Nuclear Organization Chromatin Structure and Gene Silencing

Lori L. Wallrath, John R. Danzer, Oya Yazgan and Pamela K. Geyer

University of Iowa, Iowa City, Iowa, USA

Gene silencing is the process whereby a gene is inactivated due to its chromosomal position. Chromosomal rearrangements that move a gene from a transcriptionally permissive location to a transcriptionally nonpermissive location result in gene silencing. This loss of gene expression is not due to a mutation in a gene itself, but due to the placement of a gene into a chromatin environment that is not favorable for transcription. Chromatin packaging influences whether a chromosomal location will support transcription or result in gene silencing. Eukaryotic genomes are packaged into two types of chromatin, euchromatin and heterochromatin. Euchromatin is represented by single copy, gene-rich DNA sequences located between centromeres and telomeres. These regions are packaged with acetylated histones that foster gene expression. In contrast, heterochromatin is represented by repetitive, gene-poor DNA sequences located near centromeres and telomeres. These regions are packaged with hypoacetylated histones and can cause gene silencing.

The regulation of gene expression involves complex interactions between DNA sequences and protein factors. One mechanism for regulating access of transcription factors to DNA is through chromatin packaging. Double-stranded DNA is wound twice around a histone octamer, forming the fundamental packaging unit, the nucleosome. Nucleosomal DNA is further folded into higher-order chromatin structures that differentiate euchromatin from heterochromatin. A second mechanism for regulating access of transcription factors to DNA involves the spatial positioning of genes within the nucleus. Increasing evidence supports the notion that the nucleus is not a homogenous mixture of DNA and proteins, but is a highly organized environment. For example, chromosome labeling techniques show that certain chromosomes reproducibly localize to specific positions within the nucleus, termed chromosome territories (Figure 1). Thus, a given gene might have a specific address within the nucleus.

Nuclear zones are superimposed on chromosome territories (Figure 1). A zone is defined as a region of the nucleus that is enriched in a particular class of transcription factors. There are two types of zones, those enriched for silencing factors that do not support transcription (inactive zones), and those enriched for activating factors that do support transcription (active zones). Entire chromosomes can be located within a zone, such as the case for the inactive X chromosome (Barr body) of mammalian females that localizes to an inactive zone along the nuclear periphery. Alternatively, smaller regions along a chromosome can attract transcription factors and form a local zone (Figure 1). In addition, regions of similar transcription activity on different chromosomes can come together to form a zone. Thus, regulation of gene expression can occur at many levels involving the local chromatin environment, chromosomal territories, and the nuclear zones.

Establishing a Zone

Zones are established by increasing the local concentrations of transcriptional regulatory factors within an area of the nucleus. This nonuniform distribution of factors occurs by mechanisms involving protein-protein and protein-DNA interactions. As an example, hetero-chromatin protein 1 (HP1) can direct silent chromatin into the inactive zone at the nuclear periphery. The amino terminal chromo domain of HP1 interacts with a histone modification that is enriched in heterochromatin and the carboxy chromo shadow domain of HP1 interacts with a component of the nuclear envelope. These proteinprotein interactions promote the accumulation of hetero-chromatin and HP1 at the nuclear periphery. The role of protein-DNA interactions is illustrated by studies of yeast telomeres. Repetitive DNA sequences found at yeast telomeres attract the DNA-binding protein Rapl. This protein interacts with Sir proteins that are involved in gene silencing. Through this interaction, the concentration of Sir proteins increases at the nuclear periphery relative to the interior of the nucleus. The concentration gradient of Sir proteins within the nucleus can be "sensed" by reporter transgenes containing Sir-responsive DNA sequences. When such a reporter transgene is

FIGURE 1 Diagram of organization within the nucleus. A nucleus is depicted with chromosome territories (light gray) and silencing zones (dark gray). Histone acetylation (Ac) is shown over active chromatin. Hypoacetylation is associated with inactive chromatin, such as that found at centromeres (black circle) and telomeres. Small domains within a chromosome can acquire properties similar to heterochromatin and form a silencing zone (shaded circle). Silencing factors (green and blue shapes) are involved in chromatin compaction, gene silencing, and tethering to the nuclear periphery. Insulator elements (red triangles) partition chromosomes into transcriptonally related domains and assist in organizing chromatin into zones.

