Definition of Protein Networks using RNA Display

Principal Investigator: Jack W. Szostak

Overview

The individual steps in signal transduction pathways involve protein interactions with target molecules that may be other proteins, small molecules or DNA. Identifying all of the proteins that take part in a given class of interactions, on a genome-wide scale, remains an extremely challenging task. We propose to apply mRNA display (1, 2) technology to this problem, with the goal of developing databases of protein-ligand interactions that will add value to the existing and growing sequence databases. In mRNA display, proteins or peptides are covalently linked to their own mRNA; enrichment of a protein or peptide on the basis of an interaction such as binding to a target protein or ligand allows ready identification of the enriched protein on the basis of its attached mRNA, e.g. by sequencing or DNA microarray readout. The technology is well-suited to genomic screening for functional interactions, because complex mRNA-display libraries representing all proteins and protein domains can easily be generated. We propose to initially apply this approach to a class of signaling interactions that are important to the tissue and disease states that are the subject of this proposal - PDZ-domain::peptide interactions. These and subsequent experiments aimed at other classes of interactions will generate large networks of protein::protein interactions, and may lead to the identification of previously unknown signaling molecules that play important roles in mediating disease states.

Specific Aims:

1. To create a cellular protein-RNA fusion library from pooled mRNA from normal human and mouse tissues
2. To use isolated domains from proteins transducing stress and inflammation pathway signals to identify interaction partners of those proteins
3. To automate the detection of interactions between signal transduction proteins and their target proteins.

References:

1. Roberts, R. W., and J. W. Szostak. 1997. RNA-peptide fusions for the in vitro selection of peptides and proteins. Proc Natl Acad Sci U S A 94:12297-302.
2. Liu R, B. J., Szostak JW, Roberts RW. 2000. Optimized synthesis of RNA-protein fusions for in vitro protein selection. Methods in Enzymol. in press.

Learn More

To learn more abou the Definition of Protein Networks using RNA Display project, visit the Szostak Lab Web Site.