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▲ FIGURE 12-23 Mechanism for nuclear export of cargo proteins containing a leucine-rich nuclear-export signal (NES). In the nucleoplasm, the protein exportin 1 binds cooperatively to the NES of the cargo protein to be transported and to RanGTP After the resulting cargo complex diffuses through an NPC via transient interactions with FG repeats in FG-nucleoporins, the Ran GAP associated with the NPC cytoplasmic filaments stimulates conversion of RanGTP to RanGDP The accompanying conformational change in Ran leads to dissociation of the complex. The NES-containing cargo protein is released into the cytosol, while exportin 1 and RanGDP are transported back into the nucleus through NPCs. RanGDP is transported through its interaction with NTF2. Ran-GEF in the nucleoplasm then stimulates conversion of RanGDP to RanGTP

to Ran-GTP causes a conformational change in exportin 1 that increases its affinity for the NES so that a trimolecular cargo complex is formed. Like importins, exportin 1 interacts transiently with FG repeats in FG-nucleoporins and diffuses through the NPC. The cargo complex dissociates when it encounters the Ran-GAP in the NPC cytoplasmic filaments that stimulates Ran to hydrolyze the bound GTP, shifting it into a conformation that has low affinity for exportin 1. The released exportin 1 changes conformation to a structure that has low affinity for the NES, releasing the cargo into the cytosol. The direction of the export process is driven by this dissociation of the cargo from exportin 1 in the cytoplasm that causes a concentration gradient of the cargo complex across the NPC so that it is high in the nucleoplasm and low in the cytoplasm. Exportin 1 and the Ran • GDP are then transported back into the nucleus through an NPC. Ran. GDP is transported by NTF2, as discussed earlier in regard to nuclear import. It is then converted into Ran. GTP by the Ran-GEF in the nucleoplasm.

By comparing this model for nuclear export with that in Figure 12-21 for nuclear import, we can see one obvious dif ference: Ran-GTP is part of the cargo complex during export but not during import. Apart from this difference, the two transport processes are remarkably similar. In both processes, association of a transport signal receptor with Ran-GTP in the nucleoplasm causes a conformational change that affects its affinity for the transport signal. During import, the interaction causes release of the cargo, whereas, during export, the interaction promotes association with the cargo. In both export and import, stimulation of Ran-GTP hydrolysis in the cytoplasm by the Ran-GAP associated with NPC cytoplasmic filaments produces a confor-mational change in Ran that releases the transport signal receptor. During nuclear export, the cargo is also released. Importins and exportins both are thought to diffuse through the NPC channel by successive interactions with FG-repeats in FG-nucleoporins. Localization of the Ran-GAP and -GEF to the cytoplasm and nucleus, respectively, is the basis for the unidirectional transport of cargo proteins across the NPC.

In keeping with the similarity in function of importins and exportins, the two types of transport proteins are highly homologous in sequence and structure. The entire family is called the importin p family, or karyopherins. There are 14 karyopherins in yeast and more than 20 in mammalian cells. The NESs or NLSs to which they bind have been determined for only a fraction of them. Remarkably, some individual karyopherins function as both an importin and an exportin.

A similar shuttling mechanism has been shown to export other cargoes from the nucleus. For example, exportin-t functions to export tRNAs. Exportin-t binds fully processed tRNAs in a complex with Ran-GTP that diffuses through NPCs and dissociates when it interacts with Ran-GAP in the NPC cytoplasmic filaments, releasing the tRNA into the cytosol. A Ran-dependent process is also required for the nuclear export of ribosomal subunits through NPCs. Likewise, certain specific mRNAs that associate with particular hnRNP proteins (e.g., HIV Rev discussed later) can be exported by a Ran-dependent mechanism. However, most mRNAs are exported from the nucleus by another type of transporter that does not interact with Ran. This will be discussed after we examine gene regulation through control of transcription-factor import and export.

Control of Some Genes Is Achieved by Regulating Transport of Transcription Factors

Regulation of some genes is achieved by regulating the nuclear transport of specific transcription factors that control their transcription. For example, in Chapter 11 we learned that homodimeric nuclear receptors such as the glucocorti-coid receptor are sequestered in the cytoplasm in the absence of ligand. Binding of ligand stimulates their translocation into the nucleus, where they can bind to hormone response elements in target genes and activate transcription (see Figures 11-43 and 11-44). The ligand-binding domain of these transcription factors contains an NLS, but in the absence of ligand, the NLS is masked by interactions with a cytoplasmic tethering complex. The conformational change that results

from binding hormone releases the domain from the tethering complex, exposing the NLS so that it is free to interact with its cognate importin and undergo nuclear import, as discussed above. Other examples of regulated nuclear import of transcription factors through controlled masking and exposure of their associated NLS are discussed in subsequent chapters on signal transduction.