FIGURE 1 Diagram of organization within the nucleus. A nucleus is depicted with chromosome territories (light gray) and silencing zones (dark gray). Histone acetylation (Ac) is shown over active chromatin. Hypoacetylation is associated with inactive chromatin, such as that found at centromeres (black circle) and telomeres. Small domains within a chromosome can acquire properties similar to heterochromatin and form a silencing zone (shaded circle). Silencing factors (green and blue shapes) are involved in chromatin compaction, gene silencing, and tethering to the nuclear periphery. Insulator elements (red triangles) partition chromosomes into transcriptonally related domains and assist in organizing chromatin into zones.

placed at positions close to the telomere, gene silencing is strong. When the same transgene is positioned further from the telomere, gene silencing is weak. Overexpression of Sir proteins produces silencing at internal sites, presumably due to increased levels of Sir proteins within the interior of the nucleus. These studies demonstrate that gene silencing can be modulated by the position of a gene within the nucleus, as well as, the local concentration of regulatory factors.

Organizing DNA into Zones

What mechanisms ensure that chromosome domains remain independent? Specialized DNA elements called insulators may be responsible for maintaining domain autonomy. These DNA elements, 0.2-3 kb in length, associate with specific proteins that limit the scope of regulatory interactions. An insulator located between an enhancer and promoter blocks activated gene expression. Insulator elements flanking a gene "insulate" it from neighboring positive and negative regulatory elements. Several models have been proposed to explain the mechanism of insulator action. One model states that attachment of insulators to the nuclear envelope could result in the formation of looped domains that compartmentalize gene activity (Figure 1). Physical associations with architectural components of the nucleus could serve to anchor chromosomal sites leading to spatial positioning of insulated domains within a zone.

Changes in Gene Expression Correlate with Shifting Zones

The nucleus is a highly dynamic environment in which protein factors and chromosomal domains can change positions within seconds. This mobility allows gene expression to be regulated by shifting zones. Studies on the expression and localization of lymphocyte specific genes demonstrate this point. A set of lymphocyte-specific genes in mice associate with Ikaros, a zinc-finger DNA-binding protein that also associates with centric heterochromatin. When these genes are silenced they localize to discrete foci within B-cell nuclei that correspond to the location of centromeres. Expression of these genes correlates with movement away from the centric foci. A direct role for Ikaros in this process was demonstrated by examining cells that had low levels of Ikaros. In such cells, these genes do not colocalize with centromeres when silenced. These data suggest that Ikaros is not required for silencing but plays a critical role in directing silent genes to an inactive zone. However, it appears that in these examples movement into an inactive zone is not the cause of gene silencing. Therefore, positioning a gene within a silent zone might be a consequence of silencing or assist in the maintenance of the silent state.

Additional studies correlating nuclear position with gene expression have focused on the brown gene in the fruit fly Drosophila. The brown gene is normally positioned at a site distant from centric heterochromatin.

A mutant allele designated brown Dommant contains an insertion of a large block of heterochromatin (> 1 Mb) in the coding region. Heterochromatin proteins associate with the insertion and are involved in positioning brownDominant near centric heterochromatin. Surprisingly, the wild-type brown allele also becomes silenced. This occurs due to a process called "trans-silencing" that results from homologous pairing along the length of Drosophila chromosome arms. Silencing of the wild-type brown gene demonstrates the impact of positioning within an inactive zone.

In some cases it appears that localization within an inactive zone occurs as the default state. Studies using a mouse b-globin transgene demonstrate that a complete enhancer element is required for both gene expression and localization away from inactive zones. Point mutations within the enhancer element that disrupt binding of a transcription factor eliminate gene expression and cause the b-globin transgene to localize to inactive zones near centromeres. Interactions between factors bound at enhancer elements and the transcrip-tional machinery housed within the interior of the nucleus might be responsible for localization within an active zone. Alternatively, factors that bind to enhancer elements might recruit chromatin remodeling machines and/or histone acetyltransferases, generating a chroma-tin environment that disrupts interactions with silencing proteins in the inactive zone.

Multiple Determinants for Establishing the Activity State of a Gene

The emerging picture is that both nuclear zones and local chromatin structure play essential roles in establishing the transcriptional state of a gene. Silencing at the yeast mating type locus HMR-E is dependent upon the recruitment of specific trans-acting factors. Deletion of some of their binding sites generates a defective silencer, leading to transcriptional derepression. Tethering an HMR-E with a defective silencer to the nuclear periphery restores gene silencing. However, tethering an HMR-E in which all binding sites had been removed has no effect on gene expression. These experiments demonstrate that the local chromatin structure, not just the position within a nuclear zone, is necessary to establish the transcriptionally inactive state.

