Identification of different roles for RanGDP and RanGTP in nuclear protein import.

The importin-alpha/beta heterodimer and the GTPase Ran play key roles in nuclear protein import. Importin binds the nuclear localization signal (NLS). Translocation of the resulting import ligand complex through the nuclear pore complex (NPC) requires Ran and is terminated at the nucleoplasmic side by its disassembly. The principal GTP exchange factor for Ran is the nuclear protein RCC1, whereas the major RanGAP is cytoplasmic, predicting that nuclear Ran is mainly in the GTP form and cytoplasmic Ran is in the GDP-bound form. Here, we show that nuclear import depends on cytoplasmic RanGDP and free GTP, and that RanGDP binds to the NPC. Therefore, import might involve nucleotide exchange and GTP hydrolysis on NPC-bound Ran. RanGDP binding to the NPC is not mediated by the Ran binding sites of importin-beta, suggesting that translocation is not driven from these sites. Consistently, a mutant importin-beta deficient in Ran binding can deliver its cargo up to the nucleoplasmic side of the NPC. However, the mutant is unable to release the import substrate into the nucleoplasm. Thus, binding of nucleoplasmic RanGTP to importin-beta probably triggers termination, i.e. the dissociation of importin-alpha from importin-beta and the subsequent release of the import substrate into the nucleoplasm.     

Nuclear envelope localization of human UNC84A does not require nuclear lamins

The SUN proteins are a conserved family of proteins in eukaryotes. Human UNC84A (Sun1) is a homolog of Caenorhabditis elegans UNC-84, a protein involved in nuclear anchorage and migration. We have analyzed targeting of UNC84A to the nuclear envelope (NE) and show that the N-terminal 300 amino acids are crucial for efficient NE localization of UNC84A whereas the conserved C-terminal SUN domain is not required. Furthermore, we demonstrate by combining RNA interference with immunofluorescence and fluorescence recovery after photobleaching analysis that localization and anchoring of UNC84A is not dependent on the lamin proteins, in contrast to what had been observed for C. elegans UNC-84.     

