Regulation of alternative splicing of the small GTPase Rac1

• Author(s): Gonçalves, Vânia Marina Cristóvão, 1980-
• Date: 2009
• Persistent ID: http://hdl.handle.net/10451/1628
• Origin: Repositório da Universidade de Lisboa
• Subject(s): Biologia molecular; Teses de doutoramento
• Document Type: Doctoral thesis
• Language: English
• Advisor(s): Jordan, Peter M.; Gomes, Rui Artur Paiva Loureiro, 1958-
• Contributor(s): Repositório da Universidade de Lisboa

Description

Tese de doutoramento, Biologia (Biologia Molecular), 2009, Universidade de Lisboa, Faculdade de Ciências

Tumours develop through the stepwise acquisition of genetic changes including those affecting signalling pathways that control cell proliferation and survival. The small GTPase Rac1 regulates signalling pathways controlling actin filament dynamics and transcriptional activation. An alternative splicing variant Rac1b contains 19 additional amino acids due to inclusion of a usually skipped exon 3b and is overexpressed in a subset of colorectal tumours. Rac1b is required to sustain colorectal tumour cell survival and understanding the molecular mechanism behind this alternative splicing event is of therapeutic interest. Here we describe that antagonistic SR proteins ASF/SF2 and SRp20 regulate Rac1 alternative splicing in colorectal cells. Using a Rac1 minigene we identified that SRp20 increased skipping of alternative exon 3b in HT29 cells, while ASF/SF2 increased its inclusion. Depletion of endogenous expression of these splicing factors by specific siRNAs confirmed that ASF/SF2 enhances, whereas SRp20 silences endogenous Rac1b splicing. Moreover, we found that both splicing factors bound to Rac1 exon 3b sequences and were regulated by upstream signalling pathways: inhibition of PI3-kinase pathway increased ASF/SF2 expression and promoted Rac1b, whereas activation of "-catenin/TCF4 increased SRp20 expression and inhibited Rac1b generation. We further found that "-catenin/TCF4 directly stimulates gene transcription of SRp20 and generates a subset of transcript variants through alternative splicing. This supports the recent notion that transcription and alternative splicing represent two different layers of gene expression and that signalling pathways act upon a coordinated network of transcripts in each layer. A major contribution of this work is the demonstration that different cellular signalling pathways act in concert to regulate a specific alternative splicing event. The results predict that overexpression of Rac1b can occur in tumours without enhanced stimulation of PI3-kinase and Wnt pathways, which synchronize the expression of two antagonistic SR proteins, ASF/SF2 and SRp20, regulating alternative splicing of the Rac1 pre-mRNA.