Determining the Binding Between SAGA Subunits and Spliceosomal Components

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

Analyzing Mutations of Spt7 Protein That Disrupt Interaction with SF3B Subunits

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

The Spliceosomal Protein SF3B5 is a Novel Component of Drosophila SAGA that Functions in Gene Expression Independent of Splicing

The interaction between splicing factors and the transcriptional machinery provides an intriguing link between the coupled processes of transcription and splicing. Here, we show that the two components of the SF3B complex, SF3B3 and SF3B5, that form part of the U2 small nuclear ribonucleoprotein particle (snRNP) are also subunits of the Spt-Ada-Gcn5 acetyltransferase (SAGA) transcriptional coactivator complex in Drosophila melanogaster. Whereas SF3B3 had previously been identified as a human SAGA subunit, SF3B5 had not been identified as a component of SAGA in any species. We show that SF3B3 and SF3B5 bind to SAGA independent of RNA and interact with multiple SAGA subunits including Sgf29 and Spt7 in a yeast two-hybrid assay. Through analysis of sf3b5mutant flies, we show that SF3B5 is necessary for proper development and cell viability but not for histone acetylation. Although SF3B5 does not appear to function in SAGA\u27s histone-modifying activities, SF3B5 is still required for expression of a subset of SAGA-regulated genes independent of splicing. Thus, our data support an independent function of SF3B5 in SAGA\u27s transcription coactivator activity that is separate from its role in splicing

High-pH structure of EmrE reveals the mechanism of proton-coupled substrate transport

AbstractThe homo-dimeric bacterial membrane protein EmrE effluxes polyaromatic cationic substrates in a proton-coupled manner to cause multidrug resistance. We recently determined the structure of substrate-bound EmrE in phospholipid bilayers by measuring hundreds of protein-ligand HN–F distances for a fluorinated substrate, 4-fluoro-tetraphenylphosphonium (F4-TPP+), using solid-state NMR. This structure was solved at low pH where one of the two proton-binding Glu14 residues is protonated. Here, to understand how substrate transport depends on pH, we determine the structure of the EmrE-TPP complex at high pH, where both Glu14 residues are deprotonated. The high-pH complex exhibits an elongated and hydrated binding pocket in which the substrate is similarly exposed to the two sides of the membrane. In contrast, the low-pH complex asymmetrically exposes the substrate to one side of the membrane. These pH-dependent EmrE conformations provide detailed insights into the alternating-access model, and suggest that the high-pH conformation may facilitate proton binding in the presence of the substrate, thus accelerating the conformational change of EmrE to export the substrate.</jats:p