No abstract

Here, we report the first evidence that the Ran GTPase cycle is required for nuclear pore complex (NPC) assembly. Using a genetic approach, factors required for NPC assembly were identified in Saccharomyces cerevisiae. Four mutant complementation groups were characterized that correspond to respective mutations in genes encoding Ran (gsp1), and essential Ran regulatory factors Ran GTPase–activating protein (rna1), Ran guanine nucleotide exchange factor (prp20), and the RanGDP import factor (ntf2). All the mutants showed temperature-dependent mislocalization of green fluorescence protein (GFP)-tagged nucleoporins (nups) and the pore-membrane protein Pom152. A decrease in GFP fluorescence associated with the nuclear envelope was observed along with an increase in the diffuse, cytoplasmic signal with GFP foci. The defects did not affect the stability of existing NPCs, and nup mislocalization was dependent on de novo protein synthesis and continued cell growth. Electron microscopy analysis revealed striking membrane perturbations and the accumulation of vesicles in arrested mutants. Using both biochemical fractionation and immunoelectron microscopy methods, these vesicles were shown to contain nups. We propose a model wherein a Ran-mediated vesicular fusion step is required for NPC assembly into intact nuclear envelopes.

The alternative exon 5 of the striated muscle-specific cardiac troponin T (cTNT) gene is included in mRNA from embryonic skeletal and cardiac muscle and excluded in mRNA from the adult. The embryonic splicing pattern is reproduced in primary skeletal muscle cultures for both the endogenous gene and transiently transfected minigenes, whereas in nonmuscle cell lines, minigenes express a default exon skipping pattern. Using this experimental system, we previously showed that a purine-rich splicing enhancer in the alternative exon functions as a constitutive splicing element but not as a target for factors regulating cell-specific splicing. In this study, we identify four intron elements, one located upstream and three located downstream of the alternative exon, which act in a positive manner to mediate the embryonic splicing pattern of exon inclusion. Synergistic interactions between at least three of the four elements are necessary and sufficient to regulate splicing of a heterologous alternative exon and heterologous splice sites. Mutations in these elements prevent activation of exon inclusion in muscle cells but do not affect the default level of exon inclusion in nonmuscle cells. Therefore, these elements function as muscle-specific splicing enhancers (MSEs) and are the first muscle-specific positive-acting splicing elements to be described. One MSE located downstream from the alternative exon is conserved in the rat and chicken cTNT genes. A related sequence is found in a third muscle-specific gene, that encoding skeletal troponin T, downstream from an alternative exon with a developmental pattern of alternative splicing similar to that of rat and chicken cTNT. Therefore, the MSEs identified in the cTNT gene may play a role in developmentally regulated alternative splicing in a number of different genes.A large number of genes are remarkable for their ability to generate multiple mRNAs via pre-mRNA alternative splicing (27,46). Alternative splicing is often regulated according to cell-specific pathways (such as cell type, developmental stage, or sex) to generate mRNAs that differ in protein coding potential, stability, or translation efficiency. Genetic and biochemical studies have established several paradigms for regulated splicing in Drosophila melanogaster (27); however, little is known regarding the cis elements or trans-acting factors that mediate cell-specific regulation in vertebrates.The cis elements required for the regulation of alternative splicing in vetebrates have been investigated primarily by transient transfection of cloned genes into homologous and heterologous cell types (22, 46). In heterologous cells, which presumably lack appropriate regulatory factors, an alternatively spliced pre-mRNA is spliced according to a default pathway determined by the efficiency with which the constitutive splicing machinery recognizes the constitutive splicing signals.Work from many laboratories has demonstrated that the default splicing pathway is determined by a balance of several pre-mRNA features, such a...

