Spliceosomal small nuclear ribonucleoprotein particles repeatedly cycle through Cajal bodies

The Cajal body (CB) is a nuclear structure closely associated with import and biogenesis of small nuclear ribonucleoprotein particles (snRNPs). Here, we tested whether CBs also contain mature snRNPs and whether CB integrity depends on the ongoing snRNP splicing cycle. Sm proteins tagged with photoactivatable and color-maturing variants of fluorescent proteins were used to monitor snRNP behavior in living cells over time; mature snRNPs accumulated in CBs, traveled from one CB to another, and they were not preferentially replaced by newly imported snRNPs. To test whether CB integrity depends on the snRNP splicing cycle, two human orthologues of yeast proteins involved in distinct steps in spliceosome disassembly after splicing, hPrp22 and hNtr1, were depleted by small interfering RNA treatment. Surprisingly, depletion of either protein led to the accumulation of U4/U6 snRNPs in CBs, suggesting that reassembly of the U4/U6.U5 tri-snRNP was delayed. Accordingly, a relative decrease in U5 snRNPs compared with U4/U6 snRNPs was observed in CBs, as well as in nuclear extracts of treated cells. Together, the data show that particular phases of the spliceosome cycle are compartmentalized in living cells, with reassembly of the tri-snRNP occurring in CBs.     

Thymosins: both nuclear and cytoplasmic proteins

A simple procedure based on perchloric acid extraction has been developed for the preparation and purification of bovine prothymosin alpha and thymosins beta 4 and beta 9 in high yields. Spectroscopic observations show these proteins to be non-folding at neural pH. The cellular locations of human prothymosin alpha, rat parathymosin and calf thymosin beta 4, all so-called 'thymic hormones', have been studied by injection into the cytoplasm of Xenopus oocytes, followed by separate monitoring of nuclear and cytoplasmic concentrations. It is shown that human prothymosin alpha and rat parathymosin both migrate to the nucleus whilst thymosin beta 4 remains in the cytoplasm. The peptide (1-88) of calf prothymosin alpha is shown not to accumulate in the Xenopus nucleus, demonstrating that the C-terminal 21 residues, which include a KKQK sequence, are required for nuclear migration. The present data, in association with existing evidence of wide tissue distribution and the lack of signal peptides, indicate that these proteins do not behave as hormones in the usual sense of the word. It is suggested that thymosin beta 4 should be grouped separately from the pro- and parathymosins.     

The herpes simplex virus 1 UL11 proteins are associated with cytoplasmic and nuclear membranes and with nuclear bodies of infected cells.

Earlier studies have shown that the UL11 gene of herpes simplex virus encodes a myristylated virion protein and that the UL11 gene enables efficient virion envelopment and export from infected cells. A rabbit polyclonal antibody directed against an affinity-purified UL11-glutathione-S-transferase fusion protein was made and used to study the properties of the UL11 protein and its distribution in infected cells. We report the following: (i) UL11 protein formed up to five bands (apparent M(r)s, 17,000 to 22,000) in denaturing polyacrylamide gels; (ii) fluorescent-antibody studies revealed the presence of UL11 protein in the perinuclear space and in sites within the nucleus; (iii) immune electron microscopic studies indicated that the UL11 gene products were associated with the inner nuclear membrane, with cytoplasmic membranes and ribbon-like cytoplasmic structures resembling membranous organelles, with nuclear bodies shown by fluorescence microscopy to be different from nucleoli in which US11 protein accumulates, and with enveloped virions but not with nuclear capsids; and (iv) the nuclear bodies containing UL11 protein were reminiscent both of type IV morphotypes consisting of an electron-dense core containing the UL11 proteins surrounded by a more electron-transluscent core and of type V morphotypes consisting of material homogenous in electron opacity. We conclude that (i) the UL11 protein is processed after synthesis; (ii) the localization of UL11 protein with virions and membranes is consistent with the hypothesis that UL11 plays a role in the transport of virions to the extracellular space; and (iii) although the significance of the association of UL11 proteins with nuclear bodies is unknown, the results indicate that nuclear bodies differ with respect to their morphologies and contents of viral protein and suggest that UL11 protein may have more than one function in the infected cell.     

Cajal bodies and coilin--moving towards function

Many nuclear factors are concentrated within nonmembrane-bound subnuclear bodies. The Cajal body is an example of a conserved nuclear compartment that has been linked to molecular disease. Recent studies have shown Cajal bodies to be surprisingly mobile and offer clues about their function in the cell.     

