Nuclear mRNA Export Requires Complex Formation between Mex67p and Mtr2p at the Nuclear Pores

We have identified between Mex67p and Mtr2p a complex which is essential for mRNA export. This complex, either isolated from yeast or assembled in Escherichia coli, can bind in vitro to RNA through Mex67p. In vivo, Mex67p requires Mtr2p for association with the nuclear pores, which can be abolished by mutating either MEX67 or MTR2. In all cases, detachment of Mex67p from the pores into the cytoplasm correlates with a strong inhibition of mRNA export. At the nuclear pores, Nup85p represents one of the targets with which the Mex67p-Mtr2p complex interacts. Thus, Mex67p and Mtr2p constitute a novel mRNA export complex which can bind to RNA via Mex67p and which interacts with nuclear pores via Mtr2p.     

A versatile interaction platform on the Mex67-Mtr2 receptor creates an overlap between mRNA and ribosome export

The transport receptor Mex67-Mtr2 functions in mRNA export, and also by a loop-confined surface on the heterodimer binds to and exports pre-60S particles. We show that Mex67-Mtr2 through the same surface that recruits pre-60S particles interacts with the Nup84 complex, a structural module of the nuclear pore complex devoid of Phe-Gly domains. In vitro, pre-60S particles and the Nup84 complex compete for an overlapping binding site on the loop-extended Mex67-Mtr2 surface. Chemical crosslinking identified Nup85 as the subunit in the Nup84 complex that directly binds to the Mex67 loop. Genetic studies revealed that this interaction is crucial for mRNA export. Notably, pre-60S subunit export impaired by mutating Mtr2 or the 60S adaptor Nmd3 could be partially restored by second-site mutation in Nup85 that caused dissociation of Mex67-Mtr2 from the Nup84 complex. Thus, the Mex67-Mtr2 export receptor employs a versatile binding platform on its surface that could create a crosstalk between mRNA and ribosome export pathways.     

The GLFG regions of Nup116p and Nup100p serve as binding sites for both Kap95p and Mex67p at the nuclear pore complex

Our previous studies have focused on a family of Saccharomyces cerevisiae nuclear pore complex (NPC) proteins that contain domains composed of repetitive tetrapeptide glycine-leucine-phenylalanine-glycine (GLFG) motifs. We have previously shown that the GLFG regions of Nup116p and Nup100p directly bind the karyopherin transport factor Kap95p during nuclear protein import. In this report, we have further investigated potential roles for the GLFG region in mRNA export. The subcellular localizations of green fluorescent protein (GFP)-tagged mRNA transport factors were individually examined in yeast cells overexpressing the Nup116-GLFG region. The essential mRNA export factors Mex67-GFP, Mtr2-GFP, and Dbp5-GFP accumulated in the nucleus. In contrast, the localizations of Gle1-GFP and Gle2-GFP remained predominantly associated with the NPC, as in wild type cells. The localization of Kap95p was also not perturbed with GLFG overexpression. Coimmunoprecipitation experiments from yeast cell lysates resulted in the isolation of a Mex67p-Nup116p complex. Soluble binding assays with bacterially expressed recombinant proteins confirmed a direct interaction between Mex67p and the Nup116-GLFG or Nup100-GLFG regions. Mtr2p was not required for in vitro binding of Mex67p to the GLFG region. To map the Nup116-GLFG subregion(s) required for Kap95p and/or Mex67p association, yeast two-hybrid analysis was used. Of the 33 Nup116-GLFG repeats that compose the domain, a central subregion of nine GLFG repeats was sufficient for binding either Kap95p or Mex67p. Interestingly, the first 12 repeats from the full-length region only had a positive interaction with Mex67p, whereas the last 12 were only positive with Kap95p. Thus, the GLFG domain may have the capacity to bind both karyopherins and an mRNA export factor simultaneously. Taken together, our in vivo and in vitro results define an essential role for a direct Mex67p-GLFG interaction during mRNA export.     