A Novel Class of RanGTP Binding Proteins

The importin-α/β complex and the GTPase Ran mediate nuclear import of proteins with a classical nuclear localization signal. Although Ran has been implicated also in a variety of other processes, such as cell cycle progression, a direct function of Ran has so far only been demonstrated for importin-mediated nuclear import. We have now identified an entire class of ∼20 potential Ran targets that share a sequence motif related to the Ran-binding site of importin-β. We have confirmed specific RanGTP binding for some of them, namely for two novel factors, RanBP7 and RanBP8, for CAS, Pse1p, and Msn5p, and for the cell cycle regulator Cse1p from Saccharomyces cerevisiae. We have studied RanBP7 in more detail. Similar to importin-β, it prevents the activation of Ran''s GTPase by RanGAP1 and inhibits nucleotide exchange on RanGTP. RanBP7 binds directly to nuclear pore complexes where it competes for binding sites with importin-β, transportin, and apparently also with the mediators of mRNA and U snRNA export. Furthermore, we provide evidence for a Ran-dependent transport cycle of RanBP7 and demonstrate that RanBP7 can cross the nuclear envelope rapidly and in both directions. On the basis of these results, we propose that RanBP7 might represent a nuclear transport factor that carries an as yet unknown cargo, which could apply as well for this entire class of related RanGTP-binding proteins.The nuclear pore complexes (NPC)¹ are the sites where the exchange of macromolecules between nucleus and cytoplasm occurs (Feldherr et al., 1984). Transport through the NPCs is bidirectional and comprises a multitude of substrates. Small molecules can passively diffuse through the NPC. The transport of proteins and RNAs >40–60 kD is, however, generally an active process, i.e., it is energy dependent (Newmeyer et al., 1986) and mediated by saturable transport receptors (Goldfarb et al., 1986; Michaud and Goldfarb, 1991; Jarmolowski et al., 1994). To accomplish multiple rounds of transport, these transport receptors are thought to shuttle between nucleus and cytoplasm (Goldfarb et al., 1986). An import receptor, for example, has to bind its import substrate initially in the cytoplasm, release it after NPC passage into the nucleus, and return to the cytoplasm without the cargo. Conversely, an export factor has to bind the export substrate only in the nucleus and on the way out. This model predicts asymmetry in these transport cycles and implies that the binding of the transport receptor to its cargo is regulated by the different environments of nucleus and cytoplasm.The nuclear import of proteins with a classical nuclear localization signal (NLS) is to date the best characterized nucleocytoplasmic transport pathway (for reviews see Görlich and Mattaj, 1996; Koepp and Silver, 1996; Schlenstedt, 1996). The signal contains one or more clusters of basic amino acids (for review see Dingwall and Laskey, 1991) and is recognized by the importin-α/β complex (for references see Sweet and Gerace, 1995; Panté and Aebi, 1996). The α subunit provides the NLS binding site (Imamoto et al., 1995; Weis et al., 1995) and is therefore also called the NLS receptor (Adam and Gerace, 1991). The β subunit accounts for the interaction with the NPC (Görlich et al., 1995; Moroianu et al., 1995) and carries importin-α with the NLS substrate into the nucleus. The translocation into the nucleus is terminated by the disassembly of the importin complex, and both subunits are returned probably separately to the cytoplasm. Importin-α interacts with -β via its importin-β binding domain (IBB domain; Görlich et al., 1996a ; Weis et al., 1996a ). Binding to importin-β with an IBB domain is sufficient for nuclear entry, and the IBB domain can therefore be regarded as the nuclear targeting signal of importin-α. The export domain of importin-α has not yet been identified, but it is distinct from the IBB domain.The small GTPase Ran (Drivas et al., 1990; Bischoff and Ponstingl, 1991b ; Belhumeur et al., 1993) plays a key role in NLS-dependent protein import (Melchior et al., 1993; Moore and Blobel, 1993). GTP hydrolysis by Ran is absolutely essential for import (Melchior et al., 1993; Moore and Blobel, 1993; Schlenstedt et al., 1995a ; Palacios et al., 1996) and is possibly even its sole source of energy (Weis et al., 1996b ). Although the molecular mechanism of import is far from being understood, it appears that Ran fulfils at least two distinct functions in this process: first, Ran''s GTP cycle probably drives translocation into the nucleus (Melchior et al., 1993; Moore and Blobel, 1993; Weis et al., 1996b ), which appears to involve the binding of (cytoplasmic) RanGDP to the NPC, followed by nucleotide exchange and GTP hydrolysis, but it does not involve binding of RanGTP to importin-β (Görlich et al., 1996b ). Unfortunately, nothing is known of how Ran''s GTP cycle would translate into a directed movement through the NPC. Secondly, Ran regulates the interaction between importin-α and -β (Rexach and Blobel, 1995; Chi et al., 1996; Görlich et al., 1996b ). Binding of RanGTP to importin-β disassembles the importin-α/β complex at the nuclear side of the NPC, thereby terminating translocation (Görlich et al., 1996b ). The asymmetric distribution of Ran''s principal GDP/GTP exchange factor (RCC1; Bischoff and Ponstingl, 1991a ) and GTPase activating protein (RanGAP1, or