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. 2013 Apr;10(4):579-89.
doi: 10.4161/rna.24341. Epub 2013 Apr 1.

Distinct binding properties of TIAR RRMs and linker region

Henry S Kim  1 , Stephen J Headey, Yano M K Yoga, Martin J Scanlon, Myriam Gorospe, Matthew C J Wilce, Jacqueline A Wilce
Affiliations

Distinct binding properties of TIAR RRMs and linker region

Henry S Kim et al. RNA Biol. 2013 Apr.

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.

Keywords: C-terminal extension; NMR; RNA-binding protein; RRM; TIA-1; TIAR; surface plasmon resonance (SPR); translational regulation.

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Figures

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Figure 1. TIA protein sequence alignment and domain structure. (A) Sequence alignment of TIA protein isoforms highlighting secondary structural elements within the RRMs as derived from structural information (in white font) and positions of RNP motifs (boxed). (B) Cartoon representations of TIAR RRM domains determined by NMR (PDB IDs: 2CQI; 2DH7, 1X4G) showing labeled secondary structural elements. (C) Schematic showing the bounds of TIAR constructs used in the current and a previous study.
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Figure 2. Kinetic analysis of the interactions of different TIAR constructs with U-rich RNA using SPR. The binding of (A) TIAR1, (B) TIAR2S, (C) TIAR12S and (D) TIAR2L to a U-rich RNA is shown. Biotinylated RNA was captured on SA-coated sensor chip and increasing concentrations of protein were injected over the surface. Injections were performed for 120 sec (association phase), followed by a 300 sec flow of running buffer to assess dissociation. The experiments were conducted in duplicate and showed good overlap. The red line represents the binding responses for injections of protein analyte at specified concentration over the RNA surface. The kinetic data were fit by a 1:1 Langmuir binding model. Mass transport effects were not evident. The black curves superimposed on top of the sensor grams represent the model fitted curves. The rate constants ka and kd were determined simultaneously as global fitting parameters from which the KD was determined. The resulting parameter values are given in Table 1.
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Figure 3. Kinetic analysis of the interactions of different TIAR constructs with T-rich DNA using SPR. The binding of (A) TIAR1, (B) TIAR2S, (C) TIAR12S and (D) TIAR2L to a T-rich DNA is shown. Biotinylated DNA was captured on SA-coated sensor chip and increasing concentrations of protein were injected over the surface. Injections were performed for 120 sec (association phase), followed by a 300 sec flow of running buffer to assess dissociation. The experiments were conducted in duplicate and showed good overlap. The red line represents the binding responses for injections of protein analyte at specified concentration over the DNA surface. The kinetic data were fit by a 1:1 Langmuir binding model. Mass transport effects were not evident. The black curves superimposed on top of the sensor grams represent the model fitted curves. The rate constants ka and kd were determined simultaneously as global fitting parameters from which the KD was determined. The resulting parameter values are given in Table 2.
None
Figure 4. Perturbations of TIAR2L chemical shifts upon RNA binding. (A) An overlay of the assigned (15N,1H)-HSQC spectra of TIAR2L alone and in complex with different concentrations of 6-nt AU-rich RNA (5′-UUAUUU-3′). The molar ratio of TIAR2L to RNA is 1:0 (black), 1:0.4 (red), 1:0.8 (pink) and 1:1.2 (blue). (B) Chemical shift perturbation values for each residue were calculated at the TIAR2L to RNA 1:1.2 ratio as Δδ = {[0.154 (δ15N)2+ 0.25 (δ13Cα)2+ 0.25 (δ13Cβ)2+ (δ1H)2]/4}1/2 and plotted against the amino acid sequence of TIAR2L. The 20 residues showing the largest perturbations upon RNA-binding are colored (red > 0.20 ppm; orange > 0.14 ppm; yellow > 0.08 ppm). (C) Cartoon representation of the TIAR RRM2 structure (PDB ID: 2DH7) with residues affected by RNA-binding colored as in (B) with residues that showed lesser chemical shift perturbations upon RNA binding colored gray.
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