The heterochronic genes act in a temporal cascade to permit the normal succession and synchrony of developmental events through the larval stages. Two of these genes,
lin-14 and
lin-28, act early in development to cause L1- and L2-specific events to occur. Both must be repressed to allow subsequent stage-specific events to occur in the L3 and later.
lin-4, which encodes a small RNA, is needed for the repression of both of these genes. When
lin-4 is absent, both
lin-14 and
lin-28 expression remain high in late stages, causing a reiteration of early larval fates and retarded development. However, if either
lin-14 or
lin-28 is removed by mutation in the absence of
lin-4, the other is gene is repressed. We have referred to this as the mutual positive feedback loop between
lin-14 and
lin-28. The existence of this feedback loop indicates that repression of
lin-14 and
lin-28 can take place independently of the
lin-4 RNA. We have characterized the
lin-4-independent repression of
lin-28. The repression of
lin-28 by the
lin-4-dependent and
lin-4-independent mechanisms appears to occur in the same time frame. LIN-28 decreases after the L1 in wildtype and in a
lin-4;
lin-14 double mutant, but not in a
lin-4 mutant. Repression of
lin-28 fails to occur in a
lin-14(gf) mutant that is insensitive to
lin-4 due to a rearrangement of its 3 UTR this is true even in with a functional
lin-4 gene. Therefore, the
lin-4 RNA is neither necessary nor sufficient to cause the temporal repression of
lin-28. We have determined that the
lin-4-independent repression of
lin-28 is mediated through the genes 3 UTR through a site that is distinct from the
lin-4-complementary element. We have also determined that the
lin-28 mRNA is polysome-associated in the repressed state, as is the case for the
lin-4-dependent repression, suggesting the two mechanisms of repression are similar, if not identical. It may be that
lin-4 exerts its effect by acting simultaneously on
lin-14 and
lin-28 to enhance the feedback loop repression mechanism. Using a new genetic screen we are seeking mutations in trans-acting factors that are needed for the repression of
lin-28.