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The SWI/SNF complex: Roles in transcription and pre-mRNA processing
Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute. (Neus Visa)
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 [en]
Splicing, Gene regulation, SWI/SNF, polyadenylation
National Category
Biological Sciences
Research subject
Molecular Biology
Identifiers
URN: urn:nbn:se:su:diva-94162ISBN: 978-91-7447-752-8 (print)OAI: oai:DiVA.org:su-94162DiVA: diva2:653988
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
List of papers
1. SWI/SNF regulates the alternative processing of a specific subset of pre-mRNAs in Drosophila melanogaster
Open this publication in new window or tab >>SWI/SNF regulates the alternative processing of a specific subset of pre-mRNAs in Drosophila melanogaster
Show others...
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.

National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biology
Identifiers
urn:nbn:se:su:diva-70101 (URN)10.1186/1471-2199-12-46 (DOI)000297246000001 ()
Available from: 2012-01-17 Created: 2012-01-17 Last updated: 2017-12-08Bibliographically approved
2. Brahma regulates a specific trans-splicing event at the mod(mdg4) locus of Drosophila melanogaster
Open this publication in new window or tab >>Brahma regulates a specific trans-splicing event at the mod(mdg4) locus of Drosophila melanogaster
2014 (English)In: RNA Biology, ISSN 1547-6286, E-ISSN 1555-8584, Vol. 11, no 2, 134-145 p.Article in journal (Refereed) Published
Abstract [en]

The mod(mdg4) locus of Drosophila melanogaster contains several transcription units encoded on both DNA strands. The mod(mdg4) pre-mRNAs are alternatively spliced, and a very significant fraction of the mature mod(mdg4) mRNAs are formed by trans-splicing. We have studied the transcripts derived from one of the anti-sense regions within the mod(mdg4) locus in order to shed light on the expression of this complex locus. We have characterized the expression of anti-sense mod(mdg4) transcripts in S2 cells, mapped their transcription start sites and cleavage sites, identified and quantified alternatively spliced transcripts, and obtained insight into the regulation of the mod(mdg4) trans-splicing. In a previous study, we had shown that the alternative splicing of some mod(mdg4) transcripts was regulated by Brahma (BRM), the ATPase subunit of the SWI/SNF chromatin-remodeling complex. Here we show, using RNA interference and overexpression of recombinant BRM proteins, that the levels of BRM affect specifically the abundance of a trans-spliced mod(mdg4) mRNA isoform in both S2 cells and larvae. This specific effect on trans-splicing is accompanied by a local increase in the density of RNA polymerase II and by a change in the phosphorylation state of the C-terminal domain of the large subunit of RNA polymerase II. Interestingly, the regulation of the mod(mdg4) splicing by BRM is independent of the ATPase activity of BRM, which suggests that the mechanism by which BRM modulates trans-splicing is independent of its chromatin-remodeling activity.

Keyword
splicing, SWI/SNF, chromatin remodeling, RNA polymerase II, ATPase activity
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biology
Identifiers
urn:nbn:se:su:diva-102486 (URN)10.4161/rna.27866 (DOI)000332213000006 ()
Funder
Swedish Research Council, VR-NT
Note

AuthorCount:4;

Available from: 2014-04-07 Created: 2014-04-07 Last updated: 2017-12-05Bibliographically approved
3. SWI/SNF can activate transcription through an alternative mechanism that is independent of the nucleosome remodeling activity of Brahma
Open this publication in new window or tab >>SWI/SNF can activate transcription through an alternative mechanism that is independent of the nucleosome remodeling activity of Brahma
(English)Manuscript (preprint) (Other academic)
Abstract [en]

SWI/SNF is a chromatin-remodeling complex with a well-established role in transcription regulation. SWI/SNF facilitates nucleosome remodeling in an ATPase-dependent manner, and regulates the recruitment of transcription regulators to gene promoters. We show here that the SWI/SNF complex of Drosophila melanogater has the ability to regulate transcription through an alternative mechanism that does not require the chromatin remodeling capacity of the core ATPase subunit Brahma (BRM). The expression of the genes CG44250 and CG44251 is regulated by BRM in Drosophila melanogaster S2 cells, as shown by RNA interference experiments and over-expression of recombinant BRM. ChIP data confirm that the cellular levels of BRM influence the density and the phosphorylation state of RNA polymerase II at the CG44250/51. Interestingly, a mutant BRM with an inactive ATPase domain does mimic the effect of the active recombinant BRM on the expression of CG44250/51, which suggests that the regulation of CG44250/51 by BRM does not require the chromatin remodeling activity of SWI/SNF. There are two main SWI/SNF complexes in Drosophila melanogaster: BAP and PBAP. They both harbor the ATPase subunit BRM and additional core subunits, and they are characterized by specific signature subunits that confer differential substrate preferences. Data from microarray experiments in SWI/SNF knock- down S2 cells suggest that the PBAP complex is specifically involved in the regulation of the transcription of the CG44250/51 genes. Taken together, our results show that the PBAP complex regulates gene expression by a mechanism different from chromatin remodeling. 

National Category
Biological Sciences
Research subject
Molecular Biology
Identifiers
urn:nbn:se:su:diva-94621 (URN)
Available from: 2013-10-08 Created: 2013-10-08 Last updated: 2013-10-08Bibliographically approved

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