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 . Signal inputs from cytoplasmic protein-protein interactions or protein localization can be directly utilized as an input for translational regulation , , . 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 . "
[Show abstract][Hide abstract] ABSTRACT: In contrast to the versatility of regulatory mechanisms in natural systems, synthetic genetic circuits have been so far predominantly composed of transcriptionally regulated modules. This is about to change as the repertoire of foundational tools for post-transcriptional regulation is quickly expanding. We provide an overview of the different types of translational regulators: protein, small molecule and ribonucleic acid (RNA) responsive and we describe the new emerging circuit designs utilizing these tools. There are several advantages of achieving multilevel regulation via translational switches and it is likely that such designs will have the greatest and earliest impact in mammalian synthetic biology for regenerative medicine and gene therapy applications.
IEEE Transactions on Biomedical Circuits and Systems 08/2015; 9(4). DOI:10.1109/TBCAS.2015.2451707 · 2.48 Impact Factor
"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). "
[Show abstract][Hide abstract] ABSTRACT: Sequence-specific RNA-protein interactions, though commonly used in biological systems to regulate translation, are challenging to selectively modulate. Here, we demonstrate the use of a chemically-inducible RNA-protein interaction to regulate eukaryotic translation. By genetically encoding Tet Repressor protein (TetR)-binding RNA elements into the 5'-untranslated region (5'-UTR) of an mRNA, translation of a downstream coding sequence is directly controlled by TetR and tetracycline analogs. In endogenous and synthetic 5'-UTR contexts, this system efficiently regulates the expression of multiple target genes, and is sufficiently stringent to distinguish functional from non-functional RNA-TetR interactions. Using a reverse TetR variant, we illustrate the potential for expanding the regulatory properties of the system through protein engineering strategies.
Nucleic Acids Research 01/2012; 40(9):e64. DOI:10.1093/nar/gks028 · 9.11 Impact Factor
"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. "
[Show abstract][Hide abstract] ABSTRACT: To determine whether sequence-specific RNA-protein interaction at the 5'-untranslated region (5'-UTR) can potently repress translation in mammalian cells, a bicistronic translational repression assay was developed to permit direct assessment of RNA-protein interaction and translational repression in transiently transfected living mammalian cells. Changes in cap-dependent yellow fluorescent protein (YFP) and internal ribosome entry sequence (IRES)-dependent cyan fluorescent protein (CFP) translation were monitored by fluorescence microscopy. Selective repression of YFP or coordinate repression of both YFP and CFP translation occurred, indicating two distinct modes by which RNA-binding proteins repress translation through the 5'-UTR. Interestingly, a single-stranded RNA-binding protein from Bacillus subtilis, tryptophan RNA-binding attenuation protein (TRAP), showed potent translational repression, dependent on the level of TRAP expression and position of its cognate binding site within the bicistronic reporter transcript. As the first of its class to be examined in mammalian cells, its potency in repression of translation through the 5'-UTR may be a general feature for this class of single-stranded RNA-binding proteins. Finally, a one-hybrid screen based on translational repression through the 5'-UTR identified linkers supporting full-translational repression as well as a range of partial repression by TRAP within the context of a fusion protein.
Nucleic Acids Research 02/2006; 34(19):5528-40. DOI:10.1093/nar/gkl584 · 9.11 Impact Factor