| dc.description.abstract | In order for appropriate gene expression to be carried out in eukaryotes, there must be strict regulation between steps in gene expression. Two key steps are transcription, in which an RNA copy of the DNA gene is created, and RNA splicing, the process that removes non-protein coding regions of RNA and fuses together the pieces. Previous research has indicated that these two processes occur simultaneously. However, the mechanisms and connections by which transcription and pre-mRNA splicing occur are still unknown. Inside the cell, DNA is wrapped around histone proteins. These histones can be chemically modified to make DNA more or less accessible to the transcription enzyme. For example, acetylated histones loosens the DNA. Splicing therefore also takes place in this dynamic context and has the potential to be regulated by histone modification. Our experiment aims to elucidate these mechanisms by proposing two potential models: kinetic and recruitment. The recruitment model involves the physical recruitment of splicing factors during transcription via histone modification. The kinetic model is based on the “window of opportunity” for splicing to take place, which is determined by the speed at which transcription occurs. In an effort to determine the mechanism, our lab has made use of a panel of point mutations in the H4 histone in the model yeast Saccharomyces cerevisiae. The amino acid lysine was mutated to either glutamine or alanine, which mimic the acetylated state or arginine, which mimics the deacetylated state. These mutations can alter the speed of transcription. Genetic analyses identified interactions between these mutants and splicing factor genes, thus linking histone acetylation to splicing. Furthermore, using RT-qPCR we found that abolishing histone acetylation alters RNA splicing patterns. In other words, splicing improved or worsen with the loss of acetylation. In the future, we plan to perform chromatin immunoprecipitation to test whether histone acetylation is important for recruiting splicing proteins during transcription. This can help us further determine which model, kinetic, recruitment, or both, the cell is following during this process. | en_US |
