Volume 1, Issue 5, Pages 649-659 (April 1998) TAP, the Human Homolog of Mex67p, Mediates CTE-Dependent RNA Export from the Nucleus  Patric Grüter, Carlos Tabernero, Cayetano von Kobbe, Christel Schmitt, Claudio Saavedra, Angela Bachi, Matthias Wilm, Barbara K Felber, Elisa Izaurralde  Molecular Cell  Volume 1, Issue 5, Pages 649-659 (April 1998) DOI: 10.1016/S1097-2765(00)80065-9

Figure 1 Structure of the SRV-1 CTE The CTE folds into an extended RNA stem-loop structure. This computer-predicted model is supported by previously reported structure-function studies. The internal loops A and B and the AAGA bulge are hallmarks of this class of posttranscriptional control elements (Tabernero et al. 1996; Tabernero et al. 1997; Ernst et al. 1997a; Ernst et al. 1997b). Arrows indicate similar sequences in loops A and B. Molecular Cell 1998 1, 649-659DOI: (10.1016/S1097-2765(00)80065-9)

Figure 2 Nuclear Export of Excised Intron-Lariats Harboring SRV-1 CTE Mutants in Xenopus laevis Oocytes Xenopus oocyte nuclei were injected with a mixture of in vitro transcribed 32P-labeled U6Δss RNA and the precursor RNAs indicated above the lanes. CTE mutants are described in the Experimental Procedures. Synthesis of precursor RNAs was primed with the m7GpppG cap dinucleotide, whereas synthesis of U6Δss RNA was primed with γ-mGTP. RNA samples from total oocytes (T), or cytoplasmic (C) and nuclear (N) fractions were collected 3 hr after injection. Products of the splicing reaction were resolved on 10% acrylamide/7 M urea denaturing gels. The mature products and intermediates of the splicing reaction are indicated diagrammatically on the left of the panels. The closed triangle represents the CTE. The asterisk on the left of the panel indicates the position of RNA molecules that are likely to be originated by degradation of the intron lariat and of the precursor RNA. Molecular Cell 1998 1, 649-659DOI: (10.1016/S1097-2765(00)80065-9)

Figure 3 A CTE-Binding Activity Is Present in HeLa Nuclear Extracts An electrophoretic mobility retardation assay was performed with a labeled CTE RNA probe and HeLa nuclear proteins eluted from a single-stranded DNA column. Lanes 1: free RNA. Lanes 2: CTEBP/CTE RNA complexes formed in the presence 0.02 mg/ml of M36 competitor RNA. In lanes 3–23 of (A) and 3–11 of (B), increasing amounts of the competitor RNAs indicated above the lanes were added to the reaction mixtures. The positions of the free RNA probe and of the CTEBP/RNA complex (CTEBP) are indicated on the left. Molecular Cell 1998 1, 649-659DOI: (10.1016/S1097-2765(00)80065-9)

Figure 4 Affinity Selection of CTEBP on Biotinylated RNA-Streptavidin Beads (A) Protein fractions eluted from a single-stranded DNA column were incubated with magnetic beads precoated with CTE or M36 RNAs as indicated above the lanes. CTEBP activity was assayed by the gel mobility retardation assay described in Figure 3. Lane 1: free RNA. Lane 2: CTEBP in the input protein fraction. Lanes 3 and 6: Flowthroughs from the CTE and M36 RNA beads. Lanes 4 and 7: Washes. Lanes 5 and 8: Selected proteins. The positions of the free RNA probe and of the CTEBP/RNA complex (CTEBP) are indicated on the left. (B) Protein fractions selected on the CTE or M36 RNA were analyzed by SDS–PAGE. The position of CTEBP is indicated on the right. Molecular Cell 1998 1, 649-659DOI: (10.1016/S1097-2765(00)80065-9)

Figure 5 CTEBP Corresponds to Human TAP A gel mobility retardation assay was performed with protein fractions eluted from a single-stranded DNA column (lanes 2–3), reticulocyte lysates programmed with TAP cDNA (lanes 4–5), or control reticulocyte lysates (lanes 6–7). In lanes 2, 4, and 6, unlabeled M36 competitor RNA was added. In lanes 3, 5, and 7, CTE competitor RNA was included in the reaction mixtures. The positions of the free RNA probe and of the CTEBP/RNA complex (CTEBP) are indicated on the left. Molecular Cell 1998 1, 649-659DOI: (10.1016/S1097-2765(00)80065-9)

