex that specifically binds structured nucleic acids. Furthermore, pull-down assays demonstrated that Rrp47p interacts directly with the N-terminal region of Rrp6p that contains the functionally uncharacterized PMC2NT domain. Strains expressing a mutant form of Rrp6p lacking the N-terminal region failed to accumulate Rrp47p at normal levels, exhibited a slow growth phenotype characteristic of rrp47-Delta mutants and showed RNA processing defects consistent with loss of Rrp47p function. These findings suggest Rrp47p promotes Rrp6p activity by facilitating binding via the PMC2NT domain to structural elements within RNA. Notably, characterized Rrp6p substrates such as the 5.8S+30 species are predicted to contain helices at their 3' termini, while others such as intergenic or antisense cryptic unstable transcripts could potentially form extensive double-stranded molecules with overlapping mRNAs.
Asunto(s)
Exorribonucleasas/química , Exorribonucleasas/metabolismo , Proteínas Nucleares/metabolismo , Procesamiento de Término de ARN 3' , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , ADN/metabolismo , Proteínas de Unión al ADN , Complejo Multienzimático de Ribonucleasas del Exosoma , Estructura Terciaria de Proteína , ARN/metabolismo , ARN Ribosómico 5.8S/metabolismo , ARN Nucleolar Pequeño/metabolismo , Proteínas de Unión al ARN , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/metabolismoRESUMEN
Rrp6 is a conserved catalytic subunit of the eukaryotic nuclear exosome ribonuclease complex that functions in the productive 3' end maturation of stable RNAs, the degradation of transiently expressed noncoding transcripts and in discard pathways that eradicate the cell of incorrectly processed or assembled RNAs. The function of Rrp6 in these pathways is at least partially dependent upon its interaction with a small nuclear protein called Rrp47/Lrp1, but the underlying mechanism(s) by which Rrp47 functions in concert with Rrp6 are not established. Previous work on yeast grown in rich medium has suggested that Rrp6 expression is not markedly reduced in strains lacking Rrp47. Here we show that Rrp6 expression in rrp47∆ mutants is substantially reduced during growth in minimal medium through effects on both transcript levels and protein stability. Exogenous expression of Rrp6 enables normal levels to be attained in rrp47∆ mutants. Strikingly, exogenous expression of Rrp6 suppresses many, but not all, of the RNA processing and maturation defects observed in an rrp47∆ mutant and complements the synthetic lethality of rrp47∆ mpp6∆ and rrp47∆ rex1∆ double mutants. Increased Rrp6 expression in the resultant rrp47∆ rex1∆ double mutant suppresses the defect in the 3' maturation of box C/D snoRNAs. In contrast, increased Rrp6 expression in the rrp47∆ mpp6∆ double mutant diminishes the block in the turnover of CUTs and in the degradation of the substrates of RNA discard pathways. These results demonstrate that a principal function of Rrp47 is to facilitate appropriate expression levels of Rrp6 and support the conclusion that the Rrp6/Rrp47 complex and Rex1 provide redundant exonuclease activities for the 3' end maturation of box C/D snoRNAs.