RESUMEN
Rrp44/Dis3 is a conserved eukaryotic ribonuclease that acts on processing and degradation of nearly all types of RNA. It contains an endo- (PIN) and an exonucleolytic (RNB) domain and, its depletion in model organisms supports its essential function for cell viability. In Trypanosoma brucei, depletion of Rrp44 (TbRRP44) blocks maturation of ribosomal RNA, leading to disruption of ribosome synthesis and inhibition of cell proliferation. We have determined the crystal structure of the exoribonucleolytic module of TbRRP44 in an active conformation, revealing novel details of the catalytic mechanism of the RNB domain. For the first time, the position of the second magnesium involved in the two-metal-ion mechanism was determined for a member of the RNase II family. In vitro, TbRRP44 acts preferentially on non-structured uridine-rich RNA substrates. However, we demonstrated for the first time that both TbRRP44 and its homologue from Saccharomyces cerevisiae can also degrade structured substrates without 3'-end overhang, suggesting that Rrp44/Dis3 ribonucleases may be involved in degradation of a wider panel of RNA than has been assumed. Interestingly, deletion of TbRRP44 PIN domain impairs RNA binding to different extents, depending on the type of substrate.
Asunto(s)
Trypanosoma brucei brucei , Complejo Multienzimático de Ribonucleasas del Exosoma/genética , ARN/química , Saccharomyces cerevisiae/enzimología , Trypanosoma brucei brucei/enzimologíaRESUMEN
Dyskeratosis congenita (DC) is a pediatric bone marrow failure syndrome caused by germline mutations in telomere biology genes. Mutations in DKC1 (the most commonly mutated gene in DC), the 3' region of TERC, and poly(A)-specific ribonuclease (PARN) cause reduced levels of the telomerase RNA component (TERC) by reducing its stability and accelerating TERC degradation. We have previously shown that depleting wild-type DKC1 levels by RNA interference or expression of the disease-associated A353V mutation in the DKC1 gene leads to decay of TERC, modulated by 3'-end oligoadenylation by noncanonical poly(A) polymerase 5 (PAPD5) followed by 3' to 5' degradation by EXOSC10. Furthermore, the constitutive genetic silencing of PAPD5 is sufficient to rescue TERC levels, restore telomerase function, and elongate telomeres in DKC1_A353V mutant human embryonic stem cells (hESCs). Here, we tested a novel PAPD5/7 inhibitor (RG7834), which was originally discovered in screens against hepatitis B viral loads in hepatic cells. We found that treatment with RG7834 rescues TERC levels, restores correct telomerase localization in DKC1 and PARN-depleted cells, and is sufficient to elongate telomeres in DKC1_A353V hESCs. Finally, treatment with RG7834 significantly improved definitive hematopoietic potential from DKC1_A353V hESCs, indicating that the chemical inhibition of PAPD5 is a potential therapy for patients with DC and reduced TERC levels.
Asunto(s)
Disqueratosis Congénita , Telomerasa , Proteínas de Ciclo Celular/genética , Niño , Proteínas Cromosómicas no Histona , ADN Polimerasa Dirigida por ADN , Disqueratosis Congénita/genética , Disqueratosis Congénita/terapia , Exorribonucleasas , Complejo Multienzimático de Ribonucleasas del Exosoma/metabolismo , Hematopoyesis , Humanos , Mutación , Proteínas Nucleares/genética , ARN Nucleotidiltransferasas , Telomerasa/genética , Telomerasa/metabolismo , Telómero/metabolismoRESUMEN
The RNA exosome is a multisubunit protein complex involved in RNA surveillance of all classes of RNA, and is essential for pre-rRNA processing. The exosome is conserved throughout evolution, present in archaea and eukaryotes from yeast to humans, where it localizes to the nucleus and cytoplasm. The catalytically active subunit Rrp44/Dis3 of the exosome in budding yeast (Saccharomyces cerevisiae) is considered a protein present in these two subcellular compartments, and here we report that it not only localizes mainly to the nucleus, but is concentrated in the nucleolus, where the early pre-rRNA processing reactions take place. Moreover, we show by confocal microscopy analysis that the core exosome subunits Rrp41 and Rrp43 also localize largely to the nucleus and strongly accumulate in the nucleolus. These results shown here shed additional light on the localization of the yeast exosome and have implications regarding the main function of this RNase complex, which seems to be primarily in early pre-rRNA processing and surveillance.
