C. elegans seam cells are lateral epidermal cells that divide with a stem cell-like lineage during larval development. It is known that the development timing of seam cells is strictly regulated by the heterochronic pathway. LIN-28, an RNA-binding protein and the
let-7 microRNA (miRNA) play pivotal roles in controlling the timing of seam cell development. However, the mechanisms of action and the downstream affecters of
lin-28/let-7 are poorly understood. Thus, the purpose of this project is to identify novel target genes in the
lin-28/let-7 pathway in C. elegans. In our lab, we reported the use of CLIP-Seq to identify 2000 mRNAs interacting directly with the LIN-28 RNA binding protein. To identify functional LIN-28 targets, we first looked for the overlap among our set of LIN-28 CLIP hits, the set of 201 known
let-7 suppressors and 213
let-7 enhancers, and our unpublished RNA-seq data from staged L2
lin-28 mutant animals. We found 16 candidate genes at the intersection of these groups. We validated these 16 candidate genes via qRT-PCR experiments in WT,
lin-28 mutant and
lin-28(gf) animals. From these experiments, we identified 8 candidate genes whose mRNA levels are dependent on
lin-28 levels. Of these genes, we found PPK-1, the Caenorhabditis elegans homolog phosphatidylinositol-4-phosphate 5' kinase (PIP5K), functions in the
lin-28/let-7 pathway. As we expected
lin-28 mutants showed decreased
ppk-1 mRNA levels.
ppk-1(RNAi) worms showed weak precocious phenotypes and increased mature
let-7 expression. In vitro, we found that PPK-1 has a physical interaction with nuclear export protein XPO-1, which is already known as a heterochronic gene that regulates mature
let-7 expression. In vivo, we also found that PPK-1 and XPO-1 co-localize in seam cells. Taken together, this indicates that PPK-1 interacts with XPO-1 to regulate mature
let-7 expression. As
lin-28/let-7 also acts in human cancer cells, we are exploring the functions of PPK-1/XPO-1 homologues in human cancer.