Structure-function studies of nucleocytoplasmic transport of retroviral genomic RNA by mRNA export factor TAP

Abstract

mRNA export is mediated by the TAP-p15 heterodimer, which belongs to the family of NTF2-like export receptors. TAP-p15 heterodimers also bind to the constitutive transport element (CTE) present in simian type D retroviral RNAs, and they mediate the export of viral unspliced RNAs to the host cytoplasm. We have solved the crystal structure of the RNA recognition and leucine-rich repeat motifs of TAP bound to one symmetrical half of the CTE RNA. L-shaped conformations of protein and RNA are involved in a mutual molecular embrace on complex formation. We have monitored the impact of structure-guided mutations on binding affinities in vitro and transport assays in vivo. Our studies define the principles by which CTE RNA subverts the mRNA export receptor TAP, thereby facilitating the nuclear export of viral genomic RNAs, and, more generally, provide insights on cargo RNA recognition by mRNA export receptors.

Figure 1

Structure of TAP-NTD bound to the hCTE RNA. (a) Domain architecture of full-length TAP and TAP-NTD construct (96 to 362) consisting of the RRM domain (in green) and the LRR domain (in blue) used for crystallization of the complex. (b) The full-length CTE RNA consisting of two large internal loops whose sequences are related by two-fold symmetry (indicated by thick and thin arrows). (c) Sequence of one symmetrical half of CTE (half CTE, designated hCTE). The 1 to 62 sequence of hCTE corresponds to the half containing the hairpin loop (top half) of the full-length CTE in panel B, with the hairpin loop replaced by a stable GAAA RNA loop (in red) in the construct used for crystallization. The bases colored in red are different from the wild-type CTE sequence and were incorporated to facilitate efficient in vitro transcription (5'-GG), hammerhead ribozyme cleavage site (UC-3') and improve crystal quality (GAAA loop with GC closing pair). The residues are numbered from 1 to 62. (d, e) Two alternate views of the crystal structure of the 1:1 complex of TAP-NTD bound to hCTE. The RRM domain is in green, the LRR domain is in blue, and the RNA is in wheat. Flipped out RNA residues A13, G14 and A15 are in yellow. The N and C termini of TAP-NTD and the 5' and 3' ends of the RNA are labeled.

Figure 2

Fold of the internal loop and bulged bases in the hCTE-TAP complex. (a) The sequence and numbering of the internal loop and bulged bases of hCTE, with dashed red lines indicating formation of four non-canonical pairs on complex formation. (b) A stereo pair of the structure of the zippered up internal loop of hCTE in the complex. The RNA is in wheat and flipped out A13, G14 and A15 are in yellow. The core of the fold contains four stacked pairs, with junctional U11•G50 and G18-C43 pairs aligned at right angles to each other. (c–f) Pairing alignments of non-canonical pairs within the zippered up internal loop of the complex. The non-canonical pairing alignments are U11•G50 (panel c), A12•A48 (panel d), C16•C49 (panel e) and A17•G47 (panel f). (g, h) Conformation of the A-A bulges in the upper (A39–A40) and lower (A54–A55) stems of the hCTE RNA in the complex. The A39–A40 bulge in the upper stem of the complex is shown in panel g, with A39 flipped out, while A40 is stacked within the duplex and forms an A40•(G21•U38) triple. The A54–A55 bulge in the lower stem of the complex is shown in panel h, with both A54 and A55 flipped out, but mutually stacked on each other.

Figure 3

Key interactions between hCTE RNA and the TAP-NTD in the complex. (a) Schematic highlighting intermolecular hydrogen bond and hydrophobic protein-RNA contacts in the complex. hCTE RNA residues involved in base-specific and sugar-phosphate specific recognition are represented as shaded and red lined boxes, respectively. Amino acid residues of RRM (in green) and LRR (in blue) involved in hydrogen-bonding and hydrophobic/stacking interactions are shown by solid and dashed arrows, respectively. (b) Intermolecular contacts between residues of hCTE RNA (A20, A31-U34 and G50–G51) and the RRM domain of TAP-NTD in the complex. (c) Intermolecular contacts between residues of hCTE RNA (A13-G14 and A54–A55) and the LRR domain of TAP-NTD in the complex. (d–f) Intermolecular hydrogen bonds involving flipped out A13, which is inserted between Arg276 and Arg233 (panel d), flipped out G14, which is positioned on the surface patch involving Tyr278, Arg279 and Lys304 (panel e), and flipped out A15, which is inserted between Glu151 and the sugar of G51 (panel f).

