Site-directed mutations used to investigate TIAR nuclear localization mapped on the TIA-1–RRM2 structure. Side chains of solvent-exposed mutated residues are shown with ball-and-stick representation and colored orange. The mutated glycines are indicated by spheres. Leu102 of the RNP2 motif (blue) and Val141 of the RNP1 motif (red) are buried within the RRM interior. Residues that contact Leu102 are shown with ball-and-stick representation, and colored salmon. Residues that contact Val141 are also shown, and colored light blue. Residues that contact both Leu102 and Val141 are colored violet.

Domain structure of human TIA-1 above a sequence alignment of TIA-1–RRM2 from representative homologs. Sequences are colored by percent identity: 100%=green, <90%=sea green, <80%=lime, <70%=bright green, <60%=yellow, and <50%=white. Secondary structure elements are indicated above the alignment [45]. The RNP1 and RNP2 are boxed and labeled below the alignment. Surface residues of human Tia-1 mutated to facilitate crystallization are highlighted by blue filled circle. Residues that interact with RNA in both PAB and SXL complexes are marked with red filled circle. Accession numbers for aligned sequences include: Homo sapiens (NP_071320), Mus musculus (NP_035715), Gallus gallus (XP_001233215), Caenorhabditis elegans (NP_495121), Neurospora crassa (XP_962723), Arabidopsis thaliana (NP_188026), Bombyx mori (NP_001036895), Entamoeba histolytica (XP_649094), Saccharomyces cerevisiae (CAA46011).

Comparisons with the TIA-1 RNP motifs. (A) Symmetry-related interactions between two TIA-1–RRM2 molecules, respectively colored yellow and green. Key interacting residues are shown with ball-and-stick representation, and consensus residues of the RNP motifs responsible for mediating the intermolecular interaction are colored magenta. Primed italics distinguish residue labels of the symmetry-related molecule. (B) View of the Y14 (red)-mago (blue) complex (PDB-code 1OO0). Regions of the mago subunit outside the α-helical interactions with the Y14 RRM are omitted for clarity. (C) Superposition of TIA-1–RRM2 with PAB residues 11-88 (PDB-code 1CVJ). (D) Superposition of TIA-1–RRM2 with SXL residues 125-202 (PDB-code 1B7F). TIA-1 is colored yellow-orange, PAB is blue, and SXL is green. The RNA strands are colored magenta. The view is rotated 90° clockwise about the y-axis relative to (A). Residues of TIA-1 (Phe98, Arg125, Asp129, Tyr138, Phe140, Arg167) are labeled. Corresponding residues of PAB (Tyr14, Arg41, Asp45, Tyr54, Tyr56, Arg83) and SXL (Ile128, Arg155, Asp159, Tyr168, Phe170, Lys197) are shown as ball-and-stick representations.

Overall structure of TIA-1–RRM2. (A) Ribbon diagram of the two TIA-1–RRM2 molecules in the asymmetric unit of the crystal. Molecules ‘A’ and ‘B’ are distinguished by subscripts of the residues labels. Secondary structure elements of molecule ‘A’ are labeled. Composite omit electron density (light blue) at 1σ contour level surrounds key aromatic residues of the RNP motifs. Anomalous difference Fourier maps (magenta) at 3σ contour level indicate the iodide positions. (B) Superposition of the top five related structures identified using DALI [46]: TIA-1–RRM2 (orange), PAB (PDB-code=1CVJ, cyan), SXL (PDB-code=1B7F, magenta), hnRNP A1 (PDB-code=1HA1, green), PRP24 (PDB-code=2GHP, marine), and nuclear cap binding protein (PDB-code=1H6K, red). (C) Noncrystallographic contacts mediated by the SER mutations, viewed 180° about the y-axis relative to (A). Ball-and-stick diagrams are shown for SER residues (green) and other residues of the iodide binding sites (orange). The iodides bound at the interface are shown as magenta spheres, and the water molecules directly coordinated to these iodides are shown as blue spheres. (D) Comparison of TIA-1 RRM and SER TIA-1–RRM2 binding RNA using fluorescence anisotropy. Overlay of nonlinear least square fits of TIA-1–RRM2 (orange) and SER TIA-1–RRM2 (green) binding 5′ fluorescein-labeled 20mer polyuridine. The average apparent equilibrium dissociation constants (K_D) and standard deviations of 2–3 experiments are inset.