11.4 Signal-Mediated Transport through Nuclear Pore Complexes -- Molecular Cell Biology -- NCBI Bookshelf

Nuclear Pore Complexes Actively Transport Macromolecules between the Nucleus and Cytoplasm

Figure 11-28
Nuclear pore complex

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Figure 11-28

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Nuclear pore complex

(a) Nuclear envelopes of Xenopus oocytes visualized by field emission in-lens scanning electron microscopy. Left: Cytoplasmic face of nuclear pore complexes (NPCs). Middle: Nucleoplasmic face of NPCs, showing the “basket” structure. Right: Nucleoplasmic face of the nuclear envelope after removal of the nuclear membrane by mild detergent treatment. The nuclear lamin network, which inserts into the nuclear ring of the NPC, is exposed by this treatment. (b) Cut-away model of the NPC. [Part (a) from V. Doye and E. Hurt, 1997, Curr. Opin. Cell Biol. 9401; courtesy of M. W. Goldberg and T. D. Allen. Part (b) adapted from M. Ohno et al., 1998, Cell 92:327.]

The nuclear pore complex is immense by molecular standards, ≈125 million daltons in vertebrates, or about 30 times larger than a ribosome. It is made up of multiple copies of some 50 – 100 different proteins. Nuclear pore complexes are well visualized in electron micrographs of the nuclear envelope microdissected from the large nuclei of amphibian oocytes (Figure 11-28a

). Electron micrographs such as these have led to the model for the nuclear pore complex shown in Figure 11-28b

. As illustrated in this model, the NPC nuclear ring supports eight ≈100-nm-long filaments whose distal ends are joined by the terminal ring, forming a structure called the nuclear basket. The nuclear ring is also attached directly to the , a network of that extends over the inner surface of the nuclear envelope (Section 19.6).

Ions, small metabolites, and globular proteins up to ≈60 kDa can diffuse through water-filled channels in the nuclear pore complex; these channels behave as if they are ≈9 Å in diameter. However, large proteins and ribonucleoprotein complexes, up to ≈25 nm in diameter, cannot diffuse in and out of the nucleus; rather, they are actively transported through the central plug of the nuclear pore complex (see Figure 11-28b

). All RNAs synthesized in the nucleus must be exported to the cytosol before they can function in protein synthesis. Conversely, all proteins found in the nucleus must be imported from the cytoplasm where they are synthesized on ribosomes. The nuclear pore complex acts as a gated channel through which these macromolecules are selectively transported in and out of the nucleus.

When nuclear pores were first visualized by electron microscopy, researchers assumed that they served as portals of entry into and exit from the nucleus. The first studies of transport through nuclear pore complexes analyzed the import of proteins into the nucleus. These early experiments made use of nucleoplasmin, an abundant nuclear protein in Xenopus oocytes that assists in the assembly of chromatin during the rapid cell replication that follows fertilization. In one key study, small gold particles coated with nucleoplasmin or with a non-nuclear protein were microinjected into the cytosol of frog oocytes. The location of the gold particles was determined by electron microscopy after sectioning the oocytes. Shortly after injection, the nucleoplasmin-coated gold particles clustered at the nuclear pore complexes; later, they accumulated in the nucleus after passing through the pores. Gold particles coated with non-nuclear proteins, by contrast, remained in the cytoplasm and did not bind to nuclear pores complexes. These experiments not only demonstrated definitively that nuclear pores are routes for protein import but also suggested that some signal present in proteins like nucleoplasmin is required for transport to occur.

Figure 11-29
Formation of coiled heterogeneous ribonucleoprotein (more...)