In some cases, regulated nuclear transport of transcription factors is achieved by expressing them as fusions to domains that localize them to cytoplasmic membranes, such as the plasma membrane or ER. In response to specific signals, specific proteases are activated that cleave them, releasing the transcription-factor domains so that they can be transported into the nucleus by an importin. In other cases, transcription-factor nuclear export is regulated by post-translational modifications such as phosphorylation that increase their affinity for a specific exportin. Regulation of nuclear import or export is a powerful mechanism for controlling transcription-factor activity, since a transcription factor can bind to its cognate sites in the genes it regulates only when it is in the nucleus.

Most mRNPs Are Exported from the Nucleus with the Aid of an mRNA-Exporter

Recent studies of temperature-sensitive yeast mutants defective in nuclear transport identified a heterodimeric mRNA-exporter protein that appears to direct most mRNPs through nuclear pores. Yeast cells with mutations in the subunits of this protein accumulate polyadenylated RNA in the nucleus at the nonpermissive temperature, indicating that the protein is required for the transport of most mRNPs. Highly conserved homologs of the large and small subunits of the yeast mRNA-exporter are found in all eukaryotes, and several lines of evidence indicate that the mRNA-exporter is required for the export of most mRNPs in vertebrate cells as well as in yeast cells. Surprisingly, as mentioned earlier, experiments in both yeast and vertebrate cells indicate that Ran is not required for export of the vast majority of mRNPs.

The small subunit of the mRNA-exporter is homologous to NTF2 and interacts with a region of the larger subunit that also shares homology to NTF2. Together they form a domain that interacts with FG repeats in FG-nucleoporins similarly to NTF2, which is a dimer. A C-terminal domain of the large subunit also interacts with FG repeats. Although several other proteins are also required for mRNP transport, researchers consider this mRNP-exporter to be the primary transport protein because of the importance of FG-repeat binding for the mechanism of cargo transport by karyopherins. The large mRNA-exporter subunit also contains RNA-binding domains that appear to bind to RNA cooperatively with specific mRNP proteins. The finding that the mRNA-exporter is associated with mRNPs and binds directly to FG repeats led to the model of mRNP transport depicted in Figure 12-24. This model proposes that the mRNA-exporter translocates mRNPs through the nuclear pore channel similarly to the karyopherins, that is, by binding transiently to successive individual FG-repeats as it diffuses through the channel.

▲ FIGURE 12-24 Proposed mechanism of mRNP transport from the nucleus via the mRNA-exporter. The large subunit (light purple) of the mRNA-exporter contains three key domains: a middle domain (M) and a carboxyl domain (C), both of which bind to hydrophobic phenylalanine-glycine (FG) repeats in FG-nucleoporins, and an N-terminal region that has weak RNA-binding activity for most sequences. Binding to most mRNPs requires cooperative binding with other specific hnRNP proteins. The small subunit (dark purple) binds to the middle domain of the large subunit and contributes to the binding of FG repeats. For simplicity, FG repeats are not shown on the lower half of the nuclear pore complex. Note that the mRNA-exporter is not drawn to scale; it is far smaller than the nuclear pore complex. The mRNA-exporter is proposed to diffuse through the pore by making transient interactions with adjacent FG-nucleoporins as it progresses. [Adapted from R. Reed and E. Hurt, 2002, Cell 108:523.]

Currently, it is not clear what provides directionality to this mechanism of mRNP transport through nuclear pores. By analogy with karyopherin transport, one hypothesis is that the mRNA-exporter-mRNP complex dissociates as it reaches the cytoplasm. This would result in a high concentration of the mRNA-exporter-mRNP complex at the nuclear basket, where it associates with FG-nucleoporins, and a low concentration of the complex in the cytoplasm, where it dissociates. As for karyopherin transport, such a concentration gradient could drive vectorial mRNP translocation. However, much remains to be learned about the hnRNP proteins associated with mRNPs during transport through the pore and the mechanism by which they dissociate from the mRNP on the cytoplasmic side of the pore.

Other Proteins That Assist in mRNP Export In addition to the mRNA-exporter and FG-nucleoporins, several other types of proteins are involved in the transport of mRNPs by this mechanism. As mentioned earlier, the mRNA-exporter is thought to bind to mRNAs cooperatively with specific mRNP proteins. For example, SR proteins associated with exons appear to stimulate the binding of the mRNA-exporter to processed mRNAs in mRNPs. Thus SR proteins not only function to define exons during RNA splicing (see Figure 12-11) but also participate in the export process that translocates most mRNPs into the cytoplasm.

