CRM1 is distantly related to receptors that mediate nuclear protein import and was previously shown to interact with the nuclear pore complex. Overexpression of CRM1 in Xenopus oocytes stimulates Rev and U snRNA export from the nucleus. Conversely, leptomycin B, a cytotoxin that is shown to bind to CRM1 protein, specifically inhibits the nuclear export of Rev and U snRNAs. In vitro, CRM1 forms a leptomycin B-sensitive complex involving cooperative binding of both RanGTP and the nuclear export signal (NES) from either the Rev or PKI proteins. We conclude that CRM1 is an export receptor for leucine-rich nuclear export signals and discuss a model for the role of RanGTP in CRM1 function and in nuclear export in general.

Nonsense-mediated mRNA decay (NMD) is a surveillance mechanism that degrades mRNA containing premature termination codons (PTCs). In mammalian cells, recognition of PTCs requires translation and depends on the presence on the mRNA with the splicing-dependent exon junction complex (EJC). While it is known that a key event in the triggering of NMD is phosphorylation of the trans-acting factor, Upf1, by SMG-1, the relationship between Upf1 phosphorylation and PTC recognition remains undetermined. Here we show that SMG-1 binds to the mRNA-associated components of the EJC, Upf2, Upf3b, eIF4A3, Magoh, and Y14. Further, we describe a novel complex that contains the NMD factors SMG-1 and Upf1, and the translation termination release factors eRF1 and eRF3 (SURF). Importantly, an association between SURF and the EJC is required for SMG-1-mediated Upf1 phosphorylation and NMD. Thus, the SMG-1-mediated phosphorylation of Upf1 occurs on the association of SURF with EJC, which provides the link between the EJC and recognition of PTCs and triggers NMD.[Keywords: SMG-1; eRF; Upf; phosphorylation; EJC; NMD] Supplemental material is available at http://www.genesdev.org.

In metazoa, assembly of spliceosomal U snRNPs requires nuclear export of U snRNA precursors. Export depends upon the RNA cap structure, nuclear cap-binding complex (CBC), the export receptor CRM1/Xpo1, and RanGTP. These components are however insufficient to support U snRNA export. We identify PHAX (phosphorylated adaptor for RNA export) as the additional factor required for U snRNA export complex assembly in vitro. In vivo, PHAX is required for U snRNA export but not for CRM1-mediated export in general. PHAX is phosphorylated in the nucleus and then exported with RNA to the cytoplasm, where it is dephosphorylated. PHAX phosphorylation is essential for export complex assembly while its dephosphorylation causes export complex disassembly. The compartmentalized PHAX phosphorylation cycle can contribute to the directionality of export.

only the receptor but also the adapter has to be recycled after transport of the substrate and therefore also shuttles between the nuclear and cytoplasmic compart-

CRM1 is an export receptor mediating rapid nuclear exit of proteins and RNAs to the cytoplasm. CRM1 export cargoes include proteins with a leucine-rich nuclear export signal (NES) that bind directly to CRM1 in a trimeric complex with RanGTP. Using a quantitative CRM1-NES cargo binding assay, significant differences in affinity for CRM1 among natural NESs are demonstrated, suggesting that the steady-state nucleocytoplasmic distribution of shuttling proteins could be determined by the relative strengths of their NESs. We also show that a trimeric CRM1-NES-RanGTP complex is disassembled by RanBP1 in the presence of RanGAP, even though RanBP1 itself contains a leucine-rich NES. Selection of CRM1-binding proteins from Xenopus egg extract leads to the identification of an NES-containing DEAD-box helicase, An3, that continuously shuttles between the nucleus and the cytoplasm. In addition, we identify the Xenopus homologue of the nucleoporin CAN/Nup214 as a RanGTP-and NES cargo-specific binding site for CRM1, suggesting that this nucleoporin plays a role in export complex disassembly and/or CRM1 recycling.Nuclear export of proteins and RNAs is mediated by soluble, saturable factors. The existence of distinct soluble factors for different classes of export substrates was originally deduced from competition studies (32), and significant progress in their identification has recently been made (for reviews see references 28, 49, and 68).One class of export substrate carries a short, leucine-rich signal that mediates rapid transport to the cytoplasm, exemplified by the human immunodeficiency virus type 1 (HIV-1) Rev protein that uses its nuclear export signal (NES) to mediate export of genomic and subgenomic HIV-1 mRNAs out of the nucleus (37, 60). We and others identified CRM1 as an export receptor for such leucine-rich NESs, based on several lines of evidence (18,20,66). In Saccharomyces cerevisiae and Xenopus laevis oocytes, CRM1 can be inactivated by very different means-through a temperature-sensitive crm1 allele (66) and through binding of the cytotoxin leptomycin B (18, 74), respectively. In both cases, CRM1 inactivation leads to the accumulation of NES-containing substrates in the nucleus, an effect that in Xenopus oocytes can be reversed by overexpression of CRM1.Further evidence for the export function of CRM1 is its ability to directly interact with leucine-rich NESs (18,20). This binding is stabilized by cooperative binding of RanGTP (3,8,18). Like other small GTPases, Ran switches between the GDP-and GTP-bound states depending on the presence of its GTPase-activating enzyme, RanGAP, which promotes GTP hydrolysis, and its nucleotide exchange factor, RanGEF, which, because of the high GTP/GDP ratio in the cell, promotes RanGDP to RanGTP exchange (reviewed in references 12 and 49). In both vertebrate cells and yeast, RanGAP (named RanGAP1 in vertebrates and Rna1p in yeast) is found in the cytoplasm, whereas RanGEF (RCC1 in vertebrates) is chromatin bound and present in the nucleus. Therefore, a steep RanGTP-RanGDP ...