All mRNA molecules are subject to posttranscriptional gene regulation (PTGR) involving sequence-dependent modulation of splicing, cleavage and polyadenylation, editing, transport, stability, and translation. In recent years, a paradigm shift has occurred in our understanding of PTGR, triggered by discoveries of regulatory RNAs and extensive RNA processing including alternative polyadenylation and splicing. These developments have led to the realization that virtually every human gene is subject to some degree of PTGR. Understanding the mechanisms of target RNA recognition, and the function of the hundreds of mRNA-binding proteins encod- ed in the human genome, is therefore an open challenge that requires the joint effort of multidisciplinary teams. The recent introduction of deep sequencing technologies has enabled the development of new methods for precisely mapping interaction sites between RNA-binding proteins (RBPs) and their RNA target sites in human cells. It is only now possible to resolve interdependencies and redundancies of binding of RBPs and ribonucleoprotein particles (RNPs) to mRNA molecules and evaluate their contribution to gene regulation in the context of organismal development or normal and disease states. The broad aim of this application is to identify and characterize the interaction networ of mRNA-binding proteins at the sequence, structural and functional level, with a particular focus on transport and shuttling - crucial PTGR mechanisms that have received little attention. A driving concept is whether larger "chromatin"- like packaging of RNPs facilitates transport and translational regulation.
The specific aims i nclude: (1) Global identification of target RNA site for a comprehensive panel of nucleocytoplasmic transport and shuttling proteins by Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation (PAR- CLIP) involving deep sequencing of libraries for all proteins and computational analysis. (2) Integrated annotation of binding sites on transcripts across libraries, using a probabilistic graphical modeling approach to identify prevalent site configurations from heterogenenous data. PAR-CLIP data, complemented by expression as well as sequence and RNA structural features, will allow for the identification of targets for combinatorial and redundant regulation. (3) Development of experimental systems for assessment of the phenotypic outcomes of the perturbation of the transporter interaction network and elucidation of its role in normal and disease states. (4) Perform biophysical and structural studies substantiating the biochemical and computational findings. Natural, as well as designed RNA-recognition element (RRE)-representing RNA ligands, will by co-crystallized with their respective recombinant proteins placing emphasis on obtaining larger RNP structures. The proposed work will thus establish the first theoretical and experimental models that relate global maps of protein-RNA interactions to RNA transport, setting the stage for future systems-wide studies of PTGR.
The control of gene expression at the mRNA level plays a larger role than previously anticipated. This proposal aims to resolve the joint contribution of dozens of mRNA-binding proteins shuttling between nucleus and cytoplasm, as well of proteins participating in nucleocytoplasmic transport. This approach will identify numerous important regulatory regions in mRNAs, as well as uncover mRNAs and RNA-binding proteins particularly vulnerable to mutation and deregulation in disease states.
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