Other nuclear RNA-binding proteins that probably function in transport of mRNPs recently have been identified. These proteins, which initially were found associated with mRNAs spliced in vitro, bind to a region about 20 bases 5' of the exon sequences joined by splicing. Analysis of the proteins present in these exon-junction complexes revealed that several are homologous to yeast proteins that are altered in mutants defective in mRNP export. Moreover, one of these proteins has been shown to bind directly to the large subunit of the mRNA-exporter. These results suggest that exon-junction complexes deposited on mRNAs during RNA splicing stimulate mRNP transport through nuclear pores. Some of the proteins in exon-junction complexes also function as splicing factors. As we discuss in the next section, on mechanisms of cytoplasmic post-transcriptional control, the exon-junction complex also participates in a type of post-transcriptional control that prevents translation of improperly spliced mRNAs.

Another participant in mRNP transport to the cytoplasm is the nuclear cap-binding complex, mentioned earlier as protection against exonuclease attack on the 5' end of nascent transcripts and pre-mRNAs. Electron microscopy experiments discussed below have demonstrated that the 5' end of mRNAs lead the way through the nuclear pore complex. Recent experiments in yeast indicate that the 3' poly(A) tail plays an important role in mRNP transport, suggesting that a poly(A)-binding protein participates. Nucleoporins associated with the NPC cytoplasmic filaments in addition to FG-nucleoporins are required for mRNA export and may function to dissociate the mRNA-exporter and other mRNP proteins that accompany the mRNP through the pore.

Once the mRNP reaches the cytoplasm, most of the mRNP proteins that associated with the mRNA in the nucleus, the nuclear cap-binding complex, and the nuclear poly(A)-binding protein (PABPII) dissociate and are shuttled back to the nucleus. In the cytoplasm, the 5' cap of an exported mRNA is bound by the eIF4E translation initiation factor, the poly(A) tail is bound by multiple copies of the cytoplasmic poly(A)-binding protein (PABPI), and other RNA-binding proteins associate with the body of the mRNA, forming a cytoplasmic mRNP that has a lower ratio of protein to RNA than nuclear mRNPs.

Nuclear Export of Balbiani Ring mRNPs The salivary glands of larvae of the insect Chironomous tentans have provided a good model system for EM studies of the formation of hnRNPs and the 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 and are processed into coiled mRNPs with an mRNA of «75 kb (Figure 12-25a, b). These giant mRNAs encode large glue proteins that adhere the developing larvae to a leaf. After processing of the pre-mRNA in Balbiani ring hnRNPs, the resulting mRNPs move through nuclear pores to the cytoplasm. Electron micrographs of sections of these cells show mRNPs that appear to uncoil during their passage through nuclear pores and then bind to ribosomes as they enter the cytoplasm. The observation that mRNPs become associated with ribosomes during transport indicates that the 5' end leads the way through the nuclear pore complex, as mentioned earlier. Detailed electron microscopic studies of the transport of Balbiani ring mRNPs through nuclear pore complexes led to the model depicted in Figure 12-25c.

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 that might interfere with the functioning of the cell. By mechanisms that are not fully understood, pre-mRNAs associated with snRNPs in spliceosomes usually are prevented from being transported to the cytoplasm.

In one type of experiment demonstrating this restriction, a gene encoding a pre-mRNA with a single intron that normally is spliced out was mutated to introduce deviations from the consensus splice-site sequences. Mutation of either the 5' or the 3' invariant splice-site bases 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 export of the unspliced pre-mRNA, although less efficiently than for the spliced mRNA. When both splice sites were mutated, the pre-mRNAs were not efficiently bound by snRNPs, and, consequently, their export was not blocked.

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. I

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 Figures 12-24 and 12-25). 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

(c) Nuclear envelope Nucleoplasm Cytoplasm

^ FIGURE 12-25 Formation of heterogeneous ribonucleoprotein particles (hnRNPs) and export of mRNPs from the nucleus. (a) Model of a single chromatin transcription loop and assembly of Balbiani ring (BR) mRNP in Chironomous tentans. Nascent RNA transcripts produced from the template DNA rapidly associate with proteins, forming hnRNPs. The gradual increase in size of the hnRNPs reflects the increasing length of RNA transcripts at greater distances from the transcription start site. The model was reconstructed from electron micrographs of serial thin sections of salivary gland cells. (b) Schematic diagram of the biogenesis of hnRNPs. Following processing of the pre-mRNA, the resulting ribonucleoprotein particle is referred to as an mRNP (c) Model for the transport of BR mRNPs through the nuclear pore complex (NPC) based on electron microscopic studies. Note that the curved mRNPs appear to uncoil as they pass through nuclear pores. As the mRNA enters the cytoplasm, it rapidly associates with ribosomes, indicating that the 5' end passes through the NPC first. [Part (a) from C. Erricson et al., 1989, Cell 56:631; courtesy of B. Daneholt. Parts (b) and (c) adapted from B. Daneholt, 1997, Cell 88:585. See also B. Daneholt, 2001, Proc. Nat'l. Acad. Sci. USA 98:7012.]

(c) Nuclear envelope Nucleoplasm Cytoplasm mRNP

mRNP

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