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

LIN-28 is an evolutionarily conserved RNA-binding protein that plays critical roles in 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 result in precocious cell fate determination during larval development. Previous studies showed that the proper progression of stage specific cell fates during larval development depends on the progressive downregulation of LIN-28, which is negatively regulated by the lin-4 microRNA through complementary sequences located in the lin-28 3 UTR. In this study, we employ CRISPR/Cas9 editing of the endogenous lin-28 locus to demonstrate that the robust developmental downregulation of LIN-28 involves contributions from multiple inputs. These include a convergent action of the let-7 family and lin-4 microRNAs via adjacent complementary sites in the lin-28 3 UTR, in conjunction with the previously described post-translational inhibition of LIN-28 by the lep-5 long non-coding RNA, which all together account for virtually the entirety of LIN-28 repression. Furthermore, through the systematic testing of a series of truncations of the lin-28 3 UTR, we identify three positive regulatory regions that enhance LIN-28 expression, thereby counterbalancing the negative effects of the let-7 and lin-4 microRNAs and the lep-5 long non-coding RNA.