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.

Public Health Relevance

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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM059708-14
Application #
8727575
Study Section
Genetic Variation and Evolution Study Section (GVE)
Program Officer
Janes, Daniel E
Project Start
1999-08-01
Project End
2017-06-30
Budget Start
2014-07-01
Budget End
2015-06-30
Support Year
14
Fiscal Year
2014
Total Cost
$325,887
Indirect Cost
$123,543
Name
Princeton University
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
002484665
City
Princeton
State
NJ
Country
United States
Zip Code
08544
Chen, Xiao; Bracht, John R; Goldman, Aaron David et al. (2014) The architecture of a scrambled genome reveals massive levels of genomic rearrangement during development. Cell 158:1187-98
Khurana, Jaspreet S; Wang, Xing; Chen, Xiao et al. (2014) Transcription-independent functions of an RNA polymerase II subunit, Rpb2, during genome rearrangement in the ciliate, Oxytricha trifallax. Genetics 197:839-49
Fang, Wenwen; Landweber, Laura F (2013) RNA-mediated genome rearrangement: hypotheses and evidence. Bioessays 35:84-7
Swart, Estienne C; Bracht, John R; Magrini, Vincent et al. (2013) The Oxytricha trifallax macronuclear genome: a complex eukaryotic genome with 16,000 tiny chromosomes. PLoS Biol 11:e1001473
Vogt, Alexander; Goldman, Aaron David; Mochizuki, Kazufumi et al. (2013) Transposon domestication versus mutualism in ciliate genome rearrangements. PLoS Genet 9:e1003659
Bracht, John R; Fang, Wenwen; Goldman, Aaron David et al. (2013) Genomes on the edge: programmed genome instability in ciliates. Cell 152:406-16
Swart, Estienne C; Nowacki, Mariusz; Shum, Justine et al. (2012) The Oxytricha trifallax mitochondrial genome. Genome Biol Evol 4:136-54
Zoller, Stephen D; Hammersmith, Robert L; Swart, Estienne C et al. (2012) Characterization and taxonomic validity of the ciliate Oxytricha trifallax (Class Spirotrichea) based on multiple gene sequences: limitations in identifying genera solely by morphology. Protist 163:643-57
Nowacki, Mariusz; Shetty, Keerthi; Landweber, Laura F (2011) RNA-Mediated Epigenetic Programming of Genome Rearrangements. Annu Rev Genomics Hum Genet 12:367-89
Zhou, Yi; Wubneh, Helmae; Schwarz, Clayton et al. (2011) A chimeric chromosome in the ciliate oxytricha resulting from duplication. J Mol Evol 73:70-3

Showing the most recent 10 out of 41 publications