PDF file: EURASNET Annual Report 2008

8. Genome-wide Analyses of Splicing RegulationWorkpackage description (month 37-54)Workpackage 8 Start date or starting event: 0 monthnumberParticipant id 1b 5b 6 9 10 10b 11 14 15 16 18 19 21 23 24 26 28aPerson months 10 8 12 9 8 8 8 8 9 8 8 8 8 45 8 6 9Objectives• To develop and share relevant expertise, techniques and reagents.• To test the feasibility of and to optimize key techniques e.g. splicing factor ChIP, CLIP, iTRAQ analysis ofalternative splicing. Where possible, to coordinate such tests within common model systems.• To develop a co-ordinated strategy for genome-wide investigation of splicing factor activity, aiming to maximizecomplementarity and minimize overlap between participants, particularly in the area of mammalian splicing factoractivity, where the majority of participants are active. To identify and eliminate major “gaps” (e.g. important splicingfactors not currently being analyzed).Description of workExperimental model systems for analysis of splicing factors include human and mouse cells, Drosophila, C. elegansand Arabidopsis. Approaches for analyzing pre-mRNA/mRNA binding targets will include chromatinimmunoprecipitation (ChIP) followed by microarray analysis (KN), conventional immunoprecipitation and microarrayanalysis (JC, JT), cross-link immunoprecipitation (CLIP) tag analysis (DA, JC, AKr) and Genomic SELEX (JK).Functional target analysis will involve perturbation of splicing factor levels via RNAi (ABi, JC, MCF, AKr, RL, CS)by genetic depletion (JBr), sequestration by SatIII RNA overexpression (GB) and by splicing factor overexpression(JBr, SS, CS, JT). The functional consequences of alterations in splicing factor levels will be analyzed by splicesensitivemicroarrays (DA, ABi, JC, MCF/JV). In addition, the potential for direct quantitative proteomic analysis ofalternative splicing by isotope tagging for relative and absolute quantitation (iTRAQ) will be tested (CS). Given thelarge number of participating investigators a major activity of the initial 18-month period will be coordination ofsubsequent activities and transfer of optimized techniques. However, some early attempts at coordination will bedeveloped e.g. the differentiating C2C12 myogenic cell model is being used to validate alternative splice-sensitivemicroarrays (MCF/JV), and will also be used for proteomic analysis of alternative splicing by iTRAQ (CS), and forChIP analysis of SR proteins (KN).259