TAP (NXF1) Belongs to a Multigene Family of Putative RNA Export Factors with a Conserved Modular Architecture

Abstract

Vertebrate TAP (also called NXF1) and its yeast orthologue, Mex67p, have been implicated in the export of mRNAs from the nucleus. The TAP protein includes a noncanonical RNP-type RNA binding domain, four leucine-rich repeats, an NTF2-like domain that allows heterodimerization with p15 (also called NXT1), and a ubiquitin-associated domain that mediates the interaction with nucleoporins. Here we show that TAP belongs to an evolutionarily conserved family of proteins that has more than one member in higher eukaryotes. Not only the overall domain organization but also residues important for p15 and nucleoporin interaction are conserved in most family members. We characterize two of four human TAP homologues and show that one of them, NXF2, binds RNA, localizes to the nuclear envelope, and exhibits RNA export activity. NXF3, which does not bind RNA or localize to the nuclear rim, has no RNA export activity. Database searches revealed that although only one p15 (nxt) gene is present in the Drosophila melanogaster and Caenorhabditis elegans genomes, there is at least one additional p15 homologue (p15-2 [also called NXT2]) encoded by the human genome. Both human p15 homologues bind TAP, NXF2, and NXF3. Together, our results indicate that the TAP-p15 mRNA export pathway has diversified in higher eukaryotes compared to yeast, perhaps reflecting a greater substrate complexity. mRNAs are exported from the nucleus as large ribonucleoprotein complexes (mRNPs). To date, proteins directly implicated in this process include several nucleoporins and RNA binding proteins (hnRNPs), an RNA helicase of the DEADbox family (Dbp5), and the nuclear pore complex (NPC)-associated proteins Gle1p, TAP and Mex67p, and RAE1 (also called Gle2p) (reviewed in references 22, 28, and 32). Mex67p is essential for mRNA export in Saccharomyces cerevisiae, while RAE1 is essential for mRNA export in Schizosaccharomyces pombe (9,27,36). Their vertebrate homologues, TAP and RAE1, have also been implicated in the export of cellular mRNAs (6,8,12,15,20,31).We identified TAP as the cellular factor which is recruited by the constitutive transport element (CTE) of simian type D retroviruses to promote nuclear export of their genomic RNAs (12). In Xenopus oocytes, titration of TAP with an excess of CTE RNA prevents cellular mRNAs from exiting the nucleus (12,30,33). This suggests a role for this protein in the export of cellular mRNA.Considerable progress has been made in defining TAP structural and functional domains (see Fig. 1) and in identifying its binding partners. TAP partners include various nucleoporins (4, 17); p15 (also called NXT1), a protein related to nuclear transport factor 2 (NTF2) (7, 17); transportin, which mediates TAP nuclear import (4); and several mRNP-associated proteins, such as E1B-AP5, RAE1 (4), and members of the Yra1p/ REF family of proteins (37, 39). Binding of TAP to these mRNP-associated proteins is mediated by its N-terminal domain (residues 1 to 372) (4, 39). This domain includes a noncanonical RNP-type RNA binding domain (RBD)...

Karyopherin β2B participates in mRNA export from the nucleus

“…HeLa cells were transfected with a plasmid coding for TAP tagged C terminally with GFP. At 24 h after transfection the fusion protein was visible in the nuclei of most cells in agreement with previous reports (30,48). At this time point the cells were treated with digitonin, which selectively permeabilizes plasma membranes (49).…”

The Evolutionarily Conserved Kaposi's Sarcoma-associated Herpesvirus ORF57 Protein Interacts with REF Protein and Acts as an RNA Export Factor

“…Augmentation of the export activity of RRE-tethered RevM10-ORF57 could occur by RRE-independent binding of ORF57 to cat mRNA or via ORF57 recruitment by RevM10-ORF57 as ORF57 protein is capable of self-interaction. 2 The export activity of ORF57 alone was comparable with that obtained with GFP-TAP alone (48,62) and activity of RevM10-ORF57 was comparable with that obtained with RevM10-TAP alone; both TAP export activities were dramatically increased by the addition of exogenous p15, a TAP co-factor (48). This suggests the presence of a similarly acting, as yet unidentified, ORF57 co-factor, either viral or cellular, that modulates its export ability during KSHV lytic replication.…”

“…Used extensively to study HIV-1 Rev (57,58), this assay was used to demonstrate that TAP protein increased CAT protein activity thereby inducing the cytoplasmic accumulation of intron-containing RNAs (48,62). This assay was also used to demonstrate that EBV MTA protein promotes RNA export (73).…”

“…␤-Galactosidase activity was measured using the ␤-galactosidase reporter gene system (Promega) following the manufacturer's instructions. To compensate for any differences in transfection efficiencies between experiments, CAT activities were normalized with respect to the ␤-galactosidase internal control (48,62). Standard deviations of the CAT activity values from three separate experiments are shown as error bars.…”

“…DNA Transfections, RNA Export Assays, and RNase Protection Assay-DNA transfections and CAT export assays were performed essentially as described (48,62) with the following modifications. Subconfluent 293 cells (6 ϫ 10 5 cells/well) were transfected using Polyfect (Qiagen) according to the manufacturer's instructions.…”