Sequence and structure requirements of Y RNA-derived small RNA biogenesis

Turnbull, Carly (2014) Sequence and structure requirements of Y RNA-derived small RNA biogenesis. Doctoral thesis, University of East Anglia.

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Numerous small non-coding RNAs have been identi�ed 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 e�ects 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 �nd many di�erentially 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 di�erent 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 e�ect 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

Item Type: Thesis (Doctoral)
Faculty \ School: Faculty of Science > School of Biological Sciences
Depositing User: Users 2259 not found.
Date Deposited: 09 Jul 2014 12:25
Last Modified: 09 Jul 2014 12:25


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