Identification of ribonucleoprotein (RNP)-specific protein interactions using a yeast RNP interaction trap assay (RITA)

We describe an adaptation of the yeast three-hybrid system that allows the reconstitution in vivo of tripartite (protein-ANA-protein) ribonucleoprotei ns (RNPs). To build and try this system that we called RNP interaction trap assay (RITA), we used asa model the autoantigenic Ro RNPs. hY RNAs bear di stinct binding sites for Ro60 and La proteins, and Ro RNPs are thus physiol ogically tripartite (Ro60/hY RNA/La). Using recombinant La (rLa) and Ro60 ( rRo60) proteins and recombinant hY RNAs (rhY) co-expressed in yeast, we fou nd that RNPs made of rRo60/rhY/rLa were readily reassembled. Reconstitution of tripartite RNPs was critically dependent on the presence of an appropri ate Ro60 binding site on the recombinant RNA. The RITA assay was further us ed to detect (rRo60/rhY RNP)-binding proteins from a HeLa cell cDNA library , allowing specific identification of La and of a novel Ro RNP-binding prot ein (RoBPI) in more than 70% of positive clones. RITA assay may complement already available two- and three-hybrid systems to characterize RNP-binding proteins by allowing the in vivo identification of interactions strictly d ependent upon the simultaneous presence of a protein and of its cognate RNA .