The heterochronic gene
lin-14 controls stage-specific cell lineages during development by forming a temporal gradient of the LIN-14 protein. Down-regulation 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. With the Ambros lab. we proposed that the
lin-4 RNAs base-pair with the
lin-14 3'UTR to cause down-regulation 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
lin-14 3'UTR and assayed the temporal regulation of lacZ or GFP expression from reporter genes bearing this mutant
lin-14 3'UTR. In a wild type background the transgenic worms with the triple point mutations in
lin-14 3'UTR show less down regulation than those with wild type
lin-14 3'UTR from L1 stage to L4 stage by microscopic observations. But residual down-regulation still remains. Thus the
lin-4 binding sites are necessary for full down regulation but other factors may also be involved. This observation is consistent with the partial temporal regulation of LIN-14 previously noted in a
lin-4 null mutant. Chemically synthesized
lin-4 RNA binds in vitro to a truncated
lin-14 3'UTR bearing 3
lin-4 binding sites, and a point mutation in each
lin-4 complementary region of the
lin-14 3'UTR disrupts this binding. However reporter genes bearing a truncated
lin-14 3'UTR with 3 wt or 3 mutant
lin-4 binding sites show the same level of down regulation in wild type. This result suggests that there might be some factor(s) that stabilize the
lin-14/lin-4 duplex in vivo. To find the factor(s) which detect the
lin-14/lin-4 duplex to down-regulate translation of mRNAs bearing the
lin-14 3'UTR we fused it to the easily screenable genetic marker
let-23. LET-23 transduces the LIN-3 signal for vulval development during the L3 stage. We expect that post-L1 expression of
lin-4 will down-regulate the expression of LET-23 before the L3 stage when it must function in a strain where the only source of LET-23 is the
let-23/lin-14 3'UTR transgene. Indeed the transgenic
let-23(
sy97) worms with
let-23/lin-14 3'UTR are Vul but a
let-23 transgene with its own 3'UTR completely rescues the Vul phenotype in
let-23 (
sy97) at the same DNA concentration. We are currently doing a screen for the mutations which suppress the Vul phenotype of
let-23(
sy97);
let-23/lin-14 3' UTR.