High purity NaI(Tl) scintillator to search for dark matter

A high purity and large volume NaI(Tl) scintillator was developed to search for cosmic dark matter. The required densities of radioactive impurities (RIs) such as U-chain, Th-chain are less than a few ppt to establish high sensitivity to dark matter. The impurity of RIs were effectively reduced by selecting raw materials of crucible and by performing chemical reduction of lead ion in NaI raw powder. The impurity of $^{226}$Ra was reduced less than 100 $\mu$Bq/kg in NaI(Tl) crystal. It should be remarked that the impurity of $^{210}$Pb, which is difficult to reduce, is effectively reduced by chemical processing of NaI raw powder down to less than 30 $\mu$Bq/kg. The expected sensitivity to cosmic dark matter by using 250 kg of the high purity and large volume NaI(Tl) scintillator (PICO-LON; Pure Inorganic Crystal Observatory for LOw-background Neutr(al)ino) is 7$\times$10$^{-45}$ cm$^{2}$ for 50 GeV$/c^{2}$ WIMPs.Comment: 6 pages, 2 Figures, Proceedings of International Symposium on Radiation Detectors and Their Uses (ISRD2016). Talk given on 19th Jan. 2016 by K.Fushimi. To be published in Proceedings will be published as JPS conference proceedings (2016

Proteomics uncovers novel components of an interactive protein network supporting RNA export in trypanosomes

In trypanosomatids, transcription is polycistronic and all mRNAs are processed by trans-splicing, with export mediated by noncanonical mechanisms. Although mRNA export is central to gene regulation and expression, few orthologs of proteins involved in mRNA export in higher eukaryotes are detectable in trypanosome genomes, necessitating direct identification of protein components. We previously described conserved mRNA export pathway components in Trypanosoma cruzi, including orthologs of Sub2, a component of the TREX complex, and eIF4AIII (previously Hel45), a core component of the exon junction complex (EJC). Here, we searched for protein interactors of both proteins using cryomilling and mass spectrometry. Significant overlap between TcSub2 and TceIF4AIII-interacting protein cohorts suggests that both proteins associate with similar machinery. We identified several interactions with conserved core components of the EJC and multiple additional complexes, together with proteins specific to trypanosomatids. Additional immunoisolations of kinetoplastid-specific proteins both validated and extended the superinteractome, which is capable of supporting RNA processing from splicing through to nuclear export and cytoplasmic events. We also suggest that only proteomics is powerful enough to uncover the high connectivity between multiple aspects of mRNA metabolism and to uncover kinetoplastid-specific components that create a unique amalgam to support trypanosome mRNA maturation

An Essential Nuclear Protein in Trypanosomes Is a Component of mRNA Transcription/Export Pathway

In eukaryotic cells, different RNA species are exported from the nucleus via specialized pathways. The mRNA export machinery is highly integrated with mRNA processing, and includes a different set of nuclear transport adaptors as well as other mRNA binding proteins, RNA helicases, and NPC-associated proteins. The protozoan parasite Trypanosoma cruzi is the causative agent of Chagas disease, a widespread and neglected human disease which is endemic to Latin America. Gene expression in Trypanosoma has unique characteristics, such as constitutive polycistronic transcription of protein-encoding genes and mRNA processing by trans-splicing. In general, post-transcriptional events are the major points for regulation of gene expression in these parasites. However, the export pathway of mRNA from the nucleus is poorly understood. The present study investigated the function of TcSub2, which is a highly conserved protein ortholog to Sub2/ UAP56, a component of the Transcription/Export (TREX) multiprotein complex connecting transcription with mRNA export in yeast/human. Similar to its orthologs, TcSub2 is a nuclear protein, localized in dispersed foci all over the nuclei —except the fibrillar center of nucleolus— and at the interface between dense and non-dense chromatin areas, proposing the association of TcSub2 with transcription/processing sites. These findings were analyzed further by BrUTP incorporation assays and confirmed that TcSub2 is physically associated with active RNA polymerase II (RNA pol II), but not RNA polymerase I (RNA pol I) or Spliced Leader (SL) transcription, demonstrating participation particularly in nuclear mRNA metabolism in T. cruzi. The double knockout of the TcSub2 gene is lethal in T. cruzi, suggesting it has an essential function. Alternatively, RNA interference assays were performed in Trypanosoma brucei. It allowed demonstrating that besides being an essential protein, its knockdown causes mRNA accumulation in the nucleus and decrease of translation levels, reinforcing that Trypanosoma-Sub2 (Tryp-Sub2) is a component of mRNA transcription/export pathway in trypanosomes

Caracterização funcional de TcDbp5 como componente da via de exportação de mRNA em Trypanosoma Cruzi

