This project is designed to systematically decode functional RNA elements in the human genome. The proposal integrates several recent technological advances and capitalizes the availability of the Solexa system in our lab. Drs. Fu and Yeo have extensive experimental and computational expertise in RNA research and will jointly lead the team to carry out the proposed project in three specific aims: (1) We propose to couple the CLIP (Crosslinking ImmunoPrecipitation) technology with high throughput sequencing to identify in vivo binding sites for a large number of RNA binding proteins, focusing initially on RNA binding proteins implicated in both constitutive and regulated pre-mRNA processing. The results are expected to generate novel biological and mechanistic insights into regulated RNA processing in humans. To prepare future expansion of the project, we are working with an antibody production company to systematically generate antibodies against RNA binding proteins encoded in the human genome. (2) In parallel to physical mapping, we will couple the oligo selection/ligation strategy previously developed in our lab with high throughput sequencing to perform digital analysis of alternative splicing. Having experienced all array-based systems for detecting mRNA isoforms, we believe that the current experimental design will generate quantitative information on regulated splicing with both high sensitivity and specificity by overcoming all technical problems associated with array-based approaches. (3) We will perform bioinformatics analysis of both physical mapping and functional profiling data generated in aim 1 and 2 to deduce regulatory RNA elements in the human genome. The data will be further integrated with other large-scale analysis of genomic sequences, such as computationally predicted RNA elements, conservation across genomes, and coupling with known splice sites. We believe that this project will make a unique contribution to RNA genomics as well as to our understanding of post-transcriptional regulation of gene expression in general. Project Narrative: The expression of human genes is regulated at both DNA and RNA levels. Complementary to most effort devoted to decipher functional DNA elements, this proposal focuses on functional RNA elements in the human genome by physical mapping of a large panel of RNA binding proteins coupled with functional analysis of alternative mRNA isoform expression. The experimentally generated data will be integrated with other large-scale analysis of computationally deduced RNA elements to understand general principle behind regulated gene expression at the RNA level, which has a high potential to reveal a code for regulated splicing in human cells and shed new light on various disease mechanisms.