RNA1 in yeast; Bischoff et al., 1995a ; Becker et al., 1995) crucially determines where the importin heterodimer can form and where it is forced to dissociate. RCC1 is a nuclear, chromatin-bound protein (Ohtsubo et al., 1987, 1989) that generates RanGTP in the nucleus, whereas free RanGTP is depleted from the cytoplasm by RanGAP1, which is excluded from the nucleoplasm (Hopper et al., 1990; Matunis et al., 1996; Mahajan et. al, 1997). Thus, low RanGTP levels in the cytoplasm allow importin-α to bind -β, and the high RanGTP concentration in the nuclear compartment dissociates the importin complex. The concentration of free RanGTP can, in this model, be regarded as a marker for cytoplasmic identity (low RanGTP) and nuclear identity (high RanGTP), which is “sensed” by the Ran-binding site in importin-β.It is likely that at least some properties of importin-β are shared by the mediators of the other nucleocytoplasmic transport pathways. This is emphasized by the recent identification of the importin-β–related transportin (Pollard et al., 1996) as an import receptor recognizing the M9 domain, the nuclear targeting signal in hnRNP A1 (Michael et al., 1995), and of yeast transportin (Kap 104p) as an import receptor for mRNA binding proteins (Aitchison et al., 1996). Furthermore, importin-β or its NPC-binding domain cross-compete with other pathways, such as M9-dependent import, NES-mediated nuclear export, and the export of mRNA and U snRNA (Kutay et al., 1997). This would suggest that these other transport receptors share at least some binding sites at the NPC and take a similar path through the nuclear pore complex as importin-β.In addition to importin-β, a number of other Ran-binding proteins are detectable in eukaryotic cells, e.g., in overlay blots using Ran γ-[³²P]GTP as a probe. These can be grouped into two classes (Lounsbury et al., 1994, 1996): first, those with a RanBP1 homology domain including the Ran binding protein 1 (RanBP1) itself (Coutavas et al., 1993; Bischoff et al., 1995b ) and the nuclear pore protein RanBP2, which has four RanBP1 homology domains (Wu et al., 1995; Yokoyama et al., 1995). Their binding to Ran can be competed by RanBP1. Second, importin-β and so far unidentified protein(s) of ∼120 kD whose Ran-binding is competed by importin-β but not by excess of RanBP1 (Lounsbury et al., 1994, 1996). Both RanBP1 and importin-β inhibit the nucleotide exchange on RanGTP (Coutavas et al., 1993; Lounsbury et al., 1994, 1996; Bischoff et al., 1995b ; Görlich et al., 1996b ). However, they do not cross-compete with each other for Ran binding but instead bind to different, nonoverlapping sites on Ran (Chi et al., 1996; Kutay et al., 1997; Lounsbury and Macara, 1997). Another striking difference is that RanBP1 facilitates the activation of Ran''s GTPase by RanGAP1 (Beddow et al., 1995; Bischoff et al., 1995b ), whereas the importin-β/RanGTP complex is entirely GAP resistant (Floer and Blobel, 1996; Görlich et al., 1996b ).Although a direct involvement of Ran has so far only been demonstrated in the importin-dependent transport pathway, perturbations in the Ran system have severe effects on a great variety of cellular functions, such as RNA processing, RNA export, regulation of chromosome structure, cell cycle progression, initiation of replication, microtubule structure, etc. (for review see Dasso, 1993; Sazer, 1996). One could argue that these effects are all secondary consequences from an impaired NLS-dependent protein import. However, it is also possible that these defects are more direct and that eukaryotic cells contain many immediate targets of Ran function.Here we describe a novel superfamily of Ran-binding proteins, which includes about a dozen factors in yeast and probably even more in higher eukaryotes. The members of this superfamily share with importin-β an NH₂-terminal sequence motif that appears to account for RanGTP binding. Indeed we could confirm the interaction with Ran for the following factors: RanBP7 and RanBP8, two novel, related proteins described here, Cse1p, a cell cycle regulator in yeast, CAS, which is required for apoptosis in cultured human cells, and for Msn5p and Pse1p from yeast. Of these we have characterized RanBP7 and RanBP8 in more detail. Both resemble closely importin-β in their interaction with Ran, and both bind directly to nuclear pore complexes. RanBP7 can cross the nuclear membrane rapidly and in both directions. We provide evidence for a transport cycle in which RanBP7 first enters the nucleus, binds RanGTP inside the nucleus as a prerequisite for rapid re-export to the cytoplasm, after which the RanBP7/RanGTP complex becomes finally disassembled by the concerted action of RanBP1 and RanGAP1 in the cytoplasm. We propose that during these transport cycles, RanBP7 would normally carry an as yet unidentified cargo. This means, RanBP7 and possibly also the other members of the RanBP7/Cse1p/ importin-β superfamily could function as transport receptors that shuttle between nucleus and cytoplasm. RanBP7 and importin-β form an abundant, heterodimeric complex in the cytoplasm that appears to have a function different from nuclear import of proteins with a classical NLS. It might be a way to regulate either RanBP7 or importin-β function. Alternatively, the RanBP7/importin-β complex might constitute an import receptor with a substrate specificity different from that of the importin-α/β complex.     

