The RAS/MAPK pathway provides the spatial cue for the proper vulval induction and morphogenesis,while the heterochronic pathway provides a temporal cue for these processes.However,how these two cues integrate to specify vulval development remains to be explored.In a genetic screen to identify additional genes regulating vulval development,we isolated several mutations that suppresses the Muv phenotype of a
lin-31(lf) allele.Three such mutations are loss of function alleles of a previously uncharacterized gene,
ain-1.
ain-1(lf) could effectively suppress Muv of
lin-31(lf) but not that of
let-60 RAS(gf) or
lin-1(lf),indicating AIN-1 is unlikely a component of the RAS/MAPK pathway.Previously,LIN-31 has been shown to have both RAS-dependent and RAS-independent functions.AIN-1 may thus be associated with a RAS-independent function of LIN-31.Further analysis showed that vulval cell division is delayed in
lin-31(lf);
ain-1(lf),suggesting the suppression is due to a temporal delay.Genetic analysis of the interaction between
ain-1 and known heterochronic genes (
lin-28 and
lin-14) demonstrated that
ain-1 acts in the heterochronic pathway to control developmental timing.We cloned
ain-1 and found that AIN-1 has homology to a candidate human disease gene.A functional translational GFP reporter revealed that AIN-1 is expressed in the cytoplasm of a various tissues including neurons and epidermis.To understand the mechanism of how AIN-1 functions, IP followed by mass spec analysis were carried out to identify its binding proteins.Both in vivo and in vitro assays indicated that AIN-1 binds to ALG-1,an argonaute protein.In addition,we found that AIN-1::GFP colocalizes with DsRed2-ALG-1.Furthermore,IP followed by Western indicated that AIN-1 also associates with DCR-1,the only Dicer homolog in C. elegans.ALG-1 and DCR-1 are core components of miRISC,which contains miRNAs.Indeed,we found that AIN-1 complex also contains miRNAs.
alg-1 and
dcr-1 have been shown to control the developmental timing in C. elegans.This reinforced our genetic data that
ain-1 functions in the heterochronic pathway.
alg-1(lf) or
dcr-1(lf) mutants displayed more severe heterchronic phenotypes than
ain-1(lf),implying that AIN-1 is involved in a subset of the functions of ALG-1 or DCR-1.We then cloned the miRNAs associated with the AIN-1 complex.Several miRNAs are enriched in the AIN-1 complex in comparison with the published general miRNA pool.This result suggested that AIN-1 only binds to a subset of miRISCs and there may be a hierachical order of miRISCs.Additional genetic and molecular experiments are underway to elucidate the cellular functions of AIN-1 and its mammalian counterpart in miRNA-mediated regulatory processes.