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SWI/SNF regulates the alternative processing of a specific subset of pre-mRNAs in Drosophila melanogaster
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. University of Würzburg, Germany.
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Number of Authors: 8
2011 (English)In: BMC Molecular Biology, ISSN 1471-2199, E-ISSN 1471-2199, Vol. 12, 46Article in journal (Refereed) Published
Abstract [en]

Background: The SWI/SNF chromatin remodeling factors have the ability to remodel nucleosomes and play essential roles in key developmental processes. SWI/SNF complexes contain one subunit with ATPase activity, which in Drosophila melanogaster is called Brahma (Brm). The regulatory activities of SWI/SNF have been attributed to its influence on chromatin structure and transcription regulation, but recent observations have revealed that the levels of Brm affect the relative abundances of transcripts that are formed by alternative splicing and/or polyadenylation of the same pre-mRNA.

Results: We have investigated whether the function of Brm in pre-mRNA processing in Drosophila melanogaster is mediated by Brm alone or by the SWI/SNF complex. We have analyzed the effects of depleting individual SWI/SNF subunits on pre-mRNA processing throughout the genome, and we have identified a subset of transcripts that are affected by depletion of the SWI/SNF core subunits Brm, Snr1 or Mor. The fact that depletion of different subunits targets a subset of common transcripts suggests that the SWI/SNF complex is responsible for the effects observed on pre-mRNA processing when knocking down Brm. We have also depleted Brm in larvae and we have shown that the levels of SWI/SNF affect the pre-mRNA processing outcome in vivo.

Conclusions: We have shown that SWI/SNF can modulate alternative pre-mRNA processing, not only in cultured cells but also in vivo. The effect is restricted to and specific for a subset of transcripts. Our results provide novel insights into the mechanisms by which SWI/SNF regulates transcript diversity and proteomic diversity in higher eukaryotes.

Place, publisher, year, edition, pages
2011. Vol. 12, 46
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biology
Identifiers
URN: urn:nbn:se:su:diva-70101DOI: 10.1186/1471-2199-12-46ISI: 000297246000001OAI: oai:DiVA.org:su-70101DiVA: diva2:479198
Available from: 2012-01-17 Created: 2012-01-17 Last updated: 2017-12-08Bibliographically approved
In thesis
1. The SWI/SNF complex: Roles in transcription and pre-mRNA processing
Open this publication in new window or tab >>The SWI/SNF complex: Roles in transcription and pre-mRNA processing
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The regulation of gene expression is fundamental to the development of complex organisms and an important driving force in this process. When and where the genes are expressed decide the fate of a cell and its physiological context in the organism. It is well established that the packaging of the DNA into a more compact but dynamic chromatin structure affects the basal regulation of gene expression. In this thesis, we will discuss how the chromatin-remodeling SWI/SNF complex influences the regulation of genes, and we will focus on the roles of SWI/SNF in transcription and pre-mRNA processing. In Paper I, we show through a genome-wide approach that the levels of the different SWI/SNF subunits affect the alternative processing of a subset of Drosophila melanogaster pre-mRNAs in S2 cells. It was previously not known whether the effects on pre-mRNA processing were attributed exclusively to the ATPase subunit Brahma or if other subunits of the SWI/SNF complex were also involved in the regulation of pre-mRNA processing. Analysis of microarray data and RT-qPCR showed that depletion of the SWI/SNF subunits Moira and SNR1 mimic to a large extent the effects of Brahma, which suggests a role for SWI/SNF in pre-mRNA processing. Moreover, RNAi experiments in larvae also provide evidence for an effect of SWI/SNF on pre-mRNA processing in vivo. In Paper II, we show that Brahma modulates the abundance of a specific trans-spliced transcript derived from the mod(mdg4) locus of D. melanogaster. We have characterized the relative expression of anti-sense mod(mdg4) transcripts in S2 cells, mapped transcription start sites and cleavage sites, identified and quantified cis-spliced and trans-spliced transcripts, and obtained insight into the regulation of the mod(mdg4) trans-splicing. Using RNA interference and over-expression of recombinant Brahma proteins, we show that the levels of Brahma affect the levels of the mod(mdg4)-RX trans-spliced mRNA isoform in S2 cells. Interestingly, the trans-splicing effect is independent of the ATPase activity of Brahma, which suggests that the mechanism by which Brahma modulates trans-splicing is independent of its chromatin‑remodeling activity. In Paper III, we show that the one of the SWI/SNF complexes, PBAP, specifically regulates the transcription of the CG44250 and CG44251 genes in S2 cells. Depletion of BRM reduced the levels of CG44250/51 transcripts, whereas BRM overexpression had the opposite effect. These changes in transcript levels were accompanied by changes in the density of Pol-II at the CG44250/51 locus. Intriguingly, the effect of BRM on the expression of the CG44250/51 genes was independent of the ATPase function of BRM, as shown by over-expression of a mutant form of BRM that lacks ATPase activity. Altogether, the results presented in this thesis confirm that SWI/SNF can regulate not only transcription but also pre-mRNA processing, and they reveal that some of the regulatory functions of SWI/SNF are independent of BRM’s nucleosome‑remodeling activity. 

