GeneCards (www.genecards.org) is a comprehensive, authoritative compendium of annotative information about human genes, widely used for nearly 15 years. Its gene-centric content is automatically mined and integrated from over 80 digital sources, resulting in a web-based deep-linked card for each of >73 000 human gene entries, encompassing the following categories: protein coding, pseudogene, RNA gene, genetic locus, cluster and uncategorized. We now introduce GeneCards Version 3, featuring a speedy and sophisticated search engine and a revamped, technologically enabling infrastructure, catering to the expanding needs of biomedical researchers. A key focus is on gene-set analyses, which leverage GeneCards’ unique wealth of combinatorial annotations. These include the GeneALaCart batch query facility, which tabulates user-selected annotations for multiple genes and GeneDecks, which identifies similar genes with shared annotations, and finds set-shared annotations by descriptor enrichment analysis. Such set-centric features address a host of applications, including microarray data analysis, cross-database annotation mapping and gene-disorder associations for drug targeting. We highlight the new Version 3 database architecture, its multi-faceted search engine, and its semi-automated quality assurance system. Data enhancements include an expanded visualization of gene expression patterns in normal and cancer tissues, an integrated alternative splicing pattern display, and augmented multi-source SNPs and pathways sections. GeneCards now provides direct links to gene-related research reagents such as antibodies, recombinant proteins, DNA clones and inhibitory RNAs and features gene-related drugs and compounds lists. We also portray the GeneCards Inferred Functionality Score annotation landscape tool for scoring a gene’s functional information status. Finally, we delineate examples of applications and collaborations that have benefited from the GeneCards suite.Database URL: www.genecards.org

The vertebrate nuclear pore complex, 30 times the size of a ribosome, assembles from a library of soluble subunits and two membrane proteins. Using immunodepletion of Xenopus nuclear reconstitution extracts, it has previously been possible to assemble nuclei lacking pore subunits tied to protein import, export, or mRNA export. However, these altered pores all still possessed the bulk of pore structure. Here, we immunodeplete a single subunit, the Nup107-160 complex, using antibodies to Nup85 and Nup133, two of its components. The resulting reconstituted nuclei are severely defective for NLS import and DNA replication. Strikingly, they show a profound defect for every tested nucleoporin. Even the integral membrane proteins POM121 and gp210 are absent or unorganized. Scanning electron microscopy reveals pore-free nuclei, while addback of the Nup107-160 complex restores functional pores. We conclude that the Nup107-160 complex is a pivotal determinant for vertebrate nuclear pore complex assembly.

