Understanding Complex Gene Editing Systems in Protists

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DESCRIPTION (provided by applicant): Protists have surprised molecular biologists with an unprecedented array of genetic organization, from the discoveries of self-splicing RNA and telomerase, to gene-sized pieces, programmed DNA deletion, and scrambled DNA rearrangements in ciliates. Therefore, these organisms are the natural place to focus a combined mechanistic, evolutionary, and computational study of programmed DNA rearrangements. In particular, the "scrambled genes" in ciliates offer a unique experimental system in which to study the origin and function of a complex genetic and computational mechanism. Scrambled genes exist in many other systems, ranging from protists to humans. Rarely, however, are the gene fragments actually 'sewn' back together at the level of DNA, to create a contiguous gene from all pieces. The complexity of DNA splicing events that take place in ciliates makes them one of the most interesting and challenging cases to study. Moreover, frequent DNA deletions and other extensive genome rearrangement events are increasingly associated with several cancers, underscoring the importance of studying such biological events. The recent discovery that RNA, normally thought of as a conduit in gene expression, has a novel mode of action in ciliated protozoa motivates the research in this proposal. The goal is to study genome rearrangements in the ciliate Oxytricha, pairing a nearly complete genome project with recent discoveries and tools that make Oxytricha a powerful model system. For example, RNA templates can provide both an organizing guide for DNA rearrangements and a template that can transmit spontaneous somatic mutations to the next generation. This opportunity for RNA-guided DNA repair is profound in Oxytricha, because this organism destroys 95% of its germline DNA through global rearrangements, and then sorts and reorders the remaining segments. The specific aims of this research all focus on questions about the processing of scrambled genes at the genomic, evolutionary, or developmental scale. The global aim is to use this model system to explore the complex rewriting mechanisms in microbial eukaryotic genomes. Specific aims include: 1. What are the minimal requirements for an RNA template to be able to program DNA rearrangement? 2. Understanding the epigenetic inheritance of nucleotide substitutions from an RNA template. 3. Small RNAs and their sequence distribution in Oxytricha and the related ciliate Stylonychia. 4. Identification and testing of candidate genes involved in macronuclear development in Oxytricha. 5. The genomic distribution of scrambled genes in O. trifallax, and significant updates to a database. 6. Comparative studies of the highly scrambled germline ribosomal RNA locus. PUBLIC HEALTH RELEVANCE: DNA rearrangements (deletions, inversions, and duplications) are a major factor contributing to genome instability associated with many human diseases, including cancer, with DNA translocations and gross deletions responsible for a significant portion of cancers and inherited diseases. Recombination often occurs between "hotspots", which may be short identical repeats that resemble "pointers" at DNA recombination junctions in Oxytricha, and such events can result in either deletion of tumor-suppressing genes, the formation of chimeric genes, or duplication and subsequent over-expression of genes that promote tumor stability. Because of its magnitude of DNA rearrangements and the recent demonstration that RNA templates can reprogram DNA rearrangements in vivo, Oxytricha is unparalleled as a model system to shed light on the complex events during DNA rearrangement and similar mechanism(s) responsible for cancer and genome instability in humans.