The heterochronic gene pathway of C. elegans ensures that developmental programs are executed in the correct sequence during larval development. Many of the genes identified as members of this pathway are conserved, including
lin-42, the C. elegans homolog of the circadian clock gene period. Strikingly, like period,
lin-42 mRNA and protein levels cycle. However,
lin-42 levels cycle with larval stages rather than the 24-hr clock.
lin-42 mutants have a precocious phenotype where adult specific fates occur too early. Also,
lin-42 regulates the molting cycle; null and strong
lin-42(lf) mutants undergo a prolonged lethargus, molt asynchronously, and are defective in ecdysis. These defects cause developmental delays and often result in larval arrest. Genetic analyses reveal that
lin-42 acts in parallel with
lin-28 and opposition to
daf-12, two genes that regulate the expression of
let-7-family miRNAs. We show by Taqman qRT-PCR that
lin-42 also regulates the levels of heterochronic miRNAs, including Let-7 and miR-48. In
lin-42 mutants, these miRNAs accumulate earlier than in wild-type animals. miRNAs are generated through multiple processing events, and there are several steps at which their levels can be regulated. LIN-42 is nuclear, and PERIOD functions as a negative transcriptional regulator; suggesting that
lin-42 could regulate (pri)mary-miRNA transcription. To determine if
lin-42(lf) affects pri-miRNA expression, we measured pri-miRNAs during larval development in wild-type and
lin-42(lf) mutants. We show that primary transcript levels of heterochronic miRNAs oscillate over larval development, as previously reported for pri-
let-7. pri-
let-7, pri-
mir-48, pri-
mir-241, pri-
mir-84, and pri-
lin-4 levels all cycle in wild-type animals. Surprisingly, although LIN-42 levels oscillate,
lin-42 is not required for pri-miRNA cycling. Notably, however, the amounts of several of these transcripts are increased in each stage in
lin-42 mutants; supporting the idea that
lin-42 may act to negatively regulate transcription. In mammals, many miRNAs are subject to regulation by the circadian clock. Investigation of C. elegans
lin-42 will provide further insight into mechanisms of temporal regulation of miRNA expression.