T7 phage display: a novel genetic selection system for cloning RNA-binding proteins from cDNA libraries

Abstract

RNA-binding proteins are central to posttranscriptional gene regulation and play an important role in a number of major human diseases. Cloning such proteins is a crucial but often difficult step in elucidating the biological function of RNA regulatory elements. To make it easier to clone proteins that specifically bind RNA elements of interest, we have developed a rapid and broadly applicable in vitro genetic selection method based on T7 phage display. Using hairpin II of U1 small nuclear RNA (U1hpII) or the 3' stem loop of histone mRNA as bait, we could selectively amplify T7 phage that display either the spliceosomal protein U1A or the histone stem loop-binding protein from a lung cDNA phage library containing more than 10(7) independent clones. The use of U1hpII mutants with various affinities for U1A revealed that this method allows the selection even of proteins that bind their cognate RNA targets with relatively weak affinities (K(d) as high as the micromolar range). Experiments with a mixture of recombinant phage displaying U1A or the closely related protein U2B" demonstrated that addition of a competitor RNA can suppress selection of a protein with a higher affinity for a given RNA target, thereby allowing the preferential amplification of a lower affinity protein. Together, these findings suggest that T7 phage display can be used to rapidly and selectively clone virtually any protein that binds a known RNA regulatory element, including those that bind with low affinity or that must compete for binding with other proteins.

Figure 1

In vitro selection of RNA-BP cDNAs by using T7 phage display. An RNA-BP cDNA is inserted into a T7 cloning vector and packaged in a phage capsid to generate a recombinant phage in which the RNA-BP is displayed on the surface as a carboxyl-terminal fusion to the T7 capsid protein 10B. The resulting phage is allowed to bind to the RNA bait, which itself is annealed to a biotinylated DNA oligonucleotide. RNA-bound T7 phage are captured on streptavidin-coated paramagnetic beads, separated from other members of the phage mixture with a magnet, and used to infect E. coli without prior release from the beads. After replication, the phage progeny can be subjected to additional rounds of selection.

Figure 2

RNA stability in phage lysates generated from different E. coli host strains. Radiolabeled U1hpII RNA (5 pmol) was mixed with either of two T7 phage lysates (5 × 10⁹ pfu) prepared from E. coli BLT5615 or RNA5615 (an isogenic strain deficient in RNase I). After incubation at room temperature for the times indicated, RNA samples were analyzed by electrophoresis on a nondenaturing polyacrylamide gel.

Figure 3

Selection of U1A-displaying phage from phage pools of various complexities. (A) Selection of recombinant U1A-displaying phage after dilution with a 10⁶-fold excess of bare phage. The phage mixture (2 × 10⁹ pfu) was incubated with each of three bait RNAs (100 nM): U1hpII, a U1hpII variant (U1hpII_mut1), or U2hpIV. Multiple rounds of in vitro selection were performed as outlined in Fig. 1. The relative abundance of U1A-displaying phage in the original phage mixture (denoted round 0) and after each round of selection was monitored by PCR analysis with primers flanking the site of cDNA insertion. Signals corresponding to the U1A-displaying phage and the bare phage are indicated. (B) Isolation of U1A-displaying phage from a human lung cDNA library comprising 1.2 × 10⁷ independent clones. In vitro selection from the phage library (2.5 × 10⁹ pfu) was performed as in A, using the same three bait RNAs (100 nM) but a different reverse primer for PCR analysis. The cDNA insert of the phage clone isolated in this manner was shown by sequence analysis to encode full-length U1A. (C) Schematic representation of the three RNA stem loop structures used in these experiments. The previously determined affinity of each for U1A (K_d) is indicated (23). The nucleotide changes in U1hpII_mut1 and U2hpIV that are responsible for the diminished affinity of U1A for these stem loops are highlighted. In addition to the stem loop structures shown, each RNA contained four or six additional base pairs at the bottom of the stem, a short 5′ leader, and a 3′ tail that had been annealed to a complementary 5′-biotinylated DNA oligonucleotide before selection.

Figure 4

Isolation of an SLBP-displaying phage from a lung cDNA phage library. (A) In vitro selection from a human lung cDNA library was performed as in Fig. 3, except that the 3′-terminal stem loop of histone mRNA (120 nM) was used as the RNA bait. Sequence analysis confirmed that the cDNA insert in the amplified phage clone encoded SLBP. (B) Structure of the 3′ histone RNA stem loop used in this experiment. In addition, this RNA bore a 5′ leader and a 3′ tail annealed to a complementary biotinylated DNA oligonucleotide (the same as in Fig. 3C). (C) Schematic representation of full-length human SLBP (13, 14) and of the amino-terminally truncated form of SLBP (amino acid residues 87–270) displayed on the surface of the isolated phage clone. The region of SLBP comprising its minimal RNA-binding domain (residues 126–198) is shown in black (13).

Figure 5

Suppression of a high affinity phage clone by the use of a competitor RNA. An equimolar mixture of recombinant phage displaying U1A or U2B" was diluted with a 10⁶-fold excess of bare phage. U1hpII_mut2 (50 nM) bearing a 3′ tail annealed to a biotinylated DNA oligonucleotide was used as the RNA bait. Wild-type U1hpII (0 or 25 nM) that had not been annealed to biotinylated DNA served as a competitor. All other conditions of in vitro selection were identical to those described for Fig. 3A. The K_d values previously determined for dissociation of U1hpII_mut2 and U1hpII from U1A and U2B" are indicated (23).