Nucleic Acids Research Advance Access published online on October 5, 2006
Nucleic Acids Research, doi:10.1093/nar/gkl584
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Molecular Biology |
Different modes and potencies of translational repression by sequence-specific RNA–protein interaction at the 5'-UTR
1 Department of Obstetrics and Gynecology, Molecular Biology Institute University of California Los Angeles-Jonsson Comprehensive Cancer Center, 22-168 CHS, David Geffen School of Medicine at UCLA, 10833 Le Conte Avenue, Box 951740, Los Angeles, CA 90095-1740, USA 2 Department of Molecular and Medical Pharmacology, Molecular Biology Institute University of California Los Angeles-Jonsson Comprehensive Cancer Center, 22-168 CHS, David Geffen School of Medicine at UCLA, 10833 Le Conte Avenue, Box 951740, Los Angeles, CA 90095-1740, USA
*To whom correspondence should be addressed. Tel: +1 310 206 3015; Fax: +1 310 206 3670; Email: hhtun{at}mednet.ucla.edu
Received June 16, 2006. Revised July 24, 2006. Accepted July 25, 2006.
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.