A BULGED LIN-4 LIN-14 RNA DUPLEX IS SUFFICIENT FOR CAENORHABDITIS-ELEGANS LIN-14 TEMPORAL GRADIENT FORMATION/

The Caenorhabditis elegans heterochronic gene lin-14 generates a tempo ral gradient of the LIN-14 proteins to control stage-specific patterns of cell lineage during development. Down-regulation of LIN-14 is medi ated by the lin-14 3' untranslated region (UTR), which bears seven sit es that are complementary to the regulatory lin-4 RNA. Here we report molecular and genetic evidence that RNA duplexes between the lin-4 and lin-14 RNAs form in vivo and are necessary for LIN-14 temporal gradie nt generation, lin-4 RNA binds in vitro to a lin-14 mRNA bearing the s even lin-4 complementary sites but not to a lin-14 mRNA bearing point mutations in these sites. In vivo, the lin-4 complementary regions are necessary for lin-14 3' UTR-mediated temporal gradient formation. Bas ed on lin-14 3' UTR sequence comparisons between C. elegans and C. bri ggsae, four of the seven lin-4/lin-14 RNA duplexes are predicted to bu lge a lin-4 C residue, and three sites are predicted to form nonbulged RNA duplexes. Reporter genes bearing multimerized bulged C lin-4 bind ing sites show almost wild-type temporal gradient formation, whereas t hose bearing multimerized nonbulged lin-4 binding sites do not form a temporal gradient. Paradoxically, lin-4 RNA binds in vitro to nonbulge d lin-14 RNA more avidly than to the bulged lin-14 RNA. This suggests that a specific secondary structure of lin-4/lin-14 RNA duplex that ma y be recognized by an accessory protein, rather than an RNA duplex per se, is required in vivo for the generation of the LIN-14 temporal gra dient.