The factors that sequester transcriptionally repressed heterochromatin at the nuclear periphery are currently unknown. In a genome-wide RNAi screen, we found that depletion of S-adenosylmethionine (SAM) synthetase reduces histone methylation globally and causes derepression and release of heterochromatin from the nuclear periphery in Caenorhabditis elegans embryos. Analysis of histone methyltransferases (HMTs) showed that elimination of two HMTs, MET-2 and SET-25, mimics the loss of SAM synthetase, abrogating the perinuclear attachment of heterochromatic transgenes and of native chromosomal arms rich in histone H3 lysine 9 methylation. The two HMTs target H3K9 in a consecutive fashion: MET-2, a SETDB1 homolog, mediates mono- and dimethylation, and SET-25, a previously uncharacterized HMT, deposits H3K9me3. SET-25 colocalizes with its own product in perinuclear foci, in a manner dependent on H3K9me3, but not on its catalytic domain. This colocalization suggests an autonomous, self-reinforcing mechanism for the establishment and propagation of repeat-rich heterochromatin.

The metazoan nuclear envelope (NE) breaks down and re-forms during each cell cycle. Nuclear pore complexes (NPCs), which allow nucleocytoplasmic transport during interphase, assemble into the re-forming NE at the end of mitosis. Using in vitro NE assembly, we show that the vertebrate homologue of MEL-28 (maternal effect lethal), a recently discovered NE component in Caenorhabditis elegans, functions in postmitotic NPC assembly. MEL-28 interacts with the Nup107-160 complex (Nup for nucleoporin), an important building block of the NPC, and is essential for the recruitment of the Nup107-160 complex to chromatin. We suggest that MEL-28 acts as a seeding point for NPC assembly.

Barrier-to-autointegration factor (BAF) is an essential, highly conserved, metazoan protein. BAF interacts with LEM (LAP2, emerin, MAN1) domain-carrying proteins of the inner nuclear membrane. We analyzed the in vivo function of BAF in Caenorhabditis elegans embryos using both RNA interference and a temperature-sensitive baf-1 gene mutation and found that BAF is directly involved in nuclear envelope (NE) formation. NE defects were observed independent of and before the chromatin organization phenotype previously reported in BAF-depleted worms and flies. We identified vaccinia-related kinase (VRK) as a regulator of BAF phosphorylation and localization. VRK localizes both to the NE and chromatin in a cell-cycle-dependent manner. Depletion of VRK results in several mitotic defects, including impaired NE formation and BAF delocalization. We propose that phosphorylation of BAF by VRK plays an essential regulatory role in the association of BAF with chromatin and nuclear membrane proteins during NE formation.

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

In metazoa, the nuclear envelope breaks down and reforms during each cell cycle. Nuclear pore complexes (NPCs), which serve as channels for transport between the nucleus and cytoplasm, assemble into the reforming nuclear envelope in a sequential process involving association of a subset of NPC proteins, nucleoporins, with chromatin followed by the formation of a closed nuclear envelope fenestrated by NPCs. How chromatin recruitment of nucleoporins and NPC assembly are regulated is unknown. Here we demonstrate that RanGTP production is required to dissociate nucleoporins Nup107, Nup153 and Nup358 from Importin beta, to target them to chromatin and to induce association between separate NPC subcomplexes. Additionally, either an excess of RanGTP or removal of Importin beta induces formation of NPC-containing membrane structures--annulate lamellae--both in vitro in the absence of chromatin and in vivo. Annulate lamellae formation is strongly and specifically inhibited by an excess of Importin beta. The data demonstrate that RanGTP triggers distinct steps of NPC assembly, and suggest a mechanism for the spatial restriction of NPC assembly to the surface of chromatin.

Nuclear pore complexes (NPCs) span the nuclear envelope and mediate communication between the nucleus and the cytoplasm. To obtain insight into the structure and function of NPCs of multicellular organisms, we have initiated an extensive analysis of Caenorhabditis elegans nucleoporins. Of 20 assigned C. elegans nucleoporin genes, 17 were found to be essential for embryonic development either alone or in combination. In several cases, depletion of nucleoporins by RNAi caused severe defects in nuclear appearance. More specifically, the C. elegans homologs of vertebrate Nup93 and Nup205 were each found to be required for normal NPC distribution in the nuclear envelope in vivo. Depletion of Nup93 or Nup205 caused a failure in nuclear exclusion of nonnuclear macromolecules of approximately 70 kDa without preventing active nuclear protein import or the assembly of the nuclear envelope. The defects in NPC exclusion were accompanied by abnormal chromatin condensation and early embryonic arrest. Thus, the contribution to NPC structure of Nup93 and Nup205 is essential for establishment of normal NPC function and for cell viability.

