The heterochronic pathway controls the appropriate timing of cell fate decisions and has been demonstrated to affect, among other tissues, lateral hypodermal-seam cell lineages by altering the timing of cell division and differentiation. In particular, three miRNA genes
mir-48, -84 and -241 (3-mirs), belonging to
let-7 family negatively regulate
hbl-1 to control the timing of L2 cell fate decisions. However, additional factors influence this regulation. Specifically,
lin-28 and
lin-46 act oppositely to control cell fates via
hbl-1. We found that
lin-28 positively regulates
hbl-1 through its 3''UTR. Our genetic analysis reveals that a negative factor other than 3-mirs acts on
hbl-1 and that
lin-28 opposes that factor in some way. In trying to identify this factor, we examined
ain-1 (ALG-1 interacting protein), which resembles GW182, a human protein involved in the miRNA pathway. An
ain-1(0) mutant highly enhances the retarded phenotype of 3-mirs(0) animals. However,
lin-28(0) precocious phenotype is epistatic to the retarded phenotype of 3-mirs(0);
ain-1(0) animals. The missing negative regulator may still be another miRNA gene. Candidates include
lin-4,
let-7, and their less well-characterized relatives (
mir-237,
mir-793 795, respectively) and the Pumillio-related gene
puf-9. Importantly, we have found that LIN-28 protein binds selectively to miRNA precursors, as was found for its human homologue, suggesting
lin-28 may inhibit miRNAs directly. Another player is
lin-46 which encodes a putative scaffolding protein. LIN-46 protein accumulates in briefly at each stage, and can form cytoplasmic foci resembling P-bodies.
lin-46 is known to enhance 3-mirs(0), and
lin-28(0) is not epistatic to that quadruple mutant phenotype. We observed that
lin-46(0) greatly enhances the retarded phenotype of
ain-1(0), and tentatively conclude that the two genes act in parallel pathways.
hbl-1(RNAi) is epistatic to
lin-46;
ain-1 double mutants and moreover, a
hbl-1::gfp::
hbl-1 reporter expression in
lin-46;
ain-1 mutants is similar to its expression in 3-mirs(0) animals. These observations suggest that
lin-46 acts upstream of
hbl-1, but whether its mechanism involves miRNAs is not yet clear.