Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Characterization of RNA exosome in Insect Cells: Role in mRNA Surveillance
Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics. (Neus Visa)
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 [en]
cell nucleus, nuclear bodies, mRNA surveillance, cotranscriptional assembly, Rrp6, Rrp4, mRNP
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biology
Identifiers
URN: urn:nbn:se:su:diva-52127ISBN: 978-91-7447-208-0 (print)OAI: oai:DiVA.org:su-52127DiVA: diva2:386816
Public defence
2011-02-11, De Geer-salen, Geovetenskapens hus, Svante Arrhenius väg 14, Stockholm, 10:00 (English)
Opponent
Supervisors
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
List of papers
1. The association of Brahma with the Balbiani ring 1 gene of Chironomus tentans studied by immunoelectron microscopy and chromatin immunoprecipitation.
Open this publication in new window or tab >>The association of Brahma with the Balbiani ring 1 gene of Chironomus tentans studied by immunoelectron microscopy and chromatin immunoprecipitation.
Show others...
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.

Keyword
immunoelectron microscopy; chromatin immunoprecipitation; gene expression; Chironomus tentans
National Category
Natural Sciences
Identifiers
urn:nbn:se:su:diva-16000 (URN)10.1111/j.1365-2583.2008.00825.x (DOI)000259270400006 ()18754808 (PubMedID)
Available from: 2008-12-12 Created: 2008-12-12 Last updated: 2017-12-13Bibliographically approved
2. The exosome Associates Cotranscriptionally with the Nascent Pre-mRNP through Interactions with Heterogeneous Nuclear Ribonucleoproteins
Open this publication in new window or tab >>The exosome Associates Cotranscriptionally with the Nascent Pre-mRNP through Interactions with Heterogeneous Nuclear Ribonucleoproteins
Show others...
2009 (English)In: Molecular Biology of the Cell, ISSN 1059-1524, E-ISSN 1939-4586, Vol. 20, no 15, 3459-3470 p.Article in journal (Refereed) Published
Abstract [en]

Eukaryotic cells have evolved quality control mechanisms to degrade aberrant mRNA molecules and prevent the synthesis of defective proteins that could be deleterious for the cell. The exosome, a protein complex with ribonuclease activity, is a key player in quality control. An early quality checkpoint takes place cotranscriptionally but little is known about the molecular mechanisms by which the exosome is recruited to the transcribed genes. Here we study the core exosome subunit Rrp4 in two insect model systems, Chironomus and Drosophila. We show that a significant fraction of Rrp4 is associated with the nascent pre-mRNPs and that a specific mRNA-binding protein, Hrp59/hnRNP M, interacts in vivo with multiple exosome subunits. Depletion of Hrp59 by RNA interference reduces the levels of Rrp4 at transcription sites, which suggests that Hrp59 is needed for the exosome to stably interact with nascent pre-mRNPs. Our results lead to a revised mechanistic model for cotranscriptional quality control in which the exosome is constantly recruited to newly synthesized RNAs through direct interactions with specific hnRNP proteins.

National Category
Natural Sciences
Identifiers
urn:nbn:se:su:diva-35089 (URN)10.1091/mbc.E09-01-0079 (DOI)000268545300002 ()
Available from: 2010-01-14 Created: 2010-01-14 Last updated: 2017-12-12Bibliographically approved
3. Splice-Site Mutations Cause Rrp6-Mediated Nuclear Retention of the Unspliced RNAs and Transcriptional Down-Regulation of the Splicing-Defective Genes
Open this publication in new window or tab >>Splice-Site Mutations Cause Rrp6-Mediated Nuclear Retention of the Unspliced RNAs and Transcriptional Down-Regulation of the Splicing-Defective Genes
Show others...
2010 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 5, no 7, e11540- p.Article in journal (Refereed) Published
Abstract [en]

Background: Eukaryotic cells have developed surveillance mechanisms to prevent the expression of aberrant transcripts. An early surveillance checkpoint acts at the transcription site and prevents the release of mRNAs that carry processing defects. The exosome subunit Rrp6 is required for this checkpoint in Saccharomyces cerevisiae, but it is not known whether Rrp6 also plays a role in mRNA surveillance in higher eukaryotes.

Methodology/Principal Findings: We have developed an in vivo system to study nuclear mRNA surveillance in Drosophila melanogaster. We have produced S2 cells that express a human β-globin gene with mutated splice sites in intron 2 (mut β-globin). The transcripts encoded by the mut β-globin gene are normally spliced at intron 1 but retain intron 2. The levels of the mut β-globin transcripts are much lower than those of wild type (wt) ß-globin mRNAs transcribed from the same promoter. We have compared the expression of the mut and wt β-globin genes to investigate the mechanisms that down-regulate the production of defective mRNAs. Both wt and mut β-globin transcripts are processed at the 3′, but the mut β-globin transcripts are less efficiently cleaved than the wt transcripts. Moreover, the mut β-globin transcripts are less efficiently released from the transcription site, as shown by FISH, and this defect is restored by depletion of Rrp6 by RNAi. Furthermore, transcription of the mut β-globin gene is significantly impaired as revealed by ChIP experiments that measure the association of the RNA polymerase II with the transcribed genes. We have also shown that the mut β-globin gene shows reduced levels of H3K4me3.

Conclusions/Significance: Our results show that there are at least two surveillance responses that operate cotranscriptionally in insect cells and probably in all metazoans. One response requires Rrp6 and results in the inefficient release of defective mRNAs from the transcription site. The other response acts at the transcription level and reduces the synthesis of the defective transcripts through a mechanism that involves histone modifications.

National Category
Biological Sciences
Research subject
Molecular Biology
Identifiers
urn:nbn:se:su:diva-52136 (URN)10.1371/journal.pone.0011540 (DOI)000279781600019 ()
Available from: 2011-01-13 Created: 2011-01-13 Last updated: 2017-12-11Bibliographically approved
4. The Intranuclear Localization of the Exoribonuclease Rrp6 in Insect Cells
Open this publication in new window or tab >>The Intranuclear Localization of the Exoribonuclease Rrp6 in Insect Cells
(English)Manuscript (preprint) (Other academic)
Identifiers
urn:nbn:se:su:diva-52261 (URN)
Available from: 2011-01-13 Created: 2011-01-13 Last updated: 2011-01-13

Open Access in DiVA

fulltext(1701 kB)765 downloads
File information
File name FULLTEXT01.pdfFile size 1701 kBChecksum SHA-512
0687c0e79440026387ef91258f1836a86f9da6c8ab5d84d3bd97819c89492be22411af4343efc59ca7885e55d96b83aae1bea7e4c9b1cf98165335fae550e44b
Type fulltextMimetype application/pdf

Search in DiVA

By author/editor
Hessle, Viktoria
By organisation
Department of Molecular Biology and Functional Genomics
Biochemistry and Molecular Biology

Search outside of DiVA

GoogleGoogle Scholar
Total: 765 downloads
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

isbn
urn-nbn

Altmetric score

isbn
urn-nbn
Total: 460 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf