LIN-67 functionally interacts with heterochronic miRNAs and regulates developmental timing in Caenorhabditis elegans

Temporal regulation of gene expression is required for developmental transitions, including differentiation, proliferation, and morphogenesis. In the nematode Caenorhabditis elegans, heterochronic microRNAs (miRNAs) regulate the temporal expression of genes that promote animal development. The heterochronic miRNAs lin-4 and let-7 are required during different stages of larval development and are associated with the miRNA-specific Argonaute ALG-1. In this study, we have identified lin-67 as a heterochronic gene that negatively regulates lin-4, let-7, and alg-1. Loss of lin-67 function restores proper developmental timing and stage-specific gene expression to hypomorphic lin-4 and let-7 mutants. We found that loss of lin-67 resulted in a reduced number of seam cells, defects in alae formation, precocious expression of an adult-specific gene reporter, and sterility. LIN-67 contains a K homology (KH) RNA-binding domain and is a homolog of the Sam68-like splicing factor KHDRBS2. We show that LIN-67 localizes to the nucleus throughout animal development and is enriched in nuclear foci. Mutating the KH domain of LIN-67 abolished the nuclear localization of LIN-67, suggesting that the localization of LIN-67 is likely dependent on RNA-binding activity. We show that LIN-67 negatively regulates lin-4 miRNA levels and restores normal levels of let-7 to alg-1 mutants, which can, at least in part, explain how lin-67 suppresses alg-1. Our data indicate that lin-67 is a novel heterochronic gene that regulates developmental timing and miRNA-dependent gene regulation in C. elegans.