Place, publisher, year, edition, pages
Stockholm: Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, 2013. 60 p.
Keyword
Splicing, Gene regulation, SWI/SNF, polyadenylation
National Category
Biological Sciences
Research subject
Molecular Biology
Identifiers
urn:nbn:se:su:diva-94162 (URN)978-91-7447-752-8 (ISBN)
Public defence
2013-11-08, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 13:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Manuscript. Paper 3: Manuscript.

Available from: 2013-10-17 Created: 2013-09-30 Last updated: 2014-04-24Bibliographically approved
2. ATPase dependent and independent roles of Brahma in transcription and pre-mRNA processing
Open this publication in new window or tab >>ATPase dependent and independent roles of Brahma in transcription and pre-mRNA processing
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

SWI/SNF is a chromatin-remodeling complex and Brahma (BRM) is the ATPase subunit of SWI/SNF. BRM regulates transcription by remodeling the nucleosomes at promoter regions. BRM is also associated with RNA and affects pre-mRNA processing together with other SWI/SNF subunits. In this thesis, I will discuss the roles of BRM in both transcription and pre-mRNA processing. In Paper I, we showed that BRM, as well as other SWI/SNF subunits SNR1 and MOR, affects the alternative processing of a subset of pre-mRNAs, as shown by microarray analysis. This observation was validated by RNAi experiments both in Drosophila S2 cells and in vivo. In Paper II, we characterized the trans-splicing of transcripts derived from the mod(mdg4) gene. RNA interference (RNAi) and overexpression experiments revealed that BRM regulates the trans-splicing of mod(mdg4)-RX in an ATPase independent manner. In Paper III, we analyzed the expression of two BRM-target genes identified in Paper I, CG44250 and CG44251. RNAi and overexpression experiments showed that the expression levels of these two genes were affected by BRM in a manner that is independent of its ATPase activity. Transcriptome analysis further proved that BRM affects gene expression both in ATPase dependent and independent manners. In Paper IV, we showed that BRM is present at the 3’-end of two analyzed genes, CG5174 and CG2051. BRM facilitates the recruitment of the cleavage and polyadenylation machinery to the cleavage sites through protein-protein interactions that do not require the ATPase activity of BRM. Morevoer, BRM promotes the cleavage of the CG5174 and CG2051 pre-mRNAs. To sum up, SWI/SNF plays important roles not only in transcription but also in pre-mRNA processing. To regulate transcription, BRM can either act as an ATPase-dependent chromatin remodeler or in a manner that does not involve ATPase activity. Additionally, BRM interacts with RNA-binding proteins to regulate the processing of a subset of pre-mRNAs, and this function of BRM is independent of its chromatin remodeling activity.

Place, publisher, year, edition, pages
Stockholm: Department of Molecular Biosciences, The Wenner-Gren Institute; Stockholm University, 2015. 59 p.
Keyword
chromatin remodeling, transcription, alternative splicing, 3'-end processing
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biology
Identifiers
urn:nbn:se:su:diva-122290 (URN)978-91-7649-245-1 (ISBN)
Public defence
2015-12-17, sal E306, Arrheniuslaboratorierna, Svante Arrhenius väg 20 C, Stockholm, 09:30 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Manuscript.

Available from: 2015-11-25 Created: 2015-10-28 Last updated: 2015-11-13Bibliographically approved

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