Effects of local chromatin structure and nuclear positioning were further demonstrated by studies involving chromosomal translocations in Drosophila. A stock possessing a reporter gene inserted within a region associated with heterochromatin-silencing factors was subjected to X-rays and translocations were recovered. The degree of silencing of the reporter gene was dependent on the location of the translocation breakpoints. Rearrangements that placed the transgene closer to centric heterochromatin showed increased levels of gene silencing. In contrast, rearrangements that placed the transgene at sites distant from centric heterochroma-tin resulted in decreased gene silencing. Contrasting observations were made when a stock containing the same transgene inserted within a chromosomal region that normally does not associate with heterochromatic silencing factors was subjected to X-rays. Resulting translocations did not alter the expression of the reporter gene, even when the transgene localized near centric heterochromatin. The transgene appeared to be insensitive to positioning in the nucleus. These data suggest that local chromatin structure can influence the response of a gene to placement within a particular nuclear zone.

Nuclear Zones and Human Disease

Studies in model organisms, such as yeast and fruit flies, have clearly demonstrated that nuclear positioning affects gene expression. There is growing evidence in humans that some diseases might result from misregula-tion of gene expression due to translocations that place a disease-causing gene into an inappropriate zone. Examples include cases of aniridia (absence of the iris), autism, and Burkitt's lymphoma. In these cases the disease-related gene is not damaged by the translocation, in fact, the breakpoints of the translocation map several kilobases or megabases from the gene affected. It is hypothesized that altered expression of the disease gene is due to placement in an inappropriate nuclear zone as a consequence of the translocation. Therefore, understanding the rules of gene regulation has become a three-dimensional problem that will require sophisticated detection of gene expression coupled with highresolution nuclear imaging.

See Also the Following Articles

Chromatin: Physical Organization • Chromosome Organization and Structure, Overview • Nucleoid Organization of Bacterial Chromosomes • Transcrip-tional Silencing

Glossary boundary elements DNA elements that bind specialized nuclear complex that attach to nuclear substructure to delimit chromosome domains.

chromosome territory An area within the nucleus that is occupied by a specific chromosome. euchromatin Regions of the genome that contain mostly single copy DNA sequence and are relatively gene-rich. These regions replicate early in S phase and decondense during interphase.

heterochromatin Regions of the genome, frequently located near centromeres and telomeres, that contain repetitive DNA sequences and are relatively gene-poor. These regions replicate late in S phase and remain condensed throughout the cell cycle.

histones Small, highly conserved, basic proteins that form a nucleo-some.

insulators Specialized DNA sequences that block the effect of enhancers or silencers when placed between a promoter and the regulatory element. When flanking both ends of a gene, they block position effects that arise from neighboring regulatory elements.

nucleosome The fundamental unit of chromatin packaging containing eight histones and ~ 165 bp of double stranded DNA.

somatic pairing Pairing of two homologous chromosomes during interphase in somatic cells.

Further Reading

Brown, K. E., Guest, S. S., Smale, S. T., Hahm, K., Merkenschlager, M., and Fisher, A. G. (1997). Association of transcriptionally silent genes with Ikaros complexes at centromeric heterochromatin. Cell 91, 845-854.

Cryderman, D. E., Morris, E. J., Biessmann, H., Elgin, S. C. R., and Wallrath, L. L. (1999). Silencing at Drosophila telomeres: Nuclear organization and chromatin structure play critical roles. EMBO J. 18, 3724-3736.

Dernberg, A. F., Broman, K. W., Fung, J. C., Marshall, W. F., Philips, J., Agard, D. A., and Sedat, J. W. (1996). Perturbation of nuclear architecture by long-distance chromosome interactions. Cell 85, 745-759.

Gasser, S. M. (2001). Positions of potential: Nuclear organization and gene expression. Cell 104, 639-642.

Ishii, K., Arib, G., Lin, C., Van Houwe, G., and Laemmli, U. K. (2002). Chromatin boundaries in yeast: The nuclear pore connection. Cell 109, 551-562.

Kleinjan, D.-J., and van Heyningen, V. (1998). Position effect in human genetic disease. Human Mol. Genet. 7, 1611-1618. Kuhn, M. J., and Geyer, P. K. (2003). Genomic insulators: Connecting properties or mechanism. Curr. Opin. Cell Biol. 15, 259-265. Mahy, N., Perry, P. E., and Bickmore, W. A. (2002). Gene density and transcription influence the localization of chromatin outside of chromosome territories detectable by FISH. J. Cell Biol. 159, 753-763.