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Figure 11-29

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Formation of coiled heterogeneous ribonucleoprotein (hnRNP) during synthesis of the Chironomous tentans Balbiani ring (BR) mRNA

(a) Electron micrograph of active transcription loops of chromatin with characteristic granules of ribonucleoprotein (arrow). The transcribed loops of chromatin extend above the dark staining, dense, nontranscribed chromatin. (b) Reconstruction of a single chromatin transcription loop from serial thin sections reveals a gradual increase in size of the associated ribonucleoprotein (RNP) particles that reflects the increasing length of the RNA transcripts being synthesized. (c) A model for the structure and biogenesis of BR hnRNP. The BR gene, which contains four exons and four introns, encodes a secreted protein that glues the insect larvae to a solid support, such as a twig, in preparation for metamorphosis. [Parts (a) and (b) from C. Erricson et al., 1989, Cell 56:631; courtesy of B. Daneholt. Part (c) adapted from B. Daneholt, 1997, Cell 88:585.]

Some of the earliest studies on nuclear export analyzed the transport of mRNA from the nucleus to the cytosol. In the nucleus, as discussed previously, nascent RNA transcripts associate with various proteins forming heterogeneous ribonucleoproteins (hnRNPs). RNPs that contain fully processed mRNAs, referred to as messenger RNPs (mRNPs), are exported to the cytosol through nuclear pore complexes. The salivary glands of larvae of the insect Chironomous tentans are a good model system for studying both the formation of hnRNPs and export of mRNPs. In these larvae, genes in large chromosomal puffs called Balbiani rings are abundantly transcribed into nascent pre-mRNAs that associate with hnRNP proteins to form 50-nm coiled hnRNPs (Figure 11-29

).

Figure 11-30
Electron micrographs of Balbiani ring mRNPs passing (more...)

Figure 11-30

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Electron micrographs of Balbiani ring mRNPs passing through nuclear pore complexes in salivary glands of a Chironomous tentans larva

The mRNPs appear to uncoil as they pass through a nuclear pore (a – d). As they enter the cytoplasm (e – g), the mRNPs appear to associate with ribosomes (arrows). [From H. Mehlin et al., 1992, Cell 69:605, courtesy of B. Daneholt.]

Figure 11-31
Model for passage of mRNAs through the nuclear pore (more...)

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Figure 11-31

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Model for passage of mRNAs through the nuclear pore complex (NPC) based on electron microscopic studies of Balbiani ring (BR) mRNA transport in Chironomous tentans (seeFigure 11-30).

After the coiled mRNP moves through the terminal ring, it uncoils as it passes through the central plug of the NPC, with the 5′ end leading the way and becoming associated with ribosomes in the cytoplasm. The NPC is thought to undergo a conformational change during transport. [Adapted from B. Daneholt, 1997, Cell 88:585.]

After processing of Balbiani ring pre-mRNA, the resulting mRNPs move through nuclear pores to the cytosol. Electron micrographs of sections of these cells show mRNPs uncoiling during their passage through nuclear pores and binding to ribosomes as they enter the cytosol (Figure 11-30

).The observation that mRNPs become associated with ribosomes during transport indicates that the 5′ end leads the way through the nuclear pore complex. Electron microscopic studies such as these have lead to the model depicted in Figure 11-31

for passage of mRNPs through nuclear pore complexes.

Subsequent experiments have revealed many details about how macromolecules are transported through nuclear pore complexes. We first discuss nuclear export and then consider nuclear import; as we will see the mechanisms of the two processes are similar in many respects.

Receptors for Nuclear-Export Signals Transport Proteins and mRNPs out of the Nucleus

Figure 11-32
Heterokaryon assay demonstrating that human hnRNP A1 (more...)

Figure 11-32

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Heterokaryon assay demonstrating that human hnRNP A1 protein can cycle in and out of the cytoplasm, but human hnRNP C protein cannot