Figure 4

In vitro ITC and direct and competitive filter binding data on TAP-NTD and hCTE RNA mutants. (a, b) Isothermal titration calorimetry (panel a) and nitrocellulose filter (panel b) binding curves for complex formation between TAP-NTD and its mutants with hCTE RNA. Wild-type protein in red circles, dual R233A R276A mutants in orange diamonds, triple Y278A R279A K304A mutants in blue pentagons, single E151A mutant in green squares and dual R128E R158E mutants in black triangles. (c) Summary of the binding constants measured from ITC and filter binding assays. (d–f) Competitive nitrocellulose filter binding assay curves for complex formation between TAP-NTD and hCTE RNA mutants. Wild-type A13 (in red circles) mutated to G13 (in blue triangles) and C13 (in green squares) (panel d), wild-type G14 (in red circles) mutated to A14 (in blue triangles) and C14 (in green squares) (panel e), and wild-type A15 (in red circles) mutated to G15 (in blue triangles) and C15 (in green squares) (panel f). (g) A summary of the binding constants from competitive filter binding assays.

Figure 5

In vivo RNA export assay. (a) Schematic representation of the pCMV128-RLuc-CTE reporter and the corresponding control without CTE. SD and SA, splicing donor and acceptor sites. (b) Human cells were transfected with a mixture of three plasmids: one expressing pCMV128-RLuc-CTE reporter or a control reporter lacking the CTE, another expressing Firefly luciferase, and a third expressing TAP (wild type or mutants). A plasmid expressing p15 was included in the transfection mixtures as indicated. Renilla luciferase activity was normalized to that of the Firefly luciferase transfection control and set to 1 in the absence of exogenous TAP. Mean values ± standard deviations are shown. (c) Human cells were transfected with the pCMV128-RLuc-CTE reporter carrying CTE wild-type or mutants. Plasmids expressing TAP and p15 were co-transfected as indicated. A plasmid expressing Firefly luciferase served as a transfection control. Luciferase activity was analyzed as described in panel a. (d) Human cells were transfected with a mixture of three plasmids: one expressing pCMV128-RLuc reporter lacking the CTE, another expressing Firefly luciferase, and a third expressing TAP (wild type or mutants). A plasmid expressing p15 was included in the transfection mixtures. Renilla and Firefly luciferase activities were measured and analyzed as described in panel b.

Figure 6

Stoichiometry of TAP-NTD binding to CTE RNA. (a) Electrophoretic mobility gel shift data for binding of TAP-NTD to full-length CTE establishing 2:1 stoichiometry of the complex. The positions of the free CTE and of the TAP-NTD-CTE complexes are indicated on the right. TAP-NTD to CTE molar ratios are listed above the lanes. CTE is fully bound at 2:1 TAP-NTD to CTE molar ratio. (b) Electrophoretic mobility gel shift data for binding of TAP-NTD to hCTE establishing 1:1 stoichiometry of the complex. (c) Gel filtration profiles (upper panel) monitoring the interaction between TAP-NTD (blue) and full-length CTE (red), and a calibration curve for an analytical gel filtration column shown with molecular mass standards (lower panel). The mixture of TAP-NTD and CTE at 2:1 protein to RNA ratio (green) migrates as a single peak corresponding to a higher molecular weight fraction. The elution volumes of TAP-NTD (blue triangle), CTE (red triangle) and TAP-NTD + CTE complex (green diamond) are denoted on the calibration curve. (d) Nitrocellulose filter binding curve for complex between TAP-NTD and full-length CTE. The apparent equilibrium binding constants (K_D) measured by non-linear least-squares fit according to equation 1 in the Online Methods section is listed together with ± fitting error. (e) Stoichiometry of TAP-NTD binding to CTE measured by filter binding assay. The total RNA concentration used in the equilibrations is listed and is 16-fold greater then the K_D of TAP-NTD for CTE determined by direct titration (panel d). The data are compared to theoretical saturation curves for 1:1, 2:1, and 4:1 protein:RNA stoichiometry. The 2:1 curve most closely approximates the data, establishing that two copies of TAP-NTD interact with a single CTE molecule. The plots in panels d and e represent mean ± standard deviation for two independent measurements.

Figure 7

Packing of two molecules of complex (TAP-NTD bound to hCTE) in the crystallographic asymmetric unit and a model of the 2:1 complex of TAP-NTD bound to full-length CTE. (a) The alignment of two hCTEs in the crystallographic asymmetric unit of the complex. The bases colored in red reflect differences from the wild-type CTE sequence. (b) The orientation of two molecules of complex (TAP-NTD bound to hCTE RNA) in the crystallographic asymmetric unit. (c) The full-length CTE RNA consisting of two large internal loops whose sequences are related by two-fold symmetry (indicated by thick and thin arrows). (d) Model of the complex of full-length CTE with two TAP-NTD molecules bound to two CTE internal loops generated based on the structure of the two molecules of complex (TAP-NTD bound to hCTE) in the crystallographic asymmetric unit. The missing parts of the full-length CTE were modeled using idealized A-form RNA duplexes and RNA structural elements from previously determined structures (see Supplementary Methods for details) followed by rounds of idealization of geometric parameters with REFMAC and Coot programs.