Asunto(s)
Nucléolo Celular/metabolismo , Complejo Multienzimático de Ribonucleasas del Exosoma/metabolismo , Exosomas/metabolismo , Precursores del ARN/metabolismo , Procesamiento Postranscripcional del ARN , ARN de Hongos/metabolismo , ARN de Transferencia/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Secuencia de Aminoácidos , Complejo Multienzimático de Ribonucleasas del Exosoma/química , Transporte de Proteínas , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Fracciones Subcelulares/metabolismoRESUMEN
Eukaryotic ribosomal biogenesis is a high-energy-demanding and complex process that requires hundreds of trans-acting factors to dynamically build the highly-organized 40S and 60S subunits. Each ribonucleoprotein complex comprises specific rRNAs and ribosomal proteins that are organized into functional domains. The RNA exosome complex plays a crucial role as one of the pre-60S-processing factors, because it is the RNase responsible for processing the 7S pre-rRNA to the mature 5.8S rRNA. The yeast pre-60S assembly factor Nop53 has previously been shown to associate with the nucleoplasmic pre-60S in a region containing the "foot" structure assembled around the 3' end of the 7S pre-rRNA. Nop53 interacts with 25S rRNA and with several 60S assembly factors, including the RNA exosome, specifically, with its catalytic subunit Rrp6 and with the exosome-associated RNA helicase Mtr4. Nop53 is therefore considered the adaptor responsible for recruiting the exosome complex for 7S processing. Here, using proteomics-based approaches in budding yeast to analyze the effects of Nop53 on the exosome interactome, we found that the exosome binds pre-ribosomal complexes early during the ribosome maturation pathway. We also identified interactions through which Nop53 modulates exosome activity in the context of 60S maturation and provide evidence that in addition to recruiting the exosome, Nop53 may also be important for positioning the exosome during 7S processing. On the basis of these findings, we propose that the exosome is recruited much earlier during ribosome assembly than previously thought, suggesting the existence of additional interactions that remain to be described.
Asunto(s)
Complejo Multienzimático de Ribonucleasas del Exosoma/metabolismo , Proteínas Nucleares/metabolismo , Precursores del ARN/metabolismo , Ribosomas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Modelos Moleculares , Proteínas Nucleares/química , Proteómica , Proteínas de Saccharomyces cerevisiae/químicaRESUMEN
OBJECTIVE: The RNA exosome is an evolutionarily conserved 3'-5' exoribonucleolytic protein complex involved in processing and degradation of different classes of nuclear and cytoplasmic RNAs, and, therefore, important for the posttranscriptional control of gene expression. Despite the extensive in vivo functional studies and the structural data on the RNA exosome, few studies have been performed on the localization and expression of exosome subunits during gametogenesis, process during which gene expression is largely controlled at the posttranscriptional level. RESULTS: We report the identification of exosome subunits in Lithobates catesbeianus and analysis of the differential subcellular localization of RNA exosome core and catalytic subunits in testis cells. In addition, we show seasonal differences in the expression levels of four exosome subunits in different organs. In addition to being part of the RNA exosome complex, its subunits might participate independently of the complex in the control of gene expression during seasonal variation in bullfrog tissues. These results may be relevant for other eukaryotic species.
