Title:
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Characterisation of the requirements for 3'end formation of human snRNAS in vitro
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Small nuclear (sn)-RNAs are short, intronless and non polyadenylated RNAs that
are required for critical cellular functions including splicing. Like protein-coding
genes snRNA genes are transcribed by Pol II. However, there are significant
differences in transcription and processing between mRNAs and snRNAs. 3' end
formation of the human snRNAs occurs in a series of steps starting with cotranscriptional
RNA processing which is directed by the snRNA gene-specific 3'
box rather than the poly(A) site as found in protein-coding genes.
Uguen and Murphy (2003) recently obtained accurate 3'end formation of
synthetic RNA substrates containing the 3' box of the human U1 RNA genes in
vitro in the absence of ongoing transcription. This system requires S1 nuclease
digestion of processing products. The first chapter of this thesis reports the
development of a faster and easier to handle in vitro processing system where
the processing products are monitored directly by the use of radiolabelled RNA.
Until recently, the factors involved in snRNA 3'end formation remained
unknown. In 2005, Baillat et a/. described a multi-protein complex called
Integrator which is necessary for snRNA 3'end formation. I have further
investigated the role of Integrator components in snRNA 3'end processing. I have
shown that a smaller complex, 240 kDa, is sufficient for in vitro processing,
suggesting that only a fraction of the Integrator complex is necessary for correct
snRNA 3'end formation in vitro. In addition, several candidate proteins were
tested and ruled out as factors involved in snRNA 3'end formation.
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