RNA, normally thought of as a conduit in gene expression, has a novel mode of action in ciliated protozoa, where long and small noncoding RNAs orchestrate an intricate process of genome remodeling, and maternal RNA templates provide both an organizing guide for DNA rearrangements and a template that can transmit spontaneous somatic mutations to the next generation. The multidisciplinary goal of this research is to understand the unusual forms of biological information processing that lead to genome rearrangement. As in the previous renewal, the focus will be mainly on ciliates, the current emphasis of the lab, pairing a complete somatic and nearly-complete germline genome assembly with recent discoveries and tools that make Oxytricha trifallax a powerful model to study genome rearrangements. The opportunity for RNA-guided DNA repair is profound in Oxytricha, which deletes 95% of its germline genome through global DNA rearrangements that severely fragment its chromosomes and then sort and reorder the hundreds of thousands of pieces remaining. The proposed research combines different approaches that together focus on the mechanism and underlying logic of massive genome reorganization in ciliates, over a range of scales, from single gene functional experiments to genome-wide and systems-level analyses. The global aim is to use this model system to explore the complex rewriting mechanisms in microbial eukaryotic genomes. Understanding the nature of gene unscrambling is the motivating force behind the current proposal, and the proposed experiments will examine the properties of both the molecules that participate in these complex rearrangement events and the machinery underlying them. The most immediate insight to be gained from the proposed research is an understanding of the cellular acrobatics and the components that execute these stunning genome rearrangements in stichotrichous ciliates, on the scales of fine-tune processing of single genes and microbial eukaryotic genomes. Longer-term insights include relevance to similar RNA-guided mechanisms that influence genome integrity, protecting eukaryotic cells from a diseased state, or that permit epigenetic inheritance of RNA-mediated states, in normal and manipulated cells.
Specific aims i nclude: 1. Improving the Oxytricha germline genome assembly through long mate-paired libraries, 2. Co-injection experiments that test the interplay between long, noncoding template RNAs and piRNAs, 3. Studying piRNA biogenesis in Oxytricha, 4. Understanding long, noncoding template RNA production and their interacting partners, 5. Creating a comprehensive database assessing RNA, DNA, and protein availability during conjugation, 6. Identifying and functionally testing regulatory motifs in somatic chromosomes, and 7. Identifying and testing candidate genes involved in Oxytricha genome rearrangement.
Genome rewiring events, through translocations, deletions, and even massive chromosomal rearrangements (chromothripsis), contribute to genome instability associated with many human diseases, including a significant portion of cancers and inherited or spontaneous diseases. Furthermore, the presence of recombination 'hotspots' in the genome that share similarities with rearrangement junctions in the ciliate Oxytricha, as well as aberrantly spliced RNA products that can template DNA recombination, may increase the frequency of genome rearrangements, resulting in either deletion of tumor-suppressing genes, formation of chimeric genes, or duplication and subsequent over-expression of genes that promote tumor stability. Because of its magnitude of RNA-guided DNA rearrangements, Oxytricha is unparalleled as a model system to shed light on the complex events during genome rearrangement and similar mechanisms that may contribute to cancer and genome instability in humans.
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