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Proper transcription, the process of converting DNA to RNA, is crucial for the health and viability of an organism. This process is regulated by many proteins, such as co-transcriptional activators; one being the protein complex known as Spt-Ada-Gcn5-acetyltransferase, or SAGA. While much is known about the roles of SAGA in cell processes, how SAGA’s subunits promote functionality is still unknown. The focus of this study is to analyze the purpose of SAGA’s SF3B subunits. These subunits are also found in the spliceosome, the compound responsible for generating mature RNA. SAGA has no known functions relating to this process, so the reason the SF3B components are in SAGA is unclear. Spt7, another SAGA subunit, interacts with both SF3B subunits. In this study, a yeast two hybrid assay was performed where different Spt7 mutants were screened. This was done by transforming yeast with Spt7 mutants, analyzing the protein interactions and sequencing the mutants to determine their mutations. A key result of this study is in the determining that the two SF3B subunits interact with different regions of Spt7. Although the overall goal is to find an Spt7 mutant that does not interact with the SF3B components but still maintains interaction with other SAGA subunits, we now have a better idea of what type of Spt7 mutant is needed. This discovery will lay the foundation for future experiments where a mutated SAGA with no SF3B components will be expressed in Drosophila melanogaster and analyzed to determine the function of SF3B subunits in SAGA.

Proper gene regulation is vital to the health and development of an organism. Determining the relationship between splicing, transcription, and chromatin structure is vital for understanding gene regulation as a whole. There have been previous studies linking these elements pairwise; however, no evidence exists for a direct link between all three. Recent data shows that splicing components of the U2 small nuclear ribonucleic protein (snRNP) co-purify with Spt-Ada-Gcn5-acetyltransferase (SAGA), a highly conserved transcriptional co-activator and chromatin modifier. We hypothesize that SAGA binds with splicing components through a multi-protein binding surface with certain core components based on preliminary yeast two-hybrid data. Here, we examine the specific binding partners between SAGA and splicing components utilizing the yeast two-hybrid system in spt7Δ Saccharomyces cerevisiae as a validation for the preliminary yeast two-hybrid performed, producing recombinant proteins through sequence and ligation-independent cloning (SLIC) and Baculovirus transfections to obtain purified proteins, and co-immunoprecipitation (co-IP) to detect specific protein-protein interactions from recombinant proteins. Yeast two-hybrid results reveal that Spt7 is necessary for the transcription of reporter genes used in this assay. Therefore, this assay cannot validate previous results or detect false positives. Currently, recombinant proteins are being produced to perform co-IPs to test direct protein interactions. The results from these experiments will demonstrate the type of binding between SAGA subunits and splicing factors and provide direct evidence of a link between all three of the elements of gene regulation.