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Protein-protein interactions of SF1 isoforms: A mass spectrometry-based approach

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Director
Denomination Master en biologie
Defense Maîtrise : Univ. Genève, 2012
Abstract The splicing factor 1 (SF1) is a 75 kDa heat stable protein, which has early been reported to be involved in the recognition of the branch point sequence (BPS), hence, leading to the formation of the spliceosomal complex E. SF1 is suggested to enhance the kinetics of spliceosome formation rather than being a constitutive splicing factor. Indeed, the yeast orthogue of SF1, BBP (Branchpoint Binding Protein), is required for the catalysis of commitment complex 2 formation, prior to yeast pre-spliceosome formation. Moreover, a structural analysis of the BPS recognition by SF1 showed that SF1 facilitates the recruitment of the U2 snRNP particle to the BPS and thus enhances A complex formation. Finally, SF1 depletion does not result in protein expression reduction as it is usually observed for constitutive splicing factors. However, upon its depletion, cell viability is greatly reduced which raises the hypothesis that SF1 fulfills other biological functions. Recent in vivo crosslinking and immune-precipitation (CLIP) experiments performed on SF1 indicated that SF1 might modulate the ratio of inclusion/skipping of alternative cassette exons. SF1 has also been reported to function as transcriptional regulator, tumor suppressor, in nuclear pre-mRNA retention and to be involved in the transduction pathway of the p53 tumor suppressor gene. Moreover, unpublished evidence from our laboratory indicates that SF1 shuttles between the nucleus and the cytoplasm. In this thesis, we focused on the characterization of protein interactions mediated by IVS1- containing and Pro-C isoforms. To depict a comprehensible inventory of proteins interacting with the IVS1 sequence and the proline-rich tail, four different fluorescent-tagged SF1 isoforms expressing either IVS1 or the proline-rich tail were analysed by mass spectrometry analyses. All isoforms were expressed in HeLa cells, co-immunoprecipitated with anti-GFP antibodies and subjected to tandem mass spectrometry analyses. A list of more than one hundred potentially interacting proteins was retrieved from MS analysis and further processed through an extensive comparative analysis of isoforms- associated protein inventories. Second, we proceed to a GST-pulldown preliminary study on potential interactions between SF1, SF3a120 (SF3A1) and SF3a60 (SF3A3). Yeast two-hybrid screen with SF1 have suggested that SF1 might target the SURP1 domain of SF3a120. In a first time, His-tagged SF1-∆Pro (expressing only constitutively spliced exons) was assayed with bacterially expressed GST-tagged SURP1- and SURP2- containing SF3a120 recombinant proteins and SF3a60 full-length proteins. GST-pulldown results were corroborated by a reverse pulldown performed with His-tagged SF1-∆Pro immobilized on Talon beads. A deeper study was further performed with in vitro translated SF3a120 proteins from six partially deleted SF3a120 clones.
Keywords Splicing Factor 1SF1SF1 isoformsSplicingMass spectrometryProteomicsMass spectrometry-based interactive proteomicsIn vitro translationGST pulldownCytoscape
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DELMOTTE, Marc Alex. Protein-protein interactions of SF1 isoforms: A mass spectrometry-based approach. Université de Genève. Maîtrise, 2012. https://archive-ouverte.unige.ch/unige:24152

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Deposited on : 2012-11-21

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