PDF file: EURASNET Annual Report 2008

Participant 22 – Angus Lamonda) Work performed during the periodWP2Dr Laura Trinkle-Mulcahy and Dr Séverine Boulon from the Lamond group further improvedthe protocol for quantitative identification of specific protein interaction partners using massspectrometry and SILAC. They generated a set of bead proteomes for use as a filter toexclude contaminants from specific interaction partners. This work was published in theJournal of Cell Biology (Trinkle-Mulcahy, L, et al, 2008).WP3Angus Lamond has acted as a mentor to YIP Dr Edouard Bertrand (Montpellier). Acollaboration was continued between Lamond and Bertrand groups using SILAC massspectrometry to study the dynamics of protein-protein interactions in the assembly of snoRNPparticles and RNA polymerases . Dr Séverine Boulon from the Lamond group has carried outSILAC MS analyses for the Bertrand group and performed data analysis.WP10David Lleres continued a collaboration with the group of Javier Caceres (Edinburgh) to applyFLIM-FRET to identify and localise protein-protein interactions between splicing factors invivo in human cells. Part of this work was published in the Journal of Cell Biology (Ellis, J etal, 2008) with a figure showing the FLIM-FRET localisation used as a cover illustration byThe Journal of Cell Biology.WP14Andrea Pawellek made further progress with the development of a solid phase pre-mRNAsplicing assay suitable for use in high throughput screening in micro tighter dishes. Andreaalso developed stable cell lines in which expression of either GFP or mCherry fluorescentproteins was splicing dependant. These cells were characterised for use in screening chemicalcompounds for splicing inhibitor effects in-vivo.WP15Further improvements were made to protocols used for studying protein-protein interactionsbetween pre-mRNA splicing factors both in vitro using quantitative mass spectrometry basedproteomics (Drs Laura Trinkle-Mulcahy and Séverine Boulon) and in-vivo using FLIM-FRETmicroscopy (Dr David Lleres).WP17Dr David Lleres (Lamond Group) provided training and assistance in the use of FLIM-FRETmicroscopy to visiting PhD student Jonathan Ellis (Caceres Group).WP18Angus Lamond acted as mentor to YIP Dr Edouard Bertrand (Montpellier) and participated intraining postdoctoral researchers in the Lamond group and PhD student Jonathan Ellis(Caceres Group) in the use of advanced fluorescence imaging.WP20Andrea Pawellek continued work on the development of a new assay format suitable forscreening HeLa cell splicing extracts in high throughput to identify chemical inhibitors.Multiple alternative assay formats were investigated and evaluated along with detailedcomparison and evaluation of extracts and reagents suitable for use in the high throughputscreen. Andrea Pawellek also constructed and characterised stable cell lines expressingseparate red and green fluorescent proteins encoded from the same transcript in vector designthat allows detection of splicing inhibition in vivo. Thus, active splicing results in expressionof a red fluorescent protein while inhibiting splicing decreases the level of red protein andconcomitantly increases the level of green protein. This provides an in vivo assay systemsuitable for screening chemical compound libraries directly in an automated fluorescencescreen. It also provides a secondary area for screening in vivo compounds identified asinhibiting splicing in vitro.179