Study into a role for Aar2p in U5 snRNP biogenesis
Cristao, Vanessa Solange Fernandes de Oliveira
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Aar2p is an essential yeast protein involved in pre-mRNA splicing and component of a U5 snRNP precursor form. It has been suggested that the mature U5 snRNP and U4/U6.U5 tri-snRNP assemble from the Aar2p-U5 core particle that is formed in the cytoplasm. After nuclear import, Aar2p would be displaced from its interaction with Prp8p in the Aar2p-U5 particle and the mature U5 snRNP would be formed by interaction of Brr2p with Prp8p. In one model, Aar2p acts as a transport factor either for nuclear import of the Aar2p-U5 particle, or for nucleocytoplasmic shuttling of Prp8p. As a non-mutually exclusive alternative, Aar2p can function as a chaperone, regulating the Prp8p/Brr2p interaction. In this thesis I investigate the role of Aar2p in U5 snRNP biogenesis. I demonstrate by fluorescence microscopy that Aar2p is not required for nuclear localisation of the U5 snRNP components Snu114p and Prp8p. By a yeast two-hybrid (Y2H) assay I establish that Prp8p1649-2413 interacts with Aar2p1-170 but not with Aar2p150-355. I also show for the first time that Aar2p is phosphorylated in five aminoacids: S253, T274, Y328, S331 and T345. In my Y2H system S253 phosphorylation disrupts the Aar2p/Prp8p interaction. This suggests a mechanism whereby formation of the mature U5 snRNP and activation of Brr2p by Prp8p may be regulated through phosphorylation. Surprisingly, when the S253A and S253E mutations are inserted into genomic AAR2 there is no change in the amount of Prp8p and U5 snRNA immunoprecipitated by Aar2p. Finally, using Aar2p1-170 as bait for a Y2H screen, a variety of new Aar2p interactors are revealed. The obtained preys include the other half of Aar2p itself, proteins involved in DNA damage repair, chromatin-binding, ubiquitin-binding and membrane proteins. Overall these results suggest that besides modulating splicing, Aar2p is involved in several other important cellular processes.