PMID: 9448266Mar 14, 1998Paper

A translational repression assay procedure (TRAP) for RNA-protein interactions in vivo

Proceedings of the National Academy of Sciences of the United States of America
E ParaskevaM W Hentze

Abstract

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...Continue Reading

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Citations

Feb 27, 1999·Current Opinion in Biotechnology·M AfsharG Varani
Jun 11, 2002·Methods : a Companion to Methods in Enzymology·Somashe NiranjanakumariMariano A Garcia-Blanco
Nov 18, 1998·Current Opinion in Chemical Biology·P B Fernandes
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Dec 18, 2009·Nature Chemical Biology·Hirohide SaitoTan Inoue
Jan 20, 2011·Nature Communications·Hirohide SaitoTan Inoue
Sep 4, 2013·Nature Communications·Kei EndoHirohide Saito
Jan 26, 2012·Nucleic Acids Research·Stephen J GoldflessJacquin C Niles
Oct 19, 2001·Proceedings of the National Academy of Sciences of the United States of America·S Danner, J G Belasco
Oct 3, 2014·Nucleic Acids Research·Andrew B KennedyChristina D Smolke
Sep 4, 2015·IEEE Transactions on Biomedical Circuits and Systems·Margarita B KopniczkyPaul S Freemont
Sep 6, 2005·Molecular and Biochemical Parasitology·Huafang ShiElisabetta Ullu
Jun 23, 2012·Nature·Simon AusländerMartin Fussenegger
Dec 7, 2007·Biotechnology & Genetic Engineering Reviews·Maung Nyan Win, Christina D Smolke
Oct 13, 2018·Synthetic Biology·Tim WeeninkTom Ellis
Apr 6, 2001·Analytical Chemistry·F LisdatR Brigelius-Flohé

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