T7 phage display: A novel genetic selection system for cloning RNA-bindingproteins from cDNA libraries

RNA-binding proteins are central to posttranscriptional gene regulation and play an important role in a number of major human diseases. Cloning such p roteins is a crucial but often difficult step in elucidating the biological function of RNA regulatory elements. To make it easier to clone proteins t hat specifically bind RNA elements of interest, we have developed a rapid a nd broadly applicable in vitro genetic selection method based on T7 phage d isplay. Using hairpin II of U1 small nuclear RNA (U1hpII) or the 3' stem lo op of histone mRNA as bait, we could selectively amplify T7 phage that disp lay either the spliceosomal protein U1A or the histone stem loop-binding pr otein 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 cog nate RNA targets with relatively weak affinities (K-d as high as the microm olar range). Experiments with a mixture of recombinant phage displaying U1A or the closely related protein U2B " demonstrated that addition of a compe titor RNA can suppress selection of a protein with a higher affinity for a given RNA target, thereby allowing the preferential amplification of a lowe r affinity protein. Together, these findings suggest that T7 phage display can be used to rapidly and selectively clone virtually any protein that bin ds a known RNA regulatory element, including those that bind with low affin ity or that must compete for binding with other proteins.