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NXT1 (p15) is a crucial cellular cofactor in TAP-dependent export of intron-containing RNA in mammalian cells - PubMed

NXT1 (p15) is a crucial cellular cofactor in TAP-dependent export of intron-containing RNA in mammalian cells

B W Guzik et al. Mol Cell Biol. 2001 Apr.

Abstract

TAP, the human homologue of the yeast protein Mex67p, has been proposed to serve a role in mRNA export in mammalian cells. We have examined the ability of TAP to mediate export of Rev response element (RRE)-containing human immunodeficiency virus (HIV) RNA, a well-characterized export substrate in mammalian cells. To do this, the TAP gene was fused in frame to either RevM10 or RevDelta78-79. These proteins are nonfunctional Rev mutant proteins that can bind to HIV RNA containing the RRE in vivo but are unable to mediate the export of this RNA to the cytoplasm. However, the fusion of TAP to either of these mutant proteins gave rise to chimeric proteins that were able to complement Rev function. Significantly, cotransfection with a vector expressing NXT1 (p15), an NTF2-related cellular factor that binds to TAP, led to dramatic enhancement of the ability of the chimeric proteins to mediate RNA export. Mutant-protein analysis demonstrated that the domain necessary for nuclear export mapped to the C-terminal region of TAP and required the domain that interacts with NXT1, as well as the region that has been shown to interact with nucleoporins. RevM10-TAP function was leptomycin B insensitive. In contrast, the function of this protein was inhibited by DeltaCAN, a protein consisting of part of the FG repeat domain of CAN/Nup214. These results show that TAP can complement Rev nuclear export signal function and redirect the export of intron-containing RNA to a CRM1-independent pathway. These experiments support the role of TAP as an RNA export factor in mammalian cells. In addition, they indicate that NXT1 serves as a crucial cellular cofactor in this process.

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Figures

FIG. 1
FIG. 1

Fusion to TAP functionally complements export-deficient Rev proteins. 293T cells were transfected with plasmids expressing the intron-containing gag-pol–RRE reporter mRNA, the indicated transactivator constructs, and a plasmid expressing SEAP from a completely spliced mRNA. At 72 h posttransfection, supernatants were collected and analyzed for p24 levels and SEAP activity. The values shown are averages of duplicate SEAP-normalized supernatant p24 levels. wt, wild type.

FIG. 2
FIG. 2

NXT1 dramatically enhances the export function of Rev-TAP fusion proteins. Transfections were performed as described in the legend to Fig. 1, except that 2 μg of pCDNA-NXT1 (empty bars) or pCDNA (black bars) was included, as indicated. wt, wild type.

FIG. 3
FIG. 3

NXT1-mediated enhancement of RevM10-TAP function occurs at the level of RNA export. Northern blot analysis of cytoplasmic (A) and nuclear (B) RNAs is shown. 293T cells were transfected with the indicated constructs. Nuclear or cytoplasmic poly(A)-selected RNA was isolated and analyzed. Blots were hybridized with probes complementary to HIV Gag (Gag/Pol) or SEAP (SEAP) coding sequences.

FIG. 4
FIG. 4

Expression of NXT1 does not alter the steady-state localization of M10-TAP or dramatically affect RevM10-TAP expression levels. (A) IP-Western blot (WB) analysis of proteins from transfected cells. Rev and RevM10-TAP fusion proteins were immunoprecipitated from lysates of transfected cells using an anti-Rev polyclonal antibody and separated using SDS-PAGE. Western blot analysis was performed with an anti-Rev monoclonal antibody (3H6) (45). Blots were visualized with ECL. Positions of commercial molecular weight standards (103 Bio-Rad) are indicated. (B) Fluorescence microscope analysis of GFP-TAP and RevM10-TAP proteins. Transfected CMT-3/COS cells were fixed and permeabilized. RevM10-TAP-expressing cells were stained using a primary anti-Rev rabbit polyclonal antibody and an Alexafluor-488-conjugated secondary antibody (Molecular Probes). Cells were visualized by fluorescence microscopy, and representative fields were photographed. wt, wild type. IF, immunofluorescence.

