Abstract
Metazoan Tap-p15 (also called Nxf1-Nxt1) and yeast Mex67-Mtr2 heterodimers are the general mRNA export receptors. The RNA binding activity of Tap-p15, which is essential for mRNA nuclear export, has been attributed to the amino-terminal RNA binding module of Tap consists of RNA recognition motif (RRM) and leucine-rich repeat. In this study, we identified a novel RNA interaction surface in the NTF2-like (NTF2L) domain of Tap, which is analogous to the rRNA binding platform of Mex67-Mtr2. Tap-p15 uses the three domains to tightly bind the retroviral constitutive transport element. The RNA binding through the NTF2L domain is functionally relevant as introduction of mutations in this region reduced CTE-containing mRNA export activity. In contrast, only when the RRM and NTF2L domains were mutated simultaneously, bulk poly (A)+ RNA export and in vivo poly (A)+ RNA binding activities of Tap-p15 were significantly attenuated. Moreover, an engineered human cell line harboring the NTF2L domain mutation in the NXF1 gene showed a synthetic growth phenotype and severe mRNA export defect under Aly/REF and Thoc5 depleted condition. These data suggest that Tap-p15 recognizes bulk mRNAs through combinatorial use of the distinct RNA binding domains.
Highlights
Soluble macromolecules, such as RNAs and proteins, are transported through the nuclear pore complexes (NPCs), huge protein assemblies that span the nuclear envelope
As has been reported previously [20], purified recombinant constitutive transport element (CTE) binding module of Tap [Tap [96–371]] (Figure 1B, lane 1) that consists of the RNA recognition motif (RRM) and leucine-rich repeat (LRR) domains bound to RNA probes encoding the pBS polylinker or the hCTE sequences in RNA gel shift assays (Figure 1C and D, lanes 2–4)
By comparing the RNA band shift patterns, we concluded that the RNA binding activity was attributable to the NTF2L domain complexed with p15 (Figures 1C and 1D, lanes 11–13)
Summary
Soluble macromolecules, such as RNAs and proteins, are transported through the nuclear pore complexes (NPCs), huge protein assemblies that span the nuclear envelope. Importin/karyopherin--type proteins constitute a major nucleo-cytoplasmic transport receptor family, which carry a variety of cargoes into or out of the nucleus. A small GTPase Ran-GTP dictates the direction of the transport by regulating the assembly (for exportins) and disassembly (for importins) of the cargotransport receptor complexes [3,4,5,6,7]. The importin/karyopherin- family members Exportin and exportin-t transport microRNAs and tRNAs from the nucleus to the cytoplasm, respectively [5]. Exportin-5, for example, along with bound Ran-GTP, forms a U-shaped conformation and recognizes the common structure of pre-miRNAs, i.e. a hairpin-like double-stranded structure with 2-nt overhang at the 3 end. A tunnel-like structure at the bottom of the U-shaped Exportin-5 accommodates the 2-nt overhang at the 3 -end of pre-miRNA [9,10]
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