The RNA binding domain within the nucleoporin Nup153 associates preferentially with single-stranded RNA

Abstract

The nuclear pore protein Nup153 is important for the transport of protein and RNA between the nucleus and cytoplasm. Recently, a novel RNA binding domain (RBD) was mapped within the N-terminal region of Nup153; however, the determinants of RNA association were not characterized. Here we have tested a range of RNAs with different general features to better understand targets recognized by this domain. We have found that the RBD associates with single-stranded RNA with little sequence preference. These results provide new information about a novel RNA binding domain and suggest new models to consider for the contribution of Nup153 to nucleocytoplasmic transport.

FIGURE 1.

The RNA binding domain of Nup153 preferentially associates with single-stranded RNA. (A) Recombinant T7-tagged RBD and GFP were immobilized on anti-T7-agarose beads followed by incubation with a panel of structured ³²P-labeled RNAs (lane 5). RNA was isolated from the supernatant (S; lanes 1,2) and the pellet (P; lanes 3,4) of each reaction. tRNA (tracer) was added to the RNA pellet fractions following the washes to monitor RNA recovery (lanes 3–5, middle panel). Protein recovery was determined by Western blotting with an anti-T7 antibody (lanes 3,4, lower panel). The size markers on the left represent 52, 34, and 28 kD, respectively. The asterisk represents a degradation product of the GFP fusion protein. (B) A binding assay was performed as above with ³²P-labeled double-stranded CAT RNA (input, lane 5). Unbound RNA (S) was compared to bound RNA (P; cf. lanes 1,2 and 3,4). Protein recovery was confirmed by T7 Western blot (lower panel). In B and C, the size markers on the left represent 48, 34, 28, and 21 kD. (C) A binding assay was performed as above with ³²P-labeled single-stranded CAT mRNA (input, lane 5). Unbound RNA (S) was compared to bound RNA (P; cf. lanes 1,2 and 3,4). Protein recovery was confirmed by T7 Western blot (lower panel). In all experiments, 50% of bound RNA, 4% of unbound RNA, and 2% of the input was loaded on each gel.

FIGURE 2.

The Nup153 RNA binding domain, whether in isolation or in its full-length context, recognizes features common to mRNAs. (A) Recombinant T7-tagged full-length Nup153 and truncated version containing a deletion of either the first 100 and 400 amino acids (Nup153ΔN100 and Nup153ΔN400) were immobilized on anti-T7 agarose beads followed by incubation with ³²P-labeled RNA cargo (lane 4). RNA remaining in the final pellet (P) is shown for each recombinant protein (lanes 1–3, upper panel). For A and B, RNA was loaded as described in Figure 1 ▶. Immobilized protein was detected using mAb414 (lanes 1–3, lower panel). The size indicators to the left represent 203, 115, and 93 kD. (B) Recombinant T7-tagged RBD and GFP were immobilized on anti-T7-agarose followed by incubation with ³²P-labeled RNA cargo (lane 3). RNA remaining in the final pellet (P) is shown in the upper panel (lanes 1,2). Immobilized protein was detected with an anti-T7 antibody (lanes 1,2, lower panel). The size indicators to the left represent 48, 34, 28, and 21 kD. (C) Shown is a schematic depicting full-length Nup153, N-terminal deletion constructs in relation to the RNA binding domain (white box labeled RBD), the zinc finger region (dark gray shading), and FG-rich region (white box with black hatch marks). The poly(G) binding and mRNA binding for these constructs is summarized at the right.

FIGURE 3.

The Nup153 RNA binding domain preferentially binds unstructured RNA. Recombinant T7-tagged Nup153ΔN100, Nup153ΔN400, Nup153 RBD, and GFP were immobilized on anti-T7 agarose. (A) ³²P-labeled RNAs, including a broad size range of single-stranded RNAs and structured RNAs (lane 8) were incubated with immobilized protein. Following washes, bound RNA (P) was isolated from the pellet (lanes 1,2,4–7). RNA samples were loaded as described in Figure 1 ▶. Two exposures of the pull-down of RNA with Nup153ΔN100 and Nup153ΔN400 are shown in lanes 1–3 (8-h exposure at room temperature labeled longer exposure) and lanes 4–8 (30 min exposure at −80°C labeled shorter exposure). (B) ³²P-labeled RNAs, including a narrow size range of single-stranded mRNAs and the structured U3 RNA were incubated with immobilized protein. Following washes, the bound RNA was isolated from the pellet (lanes 1,2,4–7). RNA samples were loaded as described in Figure 1 ▶. Two exposures of the pull-down of RNA with Nup153ΔN100 and Nup153ΔN400 are shown in lanes 1–3 (22-min exposure at −80°C labeled longer exposure) and 4–8 (15-min exposure at −80°C labeled shorter exposure).

FIGURE 4.

RNA recognition, like export, is independent of mRNA orientation. (A) Xenopus oocyte nuclei were injected with 10 nL of either sense or antisense CAT mRNA mix containing U3, U1ΔSm, and tRNA as controls (lanes 1,4). Oocytes were dissected 4 h postinjection. RNAs remaining in the nucleus (lanes 2,5) or exported to the cytoplasm are shown (lanes 3,6). (B) Xenopus oocyte nuclei were injected as above except that DHFR-sense or DHFR-antisense mRNA was added to the RNA mix. Nuclear RNAs (lanes 2,5) and RNAs exported to the cytoplasm (lanes 3,6) are shown. (C) Nup153 RBD and GFP were immobilized on anti-T7-agarose. A mix of RNAs, including either ³²P-labeled CAT-sense with DHFR-antisense mRNA (lane 3) or CAT-antisense with DHFR-sense mRNA (lane 6), were incubated with immobilized protein. After washing, the bound RNA was isolated from the pellet (P; cf. lanes 1,2 and 4,5, upper panels). RNA samples were loaded as described in Figure 1 ▶. RNA from the pellet was exposed to film for 2 h (lanes 1,2,4,5) whereas the input RNA was exposed to film for 4 h (lanes 3,6). Protein recovery was determined by Western blotting with an anti-T7 antibody (lanes 1,2,4,5, lower panel). The size markers at the left represent 90, 52, 34, and 28 kD. The asterisk on the right represents a breakdown product of the GFP fusion.