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: 9.81). 03/1998; 95(3):951-6. DOI: 10.1073/pnas.95.3.951
Source: PubMed

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 protein binding as well as the protein domains required for RNA binding, and to study effectors of RNA-protein interactions in vivo.

Download full-text


Available from: Efrosyni Paraskeva, Aug 26, 2015
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Novel RNA-binding proteins with customized specificities can be isolated by genetic selection from combinatorial libraries. Such proteins have great potential as agents for targeted manipulation of gene expression.
    Nature Structural Biology 09/1998; 5(8):665-8. DOI:10.1038/1356
  • [Show abstract] [Hide abstract]
    ABSTRACT: A variety of assay technologies continue to be developed for high-throughput screening. These include cell-based assays, surrogate systems using microbial cells such as yeast and bacterial two-hybrid and three-hybrid systems, and systems to measure nucleic acid-protein and receptor-ligand interactions. Modifications have been developed for cell-free, homogeneous assay systems, such as time-resolved fluorescence, fluorescence polarization and the scintillation proximity assay. Innovations in engineering and chemistry have led to delivery systems for nanoliter volumes and sensitive biosensors for ultra-high-throughout screening conducted in nanoliter and picoliter volumes. Spectroscopic methods have been extended to read single molecule fluorescence. Technologies are being developed to identify new targets from genomic information in order to design the next generation of screens.
    Current Opinion in Chemical Biology 11/1998; 2(5):597-603. DOI:10.1016/S1367-5931(98)80089-6 · 7.65 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The cap structure and the poly(A) tail synergistically activate mRNA translation in vivo. Recent work using Saccharomyces cerevisiae spheroplasts and a yeast cell-free translation system revealed that the poly(A) tail can function as an independent promotor for ribosome recruitment, to internal initiation sites within an mRNA. This raises the question of how regulatory upstream open reading frames and translational repressor proteins binding to the 5'UTR can function, as well as how regulated polyadenylation can support faithful activation of protein synthesis. We investigated the function of the regulatory upstream open reading frame 4 from the yeast GCN 4 gene and the effect of IRP-1 binding to an iron-responsive element introduced into the 5' UTR of reporter mRNAs. Both manipulations effectively block cap-dependent translation, whereas ribosome recruitment promoted by the poly(A) tail under non-competitive conditions can efficiently bypass both blocks. We show that the synergistic use of both, the cap structure and the poly-A tail enforced by mRNA competition reinstates the full extent of translational control by both types of 5' UTR regulatory elements. With a view towards regulated polyadenylation, we studied the function of poly(A) tails of defined length on the translation of capped mRNAs. We find that poly(A) tail elongation increases translational efficiency, particularly under competitive conditions. Our results integrate recent findings on the function of the poly(A) tail into an understanding of translational control.
    RNA 12/1998; 4(11):1321-31. DOI:10.1017/S1355838298980669 · 4.62 Impact Factor
Show more