Sass, G. L., andHenikoff, S. (1999). Pairing-dependent mislocalization of a Drosphila brown gene reporter to a heterochromatic environment. Genetics 152, 595-604. Williams, R. R. E. (2003). Transcription and the territory: The ins and outs of gene positioning. Trends Genet. 19, 298-302.

Biography

Lori L. Wallrath and Pamela K. Geyer are faculty members in the Department of Biochemistry at the University of Iowa. Their principal research interest is chromatin structure and gene expression. Dr. Wallrath's research is focused on the molecular mechanisms of gene silencing. Dr. Geyer's research is focused on the role of genomic insulators in gene expression.

John Danzer is a senior Ph.D. student in the Molecular Biology Program, working on the molecular mechanism of heterochromatin spreading.

Dr. Oya Yazgan is a Postdoctoral Fellow researching the role of proteins that associate with insulators.

Anita H. Corbett

Emory University School of Medicine, Atlanta, Georgia, USA

The hallmark of a eukaryotic cell is the presence of the nucleus, which separates the chromosomes that encode the genetic information from the protein translation machinery in the cytoplasm. This physical separation requires nuclear transport (import and export) machinery, which is capable of moving macromolecules such as proteins and RNAs rapidly but selectively into and out of the nucleus. There are many processes in eukaryotic cells that require macromolecular exchange between the nucleus and the cytoplasm. The most obvious example is the expression of a gene where the DNA is transcribed to messenger RNA (mRNA) within the nucleus and this mRNA must be transported to the ribosomes, the cytoplasmic protein translation machinery, to be decoded and translated into protein. In addition, many proteins that are created by the cytoplasmic translation machinery enter the nucleus to function in essential nuclear processes such as DNA replication, transcription, DNA repair, and many others. Thus, there is a great deal of traffic between the cytoplasm and the nucleus and these transport events are critical for the proper functioning of a eukaryotic cell.

structure that extends into the nucleoplasm. Cargoes that move through the nuclear pores probably interact first with the peripheral structures, move through the central channel, and are then released into their target compartment.

Composition

Sophisticated biochemical studies have revealed the identity of the nucleoporin proteins that make up the NPC. Surprisingly, these studies reveal that both the yeast and vertebrate nuclear pores are composed of only approximately 30 distinct proteins. The pore is large because most of these pore components are present in 16-32 copies per NPC. Many of these nucleoporins contain a characteristic repeat sequence of phenylalanine glycine repeats (FG). These FG-repeat nucleo-porins line the central channel of the pore and probably provide the conduit for movement of macromolecules through the pore complex.

Nuclear Pores

All transport between the nucleus and the cytoplasm occurs through large protein channels that are embedded in the nuclear membrane. These channels, which are called nuclear pore complexes (NPCs), are composed of proteins referred to as nucleoporins.

Structure

NPCs are large (~ 30 MDa in yeast and ~ 60 MDa in vertebrates) structures that span the double membrane, which surrounds the nucleus to provide a transport channel for very large macromolecular cargoes. Many different studies have led to a model for the structure of the NPC (Figure 1). The core of the NPC consists of a cylinder with eightfold rotational symmetry, which spans the nuclear envelope and surrounds a central channel. In addition to this central channel, there are filaments that extend into the cytoplasm and a basket

Transport Mechanisms: Protein Trafficking

Transport through nuclear pores occurs in both directions, from the nucleus to the cytoplasm and from the cytoplasm to the nucleus. The desired direction of transport generally depends on the cargo to be transported. The cell has developed sophisticated mechanisms to mark cargoes for transport and regulate the direction of the transport processes.

Targeting Signals

As with most intracellular targeting mechanisms, trafficking between the nucleus and the cytoplasm depends on amino acid sequences within the protein cargoes to be transported. However, unlike other targeting mechanisms, such as mitochondrial and ER targeting, nucleocytoplasmic trafficking signals are not

Cytoplasm

Cytoplasm

Nucleus

FIGURE 1 Schematic of the nuclear pore complex (NPC). Left: A schematic of the pore in the plane of the membrane is shown. The schematic shows the cytoplasmic filaments and the nuclear basket. Right: A schematic view of the pore looking down from the cytoplasmic face. The eightfold rotational symmetry of the pore is evident.

