Multi-dimensional regulation of LIN-28 temporal expression dynamics in the <i>C. elegans</i> heterochronic gene cascade

ABSTRACT LIN-28 is an evolutionarily conserved RNA-binding protein that is crucial for regulating pluripotency and cell fate determination during animal development. In Caenorhabditis elegans, lin-28 is an integral component of the heterochronic (developmental timing) gene regulatory cascade. Loss-of-function mutations in lin-28 cause precocious cell fate determination during larval development. Previous studies indicate that proper progression of larval stage-specific cell fates relies on the downregulation of LIN-28, which is negatively regulated by the lin-4 microRNA through complementary sequences in the lin-28 3′ untranslated region (UTR). This study employs CRISPR/Cas9 editing of the endogenous lin-28 locus to demonstrate that developmental downregulation of LIN-28 involves multiple inputs, including the action of the let-7 family and lin-4 microRNAs via adjacent complementary sites in the lin-28 3′ UTR, along with post-translational inhibition of LIN-28 by the lep-5 long non-coding RNA, collectively accounting for nearly all LIN-28 repression. Additionally, systematic testing of truncations of the lin-28 3′ UTR identifies three positive regulatory regions that enhance LIN-28 expression, counteracting the negative effects of the let-7 and lin-4 microRNAs and the lep-5 long non-coding RNA.