Heterokaryons were prepared by treating HeLa cells and cultured Xenopus cells with polyethylene glycol. The cells were treated with cycloheximide immediately after fusion to prevent protein synthesis. After 2 hours, the cells were fixed and stained with fluorescent-labeled antibodies specific for hnRNP C and hnRNP A1. These antibodies do not bind to the homologous Xenopus proteins. (a) A fixed preparation viewed by phase-contrast microscopy includes unfused HeLa cells (arrowhead) and Xenopus cells (dotted arrow), as well as fused heterokaryons (solid arrow). In the heterokaryon in this micrograph, the round HeLa-cell nucleus is to the right of the oval-shaped Xenopus nucleus. (b,c) When the same preparation was viewed by fluorescence microscopy, the stained hnRNP C protein appeared green and the stained hnRNP A1 protein appeared red. Note that the unfused Xenopus cell on the left is unstained, confirming that the antibodies are specific for the human proteins. In the heterokaryon, hnRNP C only appears in HeLa-cell nuclei (b), whereas hnRNP A1 appears in both nuclei (c). Since protein synthesis was blocked after cell fusion, some of the human hnRNP A1 must have left the HeLa-cell nucleus, moved through the cytoplasm, and entered the Xenopus nucleus in the heterokaryon. [See S. Pinol-Roma and G. Dreyfuss, 1992, Nature 355:730; courtesy of G. Dreyfuss.]

Cell-fusion experiments provided the first evidence that specific hnRNP proteins participate in the export of mRNA from the nucleus. The heterokaryon experiments, described in Figure 11-32

, demonstrated that some hnRNP proteins cycle in and out of the cytoplasm, whereas others remain localized in the nucleus.

More recent studies have revealed that certain hnRNP proteins contain a nuclear-export signal (NES) that stimulates their active transport through nuclear pores. In these studies, a gene encoding a nucleus-restricted protein such as nucleoplasmin is fused to various segments of a gene encoding a protein (e.g., human hnRNP A1) that shuttles in and out of the nucleus. The engineered gene then is transfected into HeLa cells before fusion to Xenopus cells in the heterokaryon assay. Observation of the expressed fusion protein (e.g., nucleoplasmin-hnRNP A1) in the Xenopus nucleus indicates that the short fused segment functions as a NES directing transport of the fusion protein (similar to shuttling of hnRNP A1 in Figure 11-32

). Experiments of this type have identified at least three different classes of NESs: a 38-residue sequence in hnRNP A1, one in hnRNP K, and a leucine-rich sequence found in PKI (an inhibitor of protein kinase A) and in the Rev protein of human immunodeficiency virus (HIV) discussed in a later section.

Figure 11-33
Proposed mechanism for the transport of (more...)

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Figure 11-33

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Proposed mechanism for the transport of “cargo” proteins containing a leucine-rich nuclear-export signal (NES) from the nucleus to the cytosol

In the nucleoplasm, the protein exportin 1 binds cooperatively to the NES of the cargo protein to be transported and to Ran · GTP. After the resulting cargo complex passes through a nuclear pore complex (NPC), RanGAP, localized to the cytoplasm, stimulates conversion of Ran · GTP to Ran · GDP. The accompanying conformational change in Ran leads to dissociation of the complex. The NES-containing cargo protein is left free in the cytosol, while exportin 1 and Ran · GDP are transported back into the nucleus through NPCs. RCC1, localized to the nucleus, stimulates conversion of Ran · GDP to Ran · GTP. Repetition of this cycle leads to export of multiple molecules of the cargo protein. [Adapted from M. Ohno et al., 1998, Cell 92:327.]

The mechanism of export of shuttling proteins is best understood for those containing a leucine-rich NES. According to the current model shown in Figure 11-33

, this NES promotes export of a “cargo” protein from the nucleus by binding to a specific nuclear-export receptor in the nucleus, a protein called exportin 1. Nuclear export also requires a third protein, Ran, a small GTPase that exists in two conformations, one when complexed with GTP and an alternative one when complexed with GDP. The simultaneous interaction of exportin 1 with Ran · GTP and the NES of a cargo protein in the nucleus forms a trimolecular cargo complex. Interaction of a cargo complex with proteins in the nuclear pore complex leads to movement of the cargo complex through the pore by mechanisms that are not yet understood.