Nucleus

FIGURE 1 Schematic of the nuclear pore complex (NPC). Left: A schematic of the pore in the plane of the membrane is shown. The schematic shows the cytoplasmic filaments and the nuclear basket. Right: A schematic view of the pore looking down from the cytoplasmic face. The eightfold rotational symmetry of the pore is evident.

cleaved and remain an integral part of the protein. This mechanism can allow the cargo proteins to undergo multiple rounds of import and/or export. The nuclear targeting signal within the cargo protein generally mediates a physical interaction with a soluble nuclear transport receptor, which targets the cargo to the nuclear pore for transport.

Canonical signals for both nuclear protein import and nuclear protein export have been identified and studied in some detail. Nuclear import signals are generally termed nuclear localization signals (NLS), while signals that target proteins for export from the nucleus are termed nuclear export signals (NES). The classical NLS consists of either a single cluster of basic amino acid residues (monopartite) or two clusters of basic amino acid residues separated by a 10-13 amino acid linker sequence (bipartite). The monopartite NLS sequence is typified by the NLS found in the simian virus 40 (SV40) large T antigen (PKKKRKV), whereas the bipartite NLS is typified by the nucleoplasmin NLS (KRPAATKKAGQAKKKK). The classical NES is comprised of a series of hydrophobic amino acids, generally leucine, isoleucine, or valine. Numerous variations on this theme have been identified and thus far the best consensus sequence for an NES is LxxxLxxLxL, where the spacing between the leucines can vary, and, in fact, the leucines can be substituted with virtually any hydrophobic amino acid. Although classical NLS and NES signals are typically identified through computer searches of proteins sequences, it is essential that their function be verified through experimental methods.

Transport Receptors

Targeting signals within cargo proteins are recognized by soluble receptors that direct those cargoes to the nuclear pore for transport. These receptors form a family of structurally related molecules that are generally referred to as importins (for import receptors), exportins (for export receptors), or generally as transport receptors or karyopherins. The family of related receptors is generally referred to as the importin ¡3 or karyopherin ¡-family. One feature of these transport receptors is that they are modulated in such a way that they bind tightly to their cargo in the compartment where the cargo needs to be picked up. However, following the translocation through the NPC to the compartment where the cargo needs to be delivered, the transport receptor undergoes a conformational change that leads to the efficient release and delivery of the cargo. This switch in binding to the cargo is regulated by the small GTPase Ran.

Although most transport cargoes bind directly to their cognate transport receptor, there are examples where an adaptor protein mediates this interaction. The best example of this occurs in nuclear protein import of cargoes that contain a classical NLS sequence. The NLS within these cargoes is recognized by an adaptor protein called importin a or karyopherin a. The importin/karyopherin a recognizes and binds to the cargo and then binds directly to the transport receptor to form a trimeric import complex. This complex is then translocated through the nuclear pore into the nucleus and the NLS cargo is delivered.

Once cargo is released from the transport receptors within the delivery compartment, all the receptors are recycled for another round of transport. Import receptors are recycled back to the cytoplasm and export receptors are recycled back to the nucleus. This recycling of receptors assures that a single receptor can mediate multiple rounds of transport.

The Ran GTPase Cycle

The small GTPase Ran, serves as a molecular switch that modulates the directionality of nuclear transport. As with other GTPases, Ran can exist in two forms, either bound to GDP (RanGDP) or bound to GTP (RanGTP). It is the compartmentalization of these two distinct forms of Ran that regulates cargo/receptor interactions to impart directionality on nuclear transport. This compartmentalization is achieved through the intra-cellular separation of the two important regulators of the Ran GTPase cycle. The Ran GTPase-Activating Protein (RanGAP), which enhances Ran-mediated GTP hydrolysis, is located in the cytoplasm and the Ran Guanine Nucleotide Exchange Factor (RanGEF), which facilitates exchange of GDP for GTP on Ran, is located in the nucleus. As shown in Figure 2, with the RanGAP in the cytoplasm, any RanGTP that enters the cytoplasm is rapidly converted to RanGDP. Thus, in the cytoplasm the level of RanGDP exceeds the level of RanGTP. In contrast, within the nucleus in the presence of the RanGEF, RanGDP is rapidly converted to RanGTP. Thus, in the nucleus the level of RanGTP is high compared to the level of RanGDP. Due to the asymmetric localization of the Ran regulators, high levels of RanGTP serve as a marker for the nucleus and high levels of RanGDP serve to identify the cytoplasmic compartment.

