Intellectual merit: MicroRNAs are small regulatory molecules encoded in plant and animal genomes that control genes as organisms develop to maturity. By investigating microRNAs one will gain a deeper understanding of how development proceeds from genetic information - an understanding that is relevant to growth and health of diverse animals, including humans, livestock, and wild species. The larval development of the nematode Caenorhabditis elegans is an exceptional model system for the study of microRNA function. At least five microRNAs and twelve proteins act together to control a succession of developmental events in the C. elegans larva. LIN-28 is one of these proteins and its molecular mechanism is not known. Many diverse animals possess a gene for LIN-28 which is believed to have many important developmental roles. A combination of molecular and genetic approaches will be employed to answer these specific questions: (1) Does C. elegans LIN-28 bind to and inhibit specific microRNA precursors? (2) Does LIN-28 act via these specific microRNAs to regulate development? (3) Are regulatory feedback circuits involving microRNAs part of a larval development control mechanism? The outcome of this work will be a more complete understanding of the molecular mechanisms of these regulators, which will be relevant to other investigators in medical, veterinary, and ecological sciences.
Broader impacts: The research will be performed by high school students, undergraduates, and doctoral candidates. Undergraduate students will be recruited from around the Philadelphia/Camden region to participate the research. High school students will spend afternoons working on independent aspects of the project in preparation for science fairs. The PI is committed to reaching underserved and underprivileged individuals for these opportunities. Doctoral candidates will present their findings at international scientific conferences. All strains and reagents generated will be shared freely with other investigators.
Intellectual merit: This study addressed the molecular mechanisms that govern developmental timing, that is, the way in which cells choose stage-appropriate behaviors during development. From stem cells to differentiation, cells undergo stepwise transformations that are governed in part by the internal states of their genes. Genetic analysis using the nematode has shown that those states are controlled by a dozen or more genes, called heterochronic genes. Several of these genes have homologs that exit in humans and other animals, and therefore are of general interest for developmental timing in a variety of contexts. This project showed that LIN-28, a heterochronic factor found in many diverse animals, has two separate and distinct mechanisms by which it controls timing. The project proved that one mechanism, which involves inhibiting the maturation of the microRNA let-7, accounts for only a fraction of its total function. We showed that LIN-28 protein binds to precursors of let-7 and three of its relatives, miR-48, miR-84, and miR-241, but that it blocks the maturation of only let-7. Although LIN-28's ability to block let-7 maturation appears universal, we demonstrated using animals mutant in let-7 and its three relatives that LIN-28's primary biological activity in timing events of the animals second larval stage is independent of all of these. This was surprising because it is unusual for a factor to have more than one distinct molecular mechanism by which it controls gene expression, and because it was widely assumed that let-7 explained LIN-28’s function in many contexts. Through this project we have learned that LIN-28 is able to control gene expression via a let-7-independent activity that to date remains unclear. However, through this work we determined that this let-7 independent activity works in part through the gene hbl-1 and has the hallmarks of microRNA regulation. For example, LIN-28 appears to oppose a negative regulation that works through hbl-1's 3' untranslated region. In unpublished work, we identified another microRNA, lin-4, that likely has a major role in the let-7-independent mechanism. Furthermore, we have developed a novel assay to directly measure microRNA activity independent of all other gene regulation effects. In new studies, we have used this assay and, indeed, it appears that LIN-28 directly inhibits lin-4 activity without affecting its abundance. Our theory about how the timing mechanism works is advanced by understanding that LIN-28 has two separate and sequential molecular activities. In this way it resembles another timing factor, LIN-14. We have proposed a model whereby the overlapping of pairs of timing activities give rise to the sequential ratcheting of a clockwork mechanism for developmental timing. Broader impacts: This project advanced our long term goal to explain the key regulatory events that control developmental timing in C. elegans, and to extend that understanding to other animals. Developmental timing is a fundamental issue in tissue formation, growth, and regeneration, and in the evolution of morphology and allometric variation within species. This work identified and explained key regulatory events in a potentially widespread developmental timing mechanism. The project was central to the training of six undergraduate students, a masters student, and three doctoral students. The undergraduates were from a variety of colleges and universities who took part in an organized 10-week summer program. One high school student spent 3 weeks in the lab learning to work with recombinant DNA. In addition, the PI gave a presentation to a community members who visited the campus as part of a presentation series open to the public.
