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.
Statement of Benefit to California:
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.
SYNOPSIS: Most human gene transcripts are alternatively spliced, resulting in multiple protein products from one gene. This application proposes that the relative amounts of these variant proteins may help direct ESCs to a particular developmental fate, and changing levels of splice factors may alter the ratios of alternatively spliced transcripts. Aim 1 will extend preliminary RT-PCR results, which showed changes in levels of several splicing factors during differentiation of the H9 hESC line toward a neuronal fate, to include to other (unspecified) splicing factors and seven other hESC lines. Aim 2 seeks to identify alternatively spliced products of unspecified signaling genes that show a consistent pattern in all hESC lines. Aim 3 will identify RNA-binding splicing factors that correlate with specific splice variants by using HeLa cell extracts in an in vitro splicing assay. Aim 4 tests the effect of increasing or decreasing levels of particular splicing factors on neuronal differentiation. INNOVATION AND SIGNIFICANCE: Qualitative changes in gene expression through alternative splicing certainly occur during early embryonic development and during human ESC differentiation in vitro. Despite extensive ongoing analyses of cell-cell signaling and transcriptional control mechanisms, little is known about the role of this potentially important post-transcriptional regulation. Although there is indeed an enormous gap in our understanding of alternative splicing in embryonic stem cells and during their differentiation, filling that gap is not, as stated, absolutely required before hESC can be used for therapeutic benefit. Also, the significance of the proposal is unclear -- it feels like a fishing expedition, and it is unclear what the potential benefit would be of identifying certain alternative splicing patterns/perturbations. STRENGTHS: Preliminary results demonstrate the ability to work effectively with human ESC and perform many of the necessary techniques for analyzing individual genes and splicing reactions. Indeed, levels of mRNAs for several known splicing regulators change during ESC differentiation toward a neural phenotype and changes in splicing patterns were observed for a number of candidate genes encoding components of signaling pathways. Focus on signaling makes the analysis tractable; nonetheless, showing an important function of alternate products will be difficult for more than a few candidates. Although the PI is new to the field of ES cell biology, she is an accomplished researcher in alternative splicing with extensive experience studying alternative splicing in Drosophila. The PI has begun a successful cultivation of the WiCell H9 line and analysis of splicing patterns of signaling genes. She interacts with Dr. Tim O’Connor in the Biology Division of the COH Medical Center on a regular basis. A new hire, Dr. Tiziano Barberi, will set up a hES cell core facility at the COH and conduct his own experiments on mesenchymal differentiation. The postdoctoral researcher performing these experiments attended an NIH-sponsored hESC workshop. WEAKNESSES: For aim 1 to be effective, the levels of a large fraction of all factors that can affect the choice in alternative splices must be analyzed for comparison with the broad analysis of alternative splicing. A list of these factors, let alone immunologic reagents for them, is unavailable. Without this it won’t be possible to match alternative splices with potential factors in the vast majority of cases. If, as proposed, subtle differences in the levels of splicing factors determine paths of alternative splicing, more precise quantification of factor mRNAs than the RT-PCR assay proposed is necessary. Moreover, measurements of the levels of the splicing factors themselves must be undertaken to verify that mRNA levels correlate with protein levels. For aim 2, the nature, source and specificity of neural differentiation of the hESC is not clear (ES, Ebs, neural EB/neurospheres, and neural progenitor cells). In order to correlate differences with a specific cell-fate, this eventually will be an important consideration. Aim 3 proposes to examine the splicing in cell-free HeLa splicing extracts by depleting and reconstitution with purified factors. This requires immunologic reagents for each of the splicing factors to be examined, many of these reagents are not available at this time, nor planned in the proposal. Moreover, the identity of many splicing regulators is unknown, so many candidates for regulators in human ES cells or early human development are not available and a comprehensive analysis is not possible at this time. The methods they plan to use to modulate alternative splicing described under Specific Aim 4 are very vague and appear to not be well thought out. They indicate they will use transient transfection, but don't indicate a method, or what type of vector would be used. The investigator indicates the ability to collaborate with another investigator in the same institute who has experience with hESC, but has not provided a letter confirming the willingness of that investigator to collaborate in this study. There were concerns about the likelihood of successful completion of this ambitious project given the productivity of the principal investigator. DISCUSSION: There was no further discussion following reviewers' comments.