Once the Ran · GTP/exportin 1/cargo protein complex reaches the cytosol, Ran GTPase-activating protein (RanGAP) stimulates Ran to hydrolyze its bound GTP to GDP. The resulting conformational change in Ran causes dissociation of the cargo complex, releasing the free NES-containing cargo protein (see Figure 11-33

). The free exportin 1 and Ran · GDP are transported back into the nucleus where a Ran nucleotide-exchange factor, called RCC1, causes Ran to release its GDP and rebind GTP, which is present in much higher concentration. The regenerated Ran · GTP and exportin 1 then can transport another NES-containing cargo protein to the cytosol. (As discussed in , other GTPases that cycle between GTP-bound and GDP-bound forms, such as G_sα and Ras, function in many signal-transduction pathways.) Localization of RanGAP and RCC1 to the cytosol and nucleus, respectively, is the basis for the unidirectional transport of cargo proteins containing a leucine-rich NES.

Figure 11-34
Proposed mechanism for hnRNP (more...)

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Figure 11-34

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Proposed mechanism for hnRNP protein – mediated export of mRNA from the nucleus

(a) The 5′ end of the fully processed mRNA – hnRNP protein complex (mRNP) associates with cap-binding complex (CBC), which passes through the nuclear pore complex (NPC) first. (b) Nucleusrestricted hnRNPs (orange and dark blue) are removed as a mRNP is transported through the NPC; these proteins, which lack a NES, would hold the mRNA (red) in the nucleus. NES-bearing hnRNPs, such as hnRNP A1, are transported through the NPC by the mechanism diagrammed in Figure 11-33

, carrying the associated mRNA into the cytoplasm. (c) Cytoplasmic RanGAP stimulates hydrolysis of GTP by Ran. The shuttling hnRNP proteins then dissociate from the receptor proteins (exportin 1 for leucine-rich NESs) and are transported back into the nucleus. The mRNA is then available to interact with cytosolic mRNP proteins, including poly(A)-binding protein (PABP), which binds to the 3′ poly(A) tail of mRNA. [Adapted from S. Nakielny and G. Dreyfuss, 1997, Curr. Opin. Cell Biol. 9:420.]

A similar nuclear-cytosolic “shuttle” is thought to export fully processed mRNA – hnRNP protein complexes (mRNPs) from the nucleus, as depicted in Figure 11-34

. According to this model, a nuclear cap – binding complex (CBC), which associates with the 5′ cap, leads the way through the nuclear pore complex. Receptors like exportin 1 are postulated to make successive interactions with proteins of the nuclear pore complex as they are transported through a pore. Through mechanisms that are not fully understood, the shuttling hnRNP proteins are displaced from the mRNA and then replaced with cytosolic mRNA-binding proteins. The released hnRNP proteins, nuclear-export receptor, and Ran · GDP are then transported back into the nucleus by a mechanism that is analogous to their export.

Pre-mRNAs in Spliceosomes Are Not Exported from the Nucleus

It is critical that only fully processed mature mRNAs be exported from the nucleus because translation of incompletely processed pre-mRNAs containing introns would produce defective proteins, which might interfere with the functioning of the cell. By mechanisms that are not fully understood, pre-mRNAs associated with snRNPs in spliceosomes are prevented from being transported to the cytosol. In one type of experiment, for instance, a gene encoding a pre-mRNA with a single intron that is efficiently spliced out was mutated to introduce deviations from the consensus splice-site sequences. Mutation of either the 5′ or 3′ invariant splice site at the ends of the intron resulted in pre-mRNAs that were bound by snRNPs to form spliceosomes; however, RNA splicing was blocked and the pre-mRNA was retained in the nucleus. In contrast, mutation of both the 5′ and 3′ splice sites in the same pre-mRNA resulted in efficient export of the unspliced pre-mRNA. In this case, the pre-mRNAs were not efficiently bound by snRNPs.

graphic element Many cases of thalassemia, an inherited disease that results in abnormally low levels of globin proteins, are due to mutations in globin-gene splice sites that decrease the efficiency of splicing but do not prevent association of the pre-mRNA with snRNPs. The resulting unspliced globin pre-mRNAs are retained in reticulocyte nuclei and are rapidly degraded.

