TAP binds to the constitutive transport element (CTE) through a novel RNA‐binding motif that is sufficient to promote CTE‐dependent RNA export from the nucleus

The N‐terminal extension does not affect the CTE‐binding and export functions of TAP. (A) A gel mobility retardation assay was performed with reticulocyte lysates programmed with cDNAs encoding TAP 61–619 (lanes 3–12) or 1–619 (lanes 13–22). In lanes 4 and 14, unlabelled M36 competitor RNA was added. In lanes 5–8 and 15–18, CTE competitor RNA was included in the reaction mixtures, while in lanes 9–12 and 19–22, DHFR mRNA was used as a competitor. The concentrations of the competitor RNAs are indicated above the lanes. The positions of the free RNA probe and of the TAP–CTE RNA complex are indicated on the left. The upper complex may represent two molecules of TAP bound to the CTE RNA. (B) Xenopus oocyte nuclei were injected with a mixture of in vitro‐transcribed ³²P‐labelled U1ΔSm RNA, U6Δss RNA and a precursor RNA containing the SRV‐1 CTE inserted at the intron (Ad‐CTE). Purified recombinant GST–TAP 61–619 or 1–619 was included in the injection mixtures as indicated. The concentration of the protein in the injected samples was 8 μM. RNA samples from total oocytes (T), cytoplasmic (C) and nuclear (N) fractions were collected immediately after injection (lanes 1–3) or 2.5 h after injection (lanes 4–12). Products of the splicing reaction were resolved on 10% acrylamide–7 M urea denaturing gels. One oocyte equivalent of RNA, from a pool of 10 oocytes, was loaded per lane. The mature products and intermediates of the splicing reaction are indicated diagrammatically on the left of the panels. The filled triangle represents the CTE.

The N‐terminal domain of TAP including the LRRs is sufficient to interact with the CTE RNA. An electrophoretic mobility retardation assay was performed with a labelled CTE RNA probe and the purified recombinant proteins indicated above the lanes. For each protein, binding reactions were performed in the absence of competitor or in the presence of either CTE or M36 competitor RNAs as indicated. The concentration of the competitors was 15 μg/ml for the CTE and 50 μg/ml for the M36 RNA. The concentration of the recombinant proteins in the binding reactions was 10 μg/ml, with the exception of TAP 61–619 and 61–372, which were tested at 4 μg/ml, and TAP 102–372, 61–198 and 540–619, which were added at 8 μg/ml. The position of the free RNA probe is shown on the left. The additional complexes in lanes 5–7 were not characterized further.

The β‐strand region, but not the α‐helices, of the LRRs may contact the CTE RNA directly. (A) The positions of the alanine scan mutants are shown on the primary amino acid sequence of human TAP. Mutants are designated according to the position of the first substituted residue. The predicted folding of the LRRs is indicated below the sequence. The black dots above the sequence indicate the N‐ and C‐termini of the truncations described in Figures 3 and 5. The open arrowhead below the sequence shows the N‐terminus of the CTE‐binding domain, while the filled arrowheads indicate the boundaries of the minimal CTE‐binding domain. (B) Alanine scan mutants, indicated above the lanes, were in vitro translated in the presence of [³⁵S]methionine. Proteins were analysed by SDS–PAGE followed by fluorography. (C) An electrophoretic mobility retardation assay was performed with a labelled CTE RNA probe and the in vitro‐translated proteins indicated above the lanes. Symbols are as in Figure 2A. (D) Domain organization of human TAP protein. TAP domains are highlighted with different patterns. The predicted folding within the LRRs is indicated diagrammatically. The position of alanine substitutions and the binding properties of the mutants are shown. Binding activities were defined relative to the wild‐type protein. +, binding at the wild‐type level; −, binding activity below 5% relative to the wild‐type protein. Note that although under these conditions mutant 183 was scored +, this mutant was impaired in binding in the presence of higher concentrations of competitor.

The CTE‐binding domain is sufficient to mediate CTE‐dependent export. Xenopus oocyte nuclei were injected with the recombinant proteins indicated above the lanes and a mixture of radiolabelled Ad‐CTE pre‐mRNA, U1ΔSm RNA and U6Δss RNA. RNA samples from total oocytes (T), cytoplasmic (C) and nuclear (N) fractions were collected 3.5 h after injection, or immediately after injection [t₀: lanes 1–3 in (A) and (B)]. Products of the splicing reaction were resolved on 10% acrylamide–7 M urea denaturing gels. One oocyte equivalent of RNA, from a pool of 10 oocytes, was loaded per lane. The products of the splicing reaction are indicated diagrammatically on the left of the panel. The asterisks on the left of (A) indicate the positions of RNA molecules that are likely to be due to degradation of the intron lariat and of the precursor RNA. (A) In lanes 7–24, unlabelled competitor CTE RNA was included in the injection mixtures at 0.8 μM. The concentration of the proteins in the injected samples was as follows: 61–619, 14 μM; 61–372, 18 μM; 102–372, 195–372 and 540–619, 36 μM. (B) The concentration of the proteins in the injected samples was as follows: TAP 61–619 and 61–372, 14 μM; 102–372 and 540–619, 36 μM. (C) SDS–PAGE of the purified recombinant proteins used in (A) and (B).

TAP and the minimal CTE‐binding domain enter the cytoplasm upon nuclear injection. (A) Purified recombinant TAP (61–619) or the truncations 102–372 or 195–372 were injected into oocyte nuclei together with a mixture of radiolabelled U1ΔSm, U6Δss and Ad‐CTE RNAs. In lanes 8 and 16, Ad‐CTE was replaced by Ad‐M36 RNA. RNA samples were collected 3 h after injection and analysed directly (Inputs), or subjected to immunoprecipitation using anti‐GST antibodies (Pellets). The concentration of the proteins in the injected samples was 14 μM for GST–TAP 61–619 and 36 μM for 102–372 and 195–372. RNAs in the input or the precipitates were analysed as described in Figure 2B. Symbols are as in Figure 2B. (B) Purified recombinant TAP and the truncated proteins indicated on the left of the panels were injected into Xenopus oocyte nuclei in either the absence or the presence of CTE RNA or DHFR mRNA as indicated above the lanes. Protein samples from total oocytes (T), cytoplasmic (C) and nuclear (N) fractions were collected 4.5 h after injection, and analysed by Western blotting using a rabbit anti‐GST antibody. One oocyte equivalent of proteins, from a pool of 10 oocytes, was loaded per lane. The concentration of TAP fragments in the injected samples was ∼18 μM, and of the CTE and DHFR RNAs 9 and 6 μM, respectively.