mab-2 (male abnormal) was originally identified in an EMS screen for males defective in mating ability. The phenotype is characterized by a deformed male tail which has one or more missing rays and defective V and T cell lineages. The mutation has been mapped using visible markers to the
dpy-5 unc-13 region of LGI but subsequent cosmid rescue attempts were unsuccessful. We therefore decided to attempt positional cloning by a reverse genetic approach using RNAi. We used a feeding RNAi library (from J.Ahringer) in order to quickly screen genes from the region for phenocopy of the
mab-2 mutant in a
him-8 background. A putative gene, F55F8.3 was found to give a
mab-2-like phenotype in >90% of male F1 progeny and is therefore a good candidate for the gene mutated in
mab-2 worms. This gene had been previously screened for function in hermaphrodites using the same RNAi library and been found to produce a larval arrest (Lva) phenotype and indeed we observed an approximately 2-fold slower rate of growth compared to wildtype. The feeding result has been confirmed for F55F8.3 using injection of dsRNA. However, the later progeny of these injected hermaphrodites showed an embryonic lethal (Emb) phenotype in addition to the Lva and Mab phenotypes of the earlier progeny indicating a dosage effect: it may be that a high level of RNAi causes arrest at the embryonic stage whereas lower levels cause a delay in larval development and male tail abnormalities. This data is consistent with the fact that sequencing of F55F8.3 from
mab-2 worms showed no mutation in the coding sequence implicating either the promoter region, or other sites involved in regulation of expression, as likely sites of the
mab-2 mutation and that these may be hypomorphic alleles of an essential gene. Complementation tests are therefore being conducted to see if any of the many lethal (Let) genes mapped to this region are indeed allelic with
mab-2. The other interpretation of these observations is that while giving a male tail phenotype when subjected to RNAi and mapping to exactly the same region as
mab-2, F55F8.3 is not the gene defined by the
mab-2 (
e1241) mutation. Analysis of the genomic context of F55F8.3 showed that it is clustered with five other genes each with short intergenic regions and a putative 5' trans splice site just upstream of the presumed translational start site indicating that this region may be operonic. This raises the possibility that the RNAi is targeting the polycistronic pre-mRNA for degradation. Consistent with this, two other genes in this cluster also gave an Lva phenotype and one an Emb phenotype. However, if there was global degradation of pre-mRNA then all five genes would be expected to give these phenotypes; only one of the genes gives the
mab-2 phenotype when subjected to RNAi. It may however, indicate that these genes are functionally related. In order to see if F55F8.3 is down- regulated in the
mab-2 mutants we are doing semi-quantitative RT-PCR on the putative operon genes. F55F8.3 contains WD-40 repeats, a protein-protein interaction motif, and is highly similar to the S.cerevisiae
pwp2 gene involved in growth, bud site selection and cell separation. Yeast two- hybrid screens are therefore an attractive means of revealing possible interacting proteins. It may be significant that another Lva gene in the operon also contains WD-40 repeats. Further work will also be done on characterizing the
mab-2 phenotype and the lineage defect implicated previously, using V and T cell markers.