Less than 5% of Saccharomyces cerevisiae genes have introns and most yeast intron-containing genes have only one intron. This contrasts with many other species in which the majority of genes possess multiple introns. It is possible that yeast retained only introns with an important function throughout evolution. Interestingly, the yeast intron-containing genes fall into only a few classes such as ribosomal protein genes, genes involved in sporulation and ubiquitin-like conjugation enzymes (Grate and Ares, 2002), suggesting a possible function of splicing in these pathways. In support of this, ribosomal protein gene introns accumulate upon amino acid starvation, conditions which lead translational stress (Pleiss et al., 2007a). This observation is the foundation for future experiments aiming to understand the starvation splicing regulatory mechanism as well as additional environmental stresses. All cells must respond to multiple environmental stress conditions and an improper response can lead to cellular death or disease.
The aim of this proposal is to determine if cells employ splicing regulation as an additional mechanism to deal with stress. ) Preliminary results show both cis- and trans-acting factors are involved in the starvation splicing regulatory pathway. The objective of Aim 1 is to follow up preliminary results showing ribosomal protein gene promoters and not the introns are essential for the starvation splicing response and to determine the sequences specifically regulating this response. There is also evidence that a protein kinase, casein kinase 2, is necessary for the splicing response.
Aim 2 focuses on determining additional factors involved in the regulatory pathway and will employ a genome-wide screen. And finally, the goal of Aim 3 is to assess whether other intron-rich gene classes are regulated at the level of splicing in response to environmental stress by examining additional cellular stress conditions, such as DNA damage. RNA undergoes many different processing steps, in addition to splicing, throughout gene expression. When any of these processing pathways is incorrectly regulated in mammals, diseases arise such as Retinitis pigmentosa caused by mutation of proteins involved in splicing (Cooper et al., 2009). A greater understanding of splicing regulatory pathways will not only allow for a more detailed view of splicing regulation in yeast, but also the many mechanisms altering gene expression during environmental stress.
During gene expression, RNA serves as a transient message which undergoes many molecular changes, or processing reactions, which alter its stability including 5'cap formation, RNA splicing and addition of a polyadenine tail. Cells regulate each of these reactions in response to extracellular stimuli or environmental stress and disruption of many of these processing mechanisms leads to disease. The goal of this research proposal is to understand the mechanisms regulating RNA splicing in response to environmental stress.
