RNA-protein interactions are essential in transcription, translation, RNA processing, telomere formation and other cellular activities; in some cases, specific RNA elements involved in regulating these functions have been localized. Additionally, both from classical genetic analysis and biochemical studies and from the complete Saccharomyces cerevisiae genome sequence, numerous proteins have been identified that are potentially associated with RNA and may directly bind to it. However, the function of many of these proteins is not precisely defined, and in other cases of better- characterized proteins, the precise sequence(s) of RNA recognized is not known. The three-hybrid system is a simple genetic strategy, based on the two-hybrid system to detect protein-protein interaction, that can detect RNA-protein interactions. It uses a Lex-A-MS2 coat protein hybrid to tether a hybrid RNA to HIS3 and lacZ reporter genes that are regulated by LexA binding sites. The hybrid RNA is composed of MS2 coat protein binding sites and another RNA species (RNA X). A second hybrid protein is generated that is composed of a transcriptional activation domain and an RNA- binding domain that recognizes RNA X. The interaction of the hybrid RNA with the two hybrid proteins results in activation of the reporter genes. We propose: 1) To develop the three-hybrid assay further such that it can identify specific RNAs from a library that bind to an RNA-binding protein; and to generate a library of all possible yeast genomic sequences that will be expressed as hybrid RNAs with the MS2 coat binding sites; 2) To screen this library for RNAs that can bind to all likely yeast RNA-binding proteins, beginning with the 40 proteins containing the RNA recognition motif (RRM); and to establish a database of RNA-protein interactions; 3) To choose individual cases of RNA-protein interaction for additional mutational analysis, in order to improve the technology for this type of analysis.
| Sengupta, D J; Wickens, M; Fields, S (1999) Identification of RNAs that bind to a specific protein using the yeast three-hybrid system. RNA 5:596-601 |
