Long noncoding RNAs (lncRNAs) were initially defined by a few prominent examples. Application of high-throughput genomic methods has revealed a dramatically expanded inventory of candidate lncRNAs, produced in the pervasive transcription of animal genomes. Some lncRNAs are implicated in cancer, and many others map to regions of the genome suggestive of a link to cancer. Learning how lncRNAs function thus fits with the overall mission of the NIH. The large number of predicted lncRNAs (over 9000 in humans) sets up a series of challenges for fully appreciating their functions. An outstanding model for analysis of lncRNA function is provided by the Drosophila oskar (osk) gene. Although osk RNA encodes a protein, it also has a noncoding function. The coding and noncoding functions are clearly distinct, acting at different stages of oogenesis and embryogenesis, and contributing to different processes. The lncRNA function of osk is essential for progression through oogenesis, and absence of osk RNA results in loss of certain proteins from nuage, a perinuclear structure found in germ line cells of all animals. Nuage is the site where piRNAs are produced. piRNAs act in restricting the activity of transposons, thus protecting the germ line DNA from damage. Notably, loss of osk lncRNA activity leads to a dramatic reduction in piRNA levels, and a corresponding increase in levels of transposon mRNAs. The goal of this proposal is to understand how osk RNA acts in nuage organization and piRNA production, features that are conserved among all animals including humans.
One Aim i s to complete the genetic characterization of osk mutants and of genes encoding proteins that bind to the key elements in osk RNA acting in its lncRNA function.
One Aim i s to define how nuage is affected by loss of osk RNA, examining individual components to determine if specific subsets of nuage proteins are primarily affected, and evaluating the overall organization of nuage by ultrastructural studies. A part of this Aim is to explore and test possible explanations for the requirement that osk RNA contains signals for transport out of the nurse cells (the cells with nuage) and into the oocyte. Another Aim centers on the proteins that bind to osk lncRNA functional elements. This builds on our work in defining these elements, and on identifying the factors that bind to these elements. The goal is to obtain a more complete understanding of all proteins that bind to the functional elements, to learn about the complex assembled from these proteins and the osk RNA, and thus to understand the underlying mechanisms.
The final Aim i s to search for mammalian equivalents of the critical sequence element that mediates osk lncRNA activity. Candidates will be tested in mice to determine if this feature plays a conserved role across all animals, as might be expected given the conservation of nuage, piRNAs, and many nuage components involved in piRNA production. Collectively, these studies have a high probability of revealing mechanistic details of nuage function that have direct relevance to germ line cells in humans.
Long noncoding RNAs are a large and diverse group of molecules that often act to regulate gene expression, although the underlying mechanisms are largely unknown. This proposal aims to exploit a well-developed experimental system to study the action of one such RNA organization of nuage and production of piRNAs, two processes shared by all animals.
