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

Patterns and plasticity in RNA-protein interactions enable recruitment of multiple proteins through a single site

“…Amino acids mutated to alanine by site-directed mutagenesis are indicated in Table S3. Mutant RNA sequences were expressed using the p3HR2 vector as in the study by Stumpf et al (48) and their binding assayed as described (32,34). Within an experiment, the relative β-gal levels for each protein were normalized to that of the protein binding to its canonical WT site (Puf3p to 3BE, Puf4p to 4BE, Puf5p to 5BE, and FBF-2 to FBE), which was set to 100%.…”

“…HIS3-HO 3′UTR reporter plasmids were modified based on the work of Hook et al (34) (SI Materials and Methods). The p415-glyceraldehyde-3-phosphate dehydrogenase (GPD) Puf4p is as described (3).…”

“…A PUF protein fused to the GAL4 activation domain was expressed in cells carrying a hybrid RNA with the PBE of interest. Binding of the PUF and RNA triggers LacZ expression, which correlates with the affinity of the interaction (34). Analysis of the three PUF/RNA complexes follows.…”

Unbiased selection of localization elements reveals cis-acting determinants of mRNA bud localization in Saccharomyces cerevisiae

“…Although targeted mutatgenesis had revealed weak sequence preferences in the two loop-proximal base pairs, no biases were observed by randomization͞selection ( Fig. 5c; see also Table 7, which is published as supporting information on the PNAS web site), possibly because 3-AT selection does not discriminate between modest differences in three-hybrid activity (24). Surprisingly, we recovered a bias toward the C 1283 C dinucleotide in the 5Ј strand of the stem and a weaker bias for G 1300 ( Fig.…”

Pyrrolysine is not hardwired for cotranslational insertion at UAG codons

“…The yeast three-hybrid experiments were performed as described (29). The effect of point mutations in tRNA Pyl on PylRS binding was measured by using the in vivo yeast three-hybrid method.…”