Protein-RNA interactions make a profound contribution to the regulatory logic of post- transcriptional control of gene expression. Tools to decipher this intricate layer of molecular interactions on a global scale are coming to the forefront of molecular biology and genomics. The most wide spread methodology to examine interactions of RNA binding proteins (RBPs) with their targets is an IP-microarray plagued by several experimental pitfalls. The cross-linking immunoprecipitation (CLIP) method overcomes many of these pitfalls while facilitating identification of RNA targets as well as the in situ bindng sites. Developing a novel tool to decipher this intricate layer of molecular interaction is a focusof this proposal. During development, we will identify noise arising from clonal reads and improve the sensitivity of the procedure. Analytical techniques will be developed to allow modeling of ligation efficiency and estimate clonal biases accurately. The ultimate goal of the project is to create minimally biased CLIP-Seq based libraries that are easily and fairly interrogated. Our proposal to develop technology to study these RNAs in an unbiased, high-throughput manner should make future clinical applications a reality and propel research in comparative tissue disease profiles, further un-locking transcriptional regulation.
Despite the contribution of protein-RNA interactions to the transcriptional control of gene expression and ever increasing involvement of RNA binding proteins and aberrant RNA processing decisions in human disease, powerful methods of inquiry remain inaccessible. Our goal is to develop a streamlined sequencing tool to decipher this intricate layer of molecular interaction. Such a kit will have a major impact on areas where protein-RNA interactions intersect with physiological relevant problems such as cancer and neurodegenerative diseases.