Orientadora : Profa. Drª Andréa Rodrigues ÁvilaDissertação (mestrado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Biologia Celular e Molecular. Defesa: Curitiba,28/02/2011Bibliografia: fls. 107-121Resumo: Em Trypanosoma cruzi, o agente etiológico da doença de Chagas, o estudo de processos pós-transcricionais denota-se fundamentalmente importante, pois muitas das modificações celulares do parasita são decorrentes de um controle na expressão de genes que remodela o repertório de proteínas em cada estágio de seu ciclo de vida. Porém, algumas vias pós-transcricionais ainda são pouco compreendidas como o caso da via de exportação nucleocitoplasmática de RNA mensageiro. Dados recentes do nosso grupo de pesquisa demonstram que a via de exportação de mRNA é a menos conservada entre eucariotos e pouquíssimas proteínas apresentaram-se altamente conservadas. Baseados nisso, a caracterização funcional de TcDbp5 como um possível componente da via foi o objeto desse estudo, visto que está entre as proteínas altamente conservadas. As homólogas são descritas em outros organismos por interagir com proteínas do poro nuclear onde então atuam liberando o mRNA para a maquinaria da tradução. Assim, a busca de nucleoporinas em T. cruzi foi um dos enfoques, uma vez que TcDbp5 apresentou localização tanto no núcleo quanto no citoplasma com enriquecimento próximo ao complexo do poro. Pelas análises de associação com polissomos, foi possível observar que a localização no citoplasma não implicou em associação de TcDbp5 com a maquinaria de tradução. Porém, dados de microscopia por FISH sugerem que TcDbp5 está associada com RNA mensageiro no citoplasma. Pode ser que esta associação ocorra já no núcleo, pois análises de ultraestrutura mostraram que a proteína se encontra em regiões características de transcrição e processamento. Além disso, foi demonstrado através do tratamento com Leptomicina B, que especificamente interrompe a via Crm1 de transporte de proteínas, a capacidade de TcDbp5 migrar do núcleo para o citoplasma. Interessantemente, abordagens de imunoprecipitação permitiram demonstrar a interação de TcDbp5 com TcMex67, o receptor de transporte de mRNA. Em resumo, pressupõe-se que a atividade de TcDbp5 possa estar envolvida em vias de transporte de mRNA e para melhor compreensão da sua atuação e identificação de novos componentes da via é necessário investir em abordagens de enética reversa e proteômica.Abstract: In Trypanosoma cruzi, the etiological agent of Chagas'disease, studies on post transcriptional processes are fundamentally important because most of cellular modifications of the parasite are due to gene expression control that changes the protein composition in each stage of its life cycle. However, some post-transcriptional pathways are poorly understood, such as the nucleocytoplasmic export of messenger RNA (mRNA). Recent data from our research group showed that mRNA export pathway is the least conserved in eukaryotes and few proteins are highly conserved. The goal of this study was the functional haracterization of TcDbp5, a possible component of this pathway, since it is one of the highly conserved proteins. The homologs are described by the interaction with proteins of nuclear pore, acting on the release of mRNA to translation. Searching for nucleoporins in T. cruzi was one of the approaches, because TcDbp5 showed nuclear and cytoplasmic localization with enrichment near to the nuclear pore complex. By olysomes association approach, it was observed that the cytoplasmic localization of TcDbp5 is not associated with the translation machinery. Nevertheless, microscopy analyses using FISH suggest that TcDbp5 is associated with mRNA in the cytoplasm. Probably, this association starts already inside the nucleus, since ultrastructural analyses showed that the protein is present in regions that are likely to be transcription and processing sites. Besides, it was demonstrated after Leptomycin B treatment, which blocks specifically the Crm1 transport protein, the ability of TcDbp5 in shuttling from nucleus to the cytoplasm. Interestingly, immnuoprecipitation showed the interaction of TcDbp5 with TcMex67, an mRNA export receptor. Taking together, it is assumed that the activity of TcDbp5 is related with mRNA transport pathways and to get urther into its function, and identification of novel components, is necessary to invest in genetic reverse and proteomic approaches

Identification of a novel nucleocytoplasmic shuttling RNA helicase of Trypanosomes

Gene expression in trypanosomes is controlled mostly by post-transcriptional pathways. Little is known about the components of mRNA nucleocytoplasmic export routes in these parasites. Comparative genomics has shown that the mRNA transport pathway is the least conserved pathway among eukaryotes. Nonetheless, we identified a RNA helicase (Hel45) that is conserved across eukaryotes and similar to shuttling proteins involved in mRNA export. We used in silico analysis to predict the structure of Trypanosoma cruzi Hel45, including the N-terminal domain and the C-terminal domain, and our findings suggest that this RNA helicase can form complexes with mRNA. Hel45 was present in both nucleus and cytoplasm. Electron microscopy showed that Hel45 is clustered close to the cytoplasmic side of nuclear pore complexes, and is also present in the nucleus where it is associated with peripheral compact chromatin. Deletion of a predicted Nuclear Export Signal motif led to the accumulation of Hel45ΔNES in the nucleus, indicating that Hel45 shuttles between the nucleus and the cytoplasm. This transport was dependent on active transcription but did not depend on the exportin Crm1. Knockdown of Mex67 in T. brucei caused the nuclear accumulation of the T. brucei ortholog of Hel45. Indeed, Hel45 is present in mRNA ribonucleoprotein complexes that are not associated with polysomes. It is still necessary to confirm the precise function of Hel45. However, this RNA helicase is associated with mRNA metabolism and its nucleocytoplasmic shuttling is dependent on an mRNA export route involving Mex67 receptor