FIG. 4
FIG. 4

Expression of NXT1 does not alter the steady-state localization of M10-TAP or dramatically affect RevM10-TAP expression levels. (A) IP-Western blot (WB) analysis of proteins from transfected cells. Rev and RevM10-TAP fusion proteins were immunoprecipitated from lysates of transfected cells using an anti-Rev polyclonal antibody and separated using SDS-PAGE. Western blot analysis was performed with an anti-Rev monoclonal antibody (3H6) (45). Blots were visualized with ECL. Positions of commercial molecular weight standards (103 Bio-Rad) are indicated. (B) Fluorescence microscope analysis of GFP-TAP and RevM10-TAP proteins. Transfected CMT-3/COS cells were fixed and permeabilized. RevM10-TAP-expressing cells were stained using a primary anti-Rev rabbit polyclonal antibody and an Alexafluor-488-conjugated secondary antibody (Molecular Probes). Cells were visualized by fluorescence microscopy, and representative fields were photographed. wt, wild type. IF, immunofluorescence.

FIG. 5
FIG. 5

Schematic diagram of RevM10-TAP deletion mutant proteins. The indicated fragments of TAP-encoding DNA were fused to the end of the ORF encoding RevM10 using SOE-PCR. Solid lines indicate ORFs, and dotted lines represent deleted regions. These mutant DNAs were cloned into the mammalian expression vector pCMV (35). Functional data generated with these mutant constructs are shown in Fig. 6 and 7. NUP, nucleoporin.

FIG. 6
FIG. 6

The C-terminal region of TAP is essential for export activity. Plasmids expressing the indicated portions of TAP (Fig. 5) fused to RevM10 were transfected into 293T cells and tested for activity as described in the legend to Fig. 1. A plasmid expressing NXT1 was cotransfected as indicated. Plasmids expressing Rev or RevM10 were also tested as controls. The values shown are averages of duplicate SEAP-normalized p24 levels.

FIG. 7
FIG. 7

The NXT1- and nucleoporin-binding domains of TAP are required for export activity. (A) Plasmids expressing the indicated portions of TAP (Fig. 5) fused to RevM10 were transfected into 293T cells and tested for activity as described in the legend to Fig. 1. A plasmid expressing NXT1 was cotransfected as indicated. Plasmids expressing Rev or RevM10 were also tested as controls. The values shown are averages of duplicate SEAP-normalized p24 levels. (B) Lysates of 293T cells transfected with plasmids expressing C-terminal deletion mutant RevM10-TAP were subjected to IP-Western blot analysis as described in the legend to Fig. 4A. The values on the right are molecular weights in thousands. (C) FLAG-RevM10-TAP and NXT1 binding in vitro. Full-length RevM10-Tap (61-619) and the indicated RevM10-TAP deletion mutant proteins were assayed for in vitro NXT1-binding activity. 35S-labeled FLAG-RevM10-TAP fusion proteins and NXT1 protein were synthesized in vitro in rabbit reticulocyte lysates. FLAG-RevM10-TAP lysates were mixed with NXT1 lysates and allowed to bind. FLAG-RevM10-TAP proteins were immunoprecipitated with M2 anti-FLAG monoclonal antibody or a control irrelevant antibody, resolved by SDS-PAGE, and visualized by autoradiography. The bands corresponding to the proteins are indicated.

FIG. 8
FIG. 8

TAP-mediated export uses a CRM1-independent pathway. Duplicate transfections of 293T cells were performed with plasmids expressing the indicated Rev or Rev-TAP fusion proteins. pcDNA-NXT1 and pCMV-SEAP were included in all transfections. At 24 h posttransfection, the medium was replaced with a medium containing 2 nM LMB (black bars) or an equivalent amount of ethanol solvent (white bars). Samples were collected 12 h later. Fold inhibition was calculated taking the ratio of the SEAP-adjusted p24 value obtained with no drug to the SEAP-adjusted p24 value of the LMB-treated sample. N/A, not applicable. wt, wild type.

FIG. 9
FIG. 9

RevM10-TAP-mediated export is sensitive to the transdominant negative nucleoporin ΔCAN. Duplicate transfections of 293T cells were performed with increasing amounts of a plasmid expressing ΔCAN, and the total amount of plasmid DNA was held constant with the parental pBC12 plasmid. This was done in combination with either pCMVgagpol-CTE or pCMVgagpol-RRE and the pCMV-Rev or pCMV-RevM10-TAP and pcDNA-NXT1 plasmids, as indicated pCMV-SEAP was included in all transfections. Samples were collected and analyzed as described in the legend to the Fig. 1. The CTE, Rev, and RevM10-TAP values shown are SEAP-normalized averages of duplicate transfections expressed as percentages of the value obtained in the absence of ΔCAN. The SEAP curve was generated by using the average of all of the SEAP values from the CTE, Rev, and RevM10-TAP transfections.

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