Figure 6 Purified Recombinant GST-TAP Exhibits the Same RNA Binding Specificity as CTEBP An electrophoretic mobility retardation assay, as described in Figure 3, was performed with purified recombinant TAP expressed in E. coli as a glutathione S-transferase fusion. Lanes 1: free RNA. Lanes 2: GST-TAP/CTE RNA complexes formed in the presence 0.02 mg/ml M36 competitor RNA. In lanes 3–23 of (A) and 3–11 of (B), increasing amounts of the competitor RNAs indicated above the lanes were added to the reaction mixtures. The positions of the free RNA probe and of the GST-TAP/RNA complex (GST-TAP) are indicated on the left. Molecular Cell 1998 1, 649-659DOI: (10.1016/S1097-2765(00)80065-9)

Figure 7 Human TAP Directly Stimulates CTE-Dependent Export (A) Purified recombinant GST-TAP fusion protein was injected into Xenopus laevis oocytes either in the nucleus (lanes 7–9) or in the cytoplasm (lanes 10–12). After 1 hr of incubation, a second microinjection was performed with a mixture of the following radioactively labeled RNAs: U1ΔSm, U6Δss, and Ad-CTE. RNA samples from total oocytes (T), or cytoplasmic (C) and nuclear (N) fractions were collected 3 hr after injection in lanes 4–12 or immediately after injection in lanes 1–3. Symbols are as in Figure 2. (B) Xenopus oocyte nuclei were injected with recombinant GST-TAP. After 1 hr, a second nuclear injection was performed with a mixture of the following in vitro transcribed 32P-labeled RNAs: U1ΔSm, U6Δss, and precursor Ad-M36 (lanes 1–6) or Adcte(as) (lanes 7–9) as indicated above the lanes. RNA nuclear export was analyzed after 3 hr. Symbols are as in Figure 2. (C) Xenopus oocyte nuclei were injected with recombinant GST-TAP (lanes 7–9). After 1 hr of incubation, a second microinjection was performed with a mixture of radiolabeled Ad-CTE precursor RNA, U6Δss RNA, and unlabeled competitor CTE RNA. RNAs were extracted 3 hr after injection in lanes 4–9 or immediately after injection in lanes 1–3. Symbols are as in Figure 2. Molecular Cell 1998 1, 649-659DOI: (10.1016/S1097-2765(00)80065-9)

Figure 8 Recombinant TAP Localizes to the Oocyte Nucleus Purified recombinant GST-TAP fusion protein was injected into Xenopus laevis oocytes either in the cytoplasm (lanes 1–6) or in the nucleus (lanes 7–12). Protein samples from total oocytes (T), or cytoplasmic (C) and nuclear (N) fractions were collected 4 hr after injection in lanes 4–6 and 10–12 or immediately after injection in lanes 1–3 and 7–9. The recombinant protein was detected by Western blotting with a rabbit anti-GST antibody. The asterisks on the left of the panel indicate the position of GST-TAP degradation products. Molecular Cell 1998 1, 649-659DOI: (10.1016/S1097-2765(00)80065-9)

Figure 9 TAP Defines a Late Step in the mRNA Export Pathway In this model, the mRNA export pathway is shown to proceed by a series of consecutive steps (arrows). Saturation of a specific step is indicated by a cross over the arrows. DHFR mRNA export includes a specific step that is saturated by an excess of hnRNP A1 protein. An excess of CTE RNA saturates its own export and the export of both DHFR mRNA and of the spliced Ad-mRNA. This provides support for the existence of a late step on the mRNA export pathway that is commonly used by DHFR mRNA, the spliced Ad-mRNA, and the CTE RNA. Here, we provide evidence that TAP defines this late common step. The CTE directly interacts with TAP and allows intron-containing RNAs to access this late step of the mRNA export pathway. Arrows between TAP and the NPC indicate that TAP may be loosely associated with nucleoporins. Molecular Cell 1998 1, 649-659DOI: (10.1016/S1097-2765(00)80065-9)