Different modes and potencies of translational repression by sequence-specific RNA–protein interaction at the 5′-UTR

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.