T7 phage display: a novel genetic selection system for cloning RNA-binding proteins 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 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.
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Skirball Institute of Biomolecular Medicine and Department of Microbiology, New York University School of Medicine, New York, NY 10016, USA.
Belasco
J G
JG
D017123:Bacteriophage T7 / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D018076:DNA, Complementary; D006801:Humans; D008168:Lung / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism; D011995:Recombination, Genetic; D012641:Selection, Genetic
D017931:DNA Primers; D018076:DNA, Complementary; D016601:RNA-Binding Proteins