The nucleoporin nup153 plays a critical role in multiple types of nuclear export.
(9/1744)
The fundamental process of nucleocytoplasmic transport takes place through the nuclear pore. Peripheral pore structures are presumably poised to interact with transport receptors and their cargo as these receptor complexes first encounter the pore. One such peripheral structure likely to play an important role in nuclear export is the basket structure located on the nuclear side of the pore. At present, Nup153 is the only nucleoporin known to localize to the surface of this basket, suggesting that Nup153 is potentially one of the first pore components an RNA or protein encounters during export. In this study, anti-Nup153 antibodies were used to probe the role of Nup153 in nuclear export in Xenopus oocytes. We found that Nup153 antibodies block three major classes of RNA export, that of snRNA, mRNA, and 5S rRNA. Nup153 antibodies also block the NES protein export pathway, specifically the export of the HIV Rev protein, as well as Rev-dependent RNA export. Not all export was blocked; Nup153 antibodies did not impede the export of tRNA or the recycling of importin beta to the cytoplasm. The specific antibodies used here also did not affect nuclear import, whether mediated by importin alpha/beta or by transportin. Overall, the results indicate that Nup153 is crucial to multiple classes of RNA and protein export, being involved at a vital juncture point in their export pathways. This juncture point appears to be one that is bypassed by tRNA during its export. We asked whether a physical interaction between RNA and Nup153 could be observed, using homoribopolymers as sequence-independent probes for interaction. Nup153, unlike four other nucleoporins including Nup98, associated strongly with poly(G) and significantly with poly(U). Thus, Nup153 is unique among the nucleoporins tested in its ability to interact with RNA and must do so either directly or indirectly through an adaptor protein. These results suggest a unique mechanistic role for Nup153 in the export of multiple cargos. (+info)
Conserved loop I of U5 small nuclear RNA is dispensable for both catalytic steps of pre-mRNA splicing in HeLa nuclear extracts.
(10/1744)
The function of conserved regions of the metazoan U5 snRNA was investigated by reconstituting U5 small nuclear ribonucleoprotein particles (snRNPs) from purified snRNP proteins and HeLa or Xenopus U5 snRNA mutants and testing their ability to restore splicing to U5-depleted nuclear extracts. Substitution of conserved nucleotides comprising internal loop 2 or deletion of internal loop 1 had no significant effect on the ability of reconstituted U5 snRNPs to complement splicing. However, deletion of internal loop 2 abolished U5 activity in splicing and spliceosome formation. Surprisingly, substitution of the invariant loop 1 nucleotides with a GAGA tetraloop had no effect on U5 activity. Furthermore, U5 snRNPs reconstituted from an RNA formed by annealing the 5' and 3' halves of the U5 snRNA, which lacked all loop 1 nucleotides, complemented both steps of splicing. Thus, in contrast to yeast, loop 1 of the human U5 snRNA is dispensable for both steps of splicing in HeLa nuclear extracts. This suggests that its function can be compensated for in vitro by other spliceosomal components: for example, by proteins associated with the U5 snRNP. Consistent with this idea, immunoprecipitation studies indicated that several functionally important U5 proteins associate stably with U5 snRNPs containing a GAGA loop 1 substitution. (+info)
Nop58p is a common component of the box C+D snoRNPs that is required for snoRNA stability.
(11/1744)
Eukaryotic nucleoli contain a large family of box C+D small nucleolar RNA (snoRNA) species, all of which are associated with a common protein Nop1p/fibrillarin. Nop58p was identified in a screen for synthetic lethality with Nop1p and shown to be an essential nucleolar protein. Here we report that a Protein A-tagged version of Nop58p coprecipitates all tested box C+D snoRNAs and that genetic depletion of Nop58p leads to the loss of all tested box C+D snoRNAs. The box H+ACA class of snoRNAs are not coprecipitated with Nop58p, and are not codepleted. The yeast box C+D snoRNAs include two species, U3 and U14, that are required for the early cleavages in pre-rRNA processing. Consistent with this, Nop58p depletion leads to a strong inhibition of pre-rRNA processing and 18S rRNA synthesis. Unexpectedly, depletion of Nop58p leads to the accumulation of 3' extended forms of U3 and U24, showing that the protein is also involved in snoRNA synthesis. Nop58p is the second common component of the box C+D snoRNPs to be identified and the first to be shown to be required for the stability and for the synthesis of these snoRNAs. (+info)
Structures of endogenous nonecotropic murine leukemia virus (MLV) long terminal repeats in wild mice: implication for evolution of MLVs.
