During the juvenile-to-adult transition at the L4 stage, the tail tip of C. elegans males changes shape from long and pointed to short and round. The timing of this tail tip morphogenesis (TTM) is under the control of the heterochronic pathway, best known for scheduling seam cell development. Previous work showed that let-7 and lin-41 play a role in the timing of TTM: in loss-of-function mutants of let-7 and gain-of-function mutants of lin-41, TTM is delayed into adulthood. We identified two new genes involved in the timing of TTM, lep-2 and lep-5 (H36L18.2). Males mutant in either gene have fully un-retracted tail tips that undergo TTM as adults. Also, mutant adults undergo an additional molt and mutant lep-2 males show defective mating behavior, suggesting that the gene products of lep-2 and lep-5 act not only in the tail tip but also in the body epidermis and nervous system. Unexpectedly, neither mutant has defects in the development of the lateral seam. lep-2 encodes a Makorin, a member of a family of conserved proteins with putative nucleic acid binding and E3 ubiquitin ligase activities. lep-5 is a long noncoding RNA (lncRNA), highly conserved within Caenorhabditis but not identifiable outside of this genus. SL1-spliced lep-5 lncRNA is predicted to adopt a complex secondary structure with multiple stem-loops, at least two of which are necessary for its function.
By testing genetic interactions, we determined that lep-2 and lep-5 repress lin-28. It is known that lin-28 is downregulated transcriptionally (by lin-14) and post-transcriptionally (by daf-12, lin-4, lin-66, and sea-2) after the L2 stage to ensure that mature let-7 microRNA is present during L3. We found that both LEP-2 and lep-5 lncRNA also down-regulate LIN-28 protein levels post-transcriptionally without affecting lin-28 mRNA levels. Using a photo-convertible LIN-28::Dendra2 fusion, we determined that LEP-2 and lep-5 lncRNA promote degradation of LIN-28 protein. This is consistent with the predicted E3 ubiquitin ligase activity of LEP-2. The mechanism for the activity of lep-5 lncRNA is still unknown. Interestingly, the human homolog of LEP-2—Makorin3—as well as human LIN28b, are involved in the regulation of pubertal timing. Together with our findings in C. elegans, this suggests a conserved role of Makorins and LIN-28 in timing of the juvenile-to-adult transition.
The fact that seam cell development is unaffected by mutations in lep-2 and lep-5 suggest that developmental timing is regulated in a tissue-specific manner. Therefore, the male tail tip is a valuable model for studying the heterochronic pathway in all its complexity.