The mRNA export in Caenorhabditis elegans is mediated by Ce-NXF-1, an ortholog of human TAP/NXF and Saccharomyces cerevisiae Mex67p

Human TAP and Saccharomyces cerevisiae Mex67p belong to a family of proteins that mediate mRNA export. Computer searches identified previously two Caenorhabditis elegans genes, C15H11.3 and C115H11.6, that encode putative homologs of hTAP and Mex67p (Segref et al., EMBO J, 1997, 16:3256-3271). Using RNA interference experiments in C. elegans, we found that functional knockout of C15H11.3 resulted in nuclear accumulation of poly(A)-containing RNAs and was lethal for both embryos and adult nematodes. No embryonic or progeny abnormality was observed in functional knockout of C15H11.6. Taken together, these data established that the C15H11.3 gene product is an ortholog of hTAP and Mex67p; thus, it was named Ce-NXF-1. Ce-NXF-1 binds RNA directly and is a nucleocytoplasmic shuttle protein accumulating in the nucleoplasm and at the nuclear rim. The rim association is mediated via unique signals present in the C-terminal portion of all TAP/NXF and Mex67p proteins. This region was shown to interact with the FG-repeat domains of nucleoporins Nup98, Nup153, and Nup214, indicating that the rim association occurs through components of the nuclear pore complex. In summary, Ce-NXF-1 belongs together with hTAP and Mex67p to a family of proteins that participate in mRNA export and can provide a direct molecular link between mRNAs and components of the nuclear pore complex. Therefore, despite differences in mRNA metabolism between these species, they utilize a conserved mRNA transport mechanism.     

Mex67p, a novel factor for nuclear mRNA export, binds to both poly(A)+ RNA and nuclear pores.

An essential cellular factor for nuclear mRNA export called Mex67p which has homologous proteins in human and Caenorhabditis elegans was identified through its genetic interaction with nucleoporin Nup85p. In the thermosensitive mex67-5 mutant, poly(A)+ RNA accumulates in intranuclear foci shortly after shift to the restrictive temperature, but NLS-mediated nuclear protein import is not inhibited. In vivo, Mex67p tagged with green fluorescent protein (GFP) is found at the nuclear pores, but mutant mex67-5-GFP accumulates in the cytoplasm. Upon purification of poly(A)+ RNA derived from of UV-irradiated yeast cells, Mex67p, but not nucleoporins Nup85p and Nup57p, was crosslinked to mRNA. In a two-hybrid screen, a putative RNA-binding protein with RNP consensus motifs was found to interact with the Mex67p carboxy-terminal domain. Thus, Mex67p is likely to participate directly in the export of mRNA from the nucleus to the cytoplasm.     

Novel vertebrate nucleoporins Nup133 and Nup160 play a role in mRNA export.

RNA undergoing nuclear export first encounters the basket of the nuclear pore. Two basket proteins, Nup98 and Nup153, are essential for mRNA export, but their molecular partners within the pore are largely unknown. Because the mechanism of RNA export will be in question as long as significant vertebrate pore proteins remain undiscovered, we set out to find their partners. Fragments of Nup98 and Nup153 were used for pulldown experiments from Xenopus egg extracts, which contain abundant disassembled nuclear pores. Strikingly, Nup98 and Nup153 each bound the same four large proteins. Purification and sequence analysis revealed that two are the known vertebrate nucleoporins, Nup96 and Nup107, whereas two mapped to ORFs of unknown function. The genes encoding the novel proteins were cloned, and antibodies were produced. Immunofluorescence reveals them to be new nucleoporins, designated Nup160 and Nup133, which are accessible on the basket side of the pore. Nucleoporins Nup160, Nup133, Nup107, and Nup96 exist as a complex in Xenopus egg extracts and in assembled pores, now termed the Nup160 complex. Sec13 is prominent in Nup98 and Nup153 pulldowns, and we find it to be a member of the Nup160 complex. We have mapped the sites that are required for binding the Nup160 subcomplex, and have found that in Nup98, the binding site is used to tether Nup98 to the nucleus; in Nup153, the binding site targets Nup153 to the nuclear pore. With transfection and in vivo transport assays, we find that specific Nup160 and Nup133 fragments block poly[A]+ RNA export, but not protein import or export. These results demonstrate that two novel vertebrate nucleoporins, Nup160 and Nup133, not only interact with Nup98 and Nup153, but themselves play a role in mRNA export.     

