The heterochronic gene
lin-14 controls stage-specific cell lineages during development by forming a temporal gradient of the L1N-14 protein. Downregulation of LIN-14 occurs post-transcriptionally via elements in the 3'UTR of the
lin-14 mRNA, seven of which are complementary to the
lin-4 RNAs. We propose that the
lin-4 RNAs base pair with the
lin-14 3'UTR to cause downregulation of translation of the
lin-14 mRNA. To test this model, we have introduced triple point mutations (UCA to AGU) in the duplex regions of the seven elements of the
lin-14 3'UTR and assayed the temporal regulation from a lacZ reporter gene. In a wild type background, the transgenic worms show almost no downregulation. This suggests that
lin-4/lin-14 RNA duplex formation is essential for the downregulation. Many loops or bulges in RNA structures have been shown to be important for interacting with proteins. Out of the seven
lin-4 binding sites in the
lin-14 3'UTR, four
lin-14lin-4 RNA duplexes make a bulged C structure in
lin-4 RNA. We multimerized one of the bulged
lin-4 binding motif (6 copies) of
lin-14 3' UTR and put it into a transgenic construct bearing the
unc54 3'UTR. The transgene with 6 copies of the bulged lin- 14/lin-4 duplex show downregulation. However when the six bulged Cs were debulged by introducing extra Gs in the bulged
lin-4 binding sites the trangene shows much less downregulation. Chemically synthesized
lin-4 RNA binds in vitro to debulged
lin-14 RNA much stronger than to the bulged
lin-14 RNA. These results suggest that there might be some factor(s) that stabilize the lin-14lin4 duplex and recognize the bulged C of
lin-14/lin-4 duplex in vivo To identify the factor(s) we fused the
lin-14 3'UTR to the easily screenable genetic marker
let-23. LET-23 transduces the LIN-3 signal for vulval development during the L3 stage. Iet-23 mutants are Vul and we expect that post-L1 expression of
lin4 will downregulate the expression of LET-23 before the L3 stage. This construct is put into a strain where the only source of LET-23 is the
let-23/lin-14 3'UTR transgene. Therefore the transgenic animals will be still Vul. We will use this transgene for screening for mutants disrupting downregulation of LET-23 from
let-23/1in-14 3'UTR, which will show a nonVul phenotype.