Development of Genomics and Bioinformatics Tools to Elucidate Alternative Splicing and Gene Expression Networks in Human Embryonic Stem Cells
Tools and Technologies I
The hundreds of different cell types in the human body – each with its own functional characteristics – ultimately all derive from a single type of undifferentiated precursor cell in the embryo, the pluripotent human embryonic stem cell (hESC). hESCs can proliferate indefinitely while maintaining their capacity to give rise to all cell types in the body, a process known as self-renewal. Upon appropriate developmental triggers, hESCs differentiate to generate the more specialized cells of the body, such as blood cells or cells of the nervous system. Understanding the processes governing hESC self-renewal and differentiation are critical for understanding basic mechanisms of developmental biology, and moreover will illuminate the pathways that go awry in human developmental diseases. We hope to gain a comprehensive knowledge of such processes with the ultimate goal of identifying strategies for therapeutic intervention. The genetic information in the human genome resides in units of DNA known as genes. Information from the DNA is converted to an intermediate called messenger RNA (mRNA), which in turn is used as a template for synthesizing the cell’s proteins. Almost all the approximately 25,000 genes in humans are composed of discontinuous building blocks known as exons. These exons are interleaved with intervening segments called introns. In a process known as RNA splicing, the exon segments are made continuous through the removal of the intervening introns. Alternative splicing refers to a mechanism by which the exons within a given gene are mixed and matched in any number of different combinations. This process of mixing and matching of exons results in an increase in the number and diversity of proteins encoded by the genes in the human genome. The pattern in which different exons of individual genes are spliced together in different cell types underlies the diversity and function of specialized cells and tissues of the body. Importantly, 30-40% of known mutations causing human diseases are thought to involve the mRNA splicing process. Thus, understanding the nature of alternative splicing events in human pluripotent stem cells is crucial not only for understanding how alternative splicing regulates cell differentiation, but also how defects in alternative splicing cause inherited human diseases. We propose to develop new tools and computational methods for studying alternative splicing in human pluripotent stem cells. Our approach will leverage and apply the latest technological advances in studying gene expression in complex systems. The anticipated developments should be broadly applicable to a wide range of models of stem cell biology and human disease.
Statement of Benefit to California:
The proposed project will benefit the State of California in two major ways. First, the tools and technologies we plan to develop will allow new and fundamental insights into the biological processes underlying human stem cell biology. Second, these technological advances will place scientists in California in the unique and enviable position of applying this knowledge to elucidate the causes of certain inherited human diseases and devising novel strategies for their treatment.
The goal of the proposed research is to develop new tools to evaluate alternative pre-mRNA splicing in human embryonic stem cells (hESC) and induced pluripotent stem (iPS) cells, comparing undifferentiated cells with those undergoing neural differentiation. DNA microarray analysis and high-throughput DNA sequencing will be used to characterize splicing events. New statistical and informatics approaches will be developed for analyzing and comparing levels of alternatively spliced transcripts and for associating patterns of alternative splicing with genes linked to inherited human diseases. Reviewers viewed this proposal has having outstanding potential for expanding our understanding of signaling and transcriptional networks. Information gained through the techniques developed in this project could facilitate the manipulation of regulatory pathways. The research should also make an impact by developing statistical tools and bioinformatic algorithms that can have a broader applicability for computational applications. Reviewers were divided about the importance of the work for advancing stem cell research, with one reviewer suggesting that the project could contribute to new approaches for maintaining and differentiating pluripotent cells, while another reviewer questioned the rationale or need for using hESC and iPS cells to develop the proposed computational tools for alternative RNA splicing analyses. The proposal is well written and organized, describing a logical series of experiments and software design stages to develop the bioinformatics tools for RNA analyses. The feasibility is supported by preliminary results obtained by comparing alternative splicing in two breast cancer cell lines and a proof-of-concept experiment that demonstrated good agreement between the two proposed methods for detection of splice variants. Proposed milestones and a timeline for the project are explicit and entirely reasonable. Reviewers expressed serious concerns about the heterogeneity of cell populations used for analysis and also about whether splicing profiles of cellular populations may be significantly influenced by culture conditions. The well-qualified research team is a major strength of the proposal. The principal investigator (PI) has extensive experience in gene regulation and functional genomics. This is complemented by collaborator expertise in RNA processing, stem cell culture, and statistical analysis. There was some concern that the PI lacks experience as a leader of programs in stem cell research.