(12/1744)
To develop a better understanding of the interaction between retroviruses and their hosts, we have investigated the polymorphism in endogenous murine leukemia proviruses (MLVs). We used genomic libraries of wild mouse DNAs and PCR to analyze genetic variation in the proviruses found in wild mouse species, including Mus musculus (M. m. castaneus, M. m. musculus, M. m. molossinus, and M. m. domesticus), Mus spretus, and Mus spicelegus, as well as some inbred laboratory strains. In this analysis, we detected several unique forms of sequence organization in the U3 regions of the long terminal repeats of these proviruses. The distribution of the proviruses with unique U3 structures demonstrated that xenotropic MLV-related proviruses were present only in M. musculus subspecies, while polytropic MLV-related proviruses were found in both M. musculus and M. spretus. Furthermore, one unique provirus from M. spicelegus was found to be equidistant from ecotropic provirus and nonecotropic provirus by phylogenetic analysis. This provirus, termed HEMV, was thus likely to be related to the common ancestor of these MLVs. Moreover, an ancestral type of polytropic MLV-related provirus was detected in M. spretus species. Despite their "ancestral" phylogenetic position, proviruses of these types are not widespread in mice, implying more-recent spread by infection rather than inheritance. These results imply that recent evolution of these proviruses involved alternating periods of replication as virus and residence in the germ line. (+info)
Conservation of functional features of U6atac and U12 snRNAs between vertebrates and higher plants.
(13/1744)
Splicing of U12-dependent introns requires the function of U11, U12, U6atac, U4atac, and U5 snRNAs. Recent studies have suggested that U6atac and U12 snRNAs interact extensively with each other, as well as with the pre-mRNA by Watson-Crick base pairing. The overall structure and many of the sequences are very similar to the highly conserved analogous regions of U6 and U2 snRNAs. We have identified the homologs of U6atac and U12 snRNAs in the plant Arabidopsis thaliana. These snRNAs are significantly diverged from human, showing overall identities of 65% for U6atac and 55% for U12 snRNA. However, there is almost complete conservation of the sequences and structures that are implicated in splicing. The sequence of plant U6atac snRNA shows complete conservation of the nucleotides that base pair to the 5' splice site sequences of U12-dependent introns in human. The immediately adjacent AGAGA sequence, which is found in human U6atac and all U6 snRNAs, is also conserved. High conservation is also observed in the sequences of U6atac and U12 that are believed to base pair with each other. The intramolecular U6atac stem-loop structure immediately adjacent to the U12 interaction region differs from the human sequence in 9 out of 21 positions. Most of these differences are in base pairing regions with compensatory changes occurring across the stem. To show that this stem-loop was functional, it was transplanted into a human suppressor U6atac snRNA expression construct. This chimeric snRNA was inactive in vivo but could be rescued by coexpression of a U4atac snRNA expression construct containing compensatory mutations that restored base pairing to the chimeric U6atac snRNA. These data show that base pairing of U4atac snRNA to U6atac snRNA has a required role in vivo and that the plant U6atac intramolecular stem-loop is the functional analog of the human sequence. (+info)
Evidence for effective suppression of recombination in the chromosome 17q21 segment spanning RNU2-BRCA1.
(14/1744)
Characterization of associations between polymorphic sites located throughout the approximately 200-400-kb variable-length region spanning RNU2-BRCA1 reveals nearly complete linkage disequilibrium. This segment spans the RNU2 array, which includes 6-30 tandem copies of the U2 snRNA gene, and an adjacent region containing NBR1, the LBRCA1 pseudogene, NBR2, and BRCA1 in a tandemly duplicated structure. A series of biallelic polymorphisms define two common haplotypes that do not vary significantly, in structure or frequency, between populations of primarily European (n=275) or Asian (n=34) ancestry. Lower-frequency variants occurring at distantly located sites within this region also show very strong associations. The rarer haplotype classes appear to be distinguished by mutational alteration and are not recombination products of the two major classes. The two major haplotypes also exhibit significantly different allele-length distributions for local simple tandem-repeat markers. The conservation of extensive distinct chromosomal haplotypes during a long period of human population expansion and divergence indicates that selective forces or specific chromosomal mechanisms result in effective recombination suppression. The extreme degree of long-range linkage disequilibrium at this locus may be exceeded only by that reported for the human MHC locus, where allele-specific functional interactions are believed to be significant. These findings have implications for the estimation of the time of origin of BRCA1 mutations having a founder effect, the interpretation of the significance of rare allelic variants, and the study of the origins of modern populations. (+info)
Nuclear rDNA phylogeny in the fungal genus Verticillium and its relationship to insect and plant virulence, extracellular proteases and carbohydrases.