Asunto(s)
Complejo Multienzimático de Ribonucleasas del Exosoma/metabolismo , Rana catesbeiana/fisiología , Fenómenos Fisiológicos Reproductivos , Estaciones del Año , Testículo/metabolismo , Animales , Masculino , Rana catesbeiana/metabolismo , Espermatogénesis/fisiologíaRESUMEN
Ribosomal RNA precursors undergo a series of structural and chemical modifications to generate matured RNA molecules that will comprise ribosomes. This maturation process involves a large set of accessory proteins as well as ribonucleases, responsible for removal of the external and internal transcribed spacers from the pre-rRNA. Early-diverging eukaryotes belonging to the Kinetoplastida class display several unique characteristics, in particular in terms of RNA synthesis and maturation. These peculiarities include the rRNA biogenesis and the extensive fragmentation of the large ribosomal subunit (LSU) rRNA. The role of specific endo- and exonucleases in the maturation of the unusual rRNA precursor of trypanosomatids remains largely unknown. One of the nucleases involved in rRNA processing is Rrp44, an exosome associated ribonuclease in yeast, which is involved in several metabolic RNA pathways. Here, we investigated the function of Trypanosoma brucei RRP44 orthologue (TbRRP44) in rRNA processing. Our results revealed that TbRRP44 depletion causes unusual polysome profile and accumulation of the complete LSU rRNA precursor, in addition to 5.8S maturation impairment. We also determined the crystal structure of TbRRP44 endonucleolytic domain. Structural comparison with Saccharomyces cerevisiae Rrp44 revealed differences in the catalytic site and substitutions of surface residues, which could provide molecular bases for the lack of interaction of RRP44 with the exosome complex in T. brucei.
Asunto(s)
Complejo Multienzimático de Ribonucleasas del Exosoma/metabolismo , Interacciones Huésped-Parásitos/genética , Proteínas Protozoarias/metabolismo , Procesamiento Postranscripcional del ARN , ARN Ribosómico/genética , Trypanosoma brucei brucei/fisiología , Animales , Bovinos , Células Cultivadas , Complejo Multienzimático de Ribonucleasas del Exosoma/química , Modelos Moleculares , Unión Proteica , Conformación Proteica , Proteínas Protozoarias/química , ARN Ribosómico/aislamiento & purificación , Relación Estructura-Actividad , Tripanosomiasis Bovina/genética , Tripanosomiasis Bovina/parasitologíaRESUMEN
Cells release several biomolecules to the extracellular environment using them as a communication alternative with neighbor cells. Besides these molecules, cells also release more complex elements, like vesicles; structures composed of a lipidic bilayer with transmembrane proteins that protect a hydrophilic content. Exosomes are a small subtype of vesicles (30-150 nm), produced by many cell types, such as tumor cells, neurons, epithelial cells and immune cells. Included in this last group, antigen presenting cells produce exosomes that contain different types of molecules depending on their activation and/or maturation state. In recent years there has been an exponential interest in exosomes due to the recent evidences that show the immunomodulatory properties of these vesicles and therefore, their great potential in diagnostic approaches and development of therapies for different inflammation-associated pathologies.
Las células liberan biomoléculas de diversa naturaleza a su entorno para comunicarse con las células vecinas. Además de dichas moléculas, secretan también elementos más complejos como las vesículas; estructuras compuestas por bicapas lipídicas con proteínas transmembranales que encierran un contenido hidrofílico. Los exosomas son un subtipo pequeño de estas vesículas (de 30 a 150 nm), producidos por una amplia variedad de tipos celulares incluyendo las neuronas, células tumorales, células epiteliales y células del sistema inmunológico. De entre estas últimas, las células presentadoras de antígeno se han caracterizado como productoras de exosomas con contenido variable, tanto en condiciones de reposo como en aquellas que derivan de su estimulación o maduración. En los últimos años, el estudio de los exosomas ha aumentado debido a que se ha demostrado que dichas vesículas poseen propiedades inmunomoduladoras, razón por la que ostentan un gran potencial en aplicaciones de diagnóstico y desarrollo de terapias en diferentes patologías con componentes inflamatorios.