Assembly of a eukaryotic nucleus involves three distinct events: membrane recruitment, fusion to form a double nuclear membrane, and nuclear pore complex (NPC) assembly. We report that importin ␤ negatively regulates two of these events, membrane fusion and NPC assembly. When excess importin ␤ is added to a full Xenopus nuclear reconstitution reaction, vesicles are recruited to chromatin but their fusion is blocked. The importin ␤ down-regulation of membrane fusion is Ran-GTP reversible. Indeed, excess RanGTP (RanQ69L) alone stimulates excessive membrane fusion, leading to intranuclear membrane tubules and cytoplasmic annulate lamellae-like structures. We propose that a precise balance of importin ␤ to Ran is required to create a correct double nuclear membrane and simultaneously to repress undesirable fusion events. Interestingly, truncated importin ␤ 45-462 allows membrane fusion but produces nuclei lacking any NPCs. This reveals distinct importin ␤-regulation of NPC assembly. Excess full-length importin ␤ and ␤ 45-462 act similarly when added to prefused nuclear intermediates, i.e., both block NPC assembly. The importin ␤ NPC block, which maps downstream of GTP␥S and BAPTA-sensitive steps in NPC assembly, is reversible by cytosol. Remarkably, it is not reversible by 25 M RanGTP, a concentration that easily reverses fusion inhibition. This report, using a full reconstitution system and natural chromatin substrates, significantly expands the repertoire of importin ␤. Its roles now encompass negative regulation of two of the major events of nuclear assembly: membrane fusion and NPC assembly. INTRODUCTIONIn cells from yeast to mammals, importin ␣ and ␤ act together to ferry classical nuclear localization signal (NLS)-bearing proteins into the nucleus (Gorlich and Kutay, 1999;Stoffler et al., 1999;Damelin and Silver, 2000;Rout et al., 2000;Conti and Izaurralde, 2001;Vasu and Forbes, 2001;Damelin et al., 2002;Weis, 2003). Once in the nucleus the small GTPase Ran binds to importin ␤, displacing importin ␣ and the NLS cargo, thus completing import. In the nucleus, Ran is kept in a GTP state by the constant action of its chromatinbound Ran-GEF, RCC1 (Melchior and Gerace, 1998;Macara, 2001;Dasso, 2002;Kalab et al., 2002;Schwoebel et al., 2002;Steggerda and Paschal, 2002). In contrast, RanGDP is the predominant form found in the cytoplasm due to the cytoplasmic localization of RanGAP.The horizons for importin ␣ and ␤ were unexpectedly broadened when they were found to play a very different role at mitosis. In metazoans, importin ␣ and ␤ are released to the cytosol by nuclear breakdown, where they act to inhibit proteins essential for mitotic spindle assembly. However, the inhibition of spindle assembly is reversed by Ran in the vicinity of mitotic chromosomes, where RanGTP continues to be produced by chromatin-bound RCC1 (Kalab et al., 1999;Gruss et al., 2001;Nachury et al., 2001;Wiese et al., 2001;Dasso, 2002). Thus, a spindle forms only around the mitotic (ER) chromosomes and not elsewhere in the cytoplasm.At the end ...

Importin beta, once thought to be exclusively a nuclear transport receptor, is emerging as a global regulator of diverse cellular functions. Importin beta acts positively in multiple interphase roles: in nuclear import, as a chaperone for highly charged nuclear proteins, and as a potential motor adaptor for movement along microtubules. In contrast, importin beta plays a negative regulatory role in mitotic spindle assembly, centrosome dynamics, nuclear membrane formation, and nuclear pore assembly. In most of these, importin beta is counteracted by its regulator, Ran-GTP. In light of this, the recent discovery of Ran's involvement in spindle checkpoint control suggested a potential new arena for importin beta action, although it is also possible that one of importin beta's relatives, the karyopherin family of proteins, manages this checkpoint. Lastly, importin beta plays a role in transducing damage signals from the axons of injured neurons back to the cell body.

Assembly of the nuclear pore, gateway to the genome, from its component subunits is a complex process. In higher eukaryotes, nuclear pore assembly begins with the binding of ELYS/MEL-28 to chromatin and recruitment of the large critical Nup107-160 pore subunit. The choreography of steps that follow is largely speculative. Here, we set out to molecularly define early steps in nuclear pore assembly, beginning with chromatin binding. Point mutation analysis indicates that pore assembly is exquisitely sensitive to the change of only two amino acids in the AT-hook motif of ELYS. The dependence on AT-rich chromatin for ELYS binding is borne out by the use of two DNA-binding antibiotics. AT-binding Distamycin A largely blocks nuclear pore assembly, whereas GC-binding Chromomycin A 3 does not. Next, we find that recruitment of vesicles containing the key integral membrane pore proteins POM121 and NDC1 to the forming nucleus is dependent on chromatin-bound ELYS/Nup107-160 complex, whereas recruitment of gp210 vesicles is not. Indeed, we reveal an interaction between the cytoplasmic domain of POM121 and the Nup107-160 complex. Our data thus suggest an order for nuclear pore assembly of 1) AT-rich chromatin sites, 2) ELYS, 3) the Nup107-160 complex, and 4) POM121-and NDC1-containing membrane vesicles and/or sheets, followed by (5) assembly of the bulk of the remaining soluble pore subunits. INTRODUCTIONThe possession of a nuclear envelope (NE) that encompasses the genome is the defining characteristic of all eukaryotes. The envelope consists of double nuclear membranes, hundreds to thousands of nuclear pore complexes (NPCs), and in higher eukaryotes, a nuclear lamina. Bidirectional transport of protein and RNA molecules through the nuclear envelope is mediated exclusively by NPCs, large structures ϳ60 -125 MDa in size (Reichelt et al., 1990;Macara, 2001;Quimby and Corbett, 2001;Goldfarb et al., 2004;Pemberton and Paschal, 2005;Patel et al., 2007).In higher eukaryotes the nuclear envelope, including pore complexes, disassembles at mitosis as a prelude to spindle assembly and chromosome segregation (Burke and Ellenberg, 2002;Margalit et al., 2005;Prunuske et al., 2006). This disassembly then necessitates nuclear envelope reformation around each set of segregated chromosomes toward the end of mitosis, a process that involves both nuclear membrane recruitment and nuclear pore formation.Analysis of the pore subunits produced by mitotic disassembly has provided the most useful clues to nearest neighbor interactions within the vertebrate pore. Vertebrate nuclear pores are comprised of ϳ30 different proteins or nucleoporins (Nups) in 8-32 copies each, to give a 500-1000 protein structure (Cronshaw et al., 2002). At mitosis the massive vertebrate pore disassembles into ϳ14 soluble subunits, each with a distinct protein composition, whereas the integral membrane pore proteins, POM121, NDC1, and gp210, segregate into endoplasmic reticulum (ER) sheets and vesicles (Gerace et al., 1982;Wozniak et al., 1989;Greber et al., 1990;Hallberg ...

