Abstract
The NXF1:NXT1 complex (also known as TAP:p15) is a general mRNA nuclear export factor that is conserved from yeast to humans. NXF1 is a modular protein constructed from four domains (RRM, LRR, NTF2-like and UBA domains). It is currently unclear how NXF1:NXT1 binds transcripts and whether there is higher organization of the NXF1 domains. We report here the 3.4 Å resolution crystal structure of the first three domains of human NXF1 together with NXT1 that has two copies of the complex in the asymmetric unit arranged to form an intimate domain-swapped dimer. In this dimer, the linkers between the NXF1 LRR and NTF2-like domains interact with NXT1, generating a 2-fold symmetric platform in which the RNA-binding RRM, LRR and NTF2-like domains are arranged on one face. In addition to bulk transcripts, NXF1:NXT1 also facilitates the export of unspliced retroviral genomic RNA from simple type-D retroviruses such as SRV-1 that contain a constitutive transport element (CTE), a cis-acting 2-fold symmetric RNA stem–loop motif. Complementary structural, biochemical and cellular techniques indicated that the formation of a symmetric RNA binding platform generated by dimerization of NXF1:NXT1 facilitates the recognition of CTE-RNA and promotes its nuclear export.
Highlights
The export of mRNA from the nucleus through nuclear pores (NPCs) is the culmination of the nuclear phase of the eukaryotic gene expression pathway and releases mature mRNA transcripts into the cytoplasm for translation
We present here the crystal structure of the complex formed between Homo sapiens NXF1 from which the Nterminal 95 residues and the UBA domain had been removed (NXF1 N UBA) and NXT1 and show that it has a novel homodimeric domain-swapped configuration generated by a interaction between the linker between the leucinerich repeat (LRR) and nuclear transport factor 2-like domain (NTF2L) domains with NXT1
The interaction between the pre-␣1 loop and the canonical copy of NXT1 was largely dominated by hydrophobic interactions centering around residue Leu370 of NXF1 with NXT1 (mutated to Ala in the single domain structure by [12]) that dictated the spatial positioning of the LRR domain (Figure 2B, blue on yellow)
Summary
The export of mRNA from the nucleus through nuclear pores (NPCs) is the culmination of the nuclear phase of the eukaryotic gene expression pathway and releases mature mRNA transcripts into the cytoplasm for translation (reviewed by [1,2,3]). NXF1 is a modular protein comprised of four domains: an N-terminal RNA recognition motif (RRM), a leucinerich repeat (LRR), a nuclear transport factor 2-like domain (NTF2L) and an ubiquitin-associated (UBA) domain (reviewed by [7]). Structures of the individual domains [11,12,13] have shown that both the NTF2L and UBA domain have hydrophobic surface depressions to which the phenylalanine–glycine (FG) motifs present in NPC proteins (nucleoporins or ‘nups’) can bind and this interaction is crucial in enabling the complex and its associated cargo to overcome the NPC barrier function [3,7]. Efficient nuclear export of transcripts appears to require two independent nucleoporin binding domains and substitution of either the NTF2L or UBA for another NTF2L or UBA domain is capable of producing a partially functional NXF1 [14]
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