The Cellular DExD/H-Box RNA-Helicases UAP56 and URH49 Exhibit a CRM1-Independent Nucleocytoplasmic Shuttling Activity

Cellular DExD/H-box RNA-helicases perform essential functions during mRNA biogenesis. The closely related human proteins UAP56 and URH49 are members of this protein family and play an essential role for cellular mRNA export by recruiting the adaptor protein REF to spliced and unspliced mRNAs. In order to gain insight into their mode of action, we aimed to characterize these RNA-helicases in more detail. Here, we demonstrate that UAP56 and URH49 exhibit an intrinsic CRM1-independent nucleocytoplasmic shuttling activity. Extensive mapping studies identified distinct regions within UAP56 or URH49 required for (i) intranuclear localization (UAP56 aa81-381) and (ii) interaction with REF (UAP56 aa51-428). Moreover, the region conferring nucleocytoplasmic shuttling activity was mapped to the C-terminus of UAP56, comprising the amino acids 195-428. Interestingly, this region coincides with a domain within Uap56p of S. pombe that has been reported to be required for both Rae1p-interaction and nucleocytoplasmic shuttling. However, in contrast to this finding we report that human UAP56 shuttles independently from Rae1. In summary, our results reveal nucleocytoplasmic shuttling as a conserved feature of yeast and human UAP56, while their export receptor seems to have diverged during evolution

The CCR4-NOT Complex Is Implicated in the Viability of Aneuploid Yeasts

To identify the genes required to sustain aneuploid viability, we screened a deletion library of non-essential genes in the fission yeast Schizosaccharomyces pombe, in which most types of aneuploidy are eventually lethal to the cell. Aneuploids remain viable for a period of time and can form colonies by reducing the extent of the aneuploidy. We hypothesized that a reduction in colony formation efficiency could be used to screen for gene deletions that compromise aneuploid viability. Deletion mutants were used to measure the effects on the viability of spores derived from triploid meiosis and from a chromosome instability mutant. We found that the CCR4-NOT complex, an evolutionarily conserved general regulator of mRNA turnover, and other related factors, including poly(A)-specific nuclease for mRNA decay, are involved in aneuploid viability. Defective mutations in CCR4-NOT complex components in the distantly related yeast Saccharomyces cerevisiae also affected the viability of spores produced from triploid cells, suggesting that this complex has a conserved role in aneuploids. In addition, our findings suggest that the genes required for homologous recombination repair are important for aneuploid viability

Rae1 is an essential mitotic checkpoint regulator that cooperates with Bub3 to prevent chromosome missegregation

The WD-repeat proteins Rae1 and Bub3 show extensive sequence homology, indicative of functional similarity. However, previous studies have suggested that Rae1 is involved in the mRNA export pathway and Bub3 in the mitotic checkpoint. To determine the in vivo roles of Rae1 and Bub3 in mammals, we generated knockout mice that have these genes deleted individually or in combination. Here we show that haplo-insufficiency of either Rae1 or Bub3 results in a similar phenotype involving mitotic checkpoint defects and chromosome missegregation. We also show that overexpression of Rae1 can correct for Rae1 haplo-insufficiency and, surprisingly, Bub3 haplo-insufficiency. Rae1-null and Bub3-null mice are embryonic lethal, although cells from these mice did not have a detectable defect in nuclear export of mRNA. Unlike null mice, compound haplo-insufficient Rae1/Bub3 mice are viable. However, cells from these mice exhibit much greater rates of premature sister chromatid separation and chromosome missegregation than single haplo-insufficient cells. Finally, we show that mice with mitotic checkpoint defects are more susceptible to dimethylbenzanthrene-induced tumorigenesis than wild-type mice. Thus, our data demonstrate a novel function for Rae1 and characterize Rae1 and Bub3 as related proteins with essential, overlapping, and cooperating roles in the mitotic checkpoint

Functional characterization of the nuclear basket TPR nucleoporins in the fission yeast

