Parts Lists and the Isoform Problem

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

2. 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?

3. 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

4. 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)

5. 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

6. Some examples of array results P = present A = absent M = marginal Distinct probe sets Shows potential power of this approach as well as potential for false negs

7. 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)

8. Selected array results: Macrophages P = present A = absent M = marginal

9. 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

10. 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

11. 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?