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Doctoral Thesis
DOI
10.11606/T.46.2006.tde-29092006-124545
Document
Author
Full name
Juliana Silva da Luz
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2006
Supervisor
Committee
Oliveira, Carla Columbano de (President)
Castilho, Beatriz Amaral de
Farah, Shaker Chuck
Quaggio, Ronaldo Bento
Valentini, Sandro Roberto
Title in Portuguese
Análise estrutural e funcional de cofatores do exossomo em Saccharomyces cerevisiae e Pyrococcus
Keywords in Portuguese
exossomo
proteína/purificação
pyrococcus
RNA/síntese
Saccharomyces cerevisae
Abstract in Portuguese
A síntese ribossomal é uma das maiores atividades em células eucarióticas. Este processo inicia-se no nucléolo e é finalizado após a exportação das subunidades 40S e 60S para o citoplasma. Três dos RNAs ribossomais de eucariotos (18S, 5.8S e 25S) são sintetizados como um transcrito primário de 35S, o qual é processado através de uma complexa e ordenada série de modificações nucleotídicas e clivagens endo e exonucleolíticas. Estas reações dependem de aproximadamente 170 proteínas, 80 small nucleolar RNAs e de seqüências no pré-rRNA. Os fatores trans-atuantes envolvidos no processamento podem ser agrupados como RNA-helicases, endonucleases, snoRNPs (small nucleolar ribonucleoprotein complexes) e exonucleases, que incluem o complexo exossomo. O exossomo de levedura é formado por 10 proteínas essenciais que atuam na maturação de rRNAs, snRNAs, snoRNAs, além da degradação de mRNAs incorretamente processados. A estrutura do exossomo de archaea foi descrita recentemente, mas ainda não existem muitas informações sobre a regulação deste complexo e sobre a participação de cofatores que interagem de forma transiente com o exossomo. Diante disso, este trabalho visou a caracterização funcional das proteínas que formam o anel de RNases PH em Saccharomyces cerevisiae, assim como a caracterização estrutural e funcional de possíveis cofatores do exossomo de Saccharomyces cerevisiae, Nop17p e Ylr022p, e do exossomo de Pyrococcus, Pab418p, Pab1135p e aNip7p. Os dados obtidos evidenciam que a atividade exonucleolítica do exossomo de levedura, assim como o de archaea, é dependente da formação de heterodímeros; Ylr022p, uma proteína de levedura com função não caracterizada, liga inespecificamente RNA in vitro, mas mais eficientemente alguns RNAs in vivo. Dentre as proteínas de archaea, Pab418p e aNip7p também ligam RNA, e como demonstrado aqui, aNip7p influencia significativamente a atividade do exossomo de archaea.
Title in English
Structural and functional analysis of exosome cofactors in Saccharomyces cerevisiae and Pyrococcus
Keywords in English
Exosome
protein/purification
pyrococcus
RNA/synthesis
Saccharomyces cerevisiae
Abstract in English
The synthesis of ribosomes is one of the major metabolic pathways in eukaryotic cells. This process starts in the nucleolus and ends with the export and final maturation of the ribosomal subunits 40S and 60S in the cytoplasm. Three eukaryotic ribosomal RNAs (18S, 5.8S and 25S) are synthesized as a 35S primary transcript (35S pre-rRNA), which is then processed by a complex and ordered series of nucleotide modifications and endo- and exonucleolytic cleavage reactions. These processing reactions depend on 170 proteins, 80 small nucleolar RNAs and specific pre-rRNA sequences. The trans-acting factors, that take part in the processing can be grouped as RNA-helicases, endonucleases, snoRNPs (small nucleolar ribonucleoprotein complexes) and exonucleases, including the exosome. The yeast exosome is composed of 10 essential proteins that function in the processing of rRNAs, snRNAs, snoRNAs and in the degradation of aberrant mRNAs. Recently, the archaeal exosome structure was determined, but no information is yet available on the regulation of the exosome function or on the possible role of the cofactors that transiently interact with it. The main goals of this work were the functional characterization of the protein components of the Saccharomyces cerevisiae exosome RNase PH ring, as well as the structural and functional characterization of the possible cofactors of that complex, Nop17p and Ylr022p. Since the recent characterization of the Pyrococcus exosome, the study of the archaeal exosome cofactors, Pab418p, Pab1135p and aNip7p, was also included in this work, in order to correlate the data on the complex of these different organisms. Our results show that the exonucleolytic activity of the yeast exosome is dependent on the heterodimers formation, as described for archaea. Although it is not clear how Nip7p affects the exosome function in yeast, aNip7p binds RNA and inhibits a-exosome activity in vitro. Yeast Ylr022p binds RNA inespecificaly in vitro, but coprecipitates specific RNAs more efficiently from total cell extracts. Its archaeal orthologue, Pab418p, also binds RNA, but does not affect significantly a-exosome function.
 
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Publishing Date
2006-12-07
 
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