The different nucleotide bound states of Ran regulate the flow of protein cargoes into and out of the nucleus by regulating the assembly of the import and export complexes (Figure 3). Cargoes to be transported into the nucleus bind to their cognate import receptors in the

FIGURE 2 The RanGTP gradient. The departmentalization of the RanGAP to the cytoplasm and the RanGEF to the nucleus results in localized concentrations of RanGDP and RanGTP. In the cytoplasm where the RanGAP enhances the Ran-mediated GTP hydrolysis, levels of RanGDP are high. In the nucleus where the RanGEF facilitates the exchange of GTP for GDP on Ran, the levels of RanGTP are high. This compartmentalization is critical for the identification of the nuclear and cytoplasmic compartments in nuclear transport and in other cellular processes.

FIGURE 2 The RanGTP gradient. The departmentalization of the RanGAP to the cytoplasm and the RanGEF to the nucleus results in localized concentrations of RanGDP and RanGTP. In the cytoplasm where the RanGAP enhances the Ran-mediated GTP hydrolysis, levels of RanGDP are high. In the nucleus where the RanGEF facilitates the exchange of GTP for GDP on Ran, the levels of RanGTP are high. This compartmentalization is critical for the identification of the nuclear and cytoplasmic compartments in nuclear transport and in other cellular processes.

cytoplasm. These complexes are translocated into the nucleus through NPCs. Once they reach the nucleus, where levels of RanGTP are elevated, RanGTP binds to the import receptor causing a conformational change that releases the import cargo into the nucleus. In contrast, export cargoes bind to their export receptors only in the presence of RanGTP. Export complexes are trimeric complexes that consist of the export receptor, the export cargo, and RanGTP. As with import, the export complex is translocated through NPCs to the cytoplasm. In the cytoplasm, the export complex encounters the RanGAP and the bound RanGTP is converted to RanGDP. This disassembles the export complex and leads to the release of the export cargo into the cytoplasm.

Classical NLS-Mediated Protein Import

The best understood nuclear transport process is the import of protein cargoes that contain a classical basic NLS (Figure 4). Thus, this process can be used most readily to illustrate the steps that occur when a transport cargo is moved into or out of the nucleus. Historically, nuclear protein import was divided into two steps, docking at the nuclear pore, an energy-independent step, and translocation into the nucleus, an energy-dependent step. Advances in our understanding of the process and the players now lead us to define at least five distinct steps for import of a cargo that contains a classical NLS: (1) recognition and binding of the NLS cargo to the aJfi heterodimeric-import receptor in the cytoplasm; (2) targeting to the nuclear pore through interactions between the importin/karyopherin b nuclear transport receptor and the nuclear pore; (3) translocation through the pore through transient interactions between importin/karyopherin b and the FG-repeat containing nucleoporins; (4) delivery into the nucleus where RanGTP binds to importin/karyopherin b to cause a conformational change that releases a and the NLS cargo; and (5) recycling of importin/karyopherin a to the cytoplasm in a heterotrimeric complex with an importin/karyopherin a export receptor and RanGTP and importin/karyopherin b presumably in complex with RanGTP. Note that the only energy expenditure in this process occurs when the karyopherin proteins are recycled to the cytoplasm and the accompanying RanGTP is hydrolyzed.

Transport Mechanisms: RNA Trafficking

Multiple classes of RNAs, including mRNAs, tRNAs, and U snRNAs are transcribed and processed within

Karyopherin

FIGURE 3 Ran regulates the assembly of import and export complexes. Top: For transport into the nucleus (Import), cargoes are recognized in the cytoplasm by an importin/karyopherin b receptor to form an import complex. Once this complex is translocated into the nucleus, it encounters the nuclear RanGTP, which binds tightly to the importin/karyopherin b receptor and causes a conformational change that releases the import cargo. Bottom: For transport out of the nucleus (Export), cargoes are recognized in the nucleus by an importin/karyopherin b receptor. However, in contrast to the import complex, the export complex can only form in the presence of RanGTP. Thus, export complexes are obligate trimeric complexes that consist of the export cargo, the importin/karyopherin b export receptor, and RanGTP. Once the export complex is translocated to the cytoplasm, it encounters the RanGAP. In the presence of the RanGAP, the RanGTP within the complex is converted to RanGDP and the complex dissociates resulting in the release of the export cargo into the cytoplasm. Hence, by controlling complex assembly and disassembly, the different forms of Ran confer directionality on the nuclear transport machinery.

