PDF file: EURASNET Annual Report 2008

In order to develop GFP based method for determination of the stoechiometry of PTB binding to alpha-tropomyosin premRNA,Group 17 (Eperon) in collaboration with Group 23 (Smith) compared PTB and GFP-tagged PTB binding toalpha-tropomyosin pre-mRNA. To this end, RNA sequences spanning the regulated exon and the regulatory sequences in theintrons were incubated in nuclear extract, and single molecule TIRF microscopy was done to examine the complexes. Thenumber of fluorescent molecules bound to each RNA molecule was examined by statistical analysis. Cross-linking was usedto show that the binding of PTB and GFP-PTB was equally probable.169 Development of cell imaging methods and single molecule methodsGroups 17 (Eperon) and 23 (Smith) used the GFP based single molecule approach developed as described in deliverable168, to study the binding of PTB to alpha-tropomyosin pre-mRNA carrying mutations in various PTB binding sites. Theresults showed that PTB associates as a defined complex, and that the number of binding sites present in the RNA substratedoes not affect the number of bound PTB proteins but their affinity.In addition, group 17 (Eperon) uses the intensity distributions from single molecule FRET to infer information aboutconformational heterogeneity, the distance between fluorophores was tested using a simple double-stranded molecule. Theresults showed that conformational heterogeneity, which was widely reported as an explanation for the observed broaddistributions, was not the correct explanation: changes in the orientation of the fluorophore dipoles associated with minorconformational movements were found to have a much more important contribution to the broad distribution.170 Obtain 3D Structure information on splicing regulatory proteins by NMR approaches−Tra2βThe YIP Group 29 (Allain) recently determined the structure of the RRM of tra2β in complex with AAGAAU using NMRspectroscopy. The structure showed that all six nucleotides are in contact with the RRM but also with the N and C-terminalextension. Key protein-RNA contacts have been tested by affinity measurement using ITC and by splicing assays (incollaboration with the Stamm lab) on mutant proteins.-hnRNPFGroup 29 (Allain) determined the 3D solution structure of the 3 RRMs of hnRNP F in complex with AGGGAU. Thestructure reveals a new mode of RNA binding for all three RRMs, since for each of them, the β-sheet surface is not used forRNA binding as is usually the case. These data explain how a G-tract of three consecutive Gs is recognized by hnRNP F.The structure also suggests mechanisms of action for hnRNP F. Group 29 showed that hnRNP F can unfold RNA structures(RNA stem-loop or G-quadruplex). In binding G-tract, hnRNP F could free key cis-acting element in contact with the G-tract therefore activating or repressing splicing.Problems and explanations for delays, postponements etc.33 While we are aware of the fact that network efficiency may profit from focussing on a limited number of experimentalsystems, nevertheless the diversity of experimental approaches und model systems represent a network reality whichrequires a highly diverse spectrum of substrates and components.72 Taking into account the rapid evolution of technologies since the start of this network, large scale studies based on DNAchip, CLIP and proteomic methods now have to be used to pursue this deliverable which was therefore transferred to WP8.37