Alternative splicing regulation in perspective: A global analysis of exon skippin

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DESCRIPTION (provided by applicant): Alternative splicing is a cellular process that is employed by higher eukaryotes to not only regulate gene expression but to also generate protein variability. During the last decade, data derived from the Genome Project as well as sequencing information extracted from EST libraries helped to define the extension of the transcriptome that is affected by this process, highlighting its importance. Just recently, this information was compiled and used in the development of specialized microarray platforms; allowing us to perform high throughput analyses and investigate alternative splicing in ways that were not possible before. We propose to use a global approach to address important questions relevant to alternative splicing regulation. Our Aims are: Specific Aim 1. We will focus our research on a particular type of alternative splicing event, named exon skipping. In this process, an "alternative exon" is either excluded or included in the mature mRNA transcript. HeLa cells will be used as a model system and 6 nuclear RNA binding proteins (RBPs) will be selected for analysis. A custom made "exon skipping microarray" will be employed to identify the subset of exon skipping events that are regulated by each selected RBP. Specific Aim 2. The CLIP method (Crosslinking and Immunoprecipitation) will be employed to identify the RNA sequences that are bound by each RBP selected for analysis. The data derived from the both microarray analysis and CLIP experiments will be explored with different tools of Computational Biology. For each selected RBP, we will identify and characterize the cis-regulatory elements it recognizes in its target genes/RNAs. Finally, a webpage with open access will be developed to serve as repository of our data set. Specific Aim 3. Putative cis-regulatory elements identified in Specific Aim 2 will be validated by in vivo and in vitro methods. For each RBP under analysis, we will select a group of target genes/RNAs to be analyzed via mini-gene system. In each case, the genomic region containing the identified exon skipping event will be cloned in an expressing vector. Deletions and mutations of the previously identified cis-regulatory elements will be done. The splicing products will be then analyzed to confirm the direct participation of these elements in exon skipping regulation. Moreover, to establish further proof of the direct involvement of the studied RBPs in splicing regulation, in vitro binding assays will be performed to determine their direct interaction with selected target RNAs and identified cis- regulatory elements. PUBLIC HEALTH RELEVANCE: Alternative splicing is a very important molecular process implicated in gene regulation. Thanks to alternative splicing a single gene can produce different protein products; in some cases these products can have different biological functions. Alterations in the proteins and elements that are responsible for the regulation of alternative splicing can lead to cancer and diseases. We propose to use novel high throughput technology to improve our knowledge in how alternative splicing is regulated and use this information to better understand its connection to cancer and diseases.