PDF file: EURASNET Annual Report 2008

Work Package 7 Molecular Characterization of SplicingSubstratesLead Participant: Christiane Branlantdeliverables: 33 Agreement on pre-mRNA substrates, nuclear extracts andcell lines; computer analysis of secondary structures anddistributions of known preferred binding sites for SR andhnRNP proteins (month 18).72 Identification of more in vivo targets of the functionalactivity of splicing regulatory proteins (to be subsumed inWP8). (month 30)165 Identification of local pre-mRNA secondary structures, theperturbations generated by mutations found in geneticdiseases and their effects on splicing (continuation ofdeliverable 69) (month 42)166 Complete identification of components in splicingregulatory complexes and assessment of their roles in thefunctions of splicing regulatory elements and splicing sites(continuation of deliverable 70) (month 42)167 Expansion of work on detailed analysis of RNA-proteincomplexes formed with recombinant proteins and repeatsequences or pre-mRNA regulatory sequences, includingstructural analysis (continuation of deliverable 71) (month42)168 Development of methods for determination of thestoechiometry of binding of splicing factors on pre-mRNAsand to estimate their relative affinities in vitro and incellulo. (month 42)169 Development of cell imaging methods and single moleculemethods to follow the kinetics of splicing factor associationwith pre-mRNAs (continuation of deliverable 73) (month42)170 Obtain 3D Structure information on splicing regulatoryproteins by NMR approaches (Etr3 and Tra2β) by X-raycrystallography and 3D structure computer modelling(MBNL1 and CUG-BP) (continuation of deliverable N10)(month 42)monthplanned achieved18 --30 --42 3642 3642 3642 3642 3642 36Objectives of the work package• To develop a strategy with selected pre-mRNA substrates that would provide a complete description of what thespliceosomal components encounter when they come into proximity with the pre-mRNA and how they are directed to splicesites.Short description of activities33