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

European Molecular Biology Laboratory, Meyerhofstrasse 1, D-69117 Heidelberg, Germany.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 03/1998; 95(3):951-6. DOI: 10.1073/pnas.95.3.951
Source: PubMed


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.

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    • "Content is final as presented, with the exception of pagination. in carcinogenesis [57]. Signal inputs from cytoplasmic protein-protein interactions or protein localization can be directly utilized as an input for translational regulation [63], [76], [62]. Finally, the metabolic burden imposed on the cell may be alleviated via fine tuning translation, since the competition for translational resources is generally greater than the competition for transcriptional resources [145]. "
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    • "Attempts at achieving such a ligand-regulated RNA–protein interaction have been described previously (10–15). However, in these examples, the interaction either cannot be modulated in vivo (12), requires a ubiquitous and stringently regulated metabolite ligand such as iron (11), or relies on inducible transcription to control the RNA-binding protein (14,15). "
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    • "This phenomenon has also been used in a proof-of-concept experiment to screen for a RNA-binding activity in yeast cells (18). However, given typical translational repression in a reporter assay at about one order of magnitude (10,15–22,43–45), the proof-of-concept experiment required multiple rounds of cell sorting with a fluorescence-activated cell sorter on an initial pool of stable yeast transformants to isolate yeast with a particular RNA-binding activity (18). Based on the same biological phenomenon, we sought an alternative strategy that permits direct assessment of a specific RNA-binding activity and translational repression from a single pass examination of transiently transfected living mammalian cells, as normally encountered in a microscope field. "
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