NXT1 (p15) Is a Crucial Cellular Cofactor in TAP-Dependent Export of Intron-Containing RNA in Mammalian Cells

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 RevΔ78–79. 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 ΔCAN, 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.

ACKNOWLEDGMENTS

We thank Bryan Cullen and J. U. Jung for the gift of plasmids and Barbara Wolff (Novartis) for providing LMB.

This work was supported by NIH grants AI34721 to M.-L.H. and AI47008 to D.R. and American Cancer Society grant RPG-98-048-01-CSM to B.M.P. Salary support for M.-L.H. and D.R. was provided by the Charles H. Ross Jr. and Myles H. Thaler Endowments at the University of Virginia.