Selections that optimize RNA display in the yeast three-hybrid system

Abstract

The yeast three-hybrid system (Y3H) is a powerful tool to select or confirm RNA-protein interactions. Target protein recognition of an RNA insert within a test transcript depends on at least three factors: intrinsic protein affinity for the properly folded insert, retention of RNA insert tertiary structure within a longer RNA transcript, and accessibility of the RNA insert to the target protein. Y3H reporter gene readout reflects the combination of these factors. Here, we discuss RNA insert tertiary structure and accessibility in the Y3H as "RNA display." We review evidence that RNA display can sometimes be optimized during Y3H selections that do not increase the intrinsic affinity of an RNA insert for a target protein. This situation is more likely when a library of RNA inserts and heterogeneous flanking sequences is subjected to selection, and is less likely when point mutations are targeted to the insert in a fixed context. An RNA display vector with enhanced modularity has been developed to minimize sequence context effects in the Y3H.

FIGURE 1.

Schematic illustration of Y3H. (A) The RNA insert (red) is expressed in the context of RNA vector sequences (black) tethered upstream of lacZ (brown) and HIS3 reporter genes via a MS2 coat–LexA fusion protein (blue and black). Gene activation depends on binding of the Gal4 activation domain (yellow) –prey fusion protein (green).

FIGURE 2.

Theoretical contributions to Y3H readout. Y3H readout reflects the combination of the intrinsic affinity of an isolated RNA insert for a protein target as measured in a biochemical experiment (black), RNA insert tertiary structure in the context of the Y3H transcript (gray), and accessibility of the RNA insert at the reporter gene promoter in vivo (white). (A) Correlation between intrinsic affinity and Y3H readout for RNAs 1–3 because display factors are constant. (B) Lack of correlation between intrinsic target affinity and Y3H readout for RNAs 4–6 because of differing display. (C) Different Y3H readouts for RNAs 7–9 with comparable intrinsic target affinities because of differing display.

FIGURE 3.

Optimization of Y3H readout by selection. (A) An RNA insert (red) selected in isolation for proper folding to interact with target (gray polygon)/activation domain (white triangle) fusion protein. (B) Selection for flanking RNA sequences (black) that enhance proper local tertiary structure context (gray) for the RNA insert. (C) Selection for flanking RNA sequences that enhance proper global folding of display RNA. (D) Selection for flanking RNA sequences that promote accessibility of the RNA insert to target the protein-binding domain. (E) Selection for flanking RNA sequences that enhance access of bound target protein to transcriptional cofactors. (F) Selection for flanking RNA sequences that destabilize a competing complex with yeast protein “X” (black rectangle), enhancing accessibility of RNA insert.

FIGURE 4.

Steric considerations in the Y3H system. (A) Multiple LexA (black circles)/MS2 coat (gray circles) dimers are bound to yeast promoter DNA near lacZ and HIS3 reporter genes. (B) The RNA insert is displayed in the context of multiple dynamic RNA–protein complexes. Y3H readout reflects the intrinsic affinity of the target protein for the RNA insert, but also contributions of RNA display (RNA tertiary structure and accessibility).

FIGURE 5.

Anti-p50 Y3H selections and affinity analysis in vitro (Cassiday and Maher 2003). (A) Predicted secondary structure of in vitro selected anti-p50 RNA aptamer (left) compared to predicted secondary structure Y3H-selected variant from early in vitro selection RNA library (right). (B) Comparison of aptamer activities in Y3H assays. 3-AT gradient plate yeast growth image (left) and liquid β-galactosidase assays values (right) compare HIS3 and lacZ reporter gene signals. (C) Binding isotherms for isolated RNA aptamer complexes with p50₂ showing similar affinities despite different Y3H readouts.

FIGURE 6.

Anti-p65 Y3H selections and affinity analysis in vitro (Wurster et al. 2009). (A) Comparison of predicted secondary structures for R1, Y1, and Y3 anti-p65 RNA aptamers. (Circles) 5′ RNA termini. (B) In vitro binding isotherms for isolated RNA aptamer complexes with p65₂.

FIGURE 7.

Comparison of Y3H RNA expression cassettes. (A) Transcript from conventional RNA expression cassette. (B) T-cassette design (Wurster et al. 2009) to enhance modularity (folding autonomy) and accessibility of RNA inserts emanating from a four-way helical junction. (Black dots) Transcript 5′ termini. Transcript 3′ terminal sequences are truncated for clarity. Bacteriophage MS2 hairpins are labeled. (Arrows) Cloning sites.