Programa de Doctorado en Biotecnología, Ingeniería y Tecnología QuímicaLínea de Investigación: Genética de la División CelularClave Programa: DBICódigo Línea: 18Research performed over the last years has changed the view of NPCs as simple nucleo-cytoplasmic trafficking channels into a more comprehensive understanding of the multiples roles of the NPCs, which range from chromatin regulation to the maintenance of genome integrity. The most nucleoplasm-facing structure of the NPC is the nuclear basket. While in higher eukaryotes the main structural component of the nuclear basket is the translocated promoter region (TPR) nucleoporin, most yeasts possess two orthologs: Mlp1/Mlp2 in Saccharomyces cerevisiae and Nup211/Alm1 in Schizosaccharomyces pombe. Although it is known that most nuclear basket functions have been conserved along evolution, it remains unclear how TPR nucleoporins are assembled into the NPCs and the roles that they perform in the fission yeast. Previous data from our laboratory described that the absence of Alm1 leads to chromosome missegregation and altered kinetochore behaviour. In order to avoid erroneous microtubule-kinetochore attachments and chromosome segregation defects, it is required a proper centromere and kinetochore structural organization, which is regulated by proteasomal degradation. Proteasome is enriched in the nucleus, specially at the nuclear periphery, although how this localization is regulated and its biological implications are unclear. In the first part of this thesis, we have characterized the role of the nuclear basket component Alm1 in the spatial regulation of the proteasome, which is key for chromosome segregation through the regulation of the kinetochore homeostasis. The different steps of mRNA biogenesis, including transcription, processing, quality control and export are closely coordinated, and the nuclear basket has been proposed to act as a physical platform that couples such processes. nup211+ was previously described as required for mRNA export. However, it remains unknown its specific role in mRNA docking and export. In the second part of this thesis, we have characterized how the two TPR nucleoporins in the fission yeast, Nup211 and Alm1, are assembled into the nuclear basket and how they anchor to the NPC. Additionally, we have performed a genetic and functional analysis to dissect the functions of Nup211 and Alm1 in mRNA docking, quality control and export. Heat shock deeply compromise cell viability due to protein denaturation and aggregation. In order to ensure survival cells activate the evolutionary conserved heat shock response (HSR), which results in profound changes in mRNA metabolism and nuclear organization. How this switch is achieved is not fully understood. In the third part of this study, we have characterized how heat stress leads to the inhibition of bulk mRNA export and the arrest of cell growth, concomitant with the aggregation of NPC components, the mRNA processing and export machinery, cell cycle regulators, and protective chaperones and disaggregases into ring-like structures proximal to the nucleolus. We propose that these structures, named ¿nucleolar rings¿ (NuRs), are formed by the reversible aggregation of nuclear components, and constitute storage sites for those activities that are inhibited during HS and have to be protected in order to re-start cellular metabolism when normal conditions are restored.Universidad Pablo de Olavide de Sevilla. Departamento de Biología Molecular e Ingeniería BioquímicaPostprin

Exportação de mRNAs do núcleo para o Citoplasma : análise do papel da proteína sub2 em Trypanosoma cruzi e Toxoplasma gondii