Highlights d Expression of eight nucleoporins is reduced in human C9orf72 neuronal nuclei d Reduction in POM121 affects nuclear pore complex composition d Nucleoporin alterations diminish nucleocytoplasmic transport and neuronal survival d G 4 C 2 repeat RNA initiates the pathogenic cascades, leading to decreased nuclear POM121

Corals comprise a biomineralizing cnidarian, dinoflagellate algal symbionts, and associated microbiome of prokaryotes and viruses. Ongoing efforts to conserve coral reefs by identifying the major stress response pathways and thereby laying the foundation to select resistant genotypes rely on a robust genomic foundation. Here we generated and analyzed a high quality long-read based ~886 Mbp nuclear genome assembly and transcriptome data from the dominant rice coral, Montipora capitata from Hawai’i. Our work provides insights into the architecture of coral genomes and shows how they differ in size and gene inventory, putatively due to population size variation. We describe a recent example of foreign gene acquisition via a bacterial gene transfer agent and illustrate the major pathways of stress response that can be used to predict regulatory components of the transcriptional networks in M . capitata . These genomic resources provide insights into the adaptive potential of these sessile, long-lived species in both natural and human influenced environments and facilitate functional and population genomic studies aimed at Hawaiian reef restoration and conservation.

Viruses infecting bacteria (phages) are thought to greatly impact microbial population dynamics as well as the genome diversity and evolution of their hosts. Here we report on the discovery of a novel lineage of tailed dsDNA phages belonging to the family Myoviridae and describe its first representative, S-TIM5, that infects the ubiquitous marine cyanobacterium, Synechococcus. The genome of this phage encodes an entirely unique set of structural proteins not found in any currently known phage, indicating that it uses lineage-specific genes for virion morphogenesis and represents a previously unknown lineage of myoviruses. Furthermore, among its distinctive collection of replication and DNA metabolism genes, it carries a mitochondrial-like DNA polymerase gene, providing strong evidence for the bacteriophage origin of the mitochondrial DNA polymerase. S-TIM5 also encodes an array of bacterial-like metabolism genes commonly found in phages infecting cyanobacteria including photosynthesis, carbon metabolism and phosphorus acquisition genes. This suggests a common gene pool and gene swapping of cyanophage-specific genes among different phage lineages despite distinct sets of structural and replication genes. All cytosines following purine nucleotides are methylated in the S-TIM5 genome, constituting a unique methylation pattern that likely protects the genome from nuclease degradation. This phage is abundant in the Red Sea and S-TIM5 gene homologs are widespread in the oceans. This unusual phage type is thus likely to be an important player in the oceans, impacting the population dynamics and evolution of their primary producing cyanobacterial hosts.