The discovery of viroids in 1971 opened the door to a whole new field of RNA biochemistry. Viroids subsequently became the first of many facets of RNA biochemistry: the first single stranded covalently closed RNA discovered in nature, the first subviral pathogen discovered, and the first pathogen of a eukaryotic system to have its genome sequenced. Viroids are the smallest known agents of infectious disease and they represent the borders of life. They replicate autonomously within their host and since they do not code for their own proteins, they act as scavengers of the host transcriptional machinery. By doing so, viroids find ways of trafficking, localizing, and replicating within their host based on the sequence and structure of the RNA alone. Once in their hosts, viroids are incredibly resilient and can cause economic damage on several commercial crops. Apart from controlling viroids for economic reasons, the more enticing feature of viroid study is the use of viroids as model systems to study essential underlying questions about the evolution of RNA pathogens, and to use viroids as models to study non-coding RNAs. The field of non-coding RNA research has surged within the past decade and viroids are becoming important vehicles to bring insight into this field of study. The study of viroids has been extensive through the years, but several questions remain: What structural conformations do viroids employ to recruit host enzymes, and what are the enzymes that cleave and ligate viroids into mature progeny. To answer some of these questions, we have looked at processing of the potato spindle tuber viroid (PSTVd) RNA in the budding yeast Saccharomyces cerevisiae. We found that one specific construct will process into a mature viroid circle in yeast and we also found that processing in this system is distinct from other plant and non-plant based host systems. This processing is a delicate interplay of ligation and degradation by host machinery. Yeast is a great system to study viroid processing as yeast allows for use of the entire toolbox of temperature-sensitive and knockout protein mutants. By employing yeast, focus can be driven towards the mechanisms of host protein recruitment, viroid processing requirements, and degradation mechanisms from the host. We have ascertained insight into PSTVd processing using yeast. We have found methods to transform and process PSTVd, investigated enzymes that effect processing, and started to establish an in vitro yeast system. Through these studies, we have also developed a method to enrich viroid RNAs from total RNA extractives. This has been vital to assays specific around viroid transcription and cleavage. Overall, this research is further testament that viroids are minimalist scavengers of a very diverse array of cellular transcriptional machinery. They can process in higher eukaryotes (plants) and simple eukaryotes (yeast). They are shown to affect each host in distinct manners using fundamental RNA biology that all organisms share.
|Commitee:||Farber, Matthew, McEwen, Charles, Tomsho, John|
|School:||University of the Sciences in Philadelphia|
|Department:||Chemistry and Biochemistry|
|School Location:||United States -- Pennsylvania|
|Source:||DAI-B 79/04(E), Dissertation Abstracts International|
|Subjects:||Molecular biology, Biochemistry, Virology|
|Keywords:||Enrichment, PSTVd, Pathogen, Potato spindle tuber viroid, RNA processing, Viroid, Yeast|
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