Products of the LMNA gene, primarily lamin A and C, are key components of the nuclear lamina, a proteinaceous meshwork that underlies the inner nuclear membrane and is essential for proper nuclear architecture. Alterations in lamin A and C that disrupt the integrity of the nuclear lamina affect a whole repertoire of nuclear functions, causing cellular decline. In humans, hundreds of mutations in the LMNA gene have been identified and correlated with over a dozen degenerative disorders, referred to as laminopathies. These diseases include neuropathies, muscular dystrophies, lipodystrophies, and premature aging diseases. This review focuses on one of the most severe laminopathies, Hutchinson-Gilford Progeria Syndrome (HGPS), which is caused by aberrant splicing of the LMNA gene and expression of a mutant product called progerin. Here, we discuss current views about the molecular mechanisms that contribute to the pathophysiology of this devastating disease, as well as the strategies being tested in vitro and in vivo to counteract progerin toxicity. In particular, progerin accumulation elicits nuclear morphological abnormalities, misregulated gene expression, defects in DNA repair, telomere shortening, and genomic instability, all of which limit cellular proliferative capacity. In patients harboring this mutation, a severe premature aging disease develops during childhood. Interestingly, progerin is also produced in senescent cells and cells from old individuals, suggesting that progerin accumulation might be a factor in physiological aging. Deciphering the molecular mechanisms whereby progerin expression leads to HGPS is an emergent area of research, which could bring us closer to understanding the pathology of aging.

Nuclear export of intron-containing human immunodeficiency virus type 1 (HIV-1) RNA is mediated by the viral Rev protein that contains both an RNA binding domain specific for the viral Rev response element (RRE) and a nuclear export signal (NES). The cellular CRM1 (Exportin1) protein functions as a nuclear export receptor for proteins carrying a Rev-like NES in a process that also requires the GTP bound form of the Ran GTPase. Using purified recombinant factors, we show by co-precipitation, gel mobility shift and protein footprinting assays that full-length Rev protein interacts directly with CRM1 in vitro independently of both the integrity of the characteristic leucine residues of the NES and the presence of the cytotoxin leptomycin B (LMB). Addition of RanGTP induces the formation of an RRE-Rev-CRM1-RanGTP complex that is sensitive to LMB, NES mutations, and Ran being charged with GTP. Within this complex, CRM1 is readily cross-linked to Cys 89 near the NES of Rev. By protein footprinting, we demonstrate that the NES of Rev and two regions in CRM1 become inaccessible to endoproteinases upon binding suggesting that these regions are involved in protein-protein interactions. Our data are consistent with a model in which CRM1 is the nuclear export receptor for the Rev-RRE ribonucleoprotein complex and that RanGTP binds to a preformed Rev-CRM1 complex and specifies a functional interaction with the NES. Human immunodeficiency virus type 1 (HIV-1)1 encodes the regulatory protein Rev, which is absolutely required for viral replication. Rev promotes the nuclear export of incompletely spliced HIV-1 mRNA species, which are specified by the presence of a complex RNA structure, the Rev response element (RRE). Rev interacts directly with a purine-rich stem-loop called IIB within the RRE, and through oligomerization additional Rev molecules bind to lower affinity sites throughout the RRE (reviewed in Ref. 1). Rev is a 116-amino acids protein that has been shown to continuously shuttle between the nucleus and the cytoplasm (2, 3). Nuclear import of Rev is mediated by an arginine-rich nuclear localization signal (NLS) embedded in the RNA binding domain of the Rev protein (amino acids 34 -50) (4 -6). The NLS of Rev has been reported to associate with importin ␤, the large subunit of the conventional importin ␣/importin ␤ NLS receptor heterodimer, as well as with B23, a mammalian protein involved in the nuclear import of ribosomal proteins (7-9). Nuclear export of Rev is mediated by the nuclear export signal (NES), which contains a conserved stretch of characteristically spaced leucine residues (10, 11). Mutation of any of three leucines (Leu 78 , Leu 81 , or Leu 83 ) in the NES-core domain generates nonfunctional Rev proteins that loose their ability to exit the nucleus (2-4,12-16). The fact that these mutants remain active for specific binding and oligomerization on the RRE in vitro, has prompted the suggestion that they are unable to interact with a functionally important cellular export factor(s) (4). This model has gaine...