Funding opportunities

Neural and general splicing factors control self-renewal, neural survival and differentiation

Funding Type: 
Basic Biology III
Grant Number: 
RB3-05009
Principle Investigator: 
Funds requested: 
$1 372 660
Funding Recommendations: 
Recommended
Grant approved: 
Yes
Public Abstract: 
Human embryonic and patient-specific induced pluripotent stem cells have the remarkable capacity to differentiate into many cell-types, including neurons, thus enabling the modeling of human neurological diseases in vitro, and permit the screening of molecules to correct diseases. Maintaining the pluripotent state of the stem cell, directing the stem cell towards a neuronal lineage, keeping the neuronal progenitor and stem cells alive - these are all maintained by thousands of different proteins in the cell at these different "stages". Thus the levels and types of proteins are highly controlled by gene regulatory mechanisms. Genes produce pre-messenger RNA (mRNA) transcripts in the nucleus, which undergo a process of refinement called splicing, whereby long (1,000-100,000 bases) stretches of nucleotides are excised, and much shorter pieces (150 bases) are ligated together to form mature messenger RNA to eventually make proteins in the cytoplasm. Strikingly, some pieces of RNA are used in a particular cell-type, but not another, in a process called "alternative splicing". This is the most prevalent form of generating transcriptome diversity in the human genome, and is important for pushing cells from one state to another i.e. stem cells to neurons, maintaining a cell state i.e. keeping a stem cell pluripotent, or a neuron alive and functioning. Alternative splicing is highly controlled by the recognition of even smaller stretches (6-10 bases) of RNA binding sites) by proteins that bind directly to RNA called splicing factors. The goal of the proposed research is to produce a regulatory map of where these splicing factors bind within pre-mRNAs across the entire human genome with unprecedented resolution using a high-throughput biochemical strategy. Furthermore, using advanced genomic technologies, we will deduce what happens to splicing when these factors do not bind to their binding sites. Finally, using molecular and imaging methods, we will analyze what happens to survival of stem and neuronal cells when these factors are depleted or over-expressed, and if stem cells are induced to make neurons if the levels of these factors are altered. Completion of the proposed research is expected to transform our understanding of the regulatory mechanisms underlying transcriptome complexity important for neurological disease modeling, especially human neurodegeneration, and stem cell biology. In turn, this will facilitate more accurate comparisons of diseased states of neurons from stem-cell models of Amyotrophic Lateral Sclerosis (ALS), Myotonic Dystropy, Spinal Muscular Atrophy (SMA), Parkinson’s and Alzheimer’s to identify mis-spliced genes and the splicing factors responsible for therapeutic intervention.
Statement of Benefit to California: 
Our research provides the foundation for decoding the mechanisms that control the transcriptome complexity of stem cells and neurons derived from stem cells. Our work has direct application in the design of novel strategies to understand the impact of splicing factor misregulation, or mutations within the binding sites for these splicing factors in neurological diseases that heavily impact Californians, such as Amyotrophic Lateral Sclerosis (ALS), Myotonic Dystropy, Spinal Muscular Atrophy (SMA), Parkinson’s and Alzheimer’s. Our research has and will continue to serve as a basis for understanding deviations from "normal" stem and neuronal cells, enabling us to make inroards to understanding neurological disease modeling using neurons differentiated from reprogammed patient-specific lines. Such disease modeling will have great potential for California health care patients, pharmaceutical and biotechnology industries in terms of improved human models for drug discovery and toxicology testing. Our improved knowledge base will support our efforts as well as other Californian researchers to study stem cell models of neurological disease and regenerative medicine, and for the design of new diagnostics and treatments, thereby maintaining California's position as a leader in clinical and biomedical research.
Review Summary: 
Project Synopsis: This proposal focuses on investigating mechanisms of alternative splicing in human embryonic stem and neural progenitor cells (hESCs and hNPCs). Alternative splicing is a process by which genes are expressed in different forms in different cell types. It is also the most prevalent form of generating transcriptome diversity in the human genome, and is important for pushing cells from one state to another i.e. stem cells to neurons or maintaining pluripotentency. Dysregulation of alternative splicing has been associated with several neurological diseases, including amyotrophic lateral sclerosis (ALS). The applicant proposes to examine the role of alternative splicing in stem cell biology in three specific aims: (1) to evaluate the roles of known splicing factor proteins in maintaining self-renewal, survival and differentiation in hESCs and hNPCs; (2) to identify direct binding sites for these splicing factors; and (3) to perform genome-wide analysis to identify regulated targets of alternative splicing in hESCs and hNPCs. Significance and Innovation: - Reviewers agreed that this proposal addresses highly significant questions in stem cell and neuronal biology. They noted that alternative splicing is poorly understood and the proteins involved in the splicing of specific genes in human cells have not been identified. - The reviewers agreed that, if successful, this application would move the field forward and could potentially impact the field of regenerative medicine. They noted that the discovery of mutations in splicing factor binding sites could predict disease-specific alternative splicing. - Reviewers described this as a cutting-edge proposal utilizing state-of-the-art technology. Feasibility and Experimental Design: - The reviewers raised some minor concerns about the experimental design, but ultimately concluded that the research plan is feasible and a logical extension of prior work in the applicant╒s laboratory. - They appreciated the excellent preliminary data demonstrating the feasibility of the proposed techniques. - Regarding Aim 1, a reviewer raised concerns about the gain- and loss-of-function experiments. The proteins of interest are expressed at high levels, so a meaningful level of overexpression may be difficult to achieve for gain-of-function studies. Loss-of-function studies may result in cell death, which will not be informative. The reviewer noted that while these experiments alone will be difficult to interpret, the applicant further proposes to combine gain- and loss-of-function vectors. Preliminary data supporting these approaches would have been helpful. - One reviewer described the computational modeling in Aim 3 as one of the more exciting parts of the application. - Reviewers appreciated that defined and detailed milestones are provided and both potential problems and alternative approaches are addressed. Principal Investigator (PI) and Research Team: - Reviewers described the PI as a relatively junior investigator with a strong background in stem cell biology, genomics and computational neuroscience. They praised his/her strong track record of outstanding publications in high-impact journals. - The reviewers did note that the computational and bioinformatics studies are critical to the proposal and are being lead by a postdoctoral fellow in the applicant's laboratory. They wondered if an experienced senior collaborator might benefit the project in this area. Responsiveness to the RFA: - Reviewers agreed that this proposal is fully responsive to the RFA in that it utilizes human stem cells to investigate mechanisms of cellular reprogramming, differentiation and human disease.
Conflicts: 

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