Differential expression of cytoplasmic proteins during yeast bud and germ tube formation in Candida albicans

Changes in the identity and quantity of proteins synthesized during morphogenesis may result from alterations in gene expression in the dimorphic yeast Candida albicans. Stationary phase yeast cells, upon resuming growth at 25 degrees C, form budding yeast and at 37 degrees C form germ tubes. In order to identify proteins associated with morphogenesis, we compared cytoplasmic proteins synthesized during germ tube and bud formation. Proteins synthesized during this period were labeled at four intervals with either [3H]leucine or [35S]methionine and separated by two-dimensional polyacrylamide gel electrophoresis. This study shows that, of the 230 proteins resolved on each gel, 5 were specific to the yeast morphology and 2 proteins showed reduction in net synthesis in the mycelial phase. There were, however, no mycelium-specific proteins at any labeling period. The majority of proteins were common to both morphologies and showed no major shift in number during resumption of growth. The observations reported here suggest that differential gene expression occurs during morphogenesis of C. albicans.     

Functional characterization of nuclear localization signals in yeast Sm proteins

In mammals, nuclear localization of U-snRNP particles requires the snRNA hypermethylated cap structure and the Sm core complex. The nature of the signal located within the Sm core proteins is still unknown, both in humans and yeast. Close examination of the sequences of the yeast SmB, SmD1, and SmD3 carboxyl-terminal domains reveals the presence of basic regions that are reminiscent of nuclear localization signals (NLSs). Fluorescence microscopy studies using green fluorescent protein (GFP)-fusion proteins indicate that both yeast SmB and SmD1 basic amino acid stretches exhibit nuclear localization properties. Accordingly, deletions or mutations in the NLS-like motifs of SmB and SmD1 dramatically reduce nuclear fluorescence of the GFP-Sm mutant fusion alleles. Phenotypic analyses indicate that the NLS-like motifs of SmB and SmD1 are functionally redundant: each NLS-like motif can be deleted without affecting yeast viability whereas a simultaneous deletion of both NLS-like motifs is lethal. Taken together, these findings suggest that, in the doughnut-like structure formed by the Sm core complex, the carboxyl-terminal extensions of Sm proteins may form an evolutionarily conserved basic amino acid-rich protuberance that functions as a nuclear localization determinant.     

Transfer of cytoplasmic and nuclear proteins between cells in culture

Evidence is presented for transfer of proteins between cells in culture, using techniques which previously have shown RNA transfer and the lack of DNA transfer between cells in culture. These techniques involved making donor cells heavier than recipient cells by having them ingest tantalum particles. After coculture of donor and recipient cells the two cell types were separated by centri- fugation on Ficoll gradients and the recipient cells analyzed for radioactively labeled proteins that may have passed from the prelabeled donor cells.These techniques also provided evidence for passage of donor cell proteins to recipient cell nuclei. Examination of the nuclear proteins in the recipient cells revealed that histones were transferred intercellularly to a greater extent than other nuclear proteins. The histone subfractions in the recipient cell nuclei were studied by acrylamide gel electrophoresis. No major differences were found in the proportion of each histone subfraction that was transferred to the recipient cell nuclei.     

Localization of interchromatin granule cluster and Cajal body components in oocyte nuclear bodies of the hemipterans

An oocyte nucleus contains different extrachromosomal nuclear domains collectively called nuclear bodies (NBs). In the present work we revealed, using immunogold labeling electron microscopy, some marker components of interchromatin granule clusters (IGCs) and Cajal bodies (CBs) in morphologically heterogeneous oocyte NBs studied in three hemipteran species: Notostira elongata, Capsodes gothicus (Miridae) and Velia caprai (Veliidae). Both IGC and CB counterparts were revealed in oocyte nuclei of the studied species but morphological and biochemical criteria were found to be not sufficient to determine carefully the define type of oocyte NBs. We found that the molecular markers of the CBs (coilin and non-phosphorylated RNA polymerase II) and IGCs (SC35 protein) may be localized in the same NB. Anti-SC35 antibody may decorate not only a granular material representing "true" interchromatin granules but also masks some fibrillar parts of complex NBs. Our first observations on the hemipteran oocyte NBs confirm the high complexity and heterogeneity of insect oocyte IGCs and CBs in comparison with those in mammalian somatic cells and amphibian oocytes.     

Cajal bodies and histone locus bodies: Molecular composition and function

The review provides modern classification of evolutionarily conserved coilin-containing nuclear bodies of somatic and germ cells that is based on the characteristic features of their molecular composition and the nature of their functions. The main differences between Cajal bodies and histone locus bodies, which are involved in the biogenesis of small nuclear spliceosomal and nucleolar RNAs and in the 3′-end processing of histone precursor messenger RNA, respectively, are considered. It is shown that a significant contribution to the investigation of the diversity of coilin-containing bodies was made by the studies on the architecture of the RNA processing machinery in oocyte nuclei in a number of model organisms. The characteristics features of the molecular composition of coilin-containing bodies in growing oocyte nuclei (the so-called germinal vesicles) of vertebrates, including amphibians and birds, are described.