Asunto(s)
Células Presentadoras de Antígenos/citología , Células Presentadoras de Antígenos/inmunología , Exosomas/inmunología , Inmunidad Adaptativa , Complejo Multienzimático de Ribonucleasas del Exosoma/inmunología , HumanosRESUMEN
The exosome is a conserved multiprotein complex essential for RNA processing and degradation. The nuclear exosome is a key factor for pre-rRNA processing through the activity of its catalytic subunits, Rrp6 and Rrp44. In Saccharomyces cerevisiae, Rrp6 is exclusively nuclear and has been shown to interact with exosome cofactors. With the aim of analyzing proteins associated with the nuclear exosome, in this work, we purified the complex with Rrp6-TAP, identified the co-purified proteins by mass spectrometry, and found karyopherins to be one of the major groups of proteins enriched in the samples. By investigating the biological importance of these protein interactions, we identified Srp1, Kap95, and Sxm1 as the most important karyopherins for Rrp6 nuclear import and the nuclear localization signals recognized by them. Based on the results shown here, we propose a model of multiple pathways for the transport of Rrp6 to the nucleus.
Asunto(s)
Complejo Multienzimático de Ribonucleasas del Exosoma/metabolismo , Exosomas/metabolismo , Carioferinas/metabolismo , Señales de Localización Nuclear/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , beta Carioferinas/metabolismo , Transporte Activo de Núcleo Celular , Complejo Multienzimático de Ribonucleasas del Exosoma/química , Complejo Multienzimático de Ribonucleasas del Exosoma/genética , Exosomas/enzimología , Eliminación de Gen , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Carioferinas/química , Carioferinas/genética , Microscopía Confocal , Microscopía Fluorescente , Señales de Localización Nuclear/química , Señales de Localización Nuclear/genética , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/enzimología , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , beta Carioferinas/química , beta Carioferinas/genéticaRESUMEN
RNA decay pathways comprise a combination of RNA degradation mechanisms that are implicated in gene expression, development and defense responses in eukaryotes. These mechanisms are known as the RNA Quality Control or RQC pathways. In plants, another important RNA degradation mechanism is the post-transcriptional gene silencing (PTGS) mediated by small RNAs (siRNAs). Notably, the RQC pathway antagonizes PTGS by preventing the entry of dysfunctional mRNAs into the silencing pathway to avoid global degradation of mRNA by siRNAs. Viral transcripts must evade RNA degrading mechanisms, thus viruses encode PTGS suppressor proteins to counteract viral RNA silencing. Here, we demonstrate that tobacco plants infected with TMV and transgenic lines expressing TMV MP and CP (coat protein) proteins (which are not linked to the suppression of silencing) display increased transcriptional levels of RNA decay genes. These plants also showed accumulation of cytoplasmic RNA granules with altered structure, increased rates of RNA decay for transgenes and defective transgene PTGS amplification. Furthermore, knockdown of RRP41 or RRP43 RNA exosome components led to lower levels of TMV accumulation with milder symptoms after infection, several developmental defects and miRNA deregulation. Thus, we propose that TMV proteins induce RNA decay pathways (in particular exosome components) to impair antiviral PTGS and this defensive mechanism would constitute an additional counter-defense strategy that lead to disease symptoms.
Asunto(s)
Silenciador del Gen , Enfermedades de las Plantas/genética , Estabilidad del ARN/genética , Virus del Mosaico del Tabaco/genética , Complejo Multienzimático de Ribonucleasas del Exosoma/genética , Regulación de la Expresión Génica de las Plantas , Regulación Viral de la Expresión Génica , Enfermedades de las Plantas/virología , Hojas de la Planta/genética , Hojas de la Planta/virología , Plantas Modificadas Genéticamente , Interferencia de ARN , ARN de Planta/genética , ARN de Planta/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/genética , Nicotiana/genética , Nicotiana/virología , Virus del Mosaico del Tabaco/fisiologíaRESUMEN
The yeast exosome is a conserved multiprotein complex essential for RNA processing and degradation. The complex is formed by a nine-subunit core that associates with two hydrolytic 3'-5' exoribonucleases. Although catalytically inert, the assembly of this nine-subunit core seems to be essential for the exosome activity, as mutations in regions that do not directly bind RNA or are not in the active sites of the exonucleases impair the function of the complex. Previously isolated mutations in the exosome core subunit Rrp43p have been shown to negatively affect the function of the complex. With the aim of investigating the effect of these mutations on the complex stability and activity, Rrp43p and its mutant forms were purified by means of the TAP method. Mass spectrometry analyses showed that lower amounts of the exosome subunits are copurified with the mutant Rrp43p proteins. Additionally, by decreasing the stability of the exosome, other nonspecific protein interactions are favored (the data have been deposited to the ProteomeXchange with identifier PXD000580). Exosome copurified with mutant Rrp43p exhibited increased exonuclease activity, suggesting higher dissociation constants for these mutant complexes. Therefore, data reported here indicate that complexes containing a mutant Rrp43p exhibit decreased stability and provide information on additional protein interactions.