Receptors for Nuclear-Localization Signals Transport Proteins into the Nucleus

All proteins found in the nucleus are synthesized in the cytoplasm and actively imported into the nucleus. These include nucleus-restricted proteins (e.g., histones, lamins, DNA and RNA polymerases, replication and transcription factors, splicing proteins, and some hnRNPs), as well as proteins that shuttle between the nucleus and cytoplasm (e.g., hnRNP A1 protein, exportin 1). All proteins actively imported through nuclear pore complexes contain a nuclear-localization signal (NLS). Shuttling proteins contain both a nuclear-export signal (NES) and NLS.

Figure 11-35
Demonstration that the nuclear-localization signal (more...)

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Figure 11-35

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Demonstration that the nuclear-localization signal (NLS) of the SV40 large T-antigen can direct a cytoplasmic protein to the cell nucleus

(a) Normal pyruvate kinase, visualized by immunofluorescence after treating cultured cells with a specific antibody, is localized to the cytoplasm. (b) A chimeric pyruvate kinase protein, containing the SV40 NLS at its N-terminus, is directed to the nucleus. The chimeric protein was expressed from a transfected engineered gene produced by fusing a viral gene fragment encoding the SV40 NLS to the pyruvate kinase gene. [From D. Kalderon et al., 1984, Cell 39:499; courtesy Dr. Alan Smith.]

NLSs were first discovered during the analysis of mutants of simian virus 40 (SV40) that produced an abnormal form of the early viral protein called large T-antigen. The wild-type form of this protein is localized to the nucleus in virus-infected cells, whereas mutated forms of large T-antigen accumulate in the cytosol. The mutations responsible for this altered cellular localization all occur within five consecutive basic amino acids in the sequence Pro-Lys-Lys-Lys-Arg-Lys-Val. Remarkably, when this region of SV40 large T-antigen was fused to pyruvate kinase, a very large cytosolic protein involved in carbohydrate metabolism, the fusion protein was transported into nuclei (Figure 11-35

). The minimal amino acid sequence that directs pyruvate kinase to the nucleus is the seven-residue sequence shown above. Moreover, 5-nm gold particles coated with this synthetic peptide are transported through nuclear pores after microinjection into the cytoplasm of cultured cells. These experiments demonstrated that this short sequence from SV40 large T-antigen acts as a signal that causes the transport of associated macromolecules into the nucleus, analogous to the nuclear-export signals discussed above.

Similar methods have been used to identify NLS sequences in numerous other proteins imported into the nucleus. Many are similar to the SV40 large T-antigen NLS, containing several consecutive basic amino acids. Other NLSs are chemically quite different. For instance, the NLS in the hnRNP A1 protein, which shuttles between the nucleus and cytosol, is relatively hydrophobic and overlaps with the NES in this protein.

Figure 11-36
Experimental demonstration that nuclear transport in (more...)

Figure 11-36

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Experimental demonstration that nuclear transport in permeabilized cultured cells requires soluble cytosolic components and ATP

(a) Phase-contrast micrographs of untreated and digitonin-permeabilized HeLa cells. Treatment of a monolayer of cultured cells with the mild, nonionic detergent digitonin permeabilizes the plasma membrane so that cytosolic constituents leak out, but leaves the nuclear envelope and NPCs intact. (b) Fluorescence micrographs of digitonin-permeabilized HeLa cells incubated with a fluorescent protein chemically coupled to a synthetic SV40 T-antigen NLS peptide in the presence and absence of ATP and cytosol (lysate). Accumulation of this transport substrate in the nucleus occurred only when both cytosol and ATP were included in the incubation (lower left). [From S. Adam et al., 1990, J. Cell. Biol. 111:807; courtesy of Dr. Larry Gerace.]

Development of a digitonin-permeabilized cell system provided a convenient in vitro assay for demonstrating that soluble cytosolic components and ATP are required for nuclear import (Figure 11-36

). Using this assay, researchers subsequently purified and characterized four cytosolic proteins required for nuclear import of a protein containing a basic NLS: importin α; importin β; nuclear transport factor 2 (NTF2); and Ran, the same GTPase involved in nuclear export (see Figure 11-33

). Further biochemical studies showed that importin α and β form a heterodimeric nuclear-import receptor that binds to a basic NLS through the α subunit. The β subunit of dimeric importin interacts with NPC cytosolic filaments in the absence of ATP; in the presence of ATP, the β subunit is thought to interact with other components of the NPC during transport through the nuclear pore complex.