(15/1744)
Phylogenetic relationships among 18 isolates in the genus Verticillium, representing 13 species of diverse econutritional groups (pathogens of insects, plants, mushrooms, nematodes and spiders, and saprobes), were examined by using sequences from the internal transcribed spacer (ITS) and small nuclear (NS) rRNA regions. The isolates were also assessed for their abilities to infect insect larvae (Galleria mellonella) and to cause necrosis in alfalfa (Medicago sativa), and for their proteolytic, chitinolytic and pectinolytic activities. The phylogenetic data suggested that Verticillium is polyphyletic in origin and is therefore a form genus. However, the phylogenetic tree supported the plant pathogens (V. dahliae, V. albo-atrum and V. nigrescens) as a clade. The alfalfa isolate of V. albo-atrum (isolate 595) was an interesting outlier to the main body of plant pathogens as it clustered with the insect pathogen V. indicum. Strains of V. lecanii and V. indicum were able to infect insects and are present in divergent groups in the consensus tree, suggesting that the ability to infect insects may have evolved independently many times. Similarly, the nematophagous Verticillium species appear to have evolved independently along several different routes and one isolate, V. chlamydosporium, was able to infect insects. V. albo-atrum, V. nigrescens and V. dahliae all produced high levels of enzymes capable of degrading pectin, a major component of plant cell walls. The ability to excrete pectinase was a broad indicator of the ability to produce lesions on alfalfa. In the plant pathogens, the functions of a broad-spectrum protease were assumed by trypsins which degrade Bz-AA-AA-Arg-NA substrates (Bz, benzoyl; AA, various amino acids; NA, p-nitroanilide). The insect pathogens and mushroom pathogen (V. fungicola) were characterized by production of high levels of subtilisin-like proteases active against a chymotrypsin substrate (succinyl-Ala2-Pro-Phe-NA) and the inability to clear pectin. The insect and mushroom pathogens, and several nematode pathogens, were distinguishable from the plant pathogens in their ability to produce chitinases. (+info)
Coiled bodies preferentially associate with U4, U11, and U12 small nuclear RNA genes in interphase HeLa cells but not with U6 and U7 genes.
(16/1744)
Coiled bodies (CBs) are nuclear organelles involved in the metabolism of small nuclear RNAs (snRNAs) and histone messages. Their structural morphology and molecular composition have been conserved from plants to animals. CBs preferentially and specifically associate with genes that encode U1, U2, and U3 snRNAs as well as the cell cycle-regulated histone loci. A common link among these previously identified CB-associated genes is that they are either clustered or tandemly repeated in the human genome. In an effort to identify additional loci that associate with CBs, we have isolated and mapped the chromosomal locations of genomic clones corresponding to bona fide U4, U6, U7, U11, and U12 snRNA loci. Unlike the clustered U1 and U2 genes, each of these loci encode a single gene, with the exception of the U4 clone, which contains two genes. We next examined the association of these snRNA genes with CBs and found that they colocalized less frequently than their multicopy counterparts. To differentiate a lower level of preferential association from random colocalization, we developed a theoretical model of random colocalization, which yielded expected values for chi2 tests against the experimental data. Certain single-copy snRNA genes (U4, U11, and U12) but not controls were found to significantly (p < 0.000001) associate with CBs. Recent evidence indicates that the interactions between CBs and genes are mediated by nascent transcripts. Taken together, these new results suggest that CB association may be substantially augmented by the increased transcriptional capacity of clustered genes. Possible functional roles for the observed interactions of CBs with snRNA genes are discussed. (+info)