Structural view of the Ran-Importin beta interaction at 2.3 A resolution.

Transport receptors of the Importin beta family shuttle between the nucleus and cytoplasm and mediate transport of macromolecules through nuclear pore complexes. They interact specifically with the GTP-binding protein Ran, which in turn regulates their interaction with cargo. Here, we report the three-dimensional structure of a complex between Ran bound to the nonhydrolyzable GTP analog GppNHp and a 462-residue fragment from Importin beta. The structure of Importin beta shows 10 tandem repeats resembling HEAT and Armadillo motifs. They form an irregular crescent, the concave site of which forms the interface with Ran-triphosphate. The importin-binding site of Ran does not overlap with that of the Ran-binding domain of RanBP2.     

Downregulation of miR-122 in the rodent and human hepatocellular carcinomas

MicroRNAs (miRs) are conserved small non-coding RNAs that negatively regulate gene expression. The miR profiles are markedly altered in cancers and some of them have a causal role in tumorigenesis. Here, we report changes in miR expression profile in hepatocellular carcinomas (HCCs) developed in male Fisher rats-fed folic acid, methionine, and choline-deficient (FMD) diet. Comparison of the miR profile by microarray analysis showed altered expression of some miRs in hepatomas compared to the livers from age-matched rats on the normal diet. While let-7a, miR-21, miR-23, miR-130, miR-190, and miR-17-92 family of genes was upregulated, miR-122, an abundant liver-specific miR, was downregulated in the tumors. The decrease in hepatic miR-122 was a tumor-specific event because it did not occur in the rats switched to the folate and methyl-adequate diet after 36 weeks on deficient diet, which did not lead to hepatocarcinogenesis. miR-122 was also silent in a transplanted rat hepatoma. Extrapolation of this study to human primary HCCs revealed that miR-122 expression was significantly (P = 0.013) reduced in 10 out of 20 tumors compared to the pair-matched control tissues. These findings suggest that the downregulation of miR-122 is associated with hepatocarcinogenesis and could be a potential biomarker for liver cancers.     

Structure of a human pre-40S particle points to a role for RACK1 in the final steps of 18S rRNA processing

Synthesis of ribosomal subunits in eukaryotes is a complex and tightly regulated process that has been mostly characterized in yeast. The discovery of a growing number of diseases linked to defects in ribosome biogenesis calls for a deeper understanding of these mechanisms and of the specificities of human ribosome maturation. We present the 19 Å resolution cryo-EM reconstruction of a cytoplasmic precursor to the human small ribosomal subunit, purified by using the tagged ribosome biogenesis factor LTV1 as bait. Compared to yeast pre-40S particles, this first three-dimensional structure of a human 40S subunit precursor shows noticeable differences with respect to the position of ribosome biogenesis factors and uncovers the early deposition of the ribosomal protein RACK1 during subunit maturation. Consistently, RACK1 is required for efficient processing of the 18S rRNA 3′-end, which might be related to its role in translation initiation. This first structural analysis of a human pre-ribosomal particle sets the grounds for high-resolution studies of conformational transitions accompanying ribosomal subunit maturation.     

MicroRNA-122 Inhibits Tumorigenic Properties of Hepatocellular Carcinoma Cells and Sensitizes These Cells to Sorafenib

