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ATPase dependent and independent roles of Brahma in transcription and pre-mRNA processing
Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
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 [en]
chromatin remodeling, transcription, alternative splicing, 3'-end processing
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biology
Identifiers
URN: urn:nbn:se:su:diva-122290ISBN: 978-91-7649-245-1 (print)OAI: oai:DiVA.org:su-122290DiVA: diva2:865601
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
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
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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. The ATPase-dependent and ATPase-independent functions of Brahma in transcription regulation
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(English)Manuscript (preprint) (Other academic)
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biology
Identifiers
urn:nbn:se:su:diva-122286 (URN)
Available from: 2015-10-28 Created: 2015-10-28 Last updated: 2015-11-05Bibliographically approved
4. A role for SWI/SNF in pre-mRNA 3’-end processing
Open this publication in new window or tab >>A role for SWI/SNF in pre-mRNA 3’-end processing
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(English)Manuscript (preprint) (Other academic)
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biology
Identifiers
urn:nbn:se:su:diva-122288 (URN)
Available from: 2015-10-28 Created: 2015-10-28 Last updated: 2015-11-05Bibliographically approved

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