RNA–protein interactions in the yeast three-hybrid system: Affinity, sensitivity, and enhanced library screening

Abstract

The yeast three-hybrid system has become a useful tool in analyzing RNA–protein interactions. An RNA sequence is tested in combination with an RNA-binding protein linked to a transcription activation domain (AD). A productive RNA–protein interaction activates a reporter gene in vivo. The system has been used to test candidate RNA–protein pairs, to isolate mutations in each interacting partner, and to identify proteins that bind a given RNA sequence. However, the relationship between reporter gene activation and in vitro affinity of an RNA–protein interaction has not been examined systematically. This limits interpretation of the data and complicates the development of new strategies. Here, we analyze several key parameters of the three-hybrid system, using as a model the interaction of a PUF protein, FBF-1, with a range of RNA targets. We compare activation of two reporter genes as a function of the in vitro affinity of the interaction. HIS3 and LacZ expression levels are directly related to affinity over a 10-fold range of Kd. Expression of the reporter genes also is directly related to the abundance of the activation domain fusion protein. We describe a new yeast strain, YBZ1, that simplifies screening of cDNA/AD libraries. This strain possesses a tandem, head-to-tail dimer of a high-affinity variant of MS2 coat protein, fused to a monomer of the LexA DNA-binding protein. We show that the use of this strain in cDNA library screens increases the number of genuine, sequence-specific positives detected, and at the same time reduces the background of false, RNA-independent positives.

Keywords

• RNA–protein interactions
• three-hybrid system