Parts Lists and the Isoform Problem

David FrumanAfCS 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 Name WEHI 231 Primary B cells WEHI AfCS

Lyn P P P

Blk P P A

Fyn A P P

Lck P P P

Rac1 P P P

Rac2 P P P

Rac3 A A A

Akt1 P P P

Akt2 M A P

H-Ras A A A

N-Ras P P P

N-Ras P P P

Btk P P P

Btk A A A

Vav2 A A A

Vav2 A A A

Syk A A A

Syk M A A

Syk P P P

Some examples of array results

Shows potential powerof this approach as wellas potential for false negs

P = presentA = absentM = 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 Name RAW274 J774

Rac1 P P

Rac1 P P

Rac1 P A

Rac2 P P

Rac3 A A

Akt1 P P

Akt2 P P

Akt2 P P

Akt3 P A

Akt3 P M

P = presentA = absentM = 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?