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The association of Brahma with the Balbiani ring 1 gene of Chironomus tentans studied by immunoelectron microscopy and chromatin immunoprecipitation.
Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
Stockholm University, Faculty of Science, The Wenner-Gren Institute .
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2008 (English)In: Insect molecular biology (Print), ISSN 0962-1075, E-ISSN 1365-2583, Vol. 17, no 5, 505-13 p.Article in journal (Refereed) Published
Abstract [en]

Many steps of gene expression take place during transcription, and important functional information can thus be obtained by determining the distribution of specific factors along a transcribed gene. The Balbiani ring (BR) genes of the dipteran Chironomus tentans constitute a unique system for mapping the association of specific factors along a eukaryotic gene using immuno-electron microscopy (immuno-EM). The chromatin immunoprecipitation (ChIP) technique has provided an alternative, more general method for studying the association of proteins with specific genomic sequences. The immuno-EM and the ChIP methods suffer from different limitations, and thus a combination of both is advantageous. We have established optimal conditions for ChIP on chromatin extracted from the salivary glands of C. tentans, and we have analyzed the association of the SWI/SNF chromatin remodelling factor Brahma (Brm) with the BR1 gene by combined immuno-EM and ChIP. We show that Brm is not restricted to the promoter of the BR1 gene but is also associated with sequences in the middle and distal portions of the gene, which suggests that Brm has additional roles apart from regulating transcription initiation.

Place, publisher, year, edition, pages
2008. Vol. 17, no 5, 505-13 p.
Keyword [en]
immunoelectron microscopy; chromatin immunoprecipitation; gene expression; Chironomus tentans
National Category
Natural Sciences
Identifiers
URN: urn:nbn:se:su:diva-16000DOI: 10.1111/j.1365-2583.2008.00825.xISI: 000259270400006PubMedID: 18754808OAI: oai:DiVA.org:su-16000DiVA: diva2:182520
Available from: 2008-12-12 Created: 2008-12-12 Last updated: 2012-12-12Bibliographically approved
In thesis
1. Characterization of RNA exosome in Insect Cells: Role in mRNA Surveillance
Open this publication in new window or tab >>Characterization of RNA exosome in Insect Cells: Role in mRNA Surveillance
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The exosome, an evolutionarily conserved protein complex with exoribonucleolytic activity, is one of the key players in mRNA quality control. Little is known about the functions of the exosome in metazoans. We have studied the role of the exosome in nuclear mRNA surveillance using Chironomus tentans and Drosophila melanogaster as model systems. Studies of the exosome subunits Rrp4 and Rrp6 revealed that both proteins are associated with transcribed genes and nascent pre-mRNPs in C. tentans. We have shown that several exosome subunits interact in vivo with the mRNA-binding protein Hrp59/hnRNP M, and that depleting Hrp59 in D. melanogaster S2 cells by RNAi leads to reduced levels of Rrp4 at the transcription sites. Our results on Rrp4 suggest a model for cotranscriptional quality control in which the exosome is constantly recruited to nascent mRNAs through interactions with specific hnRNP proteins. Moreover, we show that Rrp6 interacts with mRNPs in transit from the gene to the nuclear pore complex, where it is released during early stages of nucleo-cytoplasmic translocation. Furthermore, we show that Rrp6 is enriched in discrete nuclear bodies in the salivary glands of C. tentans and D. melanogaster. In C. tentans, the Rrp6-rich nuclear bodies colocalize with SUMO. We have also constructed D. melanogaster S2 cells expressing human b-globin genes, with either wild type of mutated splice sites, and we have studied the mechanisms by which the cells react to pre-mRNA processing defects. Our results indicate that two surveillance responses operate co-transcriptionally in S2 cells. One requires Rrp6 and retains defective mRNAs at the transcription site. The other one reduces the synthesis of the defective transcripts through a mechanism that involves histone modifications. These observations support the view that multiple mechanisms contribute to co-transcriptional surveillance in insects.

Place, publisher, year, edition, pages
Stockholm: Department of Molecular Biology and Functional Genomics, Stockholm University, 2011. 69 p.
Keyword
cell nucleus, nuclear bodies, mRNA surveillance, cotranscriptional assembly, Rrp6, Rrp4, mRNP
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biology
Identifiers
urn:nbn:se:su:diva-52127 (URN)978-91-7447-208-0 (ISBN)
Public defence
2011-02-11, De Geer-salen, Geovetenskapens hus, Svante Arrhenius väg 14, Stockholm, 10:00 (English)
Opponent
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Note
At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript. Available from: 2011-01-20 Created: 2011-01-13 Last updated: 2011-01-13Bibliographically approved

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