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. We propose that the
lin-4 RNAs base-pair with the
lin-14 3' UTR to cause down-regulation of translation of the
lin-14 mRNA. RNA duplex formation was demonstrated in vitro and correlated with in vivo temporal gradient forming activity. Mutations in the proposed RNA duplex region of the
lin-14 mRNA that disrupt in vitro RNA duplex formation also disrupt temporal gradient formation in vivo. These cis mutations in the
lin-14 mRNA mimic the effects of a null mutation in the regulatory
lin-4 RNA. These data show that
lin-4/lin-14 RNA duplex formation is required for the generation of the LIN-14 temporal gradient. Many loops or bulges in RNA structures have been shown to be important for interacting with proteins. Four of the seven the naturally occurring
lin-4/lin-14 RNA duplexes bulge a C residue based on model building from sequence alignments, whereas three do not. Reporter genes bearing multimerized (6 copies) bulged C
lin-4 binding sites down-regulate in a wild type background but not in a
lin-4 mutant background. However similar genes bearing multimerized non-bulged
lin-4 binding sites show almost no down-regulation in wild type or
lin-4 mutant background. Interestingly
lin-4 RNA binds strongly in vitro to non-bulged
lin-14 RNA binding sites but not to the bulged C
lin-14 RNA binding sites. These results suggest that a protein factor(s) may recognize the bulged C of
lin-14/lin-4 duplex in vivo and stabilize the
lin-14/lin-4 duplex. The bulged nucleotide may form a critical feature for the assembly of a complex that ultimately regulates translation of the
lin-14 mRNA. To identify such factors genetically, we have fused the
lin-14 3' UTR to an easily screenable genetic marker and are screening for mutants which suppress the phenotype caused by
lin-14/lin-4 mediated down-regulation of the marker gene. *I. Ha is a recipient of the postdoctoral fellowship from Cancer Research Fund of the Damon Runyon-Walter Winchell Foundation (DRG-1287).