The Glc7p nuclear phosphatase promotes mRNA export by facilitating association of Mex67p with mRNA

mRNA export is mediated by Mex67p:Mtr2p/NXF1:p15, a conserved heterodimeric export receptor that is thought to bind mRNAs through the RNA binding adaptor protein Yra1p/REF. Recently, mammalian SR (serine/arginine-rich) proteins were shown to act as alternative adaptors for NXF1-dependent mRNA export. Npl3p is an SR-like protein required for mRNA export in S. cerevisiae. Like mammalian SR proteins, Npl3p is serine-phosphorylated by a cytoplasmic kinase. Here we report that this phosphorylation of Npl3p is required for efficient mRNA export. We further show that the mRNA-associated fraction of Npl3p is unphosphorylated, implying a subsequent nuclear dephosphorylation event. We present evidence that the essential, nuclear phosphatase Glc7p promotes dephosphorylation of Npl3p in vivo and that nuclear dephosphorylation of Npl3p is required for mRNA export. Specifically, recruitment of Mex67p to mRNA is Glc7p dependent. We propose a model whereby a cycle of cytoplasmic phosphorylation and nuclear dephosphorylation of shuttling SR adaptor proteins regulates Mex67p:Mtr2p/NXF1:p15-dependent mRNA export.     

The Mtr2-Mex67 NTF2-like domain complex. Structural insights into a dual role of Mtr2 for yeast nuclear export

The formation of the Mtr2-Mex67 heterodimer is essential for yeast mRNA export as it constitutes a key nuclear component for shuttling mRNA between the nuclear and cytoplasm compartments through the nuclear pore complex. We report the crystal structures of apo-Mtr2 from the human pathogen Candida albicans and of its complex with the Mex67 NTF2-like domain. Compared with other members of the NTF2 fold family, Mtr2 displays novel structural features involved in the nuclear export of the large ribosomal subunit and consistent with a dual functional role of Mtr2 during yeast nuclear export events. The structure of the Mtr2-Mex67 NTF2-like domain complex, which overall is similar to those of the human and Saccharomyces cerevisiae homologs, unveils three putative Phe-Gly repeat binding sites, of which one contributes to the heterodimer interface. These structures exemplify an unrecognized adaptability of the NTF2 building block in evolution, identify novel structural determinants associated with key biological functions at the molecular surface of the yeast Mtr2-Mex67 complex, and suggest that the yeast and human mRNA export machineries may differ.     

Complex formation between Tap and p15 affects binding to FG-repeat nucleoporins and nucleocytoplasmic shuttling.

Mammalian Tap-p15 and yeast Mex67p-Mtr2p are conserved and essential mRNA export factor complexes that transport mRNPs through the nuclear pore. Here, we report that the small subunit p15 affects the binding of the large subunit Tap to repeat nucleoporins. BIAcore measurements revealed that recombinant Tap binds with high affinity (K(d) in the nm range) to repeat nucleoporins and dissociates from them very slowly. In contrast, when recombinant Tap was bound to p15, the derived heterodimeric complex exhibited a significant lower affinity to FG-repeat nucleoporins (K(d) in the microm range). Furthermore, when recombinant Tap lacking the N-terminal nuclear localization sequences (TapDeltaNLS) was microinjected in mammalian cells, it did not shuttle; however, TapDeltaNLS with bound p15 efficiently shuttles between nucleus and cytoplasm. We conclude that heterodimerization of Tap and p15 is required for shuttling of the functional Tap-p15 mRNA exporter complex.     

Sac3 is an mRNA export factor that localizes to cytoplasmic fibrils of nuclear pore complex

In eukaryotes, mRNAs are transcribed in the nucleus and exported to the cytoplasm for translation to occur. Messenger RNAs complexed with proteins referred to as ribonucleoparticles are recognized for nuclear export in part by association with Mex67, a key Saccharomyces cerevisiae mRNA export factor and homolog of human TAP/NXF1. Mex67, along with its cofactor Mtr2, is thought to promote ribonucleoparticle translocation by interacting directly with components of the nuclear pore complex (NPC). Herein, we show that the nuclear pore-associated protein Sac3 functions in mRNA export. Using a mutant allele of MTR2 as a starting point, we have identified a mutation in SAC3 in a screen for synthetic lethal interactors. Loss of function of SAC3 causes a strong nuclear accumulation of mRNA and synthetic lethality with a number of mRNA export mutants. Furthermore, Sac3 can be coimmunoprecipitated with Mex67, Mtr2, and other factors involved in mRNA export. Immunoelectron microscopy analysis shows that Sac3 localizes exclusively to cytoplasmic fibrils of the NPC. Finally, Mex67 accumulates at the nuclear rim when SAC3 is mutated, suggesting that Sac3 functions in Mex67 translocation through the NPC.     

