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Study of Cwc24 phosphorylation as a splicing factor in Saccharomyces cerevisiae

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Author(s):
Thierry Pueblo Freitas Girotto
Total Authors: 1
Document type: Master's Dissertation
Press: São Paulo.
Institution: Universidade de São Paulo (USP). Conjunto das Químicas (IQ e FCF) (CQ/DBDCQ)
Defense date:
Examining board members:
Carla Columbano de Oliveira; Patricia Pereira Coltri; Deborah Schechtman; Cleslei Fernando Zanelli
Advisor: Carla Columbano de Oliveira
Abstract

Splicing is the name given to the intron removal reaction in pre-RNAs and binding of exons to form mature mRNAs. It is catalyzed by the spliceosome, a highly dynamic complex formed by snRNPs, snRNAs and protein associations, facilitating the process of excision of noncoding sequences by two transesterification reactions. The spliceosome is assembled on each pré-RNA that will be processed. For this, the assembly is sequential and largely coordinated by protein interactions, which are carefully regulated, often by post-translational modifications, such as phosphorylation, ubiquitination, sumoylation, among others, which may influence the functions of proteins, and consequently, steps during splicing. An essential protein in Saccharomyces cerevisiae that plays a key role in splicing is Cwc24, which protects the 5´SS (splice site) from a premature nucleophilic attack during the first transesterification reaction, and which assists in stabilizing the binding of the U2 snRNP complex to pre-RNA. Cwc24 interacts with several proteins of the splicing machinery. Phosphorylation sites in Cwc24 sequence were identified in a high throughput proteomics assay. The objective of this work was to characterize the role of the Cwc24 phosphorylation in the control of its function during splicing in Saccharomyces cerevisiae. The results showed that Cwc24 is phosphorylated in its two serine residues identified by large scale proteomics assays. By creating mutants in these two serine residues, several aspects of the protein were studied. We have seen that mutations did not affect cell viability under normal conditions and under stress growth conditions. Protein stability was tested and we saw that amino acid substitutions also had no effect on the protein. Looking at their function in the context of splicing, we saw that the mutants did not change pre-RNA processing. Cwc24 phosphorylation is present in the context of interaction with other splicing factors, but is not crucial for interaction since the phosphodeficient mutant is capable of interacting with the same proteins as wild protein. Thus, we were able to conclude that Cwc24 phosphorylation could be involved in some other cellular process that may be linked to pre-RNA splicing, or that the methodology used was not sensitive enough to detect of the mutations on Cwc24 function. (AU)

FAPESP's process: 17/11544-2 - The role of phosphorylation in protein-protein interactions in splicing regulation in Saccharomyces cerevisiae
Grantee:Thierry Pueblo Freitas Girotto
Support Opportunities: Scholarships in Brazil - Master