The heterochronic genes of C. elegans control development time during post-embryogenesis. In hypodermal seam cells, they program stage-specific temporal identities, ultimately triggering expression of adult fates during the final (L4) molt. Mutations in heterochronic genes advance or retard the timing of the adult seam cell program. Included among these genes are the first two miRNA encoding genes discovered,
lin-4 and
let-7. Extensive analysis of miRNAs in C. elegans has identified three temporally regulated
let-7 homologues, raising the possibility that some or all of them could function redundantly with
let-7 to control development time. Two of the homologues,
mir-48 and
mir-241, are situated in tandem in the interval where the heterochronic gene
lin-58 maps. The
lin-58 alleles
ve12 and
ve33 cause seam cell terminal differentiation to occur one stage early, at L3 molt. Sequence analysis revealed that each
lin-58 allele contains an independent point mutation in a GC-rich sequence at ~210 bases upstream of
mir-48, suggesting they are regulatory mutations. One possibility is that they disrupt a repressor binding site such that one or both miRNAs are expressed prematurely, resulting in precocious inactivation of target genes. Developmental northern analysis revealed that
mir-48, but not
mir-241, is expressed prematurely in
lin-58 mutants relative to wild-type. This suggests
lin-58 lesions define a
mir-48 temporal control element. To further investigate how
lin-58 mutations affect gene expression, we constructed
mir-48::gfp reporter fusions. We found that a ~1.0 kb fragment immediately upstream of
mir-48 drives GFP expression in the seam cells, vulva and nervous system. Introduction of the
ve33 lesion results in precocious and enhanced GFP expression. The same result was obtained when the GC-rich element was replaced with AT-rich sequence. These results indicate that the GC element controls temporal expression of
mir-48, and that
lin-58 mutations disrupt this element, shifting
mir-48 expression earlier. Overexpression of
mir-48 in transgenic animals causes seam cells to fuse and synthesize adult alae precociously at the L3 molt, or occasionally at the L2 molt. VPCs divide precociously during the L2 stage, resulting in vulva defects. Also, the proliferative S2 division executed by a subset of seam cells is often skipped, a phenotype observed in animals which are loss-of-function for either of two other heterochronic genes,
lin-28 and
hbl-1. Potential
mir-48 binding sites have been identified in the 3UTRs of
lin-28 and
hbl-1, suggesting that they are possible
mir-48 targets.