Figure 11-37
Proposed mechanism for the transport of (more...)

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Figure 11-37

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Proposed mechanism for the transport of “cargo” proteins containing a basic nuclear-localization signal (NLS) from the cytoplasm to the nucleus

In the cytoplasm (bottom), importin α and β interact cooperatively with the cargo protein to be transported, with the NLS binding to importin α. The importin β subunit of the resulting trimeric cargo complex interacts with components of the NPC, translocating the complex into the nucleoplasm by a poorly understood mechanism that requires ATP hydrolysis. In the nucleoplasm, Ran · GTP interacts with importin β, causing dissociation of the cargo complex, thereby delivering free cargo protein to the nucleoplasm. To support another cycle of import, monomeric importin α and the importin β – Ran · GTP complex are transported back to the cytoplasm. Ran GTP-activating protein (RanGAP) in the cytoplasm stimulates conversion of Ran · GTP to Ran · GDP resulting in a conformational change in Ran that causes it to dissociate from importin β. The free importin β can now interact with importin α and a new cargo protein bearing a basic NLS, initiating another round of nuclear import. Presumably, Ran · GDP is also transported through nuclear pores from the cytoplasm to the nucleoplasm, where the Ran nucleotide-exchange factor (RCC1) causes it to release GDP and rebind GTP.

A model for the import of cytosolic “cargo” proteins bearing a basic NLS is shown in Figure 11-37

. By comparing this model for nuclear import with that in Figure 11-33

for nuclear export, we can see the similarities in the two transport processes. Although the mechanisms differ slightly, the unidirectional nature of both import and export depends on the asymmetric distribution of Ran GTP-activating protein (RanGAP), which is restricted to the cytoplasm, and Ran nucleotide-exchange factor (RCC1), which is restricted to the nucleoplasm. The models of nuclear export and import each require that Ran · GDP be transported from the cytoplasm to the nucleus, that Ran · GTP be transported from the nucleus to the cytoplasm, and that other components (e.g., exportin 1, importin α, and importin β) be selectively transported into or out of the nucleus depending on their associations with other proteins.

There are two obvious differences in the export of leucine-rich NES-bearing proteins and import of basic-NLS – bearing proteins: (1) Ran · GTP is part of the cargo complex during export but not during import, and (2) the receptor that directs transport of a cargo protein through a nuclear pore is a monomer in the case of export (e.g., exportin 1) but a dimer in the case of import (e.g., importin αβ). Another difference between the two transport processes is that import of cargo proteins with a basic NLS also requires NTF2. This soluble cytosolic factor interacts in vitro with Ran · GDP, importin β, and NPCs, but its precise function is not clear. Deletion of the yeast homolog of NTF2 is lethal, but the effects of this mutation can be suppressed by overexpression of yeast Ran. This finding suggests that NTF2 normally functions to enhance the activities of Ran during nuclear import.

The distance between the tip of the NPC cytosolic filaments and the nuclear basket in the nucleoplasm is ≈200 nm (see Figure 11-28b

). It is likely that as nuclear-transport receptors like exportin 1 and importin β traverse this distance, they make multiple contacts with distinct NPC proteins (called nucleoporins). Many nucleoporins contain multiple short repeats of the sequences Phe-X-Phe-Gly and Gly-Leu-Phe-Gly, which have affinity for importin β and may serve as docking sites for interactions during transport. However, it is not yet known how many contacts occur between receptor proteins and nucleoporins or which nucleoporins participate in these interactions.

