Cell Biology & Molecular Genetics Theses and Dissertations
Permanent URI for this collectionhttp://hdl.handle.net/1903/2750
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Item AN INTERSECTING NETWORK OF REGULATORS IS REQUIRED FOR RNA SILENCING AND NUCLEAR INTEGRITY IN C. ELEGANS(2024) Knudsen-Palmer, Daphne R; Jose, Antony M; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Regulation of gene expression is required for an organism to develop, maintain homeostasis, and respond to environmental stimuli. While each cell in a multicellular organism contains the same genetic information, the epigenetic control of the expression of genes at different times is crucial for processes such as cell differentiation, division, and for allowing for cells to carry out different functions from one another. One type of such epigenetic regulation is mediated by small non-coding RNAs. Introduction of double-stranded RNA (dsRNA) and subsequent production of small interfering RNAs can result in sequence-specific mRNA silencing, creating the potential for highly specific therapeutics and pesticides. However, some targets are more easily silenced than others, and the mechanisms of silencing are not fully understood. Here we investigate regulators of small RNA-mediated silencing in the nematode C. elegans and find that they function in an intersecting network, allowing the potential for regulators to contribute to the silencing of any target. Quantitative modeling suggests that the production and turnover rates of a target at steady-state can affect the ease with which a target can be knocked down, and experimentally we found that changing the cis-regulatory sequences of a target can make it more susceptible to silencing. We found restricted production of RNA silencing intermediates, allowing for the recovery of a target in response to dsRNA, which we observed experimentally in non-dividing cells. In addition to roles in response to dsRNA, we report that disruption of small RNA-based regulation can result in germline nuclear defects. In the absence of the intrinsically disordered and perinuclear granule-forming protein MUT-16, some of the nuclei in the syncytial germline appear enlarged, suggesting that small RNA-based regulation may be playing an active role in maintaining nuclear size. Taken together, these findings suggest that (1) regulators of small RNA silencing can contribute to the silencing of all targets as part of an intersecting network, as opposed to operating in specialized pathways and (2) small RNA-based regulation is required for nuclear integrity, providing a paradigm for studying control of nuclear size, where enlarged nuclei can be compared with wildtype nuclei in a shared syncytium. We speculate that these findings will improve understanding of RNA silencing across species and provide insight into understanding how nuclear size is controlled, a fundamental ability of all eukaryotes.Item Mobile RNA reveal differential requirements for gene silencing in C. elegans at single-cell resolution(2018) Ravikumar, Snusha; Jose, Antony M; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Delivery of double-stranded RNA (dsRNA) into animals can silence genes of matching sequence in diverse cell types through mechanisms that have been collectively called RNA interference. In the worm C. elegans, such organism-wide silencing relies on the transport of dsRNA to most cells and requires amplification mechanisms for effective gene silencing. Amplification of silencing signals is accomplished by two tissue-specific RNA-dependent RNA polymerases - EGO-1 in the germline and RRF-1 in somatic cells. Here, we reveal instances of RRF-1-independent silencing in somatic cells, which are dictated by three variables. First, when the same intestinal target gene was silenced using ingested, intestinal, or neuronal dsRNA, only silencing by mobile RNAs derived from neuronal dsRNA was independent of RRF-1. Second, when the same source of mobile RNA was used to silence a target sequence in different genomic contexts, the requirement for RRF-1 could change. Third, measurement of silencing by mobile RNAs at single-cell resolution revealed cell-to-cell and animal-to-animal variation in the requirement for RRF-1. Therefore, the requirements for gene silencing can vary based on the source of dsRNA, the target context, and even the particular cell examined, suggesting that each C. elegans animal is a functional mosaic with respect to RNA interference