FIGURE 3 Ran regulates the assembly of import and export complexes. Top: For transport into the nucleus (Import), cargoes are recognized in the cytoplasm by an importin/karyopherin b receptor to form an import complex. Once this complex is translocated into the nucleus, it encounters the nuclear RanGTP, which binds tightly to the importin/karyopherin b receptor and causes a conformational change that releases the import cargo. Bottom: For transport out of the nucleus (Export), cargoes are recognized in the nucleus by an importin/karyopherin b receptor. However, in contrast to the import complex, the export complex can only form in the presence of RanGTP. Thus, export complexes are obligate trimeric complexes that consist of the export cargo, the importin/karyopherin b export receptor, and RanGTP. Once the export complex is translocated to the cytoplasm, it encounters the RanGAP. In the presence of the RanGAP, the RanGTP within the complex is converted to RanGDP and the complex dissociates resulting in the release of the export cargo into the cytoplasm. Hence, by controlling complex assembly and disassembly, the different forms of Ran confer directionality on the nuclear transport machinery.

the nucleus and then transported to their sites of action in the cytoplasm. As compared with proteins, RNAs require extensive processing before they reach their mature form and are ready to exit the nucleus. This means that RNA export is intimately linked to RNA processing within the nucleus.

Export of RNA via Classical Nuclear Transport Receptors

The export of tRNA, U snRNA, and rRNA follows pathways analogous to nuclear protein export. For example, tRNA is recognized directly by the importin/ karyopherin bfamily nuclear transport receptor, expor-tin-t/Los1p and is exported as a complex with RanGTP. As for any classical export process, this trimeric complex is disassembled in the cytoplasm when RanGTP is hydrolyzed to RanGDP. Preferential export of mature tRNAs seems to be achieved at least in part by the specificity of exportin-t for the mature processed, modified, and appropriately aminoacylated tRNAs. U snRNAs are synthesized in the nucleus, transported to the cytoplasm where they associate with protein components of mature small nuclear ribonucleoproteins (snRNPs), and are then reimported to the nucleus where they function in mRNA splicing. Although this export depends on Ran, it is controversial whether Ran plays a direct role in export or whether its activity may be essential for the import of components required for RNP assembly.

MRNA Export

Export of poly (A) + mRNA remains the least well understood of the RNA export mechanisms. mRNAs are not exported to the cytoplasm as naked nucleic acids, but rather as RNP complexes, and it is generally agreed that the export machinery recognizes signals within the proteins of these complexes rather than the RNA itself. For example, export of intron-containing HIV transcripts from the nucleus is mediated by the HIV protein Rev through its binding to the classical NES receptor, CRM1/exportin. While the HIV virus exploits this mechanism, none of the classical nuclear transport receptors play a central role in cellular mRNA export.

Although the mechanistic details of mRNA export have not yet been fully elucidated, it appears that there are two classes of proteins that are required to achieve export of mature messages. First, there is a family of evolutionarily conserved heterogeneous nuclear ribonu-cleoproteins (hnRNPs) that interact with poly (A) + RNA in vivo. A number of these hnRNP proteins shuttle between the cytoplasm and the nucleus and escort the poly(A) + RNAs as they are exported through the NPC. Current models suggest that at least some of the hnRNP proteins may be involved in RNA processing steps that occur cotranscriptionally. The hnRNPs that remain bound to the maturing messages may serve as markers that the different processing steps have been successfully accomplished. The second class of proteins consists of

Karyopherin

FIGURE 4 Model for classical NLS-mediated protein import into the nucleus. The best-understood transport mechanism is that of classical nuclear protein import mediated by importin/karyopherin b and the NLS binding adapter, importin/karyopherin a. This process can be divided into at least five distinct steps as shown in the model: (1) recognition and binding of the NLS cargo to the a/b heterodimeric import receptor in the cytoplasm; (2) targeting to the nuclear pore through interactions between the importin/karyopherin b nuclear transport receptor and the nuclear pore; (3) translocation through the pore through transient interactions between importin/karyopherin b and the FG-repeat containing nucleoporins; (4) delivery into the nucleus where RanGTP binds to importin/karyopherin b to cause a conformational change that releases a and the NLS cargo; and (5) recycling of importin/karyopherin a to the cytoplasm in a heterotrimeric complex with an importin/karyopherin a export receptor and RanGTP and importin/karyopherin b presumably in complex with RanGTP.