The C-terminal domain of TAP interacts with the nuclear pore complex and promotes export of specific CTE-bearing RNA substrates

Messenger RNAs are exported from the nucleus as large ribonucleoprotein complexes (mRNPs). To date, proteins implicated in this process include TAP/Mex67p and RAE1/Gle2p and are distinct from the nuclear transport receptors of the beta-related, Ran-binding protein family. Mex67p is essential for mRNA export in yeast. Its vertebrate homolog TAP has been implicated in the export of cellular mRNAs and of simian type D viral RNAs bearing the constitutive transport element (CTE). Here we show that TAP is predominantly localized in the nucleoplasm and at both the nucleoplasmic and cytoplasmic faces of the nuclear pore complex (NPC). TAP interacts with multiple components of the NPC including the nucleoporins CAN, Nup98, Nup153, p62, and with three major NPC subcomplexes. The nucleoporin-binding domain of TAP comprises residues 508-619. In HeLa cells, this domain is necessary and sufficient to target GFP-TAP fusions to the nuclear rim. Moreover, the isolated domain strongly competes multiple export pathways in vivo, probably by blocking binding sites on the NPC that are shared with other transport receptors. Microinjection experiments implicate this domain in the export of specific CTE-containing RNAs. Finally, we show that TAP interacts with transportin and with two proteins implicated in the export of cellular mRNAs: RAE1/hGle2 and E1B-AP5. The interaction of TAP with nucleoporins, its direct binding to the CTE RNA, and its association with two mRNP binding proteins suggest that TAP is an RNA export mediator that may bridge the interaction between specific RNP export substrates and the NPC.     

Conserved nuclear export sequences in Schizosaccharomyces pombe Mex67 and human TAP function in mRNA export by direct nuclear pore interactions

Mex67, the homolog of human TAP, is not an essential mRNA export factor in Schizosaccharomyces pombe. Here we show that S. pombe encodes a homolog of the TAP cofactor that we have also named p15, whose function in mRNA export is not essential. We have identified and characterized two distinct nuclear export activities, nuclear export signal (NES) I and NES II, within the region of amino acids 434-509 of Mex67. These residues map within the known NTF2-like fold of TAP (amino acids 371-551). We show that the homologs of these two NESs are present and are functionally conserved in TAP. The NES I, NES II, and NES I + II of TAP and Mex67 directly bind with -phenylalanine-glycine (-FG)-containing sequences of S. pombe Nup159 and Nup98 but not with human p62. Mutants of NES I or NES II of Mex67/TAP that do not bind -FG Nup159 and Nup98 in vitro are unable to mediate nuclear export of a heterologous protein in S. pombe and in HeLa cells. Fused with the RNA recognition motifs (RRMs) of Crp79 and green fluorescent protein (GFP) (RRM-NES-GFP), the NES I and NES II of Mex67 or TAP can suppress the mRNA export defect of the Deltap15 rae1-167 synthetic lethal S. pombe strain, suggesting that the NESs can function in the absence of p15. These novel nuclear export sequences may provide additional routes for delivering Mex67/TAP to the nuclear pore complex.     

Structure and assembly of the Nup84p complex

The Nup84p complex consists of five nucleoporins (Nup84p, Nup85p, Nup120p, Nup145p-C, and Seh1p) and Sec13p, a bona fide subunit of the COPII coat complex. We show that a pool of green fluorescent protein-tagged Sec13p localizes to the nuclear pores in vivo, and identify sec13 mutant alleles that are synthetically lethal with nup85Delta and affect the localization of a green fluorescent protein-Nup49p reporter protein. In the electron microscope, sec13 mutants exhibit structural defects in nuclear pore complex (NPC) and nuclear envelope organization. For the assembly of the complex, Nup85p, Nup120p, and Nup145p-C are essential. A highly purified Nup84p complex was isolated from yeast under native conditions and its molecular mass was determined to be 375 kD by quantitative scanning transmission electron microscopy and analytical ultracentrifugation, consistent with a monomeric complex. Furthermore, the Nup84p complex exhibits a Y-shaped, triskelion-like morphology 25 nm in diameter in the transmission electron microscope. Thus, the Nup84p complex constitutes a paradigm of an NPC structural module with distinct composition, structure, and a role in nuclear mRNA export and NPC bio- genesis.     

