[Thesis]. Manchester, UK: The University of Manchester; 2011.
The U5 snRNP is a major component of the yeast spliceosome, being part of the U4/U6.U5
tri-snRNP, the precatalytic spliceosome and the catalytically activated spliceosome.
The U5 snRNP includes, at its heart, the U5 snRNA which contains the invariant Loop
1 that functions in tethering and aligning exons during splicing. The major protein
components of the U5 snRNP are the highly conserved Prp8p, the GTPase Snu114p and
the helicase Brr2p. These proteins and the U5 snRNA are integral in forming the active
site of the spliceosome and regulating the dynamic changes of the spliceosome. The
first part of this study aimed to express and purify specific domains of Snu114p to
define the structure and function of Snu114p. The N-terminal region of Snu114p was
successfully expressed and purified from bacteria. Addition of the Snu114p N-terminal
fragment to in vitro splicing assays resulted in a first step splicing defect, indicating
a role for the N-terminus in pre-mRNA splicing. NMR studies revealed that the N-terminus
of Snu114p exists as an unstructured protein domain. Mutagenesis indicated that the
N-terminus of Snu114p is tolerant to mutation. A novel genetic interaction between
amino acids in the N-terminus of Snu114p and the 3’ side of the U5 snRNA IL1 was identified.
It is proposed here that the N-terminus of Snu114p functions to stabilise interactions
of Snu114p with other proteins or snRNAs, possibly the U5 snRNA. Alternatively, the
N-terminus of Snu114p may form intramolecular interactions with other regions of Snu114p
to regulate Snu114p function in pre-mRNA splicing.Prp8p, Snu114p and Brr2p are known
to form a stable complex but their interactions with the specific domains of the U5
snRNA are not known. The second part of this study aimed to investigate the association
of Brr2p, Snu114p and Prp8p with the U5 snRNA. Mutants of the U5 snRNA were constructed
in the conserved Loop 1 and the Internal Loop 1 (IL1). The influences of the U5 snRNA
mutations on interactions of Prp8p, Snu114p or Brr2p with the snRNA were investigated.
It was revealed that Loop 1 and both sides of IL1 of the U5 snRNA are important in
association of Brr2p, Snu114p and Prp8p. Mutations in the 3’ side of IL1 drastically
reduce association of Brr2p, Snu114p and Prp8p with the U5 snRNA, highlighting this
region as a potential ‘protein docking’ site within the U5 snRNP. Differences seen
in the associations of Brr2p, Snu114p and Prp8p with U5 snRNA mutations demonstrate
that although there are intimate interactions between Brr2p, Snu114p and Prp8p, they
do not associate with the U5 snRNA as a tri-protein complex. Genetic screening of
BRR2 and U5 snRNA mutants reveals an interaction between the N-terminal half of Brr2p
and the 3’ side of U5 snRNA IL1. This supports the proposed ‘protein docking’ site
at the 3’ side of the U5 snRNA IL1.Data presented in this study increases our understanding
of the regions in the U5 snRNA required for association of the essential U5 snRNP
proteins, Brr2p, Snu114p and Prp8p, and goes some way to elucidating the organisation
of essential proteins within the U5 snRNP.