FIGURE 4 Model for classical NLS-mediated protein import into the nucleus. The best-understood transport mechanism is that of classical nuclear protein import mediated by importin/karyopherin b and the NLS binding adapter, importin/karyopherin a. This process can be divided into at least five distinct steps as shown in the model: (1) recognition and binding of the NLS cargo to the a/b heterodimeric import receptor in the cytoplasm; (2) targeting to the nuclear pore through interactions between the importin/karyopherin b nuclear transport receptor and the nuclear pore; (3) translocation through the pore through transient interactions between importin/karyopherin b and the FG-repeat containing nucleoporins; (4) delivery into the nucleus where RanGTP binds to importin/karyopherin b to cause a conformational change that releases a and the NLS cargo; and (5) recycling of importin/karyopherin a to the cytoplasm in a heterotrimeric complex with an importin/karyopherin a export receptor and RanGTP and importin/karyopherin b presumably in complex with RanGTP.

those proteins that have been implicated more directly in the export process; including a helicase, Sub2p/ UAP56, and the heterodimeric-export receptor, Mex67p/Mtr2p (TAP/p15 in humans). TAP/Mex67p shuttles between the cytoplasm and the nucleus, and in complex with p15/Mtr2p, binds both to mRNA and to nucleoporins. Thus, it could potentially target bound RNAs directly to the NPC for export.

See Also the Following Articles mRNA Polyadenylation in Eukaryotes • mRNA Processing and Degradation in Bacteria • Neurotransmitter Transporters • Ran GTPase • RNA Editing

Glossary cargo Macromolecular substrate to be transported between the nucleus and the cytoplasm. GTPase Protein that binds to and hydrolyzes the nucleotide GTP. heterogeneous nuclear ribonucleoprotein (hnRNP) Abundant nonspecific poly(A) + RNA binding protein. HIV Human immunodeficiency virus.

messenger RNA (mRNA) Class of RNA that carries the information from the DNA to the cytoplasm and serves as the informational blueprint for protein synthesis. nuclear export signal (NES) An amino acid sequence within a protein that targets that protein for export from the nucleus. nuclear localization signal (NLS) An amino acid sequence within a protein that targets that protein for import into the nucleus.

nuclear pore complex (NPC) The large proteinaceous channel through which macromolecular cargoes are transported in and out of the nucleus.

nucleoporins Proteins that make up the NPC.

Ran A small GTPase that regulates the directionality of nuclear transport.

RanGAP Cytoplasmic protein that enhances the GTPase activity of Ran.

RanGEF Nuclear protein that facilitates the nucleotide exchange on Ran.

ribosomal RNA (rRNA) Class of RNA that serves as a structural and catalytic component of ribosomes.

transfer RNA (tRNA) Class of RNA that is charged with amino acids to decode the mRNA for protein synthesis.

transport receptor/karyopherin/importin/exportin The receptors that bind to and recognize protein and RNA cargoes that are transported into or out of the nucleus.

U snRNA Small RNAs that participate in splicing.

Further Reading

Damelin, M., Silver, P. A., and Corbett, A. H. (2002). Nuclear protein transport. Methods Enzymol. 351, 587-607.

Dasso, M. (2002). The ran GTPase: Theme and variations. Curr. Biol. 12, R502-R508.

Gorlich, D., and Kutay, U. (1999). Transport between the cell nucleus and the cytoplasm. Annu. Rev. Cell Develop. Biol. 15, 607-660.

Lei, E. P., and Silver, P. A. (2002). Protein and RNA export from the nucleus. Develop. Cell. 2, 261 -272.

Quimby, B. B., and Corbett, A. H. (2001). Nuclear transport mechanisms. Cell Mol. Life Sci. 58, 1766-1773.

Rout, M. P., and Aitchison, J. D. (2000). Pore relations: Nuclear pore complexes and nucleocytoplasmic exchange. Essays Biochem. 36, 75-88.

Strom, A. C., and Weis, K. (2001). Importin-beta-like nuclear transport receptors. Genome Biol. 2, Epub 2001 Jun 05.

Suntharalingam, M., and Wente, S. R. (2002). Peering through the pore: Nuclear pore complex structure, assembly, and function. Develop. Cell. 4, 775-789.

Weis, K. (2002). Nucleocytoplasmic transport: Cargo trafficking across the border. Curr. Opin. Cell Biol. 14, 328-335.

Biography

Anita H. Corbett, Ph.D., is an Associate Professor of biochemistry at the Emory University School of Medicine. Her principal research interest is in the area of macromolecular trafficking between the cytoplasm and the nucleus. She utilizes the budding yeast, Saccharo-myces cerevisiae as a model system for most of her studies. Dr. Corbett holds a Ph.D. in biochemistry from Vanderbilt University. She completed a postdoctoral fellowship at Harvard Medical School before moving to Emory in 1997.

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