Muneesh Tewari et al. (2001) International C. elegans Meeting "Searching for new proteins involved in dauer formation by two-hybrid protein interaction mapping."

Muneesh Tewari, Patrick Hu, Marc Vidal, Gary Ruvkun, Svetlana Busiguina

[International C. elegans Meeting, 2001]

This project aims to identify new proteins involved in dauer formation by large-scale yeast two-hybrid screens using all the known dauer gene products as baits. Open reading frames encoding each of 34 proteins implicated in dauer formation (daORFs) were cloned into yeast two-hybrid bait (DB-daORFs) and prey (AD-daORFs) vectors by Gateway recombinational cloning. First all pairwise DB-daORF/AD-daORF combinations were tested. This matrix experiment recapitulated an interaction between the DAF-3 (an ortholog of SMAD4) and DAF-5 (a relative of the SNO oncoprotein; Garth Patterson, personal communication) gene products. Individual yeast two-hybrid screens of a C. elegans AD-cDNA library were then carried out with each DB-daORF. This large-scale mapping experiment identified a total of 58 putative interactors. Among others, an interaction between AGE-1 (an ortholog of PI-3-kinase) and the worm ortholog of the p85 adaptor protein was detected. Most of the interactors identified correspond to previously uncharacterized gene products. They will be subjected to functional analysis by systematic RNA-mediated interference to test their involvement in dauer formation.

Muneesh Tewari et al. (2002) East Coast Worm Meeting "FUNCTIONAL GENOMIC ANALYSIS OF SIGNALING PATHWAYS INVOLVED IN C. ELEGANS DAUER FORMATION"

Sabrina Chaklos, Muneesh Tewari, Patrick Hu, Marc Vidal, Jin Sook Ahn, Svetlana Busiguina, Gary Ruvkun

[East Coast Worm Meeting, 2002]

Classical genetic analysis has identified about 25 genes involved in a C. elegans developmental pathway known as dauer formation. The gene products are homologous to components of human signaling pathways including the insulin signaling pathway and the transforming growth factor-beta signaling pathway. Despite vigorous genetic analysis, however, it is likely that many proteins that regulate dauer formation remain to be discovered. We are combining protein interaction mapping, large scale RNAi screening and classical genetics to identify new genes involved in dauer formation. We have undertaken yeast two-hybrid-based protein interaction mapping using all of the known dauer open reading frames (ORFs) as baits. The dauer ORFs were cloned into a yeast two-hybrid bait vector by recombination-based cloning. Each bait was used to screen a yeast two-hybrid C. elegans cDNA library. The screens identified approximately fifty novel interacting proteins. These were subjected to in vivo analysis using RNA interference (RNAi). Preliminary RNAi experiments have demonstrated that several of the novel interactors play a role in dauer formation.

Muneesh Tewari et al. (2003) International Worm Meeting "Combined functional genomic and genetic analysis of the C. elegans dauer decision."

Patrick Hu, Marc Vidal, Muneesh Tewari, Gary Ruvkun, Sabrina Chaklos, Svetlana Busiguina, Maurice Butler, Jin Sook Ahn

[International Worm Meeting, 2003]

Integrated functional genomic and proteomic maps have the potential to significantly improve our understanding of biological processes by identifying large numbers of new genes potentially involved and indicating possible functional links between them. However, it has remained unclear how the information emerging from such approaches can be combined with classical genetic analysis in high-throughput settings to provide functional directionality to the resulting networks of interaction. We have addressed this challenge in the context of the C. elegans dauer developmental decision. First, a network of potential interactions was generated using a high-throughput yeast two-hybrid system starting from most known dauer gene products as baits and subsequently, new dauer genes were identified among the potential interactors by RNAi analysis. To provide functional directionality to this integrated map, we are pursuing a systematic genetic interaction analysis that consists of testing double gene knock-downs in all pairwise combinations. This strategy has already revealed many new genes that impact the dauer decision, as well as identified new genetic interactions between these and known members of the pathway. Specific examples will be presented.