A translational repression assay procedure (TRAP) for RNA-protein interactions in vivo.
RNA-protein interactions are central to many aspects of cellular metabolism, cell differentiation, and development as well as the replication of infectious pathogens. We have devised a versatile, broadly applicable in vivo system for the analysis of RNA-protein interactions in yeast. TRAP (translational repression assay procedure) is based on the translational repression of a reporter mRNA encoding green fluorescent protein by an RNA-binding protein for which a cognate binding site has been introduced into the 5' untranslated region. Because protein binding to the 5' untranslated region can sterically inhibit ribosome association, expression of the cognate binding protein causes significant reduction in the levels of green fluorescent protein fluorescence. By using RNA-protein interactions with affinities in the micromolar to nanomolar range, we demonstrate the specificity of TRAP as well as its ability to recover the cDNA encoding a specific RNA-binding protein, which has been diluted 500,000-fold with unrelated cDNAs, by using fluorescence-activated cell sorting. We suggest that TRAP offers a strategy to clone RNA-binding proteins for which little else than the binding site is known, to delineate RNA sequence requirements for protein binding as well as the protein domains required for RNA binding, and to study effectors of RNA-protein interactions in vivo.
Paraskeva
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E
European Molecular Biology Laboratory, Meyerhofstrasse 1, D-69117 Heidelberg, Germany.
Atzberger
A
A
Hentze
M W
MW
D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002213:Capsid / Q000378:metabolism; D036022:Capsid Proteins; D002469:Cell Separation; D003001:Cloning, Molecular; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism; D005434:Flow Cytometry; D017930:Genes, Reporter; D005821:Genetic Techniques; D049452:Green Fluorescent Proteins; D035925:Iron-Regulatory Proteins; D007506:Iron-Sulfur Proteins / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D011485:Protein Binding / Q000235:genetics; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000378:metabolism*; D012342:RNA, Small Nuclear / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics; D017412:Ribonucleoprotein, U1 Small Nuclear / Q000378:metabolism; D012441:Saccharomyces cerevisiae
D036022:Capsid Proteins; D035925:Iron-Regulatory Proteins; D007506:Iron-Sulfur Proteins; D008164:Luminescent Proteins; D012333:RNA, Messenger; D012342:RNA, Small Nuclear; D016601:RNA-Binding Proteins; D017412:Ribonucleoprotein, U1 Small Nuclear; C075339:U1A protein; D049452:Green Fluorescent Proteins; C073587:MS2 polymerase; D012324:RNA-Dependent RNA Polymerase; D012321:DNA-Directed RNA Polymerases