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Title: Sequence and structure requirements of Y RNA-derived small RNA biogenesis
Author: Turnbull, Carly
ISNI:       0000 0004 5367 1713
Awarding Body: University of East Anglia
Current Institution: University of East Anglia
Date of Award: 2014
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Numerous small non-coding RNAs have been identified in mammalian cells, including microRNAs and piwi-interacting RNAs. The patterns of gene expression within cells can be altered in response to cellular stress. To examine the effects of cellular stress on all small RNA types, a number of human cell lines were treated with Poly(I:C), a mimic of viral infection, and the levels of small RNAs were examined by next generation sequencing. Surprisingly, we did not find many differentially expressed microRNAs, but we discovered a new class of small RNAs that were 30-35 nucleotides and showed up-regulation following Poly(I:C) treatment. These slightly longer small RNAs were derived from many different types of non-coding RNA and only very few small RNAs were derived directly from messenger RNAs. Small RNAs derived from various types of RNA were validated by northern blot. Further sequencing libraries were prepared for Poly(I:C)-treated and untreated MCF7 cells, as well as Poly(I:C)-treated and untreated SW1353 cells. The human Y5 RNA gene was chosen as an example of a Poly(I:C)-induced small RNA-producing gene. This gene was cloned into an expression construct and systematically mutated to alter the sequence or secondary structure of the resulting Y RNA. These mutant plasmids were expressed in mouse cells and the effect on small RNA production determined. These individual mutants together helped to determine a region vital for cleavage, and that it is the structure rather than the sequence within this region that is important. A high-throughput method was also implemented, involving the generation of large pools of plasmids containing all possible sequences within a particular region of the RNA gene. These pools were expressed in mouse cells and the mutants that were expressed and processed into small RNAs were sequenced. These experiments showed that the formation of the large internal loop determines the internal cleavage site.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available