RanGTP-Binding Protein NXT1 Facilitates Nuclear Export of Different Classes of RNA In Vitro

Abstract

To better characterize the mechanisms responsible for RNA export from the nucleus, we developed an in vitro assay based on the use of permeabilized HeLa cells. This new assay supports nuclear export of U1 snRNA, tRNA, and mRNA in an energy- and Xenopusextract-dependent manner. U1 snRNA export requires a 5′ monomethylated cap structure, the nuclear export signal receptor CRM1, and the small GTPase Ran. In contrast, mRNA export does not require the participation of CRM1. We show here that NXT1, an NTF2-related protein that binds directly to RanGTP, strongly stimulates export of U1 snRNA, tRNA, and mRNA. The ability of NXT1 to promote export is dependent on its capacity to bind RanGTP. These results support the emerging view that NXT1 is a general export factor, functioning on both CRM1-dependent and CRM1-independent pathways of RNA export.

ACKNOWLEDGMENTS

B.O.N. and C.M. contributed equally to this work.

We thank B. Wolff (Novartis) for the generous gift of leptomycin B, I. W. Mattaj, J. P. Bachellerie, B. Goud, and E. Lund for U1ΔSm, U3, rab11, and tRNA constructs, respectively, G. Dreyfuss for GST-M9, J. C. Courvalin for anti-gp210 antibodies, R. Bastos for anti-Nup, and B. Burke for anti-Nup. We are grateful to D. Tenza and G. Raposo for their help in electron microscopy.

This work was supported by grants from the Association de Recherche contre le Cancer and the Ligue contre le Cancer to C.D. and by funds from the American Cancer Society grant RPG98-048-01-CSM to B.M.P. B.O.N. is supported by Rhone Poulenc Rorer.