Here we have examined the function of Pom34p, a novel membrane protein in Saccharomyces cerevisiae, localized to nuclear pore complexes (NPCs). Membrane topology analysis revealed that Pom34p is a double-pass transmembrane protein with both the amino (N) and carboxy (C) termini positioned on the cytosolic/pore face. The network of genetic interactions between POM34 and genes encoding other nucleoporins was established and showed specific links between Pom34p function and Nup170p, Nup188p, Nup59p, Gle2p, Nup159p, and Nup82p. The transmembrane domains of Pom34p in addition to either the N-or C-terminal region were necessary for its function in different double mutants. We further characterized the pom34DN nup188D mutant and found it to be perturbed in both NPC structure and function. Mislocalization of a subset of nucleoporins harboring phenylalanine-glycine repeats was observed, and nuclear import capacity for the Kap104p and Kap121p pathways was inhibited. In contrast, the pom34D pom152D double mutant was viable at all temperatures and showed no such defects. Interestingly, POM152 overexpression suppressed the synthetic lethality of pom34D nup170D and pom34D nup59D mutants. We speculate that multiple integral membrane proteins, either within the nuclear pore domain or in the nuclear envelope, execute coordinated roles in NPC structure and function.

The yeast Saccharomyces cerevisiae nucleoporin Nup116p serves as a docking site for both nuclear import and export factors. However, the mechanism for assembling Nup116p into the nuclear pore complex (NPC) has not been resolved. By conducting a two-hybrid screen with the carboxy (C)-terminal Nup116p region as bait, we identified Nup82p. The predicted coiled-coil region of Nup82p was not required for Nup116p interaction, making the binding requirements distinct from those for the Nsp1p-Nup82p-Nup159p subcomplex (N. Belgareh, C. Snay-Hodge, F. Pasteau, S. Dagher, C. N. Cole, and V. Doye, Mol. Biol. Cell 9:3475-3492, 1998). Immunoprecipitation experiments using yeast cell lysates resulted in the coisolation of a Nup116p-Nup82p subcomplex. Although the absence of Nup116p had no effect on the NPC localization of Nup82p, overexpression of Cterminal Nup116p in a nup116 null mutant resulted in Nup82p mislocalization. Moreover, NPC localization of Nup116p was specifically diminished in a nup82-⌬108 mutant after growth at 37°C. Immunoelectron microscopy analysis showed Nup116p was localized on both the cytoplasmic and nuclear NPC faces. Its distribution was asymmetric with the majority at the cytoplasmic face. Taken together, these results suggest that Nup82p and Nup116p interact at the cytoplasmic NPC face, with nucleoplasmic Nup116p localization utilizing novel binding partners.Nuclear pore complexes (NPCs) are massive multiprotein structures embedded in the nuclear envelope (NE), which serve as portals for regulating the traffic of macromolecules between the cytoplasm and the nucleus (52). Three-dimensional structural information for yeast Saccharomyces cerevisiae and vertebrate NPCs has been recently revealed by a combination of high-resolution cryoelectron and scanning electron microscopy (EM) analysis (2,3,16,27,46,47,69). NPCs possess a central plug surrounded by eight spokes which attach to cytoplasmic and nuclear rings. These rings anchor the peripherally associated cytoplasm filaments and nuclear basket. Yeast S. cerevisiae NPCs are comprised of ϳ30 different proteins, termed nucleoporins (48). Models of NPC structure have predicted that the distinct modular structures of the NPC are formed from subsets of distinct nucleoporin subunits. In support of this, subcomplexes containing different nucleoporins have been biochemically isolated and characterized (for example, references 6, 9, 14, 21 to 23, 25, 30, 40, 41, and 58). Moreover, immunoelectron microscopy (IEM) experiments have localized some nucleoporins to exclusively the cytoplasmic filaments or the nuclear basket and others to either symmetric or asymmetric distributions on the central core structure (reviewed in references 48 and 60). This differential localization may reflect distinct roles for particular nucleoporins in mediating particular steps of the nuclear transport mechanism.One strategy for dissecting the hierarchy of protein-protein interactions that account for NPC structure and function has been to analyze yeast S. cerevisiae mutants. Mutations...