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RNA: A JACK OF ALL TRADES. Studying the regulatory role of 6S RNA in E. coli and the impact of exosomal RNA in parasite pathogenesis

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(Thesis) Ph.D.
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During the past two decades we have seen an explosion in our understanding of RNA dependent gene regulation. We now know that RNA is involved in every major event in the life of cells, from the Okazaki fragments involved in DNA replication to programmed cell death. The work described here explores two situations in which RNA plays an important role; how 6S RNA helps ensure bacterial survival and the role of RNA in helping the intracellular parasite, Leishmania, escape the immune system by taking refuge inside mammalian macrophages.6S RNA is a non-coding RNA that regulates bacterial transcription by sequestering the RNA polymerase holoenzyme (Eσ70) in low nutrient conditions. In high nutrient environments, Eσ70 is released by the synthesis of a short product RNA (pRNA) using the 6S RNA as a template. A range of 6S RNA release-defective mutants were selected and characterized from a highly diverse in vitro pool. There is complex crosstalk between regions of the 6S RNA large open bubble that interact with Eσ70 in a cooperative manner so as to ensure efficient pRNA-dependent release. When a group of 6S RNA mutants was over-expressed in E. coli, they significantly delayed growth and decreased cell survival indicating that 6S RNA release rate plays a key role in regulating normal transcriptional dynamics and ultimately cell division. Interestingly, cells resumed normal growth rates approximately 6 hours after mutant 6S RNA overexpression. This growth pattern might be correlated with the accumulation of a protein factor that binds strongly to the 6S and mutant 6S RNA, and data suggest that 6S RNA also might bind to an RNase.RNA may contribute directly to parasite pathogenesis in trypanosomatids. Leishmania spp. uses exosomes to weaken mammalian host cells. Exosomes are known to be involved in intercellular communication. We examined the use of exosomes and their RNA from two species of Leishmania and how that RNA reprograms host cells. Exosome RNA cargo is delivered to host cell cytoplasm during in vitro studies. Sequencing of exosomal RNA indicated that the majority of cargo sequences were derived from non-coding RNA, while Northern blotting confirmed the specific and selective enrichment of tRNA-derived small RNAs in exosomes. We also identified a number of novel transcripts, which appeared to be specifically enriched in exosomes compared to total cell RNA. To our knowledge this is the first report that exosomes are used by a pathogen to invade new host cells. These findings also open up a new avenue of research on non-canonical, small RNA pathways in trypanosomatid parasites, which may elucidate pathogenesis factors and identify novel therapeutic targets.
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This thesis may be printed or downloaded for non-commercial research and scholarly purposes.
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Supervisor or Senior Supervisor
Thesis advisor: Unrau, Peter J
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