Various Nuclear-Transport Systems Utilize Similar Proteins

As noted earlier, hnRNP A1 protein, which cycles between the nucleus and cytosol, has an NLS that is hydrophobic rather than basic. In the cytoplasm, hnRNP A1 interacts with a monomeric nuclear-import receptor, called transportin, that both binds the NLS and mediates interactions with the NPC resulting in transport into the nucleoplasm. The NES on hnRNP A1 has a sequence distinct from that of the leucine- rich NESs and interacts with a nuclear-export receptor different from exportin 1. The export and import of shuttling proteins like hnRNP A1 is thought to occur by mechanisms similar to those depicted in Figures 11-33

and 11-37

, and presumably requires some type of regulation of the NLS and NES signals in the same protein.

Another nuclear-transport system that has been identified and partially characterized imports snRNPs, which are critical to splicing of pre-mRNAs (see Figure 11-19). U1, U2, U4, and U5 snRNAs are transported from their site of synthesis in the nucleus to the cytosol where their 5′ methylguanylate cap is methylated twice more at specific positions and where snRNP proteins bind to form mature snRNP particles. After the mature snRNPs interact with a nuclear-import receptor that is distinct from importin β and transportin, they are transported into the nucleus through NPCs.

The three nuclear-transport receptors characterized thus far — importin β, exportin 1, and transportin — share sequence homology, which probably reflects their homologous interactions with Ran and nucleoporins. Sequencing of the yeast genome has revealed fourteen genes encoding proteins with significant homology to these three nuclear-transport receptors, including their yeast homologs. This finding suggests that several distinct receptor proteins participate in both nuclear import and export. Additional nuclear-transport systems will likely be characterized in the future.

HIV Rev Protein Regulates the Transport of Unspliced Viral mRNAs

As discussed earlier, transport of mRNPs containing mature, functional mRNAs from the nucleus to the cytoplasm entails a complex mechanism that is crucial to gene expression (see Figure 11-34

). Regulation of this transport theoretically could provide another means of gene control, although it appears to be relatively rare. Indeed, the only examples of regulated mRNA export discovered to date occur during the cellular response to conditions (e.g., heat shock) that cause protein denaturation and during viral infection when virus-induced alterations in nuclear transport maximize viral replication. Here we describe the regulation of mRNP export mediated by Rev, a protein encoded by human immunodeficiency virus (HIV).

Figure 11-38
Role of Rev protein in transport of HIV mRNAs from (more...)

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Figure 11-38

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Role of Rev protein in transport of HIV mRNAs from the nucleus to the cytoplasm

The HIV genome, which contains overlapping coding regions, is transcribed into a single 9-kb primary transcript. Several ≈4-kb mRNAs result from splicing out of one intron (blue angled line), and several ≈2-kb mRNAs from splicing out of two or more introns. After transport to the cytoplasm, each RNA species is translated into different viral proteins. Rev protein, encoded by a 2-kb mRNA, interacts with the Rev-response element (RRE) in the unspliced and singly spliced mRNAs, stimulating their transport to the cytoplasm. [Adapted from B. R. Cullen and M. H. Malim, 1991, Trends Biochem. Sci. 16:346.]

A retrovirus, HIV integrates a DNA copy of its RNA genome into the host-cell DNA (see ). The integrated viral DNA, or provirus, contains a single transcription unit. The single primary transcript produced from the HIV provirus by cellular enzymes can be spliced in alternative ways to yield three classes of mRNAs: a 9-kb unspliced mRNA; ≈4-kb mRNAs formed by removal of one intron; and ≈2-kb mRNAs formed by removal of two or more introns (Figure 11-38

). After their synthesis in the host-cell nucleus, all three classes of HIV mRNAs are transported to the cytoplasm and translated into viral proteins; some of the 9-kb unspliced RNA is used as the viral genome in progeny virions that bud from the cell surface. Since the 9-kb and 4-kb HIV mRNAs contain splice sites, they can be viewed as incompletely spliced mRNAs. However, as discussed earlier, association of such incompletely spliced mRNAs with snRNPs in spliceosomes normally blocks their export from the nucleus. Thus HIV must have evolved a mechanism for overcoming this block, permitting export of the longer HIV mRNAs.