Orientadora : Profa. Dra. Andréa Rodrigues ÁvilaCo-orientador : Prof. Dr.Stenio Perdigão FragosoTese (doutorado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Biologia Celular e Molecular. Defesa: Curitiba, 30/04/2014Inclui referênciasÁrea de concentração : Biologia celular e molecularResumo: A doença de Chagas e toxoplasmose são problemas de saúde pública no Brasil e entender com detalhes a biologia dos agentes causais, T. cruzi e T. gondii respectivamente, pode ser um ponto crucial para gerar informações relevantes voltadas para o desenvolvimento de tratamento efetivo no combate a estas doenças. No entanto, a busca de novos alvos para quimioterapia de algumas doenças parasitárias esbarra, por exemplo, na ausência de ferramentas que possibilitem o estudo da função de genes essenciais para a sobrevivência do organismo. Dentro desta problemática, o objetivo central deste estudo é a identificação de proteínas envolvidas no transporte nucleocitoplasmático de RNAs, mais especificamente a exportação de mRNAs, com a utilização de ferramentas de genética reversa para análise de proteínas essenciais envolvidas nestas vias em T. cruzi e T. gondii. Além disso, T. gondii tem sido o modelo de estudo escolhido por diversos grupos, uma vez que dispõe de várias ferramentas de genética reversa consideradas inviáveis ou laboriosas em outros parasitas (como Plasmodium ou Trypanosoma). A escolha do tema foi baseada em dois fatos principais: a) dentro do contexto de eventos moleculares essenciais, o transporte de RNA mensageiro (mRNA) ao local de tradução é uma etapa essencial e crucial na seleção de genes a serem expressos em uma célula eucariótica; b) escassez de publicações envolvendo diversos grupos de parasitas, demonstrando ser um tema da pesquisa básica em parasitologia que necessita ser explorado. Os dados gerados neste estudo permitiram identificar que a maioria das proteínas relacionadas com a exportação de mRNAs não são tão conservadas ao longo da filogenia de eucariotos, principalmente em relação aos protozoários que divergiram na base da árvore filogenética. A exceção encontrada foi Sub2/UAP56, uma proteína relacionada com exportação de mRNAs em fungos e humanos, respectivamente, que se encontra altamente conservada ao longo da filogenia de eucariotos. Em T. cruzi foi observado que TcSub2 é uma proteína nuclear, relacionada com alguns sítios de transcrição por RNA Polimerase II e essencial, não sendo possível obter o nocaute duplo do respectivo gene. Em T. brucei e T. gondii foi observado que a diminuição gradual das proteínas em questão, TbSub2 e TgUAP56 respectivamente, resulta em acúmulo de mRNAs poliadenilados nos núcleos afetando a exportação dos mRNAs e resultando em morte celular. A partir da observação da participação de proteínas ortólogas de Sub2 na exportação de mRNAs em parasitas, a outra parte deste estudo foi identificar proteínas associadas a TcSub2 para iniciar o entendimento sobre esta via bem como verificar a presença de proteínas específicas e provavelmente essenciais de parasitas para futuros estudos funcionais.Abstract: Chagas disease and toxoplasmosis are public health issues in Brazil and understanding in detail the biology of its causative agents, T. cruzi and T. gondii respectively, could generate relevant information to develop effective treatment(s) against these diseases. The search for new chemotherapy targets against parasitic disease faces a number of challenges as the lack of tools that enable the study of essential gene function in parasites. Taking this into consideration, the aim of this study was to develop reverse genetic tools to identify and analyse essential proteins in T. cruzi and T. gondii, in particular those involved in nucleocytoplasmic transport of RNAs, specifically in mRNA export. T. gondii has served as model organism because it has an ample wide repertoire of reverse genetic tools. However, transferring these tools to other parasites, i.e. Plasmodium or Trypanosoma, has not been a straightforward task. The hypothesis of this study was based on two main facts: a) the transport of mRNA to the site of translation, within the context of key molecular events, is an essential step in the selection of genes to be expressed in an eukaryotic cell and b) the lack of publications in this topic in parasites proving to be a subject of basic research in parasitology that needs to be explored. The data obtained in this study showed that most proteins related to the export of mRNAs are not well conserved throughout eukaryotic phylogeny, especially eukaryotes that diverged at the base of the phylogenetic tree, the parasites. An exception to this observation was Sub2/UAP56, a protein directly related to export of mRNAs in yeast and human respectively, which is highly conserved throughout eukaryotic phylogeny. In T. cruzi, TcSub2 is an essential nuclear protein, associated with a number of sites of RNA polymerase II transcription and, a double knockout of the gene is lethal. It has been observed in T. brucei and T. gondii that the knockdown of TbSub2 and TgUAP56 respectively, results in nuclear accumulation of polyadenylated mRNAs, effectively reducing mRNA export and leading to cell death. Extending these observations that highlight the important role of Sub2 orthologues in mRNA export, we aim to identify proteins partners of TcSub2 to better understand this pathway in T. cruzi and verify the presence of specific and likely essential proteins for future studies in this parasite