Asunto(s)
Exonucleasas/genética , Complejo Multienzimático de Ribonucleasas del Exosoma/genética , Regulación Fúngica de la Expresión Génica , Procesamiento Postranscripcional del ARN , ARN de Hongos/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Electroforesis en Gel de Poliacrilamida , Exonucleasas/metabolismo , Complejo Multienzimático de Ribonucleasas del Exosoma/metabolismo , Perfilación de la Expresión Génica , Modelos Moleculares , Anotación de Secuencia Molecular , Mutación , Estabilidad Proteica , Proteómica , ARN de Hongos/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Coloración y EtiquetadoRESUMEN
In eukaryotes, pre-rRNA processing depends on a large number of nonribosomal trans-acting factors that form intriguingly organized complexes. One of the early stages of pre-rRNA processing includes formation of the two intermediate complexes pre-40S and pre-60S, which then form the mature ribosome subunits. Each of these complexes contains specific pre-rRNAs, ribosomal proteins and processing factors. The yeast nucleolar protein Nop53p has previously been identified in the pre-60S complex and shown to affect pre-rRNA processing by directly binding to 5.8S rRNA, and to interact with Nop17p and Nip7p, which are also involved in this process. Here we show that Nop53p binds 5.8S rRNA co-transcriptionally through its N-terminal region, and that this protein portion can also partially complement growth of the conditional mutant strain Deltanop53/GAL::NOP53. Nop53p interacts with Rrp6p and activates the exosome in vitro. These results indicate that Nop53p may recruit the exosome to 7S pre-rRNA for processing. Consistent with this observation and similar to the observed in exosome mutants, depletion of Nop53p leads to accumulation of polyadenylated pre-rRNAs.
Asunto(s)
Regulación Fúngica de la Expresión Génica , Proteínas Nucleares/metabolismo , Precursores del ARN/metabolismo , Procesamiento Postranscripcional del ARN , ARN Ribosómico 5.8S/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Sitios de Unión , Nucléolo Celular/química , ADN Polimerasa Dirigida por ADN/metabolismo , Exorribonucleasas/metabolismo , Complejo Multienzimático de Ribonucleasas del Exosoma , Prueba de Complementación Genética , Proteínas Nucleares/química , Proteínas Nucleares/genética , Poliadenilación , Precursores del ARN/biosíntesis , ARN Ribosómico 5.8S/biosíntesis , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Eliminación de Secuencia , Transcripción GenéticaRESUMEN
The exosome is a complex of eleven subunits in yeast, involved in RNA processing and degradation. Despite the extensive in vivo functional studies of the exosome, little information is yet available on the structure of the complex and on the RNase and RNA binding activities of the individual subunits. The current model for the exosome structure predicts the formation of a heterohexameric RNase PH ring, bound on one side by RNA binding subunits, and on the opposite side by hydrolytic RNase subunits. Here, we report protein-protein interactions within the exosome, confirming the predictions of constituents of the RNase PH ring, and show some possible interaction interfaces between the other subunits. We also show evidence that Rrp40p can bind RNA in vitro, as predicted by sequence analysis.