Almost all genes in higher organisms are split into protein coding regions called exons, which are interrupted by non-coding regions called introns. Before a gene is translated into a functional protein, the non-coding introns must be removed and the coding exons joined together in a process called ‘splicing’. Splicing is a fundamental biological gene regulatory mechanism that operates in all higher organisms, both plants and animals. In humans (and other organisms) many genes can be spliced in several different ways such that some versions of the final product of the gene will include some coding exons but not others. This means that one gene can give rise to more than one protein. This process is called ‘alternative splicing’. Alternative splicing is also a fundamental biological gene regulatory mechanism that operates in all cells of all higher organisms. It is through alternative splicing that the human genome, containing ~30,000 genes, can produce greater than 150,000 proteins. Often, alternatively spliced variants of the same gene produce proteins with antagonistic functions, e.g. a protein that promotes the growth of cells may be produced from one spliced variant, while a protein that inhibits cell growth may be produced from another spliced variant of the same gene. These proteins with opposing functions are expressed in the same cell at the same time, but they are maintained in a delicate balance such that the cell grows just enough when necessary but can also stop growing before becoming cancerous. Human embryonic stem cells show evidence of a significant amount of alternative splicing of many genes that produce proteins that regulate cell growth and can direct the stem cells to adopt specific cell fates. Because splicing is one of the earliest steps in gene expression, alternative splicing may play a major role in controlling these types of cell fate decisions. Despite its importance in gene regulation, little is known about the role alternative splicing plays in stem cell growth or cell fate decisions. This is an enormous gap in the body of knowledge that is absolutely required before human embryonic stem cells can be used for therapeutic benefit. It is this gap that we will attempt to fill by investigating alternative splicing mechanisms in human embryonic stem cells. We will compare alternative splicing in several different human embryonic stem cell lines as they differentiate along neural pathways. During the course of these experiments we anticipate we will identify a number of new biomarkers of specific neural cell fates and may identify some gene products that direct the stem cell to adopt particular neural cell fates. We will test the role these gene products play in redirecting neural cell fates by manipulating the factors that regulate alternative splicing in human embryonic stem cells.
This grant application describes experiments that are designed to investigate and provide insight into a fundamental biological gene regulatory mechanism called ‘alternative splicing’ that operates in all cells of all higher organisms including human embryonic stem cells. Alternative splicing is a key step in determining which proteins are expressed in specific cells at particular stages of all developmental pathways. Alternative splicing is so fundamentally important in regulating gene expression that if something goes awry with alternative splicing processes of growth control genes it can and does lead to many different types of cancer including primary malignant brain tumors such as astrocytomas. Almost nothing is currently known about the role alternative splicing plays in controlling growth or specifying cell fate decisions in human embryonic stem cells. This gap in the body of knowledge of a fundamentally important gene regulatory mechanism must be addressed before human embryonic stem cells can be used to realize their full therapeutic potential. For example, before one can utilize human embryonic stem cells that have been coaxed to differentiate into dopamine-producing neurons for the treatment of Parkinson’s disease or cholinergic neurons for the treatment of Alzheimer’s disease, one must be able to insure that the therapeutically introduced neurons do not inadvertently de-differentiate into precancerous astrocytic progenitor cells that may eventually give rise to an astrocytoma. Although the research described in this grant application does not address any specific human disease, a clear understanding of the role of alternative splicing in regulating gene expression during differentiation will be absolutely crucial before human embryonic stem cells can be utilized as a potential treatment for all human diseases. This understanding of basic gene regulatory mechanisms will be of enormous benefit to the State of California and any of its citizens suffering from diseases that may eventually be treated with human embryonic stem cells. It will provide a comparative global analysis of alternative splicing patterns in several different human embryonic stem cell lines and it will identify new biomarkers for distinguishing one neural cell fate from another. It may also provide insight into mechanisms of genesis of cancer stem cells and potentially new therapeutic targets for the treatment of some cancers.