Mechanisms of nuclear mRNA export: A structural perspective

Export of mRNA from the nucleus to the cytoplasm is a critical process for all eukaryotic gene expression. As mRNA is synthesized, it is packaged with a myriad of RNA‐binding proteins to form ribonucleoprotein particles (mRNPs). For each step in the processes of maturation and export, mRNPs must have the correct complement of proteins. Much of the mRNA export pathway revolves around the heterodimeric export receptor yeast Mex67?Mtr2/human NXF1?NXT1, which is recruited to signal the completion of nuclear mRNP assembly, mediates mRNP targeting/translocation through the nuclear pore complex (NPC), and is displaced at the cytoplasmic side of the NPC to release the mRNP into the cytoplasm. Directionality of the transport is governed by at least two DEAD‐box ATPases, yeast Sub2/human UAP56 in the nucleus and yeast Dbp5/human DDX19 at the cytoplasmic side of the NPC, which respectively mediate the association and dissociation of Mex67?Mtr2/NXF1?NXT1 onto the mRNP. Here we review recent progress from structural studies of key constituents in different steps of nuclear mRNA export. These findings have laid the foundation for further studies to obtain a comprehensive mechanistic view of the mRNA export pathway.     

Structural similarity in the absence of sequence homology of the messenger RNA export factors Mtr2 and p15

The association between Mtr2 and Mex67 is essential for the nuclear export of bulk messenger RNA in yeast. In metazoans, the analogous function is carried out by the TAP–p15 heterodimer. Whereas Mex67 and TAP are highly conserved proteins, their binding partners, Mtr2 and p15, share no sequence similarity, but are nevertheless functionally homologous. Here, we report the 2.8-Å resolution crystal structure of Mtr2 in complex with the NTF2-like domain of Mex67. Mtr2 is a novel member of the NTF2-like family and interacts with Mex67, forming a complex with a similar structural architecture to that of TAP–p15. Mtr2 fulfils an analogous function to that of human p15 in maintaining the structural integrity of the heterodimer. In addition, Mtr2 presents a long internal loop, which contains residues that affect the export of the large ribosomal subunit.     

Nuclear export of ribosomal 60S subunits by the general mRNA export receptor Mex67-Mtr2

The yeast Mex67-Mtr2 complex and its homologous metazoan counterpart TAP-p15 operate as nuclear export receptors by binding and translocating mRNA through the nuclear pore complexes. Here, we show how Mex67-Mtr2 can also function in the nuclear export of the ribosomal 60S subunit. Biochemical and genetic studies reveal a previously unrecognized interaction surface on the NTF2-like scaffold of the Mex67-Mtr2 heterodimer, which in vivo binds to pre-60S particles and in vitro can interact with 5S rRNA. Crucial structural requirements for this binding platform are loop insertions in the middle domain of Mex67 and Mtr2, which are absent from human TAP-p15. Notably, when the positively charged amino acids in the Mex67 loop are mutated, interaction of Mex67-Mtr2 with pre-60S particles and 5S rRNA is inhibited, and 60S subunits, but not mRNA, accumulate in the nucleus. Thus, the general mRNA exporter Mex67-Mtr2 contains a distinct electrostatic interaction surface for transporting 60S preribosomal cargo.     

异二聚体介导的mRNA核输出机制

真核生物mRNA在细胞核内被转录,而到达细胞质内翻译成为蛋白质,因此跨越核孔的核输出过程是必须的。现在已确定异二聚体TAP/NXT(在酵母中为Mex67p/Mtr2p)在此过程中是最基本的元件,此外其他相关因子如Aly、UAP56等也参与了这个复杂的过程,包括结合、输出、解离和载体输入。本文简要介绍了mRNA输出的基本机制。