RESUMO
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.
Assuntos
Nucléolo Celular/metabolismo , Complexo Multienzimático de Ribonucleases do Exossomo/metabolismo , Exossomos/metabolismo , Precursores de RNA/metabolismo , Processamento Pós-Transcricional do RNA , RNA Fúngico/metabolismo , RNA de Transferência/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Complexo Multienzimático de Ribonucleases do Exossomo/química , Transporte Proteico , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Frações Subcelulares/metabolismoRESUMO
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.
Assuntos
Complexo Multienzimático de Ribonucleases do Exossomo/metabolismo , Proteínas Nucleares/metabolismo , Precursores de RNA/metabolismo , Ribossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Modelos Moleculares , Proteínas Nucleares/química , Proteômica , Proteínas de Saccharomyces cerevisiae/químicaRESUMO
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.
Assuntos
Complexo Multienzimático de Ribonucleases do Exossomo/metabolismo , Interações Hospedeiro-Parasita/genética , Proteínas de Protozoários/metabolismo , Processamento Pós-Transcricional do RNA , RNA Ribossômico/genética , Trypanosoma brucei brucei/fisiologia , Animais , Bovinos , Células Cultivadas , Complexo Multienzimático de Ribonucleases do Exossomo/química , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Proteínas de Protozoários/química , RNA Ribossômico/isolamento & purificação , Relação Estrutura-Atividade , Tripanossomíase Bovina/genética , Tripanossomíase Bovina/parasitologiaRESUMO
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.
Assuntos
Complexo Multienzimático de Ribonucleases do Exossomo/metabolismo , Exossomos/metabolismo , Carioferinas/metabolismo , Sinais de Localização Nuclear/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , beta Carioferinas/metabolismo , Transporte Ativo do Núcleo Celular , Complexo Multienzimático de Ribonucleases do Exossomo/química , Complexo Multienzimático de Ribonucleases do Exossomo/genética , Exossomos/enzimologia , Deleção de Genes , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Carioferinas/química , Carioferinas/genética , Microscopia Confocal , Microscopia de Fluorescência , Sinais de Localização Nuclear/química , Sinais de Localização Nuclear/genética , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , beta Carioferinas/química , beta Carioferinas/genéticaRESUMO
BRASSICA NAPUS: can be applied to other known plant sequences. The conservation of those sequences suggests a functional role for them possibly by providing recognition structures for endogenous RNases involved in the 4.5S rRNA-5S rRNA maturing process. Most of the processing signals detected here are located at single-stranded regions of a proposed secondary structure.