AuthorsRice, Nicholas P
Faculty AdvisorWilliam Theurkauf
UMass Chan AffiliationsProgram in Molecular Medicine
Document TypeDoctoral Dissertation
Genomic Stress Hypothesis
Genetics and Genomics
MetadataShow full item record
AbstractBarbara Mcclintock proposed the genomic stress hypothesis, which states that transposable element activation is an adaptive response to generate new variation. Transposons are ubiquitous selfish genetic elements that compose a large fraction of eukaryotic genomes. In Drosophila melanogaster’s germline, transposons are regulated by small RNAs called Piwi Interacting RNAs (piRNAs). piRNAs originate from clusters of repeats, whose expression is driven by a germline-specific protein Rhino, and cleavage products produced by Argonaute proteins concentrated in nuage, a phase-separated granule. The “genomic stress” model must operate in the germline, and the effect of stress on the piRNA pathway is not well understood. In my thesis, I investigated how two types of stress, temperature and DNA damage via transposon mobilization, affect the piRNA pathway. We show heat shock rapidly and reversibly disrupts the nuclear biogenesis factors required for piRNA production and the collapse of cluster transcription. This loss and recovery of Rhino and its associated factors allowed us to show that cluster assembly is independent of piRNAs. Mutations in the piRNA pathway upregulate transposons and activate the DNA damage response complicating any single piRNA mutant phenotype. We thus created double mutants with a kinase required for DNA damage signaling (Chk2) and demonstrated that Chk2 signaling alters nuage composition. Stripping away damage signaling revealed that components of the nuclear piRNA pathway are required to link nuage properly to clusters. Together we show that the piRNA pathway responds to stress in different ways and that this differential response is an additional tool for investigating the piRNA pathway.
Permanent Link to this Itemhttp://hdl.handle.net/20.500.14038/52089
RightsCopyright © 2023 Rice
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The Interplay Between Stress and the Drosophila piRNA PathwayHo, Samantha (2023-03-22)piRNAs are required to silence transposons in the germline to maintain genome stability and transmission of an intact genome to the next generation. In Drosophila, biogenesis of these small RNAs occurs in three distinct compartments: the nucleus, perinuclear nuage, and the outer mitochondria membrane. My thesis focuses on how genomic instability via transposon-initiated DNA damage and heat stress impact piRNA pathway organization and function. We show that activation of Chk2, a Checkpoint kinase required for DNA damage signaling, disrupts nuage composition and this complicates piRNA mutant phenotypes. Stripping away DNA damage signaling in piRNA mutants provided new insight into how the nuclear piRNA proteins organize the cytoplasmic nuage. Additionally, we found that localization of key components to nuage is dispensable for piRNA production and transposon silencing. piRNA pathway proteins are not only susceptible to genomic instability, but also sensitive to heat shock. Rhino is a core component of the nuclear piRNA pathway and displays drastic localization changes upon heat shock that recovers with time. piRNAs have been proposed to help localize Rhino and heat stress provided a unique platform to test this model. We show that Rhino recovery after heat shock does not require Piwi, the sole nuclear protein bound to piRNAs, and this points to piRNAs having a less significant role in Rhino localization. Taken together, we show how different types of stress can modulate the piRNA pathway in unexpected manners.
Investigating the Effects of Mutant FUS on Stress Response in Amyotrophic Lateral Sclerosis: A ThesisKaushansky, Laura J. (2015-08-14)During stress, eukaryotes regulate protein synthesis in part through formation of cytoplasmic, non-membrane-bound complexes called stress granules (SGs). SGs transiently store signaling proteins and stalled translational complexes in response to stress stimuli (e.g. oxidative insult, DNA damage, temperature shifts and ER dysfunction). The functional outcome of SGs is proper translational regulation and signaling, allowing cells to overcome stress. The fatal motor neuron disease Amyotrophic Lateral Sclerosis (ALS) develops in an age-related manner and is marked by progressive neuronal death, with cytoplasmic protein aggregation, excitotoxicity and increased oxidative stress as major hallmarks. Fused in Sarcoma/Translocated in Liposarcoma (FUS) is an RNA-binding protein mutated in ALS with roles in RNA and DNA processing. Most ALS-associated FUS mutations cause FUS to aberrantly localize in the cytoplasm due to a disruption in the nuclear localization sequence. Intriguingly, pathological inclusions in human FUSALS cases contain aggregated FUS as well as several SG-associated proteins. Further, cytoplasmic mutant FUS incorporates into SGs, which increases SG volume and number, delays SG assembly, accelerates SG disassembly, and alters SG dynamics. I posit that mutant FUS association with stress granules is a toxic gain-of-function in ALS that alters the function of SGs by interaction with SG components. Here, I show that mutant FUS incorporates in to SGs via its Cterminal RGG motifs, the methylation of which is not required for this localization. Further, I identify protein interactions specific to full-length mutant FUS under stress conditions that are potentially capable of interacting with FUS in SGs. Finally, I demonstrate a potential change in the protein composition of SGs upon incorporation of mutant FUS. These findings advance the field of ALS and SG biology, thereby providing groundwork for future investigation.
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