To enter the spliceosome, U6 snRNP must associate with the U4 snRNP to form a U4/U6 di-snRNP. This involves RNA-RNA interactions between U4 and U6 RNAs, as the two have extensive sequence complementary and form Watson-Crick base-pair interactions over their length. During splicing, the U4 snRNP dissociates from the U6 snRNP and U6 snRNA undergoes several structural rearrangements. Therefore, U4 and U6 snRNA must be reannealed following spliceosome disassembly in order to recycle the factors back into the splicing pathway. A group of seven proteins, Lsm2-Lsm8p, has previously been identified that interact with U6 snRNA and are essential for U6 snRNA stability and pre-mRNA splicing in the yeast <i>Saccharomyces cerevisiae. </i>In this thesis the Lsm (like-Sm) proteins have been produced either as recombinant protein in <i>E. coli</i> or as the product of a coupled <i>in vitro</i> transcription/translation reaction. Using these proteins it has been demonstrated that the Lsm proteins can facilitate U4/U6 RNA duplex formation. Another U6-ascoiated protein, Prp24p, did not facilitate U4/U6 duplex formation in the absence of the Lsm proteins. This was in contrast to previous reports suggesting a role for Prp24p in U4/U6 annealing. Prp24p is, however, required when the 3’ terminal Lsm binding site is deleted from U6 snRNA. Evidence is presented suggesting that the Lsm8 complex can facilitate U4/U6 duplex formation <i>in vivo</i> as well as <i>in vitro</i>. In particular, Lsm6p appears to be essential for stable U4/U6 duplex formation, whereas Lsm7p does not. The data presented, therefore, suggest a model where the Lsm8 complex, in particular Lsm6p, is sufficient and necessary for U4/U6 duplex formation. Other factors such as Prp24p may facilitate this process, but are not essential for it to occur.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:660283 |
| Date | January 2002 |
| Creators | Page, David |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/15572 |
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