Figure 5.

lin-66 regulates LIN-28 expression through the 3′UTR region of lin-28. (A) Schematic illustration of the pcol-10∷lacZ∷lin-28 3′UTR construct. (B–D) Photos showing LacZ staining of animals expressing the reporter construct (pKM50). The expression of the reporter construct is observable in the lin-66 (ku423) adult animal (D) but not in WT (B). The reporter construct is robustly expressed in WT L1 larvae (C). Bar, ∼50 μm. (E) Schematic illustration of the 3′UTR region of lin-28. Gray filled boxes indicate areas conserved between C. elegans and C. brigssae. The let-7- and lin-4-binding sites are indicated. Arrows indicate substitution or small deletion mutations made in the area, whereas bars indicate deletion mutations. The numbers indicate the plasmid shown in (F). The data for those arrows and bars without numbers are not shown, as these mutations either did not change the expression of the reporter or did not significantly change the response of the expression to lin-66, daf-12 or miRNA regulations. pKM50 is the intact 3′UTR of lin-28. pKM51 and pKM52 have a three nucleotide deletion in the let-7 (ctc) and lin-4-binding site (ggg), respectively. pKM55 has deletions of both the let-7- and lin-4-binding sites. pKM63 has a four nucleotide substitution (caaa to accc) in the indicated conserved region. (F) Percent of animals that displayed the lacZ expression of indicated construct in at least some of the hypodermal cells in various mutant backgrounds in the L4 and adult stage. Number of animals counted is indicated. Nearly 100% lacZ expression was observed in L1 animals carrying each construct and in each mutant background (data not shown).