Book of Abstracts: Albany 2009
June 16-20 2009
© Adenine Press (2008)
RNA-Protein Interactions Reveal Alternative Splicing Networks in Human Embryonic Stem Cells
Understanding regulated gene expression is vital to providing insights into disease and development. While much effort has been placed on deciphering transcriptional regulation by more than a thousand transcription factors and their interactions with functional DNA elements encoded in mammalian genomes, little is known about an equally sizable number of RNA binding proteins and their involvement in diverse aspects of RNA metabolism. A dominant function of these RNA binding proteins is to regulate alternative splicing, a major form of post-transcriptional regulation of gene expression that is thought to contribute to the structural and functional diversity of the proteome of the cell. One of the ultimate goals in the RNA field is to deduce a set of rules that govern the control of splice site selection to produce the ?splicing code?. Human embryonic stem cells (hESCs) are pluripotent cells with the capacity to self renew and differentiate into the three germ layers. Neuronal progenitor cells (NP) are multipotent cells that can theoretically generate all lineages in the central nervous system. Both are excellent models that recapitulate early neuronal development in vitro, and have motivated studies to identify gene regulatory programs that control neuronal specification. Using a combination of splicing-sensitive arrays and comparative genomics, we revealed candidate intronic cis-regulatory elements such as the Fox2 binding site GCAUG proximal to candidate alternative exons that may participate in the regulation of alternative splicing during neural differentiation of hESCs. This motivated our application of a general strategy for decoding functional RNA elements in vivo by constructing an RNA map for the cell type-specific splicing regulator Fox2 via CrossLinking-ImmunoPrecipitation coupled with high throughput sequencing (CLIP-seq) in hESCs. The map reveals that Fox2 binds to a cohort of specific RNA targets, many of which are themselves splicing regulators, and induces position-dependent exon inclusion or skipping. This finding suggests that Fox2 functions as a regulator of a network of splicing factors, and we show that Fox2 is important for the survival of human embryonic stem cells.
Gene W. Yeo*
Cellular and Molecular Medicine