Abstract
The RNA-binding protein TIAR is an mRNA-binding protein that acts as a translational repressor, particularly important under conditions of cellular stress. It binds to target mRNA and DNA via its RNA recognition motif (RRM) domains and is involved in both splicing regulation and translational repression via the formation of "stress granules." TIAR has also been shown to bind ssDNA and play a role in the regulation of transcription. Here we show, using surface plasmon resonance and nuclear magnetic resonance spectroscopy, specific roles of individual TIAR domains for high-affinity binding to RNA and DNA targets. We confirm that RRM2 of TIAR is the major RNA- and DNA-binding domain. However, the strong nanomolar affinity binding to U-rich RNA and T-rich DNA depends on the presence of the six amino acid residues found in the linker region C-terminal to RRM2. On its own, RRM1 shows preferred binding to DNA over RNA. We further characterize the interaction between RRM2 with the C-terminal extension and an AU-rich target RNA sequence using NMR spectroscopy to identify the amino acid residues involved in binding. We demonstrate that TIAR RRM2, together with its C-terminal extension, is the major contributor for the high-affinity (nM) interactions of TIAR with target RNA sequences.