MicroRNAs are negative regulators of protein coding genes. The liver-specific microRNA-122 (miR-122) is frequently suppressed in primary hepatocellular carcinomas (HCCs). In situ hybridization demonstrated that miR-122 is abundantly expressed in hepatocytes but barely detectable in primary human HCCs. Ectopic expression of miR-122 in nonexpressing HepG2, Hep3B, and SK-Hep-1 cells reversed their tumorigenic properties such as growth, replication potential, clonogenic survival, anchorage-independent growth, migration, invasion, and tumor formation in nude mice. Further, miR-122-expressing HCC cells retained an epithelial phenotype that correlated with reduced Vimentin expression. ADAM10 (a distintegrin and metalloprotease family 10), serum response factor (SRF), and insulin-like growth factor 1 receptor (Igf1R) that promote tumorigenesis were validated as targets of miR-122 and were repressed by the microRNA. Conversely, depletion of the endogenous miR-122 in Huh-7 cells facilitated their tumorigenic properties with concomitant up-regulation of these targets. Expression of SRF or Igf1R partially reversed tumor suppressor function of miR-122. Further, miR-122 impeded angiogenic properties of endothelial cells in vitro. Notably, ADAM10, SRF, and Igf1R were up-regulated in primary human HCCs compared with the matching liver tissue. Co-labeling studies demonstrated exclusive localization of miR-122 in the benign livers, whereas SRF predominantly expressed in HCC. More importantly, growth and clonogenic survival of miR-122-expressing HCC cells were significantly reduced upon treatment with sorafenib, a multi-kinase inhibitor clinically effective against HCC. Collectively, these results suggest that the loss of multifunctional miR-122 contributes to the malignant phenotype of HCC cells, and miR-122 mimetic alone or in combination with anticancer drugs can be a promising therapeutic regimen against liver cancer.     

The humoral immune response and the productivity of laying hens kept on the ground or in cages

The effects of two different keeping systems on the humoral immune response and productivity were compared for 80 laying hens, divided into four groups. Two groups each of 20 hens were kept on the ground and two were kept in cages. All the birds were immunised subcutaneously with human serum immunoglobulin G (IgG) at a dose of 100(microg per injection. The immunisations were performed twice at 4-week intervals. The lipopeptide Pam(3)Cys-Ser-(Lys)(4) was used as an adjuvant at a dose of 0.25mg per injection in one group from each housing system. In the second group from each housing system, the hens were immunised without any adjuvant (antigen control groups). The mean egg yield was significantly higher in both the antigen control group and the adjuvant group, when laying hens were kept in cages. Total egg weight remained constant in both of the housing systems. Keeping hens in cages resulted in higher mean specific antibody titres and mean immunoglobulin Y concentrations in the egg yolk.     

The beta-isoform of the BRCA2 and CDKN1A(p21)-interacting protein (BCCIP) stabilizes nuclear RPL23/uL14

BRCA2 and CDKN1A(p21,CIP1)-interacting protein (BCCIP) is an evolutionary conserved protein implicated in maintenance of genome stability and cell cycle progression. Two isoforms of BCCIP with distinct C-terminal domains exist in humans. We show that mammalian BCCIPβ, but not BCCIPα, forms a ternary complex with the ribosomal protein RPL23/uL14 and the pre-60S trans-acting factor eIF6. Complex formation is dependent on an intact C-terminal domain of BCCIPβ. Depletion of BCCIPβ reduces the pool of free RPL23, and decreases eIF6 levels in nucleoli. Overexpression of BCCIPβ leads to nucleoplasmic accumulation of extra-ribosomal RPL23 and stabilizes overexpressed RPL23, suggesting that BCCIPβ functions as nuclear chaperone for RPL23.     

Partial trisomy of chromosome no. 1 in two adult brothers due to maternal translocation (1q-;6p+)

Summary Extra chromosome material on the short arm of chromosome no. 6 (46,XY,6p+) was found in two mentally retarded adult half-brothers with mildly dysmorphic features. The phenotypically normal mother had a balanced translocation between the long arm of chromosome no. 1 and the short arm of chromosome no. 6: 46,XX,t(1;6)(q32;p25). Thus the two affected brothers were trisomic for the long arm segment of chromosome no. 1, distal to q32. These patients, with mildly dysmorphic features and mental retardation, represent the first cases of partial trisomy 1q surviving to adulthood.The clinical and cytogenetic data obtained from eight individuals with partial trisomies for different long arm segments of chromosome no. 1 suggest that partial trisomy of the distal two-thirds of the long arm is characterized by severe malformations, growth retardation, and early death. Conversely, partial trisomy for the distal one-third of the long arm is associated with milder malformations and longer survival time as well as growth and mental retardation.