Studies with HIV mutants showed that transport of unspliced 9-kb and singly spliced 4-kb viral mRNAs from the nucleus to the cytoplasm does not occur in infected cells unless Rev protein is expressed. Subsequent biochemical experiments demonstrated that Rev binds to a specific Revresponse element (RRE) present in HIV RNA. In cells infected with HIV mutants lacking the RRE, unspliced and singly spliced viral mRNAs remain in the nucleus, demonstrating that the RRE is required for Rev-mediated stimulation of transport.

Subsequent experiments, based on those described earlier with genes mutated at the 5′ and/or 3′ splice sites, have given insight into how binding of Rev protein to the RRE stimulates transport of certain HIV mRNAs. Recall that pre-mRNAs with a mutation in the 5′ or 3′ splice site are assembled into spliceosomes but cannot complete the splicing reaction; because the mutant pre-mRNAs are not released from spliceosomes, they are not transported into the cytoplasm. When a RRE is engineered into a pre-mRNA with a mutation in the 5′ or 3′ splice site, the unspliced pre-mRNA is transported into the cytoplasm in cells expressing Rev. This finding indicates that binding of Rev to a pre-mRNA somehow permits the spliceosome-associated RNA to be transported to the cytoplasm. Recently Rev has been shown to contain a leucine-rich NES that interacts with exportin 1 complexed with Ran · GTP. Consequently, Rev is thought to promote the export of unspliced and singly spliced HIV mRNAs through interactions with exportin 1 and the nuclear pore complex (see Figure 11-33

). By an unknown mechanism these interactions overcome the block to RNA export imposed by association with spliceosomes.

SUMMARY

The nuclear envelope contains numerous nuclear pore complexes (NPCs), large, complicated structures composed of multiple copies of ≈50 – 100 proteins called nucleoporins (see Figure 11-30b).
Ions, metabolites, and small proteins diffuse freely through nuclear pores, but macromolecules larger than ≈60 kDa must be actively transported by a process that requires ATP hydrolysis and probably entails substantial conformational changes in the nuclear pore complex.
In both nuclear export and import, the protein to be transported contains a specific amino acid sequence that functions as a nuclear-export signal (NES) or a nuclear-localization signal (NLS). Nucleus-restricted proteins contain a NLS but not a NES, whereas proteins that shuttle between the nucleus and cytosol contain both signals.
According to current models, the NES or NLS on a “cargo” protein interacts with a specific nucleartransport receptor protein located in the nucleus in the case of export or in the cytosol in the case of import. Both transport processes also require participation of Ran, a GTPase that exists in different conformations when bound to GTP or GDP.
Once a cargo complex is assembled, the receptor protein in the complex is thought to make multiple contacts with nucleoporins, thereby transporting the complex through a nuclear pore. After a cargo complex reaches its destination (the cytoplasm during export and the nucleus during import), it dissociates, freeing the cargo protein and other components. The latter then are transported through nuclear pores in the reverse direction to participate in transporting additional molecules of cargo protein (see Figures 11-33 and 11-37).
The unidirectional nature of both nuclear export and import is thought to result from the localization of the Ran nucleotide-exchange factor (RCC1) in the nucleus and of Ran GTPase-activating protein (RanGAP) in the cytoplasm.
During export of a nuclear mRNP, composed of a mature, functional mRNA and hnRNP proteins, nucleus- restricted hnRNPs are removed, while multiple NES-bearing hnRNPs bound to the mRNA are thought to carry it through the nuclear pore complex (see Figure 11-34). Once in the cytosol, these shuttling hnRNPs, which also contain a NLS, are removed from the mRNA and transported back into the nucleus to participate in another round of nuclear export.
Several different types of NES and NLS have been identified. Each class of nuclear-transport signal is thought to interact with a specific receptor protein. The nuclear-transport receptors characterized so far have homologous regions that interact with Ran and certain nucleoporins.
Pre-mRNAs within a spliceosome normally are not exported from the nucleus. Although the mechanism of this transport inhibition is not understood, it ensures that only properly processed, functional mRNAs are transported into the cytoplasm for translation.
The HIV Rev protein, which contains a NES, can override the restriction against transporting pre-mRNAs with unspliced splice sites (see Figure 11-38).