The recently completed Caenorhabditis elegans genome sequence allows application of high-throughput (HT) approaches for phenotypic analyses using RNA interference (RNAi). As large phenotypic data sets become available, “phenoclustering” strategies can be used to begin understanding the complex molecular networks involved in development and other biological processes. The current HT-RNAi resources represent a great asset for phenotypic profiling but are limited by lack of flexibility. For instance, existing resources do not take advantage of the latest improvements in RNAi technology, such as inducible hairpin RNAi. Here we show that a C. elegans ORFeome resource, generated with the Gateway cloning system, can be used as a starting point to generate alternative HT-RNAi resources with enhanced flexibility. The versatility inherent to the Gateway system suggests that additional HT-RNAi libraries can now be readily generated to perform gene knockdowns under various conditions, increasing the possibilities for phenome mapping in C. elegans.

The easiness of maintaining and propagating the nematode C. elegans make it a nice model organism to work with. The...

Background: Theretinoblastomatumor suppressor (Rb) acts in a conserved pathway that is deregulated in most human cancers. Inactivation of the single Rb-related gene inCaenorhabditis elegans, lin-35, has only limited effects on viability and fertility, yet causes changes in cell-fate and cell-cycle regulation when combined with inactivation of specific other genes. For instance,lin-35Rb is a synthetic multivulva (synMuv) class B gene, which causes a multivulva phenotype when inactivated simultaneously with a class A or C synMuv gene.Results: We used the ORFeome RNAi library to identify genes that interact withC. elegans lin-35Rb and identified 57 genes that showed synthetic or enhanced RNAi phenotypes inlin-35mutants as compared torrf-3anderi-1RNAi hypersensitive mutants. Based on characterizations of a deletion allele, the syntheticlin-35interactorzfp-2was found to suppress RNAi and to cooperate withlin-35Rb in somatic gonad development. Interestingly, ten splicing-related genes were found to function similar tolin-35Rb, as synMuv B genes that prevent inappropriate vulval induction. Partial inactivation of specific spliceosome components revealed further similarities withlin-35Rb functions in cell-cycle control, transgene expression and restricted expression of germline granules.Conclusion: We identified an extensive series of candidatelin-35Rb interacting genes and validatedzfp-2as a novellin-35synthetic lethal gene. In addition, we observed a novel role for a subset of splicing components inlin-35Rb-controlled processes. Our data support novel hypotheses about possibilities for anti-cancer therapies and multilevel regulation of gene expression.

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