Parts Lists and the Isoform Problem David Fruman AfCS Annual Meeting May 20, 2003

Issues with Isoforms Many signaling components are members of gene families, or exist in multiply spliced forms; should we (and how should we) determine which isoforms are expressed? If multiple isoforms are expressed, which should be studied/perturbed in different AfCS assays? What are the implications and likelihood of redundancy?

Is knowing isoform expression important? Y2H, pulldowns and localization/translocation: not as critical to know relative expression of isoforms - including everything might yield important structure- function relationships - for Y2H, reverse screening of bait/prey will determine if original bait is expressed in cell library Perturbations: critical for RNAi approach - unless we adopt integrative RNAi/microscopy strategy - not as critical for dominant negative or pharmacological approaches

Methods for measuring expression Protein level: immunoblot –protein expression is more relevant than mRNA –RNAi exps require a reliable Ab anyway (unless co-expressed tagged protein is integrated in assay) –low throughput –not all Abs are isoform-specific/splice-specific –suggest soliciting input from MP authors on best Abs –how useful is legacy/published data? –antibody arrays? (BD Biosciences/Clontech)

Methods for measuring expression mRNA level: arrays –new Affy chips have “full” coverage of mouse genome –problems: only semiquantitative (worse with 2- color platforms), also subject to false negatives and differences among replicate probe sets

Protein NameWEHI 231Primary B cellsWEHI AfCS LynPPP BlkPPA FynAPP LckPPP Rac1PPP Rac2PPP Rac3AAA Akt1PPP Akt2MAP H-RasAAA N-RasPPP PPP BtkPPP AAA Vav2AAA AAA SykAAA MAA PPP Some examples of array results Shows potential power of this approach as well as potential for false negs P = present A = absent M = marginal Distinct probe sets

Methods for measuring expression mRNA level: arrays –new Affy chips have “full” coverage of mouse genome –problems: only semiquantitative (worse with 2-color platforms), also subject to false negatives and differences among replicate probe sets –recommendation: should be done on chosen AfCS cell type to identify clear positives (data already completed for RAW and J774)

Selected array results: Macrophages Protein NameRAW274J774 Rac1PP PP PA Rac2PP Rac3AA Akt1PP Akt2PP PP Akt3PA PM P = present A = absent M = marginal

Methods for measuring expression mRNA level: Q-PCR –more sensitive and quantitative –labor intensive –perhaps best to use for checking potential false negatives and as alternative to immunoblot when Abs not available for RNAi –caveat that protein expression does not always correlate

What if multiple isoforms are expressed? Shouldn’t discourage RNAi efforts; many examples of isoform-specific functions Pan-isoform or tandem hairpins not currently feasible Y2H is fairly high-throughput assay to determine if isoforms are likely to form different signaling connections dominant negs/constitutively active constructs and pharmacological modulation

Summary Many assays do not require detailed knowledge of isoform expression RNAi is critically dependent on knowledge of expression, and requires an Ab or coexpressed tag to validate knockdown prioritize RNAi targets based on array results and availability of Abs (“targets of opportunity”), not on possible redundancy use Q-PCR mainly to examine suspected false negatives that are ULTRA or HIGH priority on parts list –note: many on B cell Ultra list are not members of isoform families long-term: develop more monospecific Abs? 2-D gels?