The spliceosomal U5 snRNP contains several proteins with well characterised functions in splicing, including: Brr2, an ATPase/RNA helicase that disrupts U4/U6 and U2/U6 snRNA base pairing during activation of the spliceosome; Snu114, a GTPase that controls the action of Brr2; and Prp8, the largest and most conserved protein considered to have a central role in the spliceosome, which interacts directly with Snu114 and Brr2. Yeast Cwc21 is one of twelve Bact complex proteins that associate with spliceosomes shortly before the first step of splicing catalysis. Cwc21 interacts directly with Prp8 and Snu114, as does its human orthologue, the SR protein SRm300/SRRM2. Although, Cwc21 is not essential for yeast cell viability, it is required for sporulation. This work aims to identify the function of Cwc21 during meiosis. PP1 is a protein phosphatase required for both steps of splicing. Multiple sequence alignments of Snu114 and Prp8 revealed the presence of putative PP1 binding motifs that are well conserved among different species. This led me to hypothesize that PP1 may interact with Snu114 and/or Prp8 to regulate these or other interacting proteins. By screening intron-containing genes that are expressed in meiosis, I found that Cwc21 is required for splicing HRB1 transcripts. In addition, I show that HRB1 is also required during meiosis. The HRB1 intron contains an unusual branchsite sequence, TACTAATG, which when changed to the consensus branchsite sequence restores sporulation in the absence of Cwc21. Therefore, it is likely that Cwc21 promotes the expression of HRB1 during an early stage of meiosis by stabilising its pre-mRNA in the catalytic centre of the spliceosome. This study demonstrates a novel function for Cwc21 during meiosis. Using yeast two hybrid assay I have identified the interacting regions of Cwc21, PP1 and Brr2 in Snu114. Through biochemical studies I provide evidence for mutually exclusive interaction of Cwc21 and PP1 in the putative PP1 binding motif situated in Snu114 domain ‘IVa’. In the case of yeast Snu114, the PP1 binding motif has a novel sequence ‘YGVQYK’. I also show that the affinity of Cwc21 and PP1 for Snu114 is influenced by the different nucleotide-bound states of Snu114. Furthermore, I show that mutations in Snu114 domain ‘IVa’ restrict Snu114 function during meiosis and affect the MER1 splicing regulatory network. Therefore, Snu114 may play a role in modulating the conformational state of the catalytic spliceosome through its interactions with Cwc21/PP1 in regulating subsets of genes during meiosis. Finally, I show that PP1 is a putative regulator of Prp8.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:693571 |
| Date | January 2013 |
| Creators | Gautam, Amit |
| Contributors | Beggs, Jean ; Walkinshaw, Malcolm |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/16185 |
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