An unusual fibrillarin gene and protein: structure and functional implications.

The diploid germinal nucleus of the ciliated protozoan Tetrahymena thermophila is unusual among eukaryotes in that it encodes a single copy of the gene for rRNA allowing identification of cis-acting mutations in rDNA affecting rRNA structure, function, and processing. The generally conserved nucleolar protein fibrillarin has been characterized from a number of systems and is involved in pre-rRNA processing. We have demonstrated that Tetrahymena has fibrillarin and have analyzed the cDNA and the genomic DNA encoding this protein. The derived amino acid sequence of the N-terminal region of Tetrahymena fibrillarin shows little similarity with the generally highly conserved glycine/arginine-rich N-terminal domain of other eukaryotic fibrillarins. The remainder of the amino acid sequence of the molecule is more conserved. Polyclonal antibodies generated against the full-length Tetrahymena fibrillarin expressed in bacteria recognize a protein of M(r) approximately 32,000 in whole-cell or nucleolar preparations. Immunocytochemistry localizes fibrillarin to nucleoli in the somatic macronuclei of vegetative cells. Transformation experiments demonstrate that fibrillarin is an essential protein in Tetrahymena. The Tetrahymena fibrillarin is expressed but does not complement a NOP1 null mutation when transformed into the yeast Saccharomyces cerevisiae, indicating less functional conservation among fibrillarins than previously suggested.     

Changes in beta-tubulin isoforms and their RNA level in synchronized tobacco cells.

Tobacco BY-2 cells were synchronized by an aphidicolin treatment, and their beta-tubulin isoforms and their mRNA were analyzed by Western, Northern and dot blottings. The relative ratio of the beta-tubulin isoforms changed with the progress of cell cycle stage. By Northern blot hybridization of poly(A)+RNAs with a cloned carrot beta-tubulin cDNA probe, a single band of about 1.6 kb was detected throughout the cell cycle. Dot blot hybridization showed that beta-tubulin mRNA existed in all stages in the cell cycle at a relatively constant level, though it accumulated slightly more than average at M phase and decreased during G1 phase.     

Nuclear-Cycle Dependence of the Development of the Preprophase Band in Tobacco BY-2 Cells

When tobacco BY-2 cells that had been treated with aphidicolinfor 24 h were cultured in the absence of aphidicolin, DNA synthesiswas initiated within 1 h. DNA synthesis was completed within4 h and then the preprophase band of microtubules (PPB) developed.However, when cells that had been treated with aphidicolin werecultured in the absence of aphidicolin for 1 h and then againin its presence, DNA synthesis, which was initiated during thehour in the absence of aphidicolin, was not completed withina further 10 h in the presence of aphidicolin. Moreover, thePPB did not develop even after the PPB had appeared and disappearedin cells that were cultured contemporaneously in the continuedabsence of aphidicolin. The development of the PPB seems to be causally associated withthe nuclear cycle of cell division in tobacco BY-2 cells. Thisprocess seems to require the completion of the replication ofall, or almost all, of the nuclear DNA. (Received July 25, 1992; Accepted November 24, 1992)     

Implication of nucleolar protein SURF6 in ribosome biogenesis and preimplantation mouse development

The step-wise assembly of a functional nucleolus, which occurs over the first few cell cycles during preimplantation development, is poorly understood. In this study, we examined the function of the evolutionary conserved nucleolar protein SURF6 in preimplantation mouse embryo development. Immunocytochemical analyses revealed that the localization of SURF6 was similar but not identical to those of fibrillarin and B23/nucleophosmin 1, which are involved in rRNA processing and ribosome biogenesis in mammalian somatic cells. Surf6 mRNA, which is expressed in oocytes and maternally inherited in the zygote, reached a peak level of expression during the 8-cell stage of embryo development, at which time rDNA is highly transcribed. Knock-down of Surf6 mRNA by RNAi led to a decrease in both the mRNA and protein levels, and resulted in developmental arrest at the 8-cell/morula stage, as well as a decrease in the level of 18S rRNA. These results suggest that Surf6 is essential for mouse preimplantation development, presumably by regulating ribosome biogenesis.     

Changes in nuclear localization of An3, a RNA helicase,during oogenesis and embryogenesis in Xenopus laevis

The immunolocalization of An3 protein, an ATP-dependent RNA helicase and a member of the DEAD box family, was compared with the localization of fibrillarin, a protein essential for rRNA processing, and snRNPs, which are involved in mRNA splicing reactions, during oogenesis and embryogenesis in Xenopus laevis. Although An3 protein was detected in the cytoplasm of all stages of oocytes, in most stages An3 protein was also present in the nucleus. Prior to stage I An3 protein was uniformly dispersed throughout the entire germinal vesicle; from stages I to V it was in nucleoli. By stage VI nucleolar labeling with anti An3 disappeared and the protein was no longer present within nuclei. An3 reactivity was also present throughout the nuclei of follicle cells surrounding prestage I to stage VI oocytes. Both cytoplasmic and nuclear An3 staining were present in cells of stages 8 to 35 embryos; however, nuclear staining was punctate and uniformly distributed throughout the nucleoplasm. Fibrillarin was diffusely distributed throughout the entire germinal vesicle prior to stage I, localized exclusively to nucleoli of oocytes between stages I and VI and in nucleoli of stages 12 and 35 embryonic cells. Reactivity for snRNPs (anti-Sm) in germinal vesicles of prestage I oocytes was diffuse, and similar to the distribution of An3 and fibrillarin; in later stage oocytes anti-Sm staining was restricted to a population of granules, much fewer in number and more heterogeneous in size than nucleoli. Anti-Sm activity was apparent in nuclei of embryonic cells of stages 8 to 35 embryos. Although colocalization of the Sm epitope and An3 was not observed in developing oocytes and in embryonic cells, Sm reactive material was frequently found in close association with An3-positive nucleoli (oocytes) and nuclear deposits (embryonic cells). In stage IV and V oocytes treated with actinomycin D (4 μg/ml) to inhibit rRNA synthesis, nucleoli, which continued to possess fibrillarin, lacked An3; staining of follicle cell nuclei for An3 was unchanged. Treatment with 200 μg/ml actinomycin D to block mRNA synthesis, inhibited An3 but not fibrillarin staining in nuclei of prestage I oocytes and follicle cells. The changing patterns of An3 reactivity and the differential effects of actinomycin D on such localizations observed here are consistent with a role for An3 in the processing/production of RNA. © 1996 Wiley-Liss, Inc.     

TONSOKU is expressed in S phase of the cell cycle and its defect delays cell cycle progression in Arabidopsis

TONSOKU(TSK)/MGOUN3/BRUSHY1 of Arabidopsis thaliana encodes a nuclear leucine-glycine-aspargine (LGN) domain protein implicated to be involved in genome maintenance, and mutants with defects in TSK show a fasciated stem with disorganized meristem structures. We identified a homolog of TSK from tobacco BY-2 cells (NtTSK), which showed high sequence conservation both in the LGN domain and in leucine-rich repeats with AtTSK. The NtTSK gene was expressed during S phase of the cell cycle in tobacco BY-2 cells highly synchronized for cell division. The tsk mutants of Arabidopsis contained an increased proportion of cells with 4C nuclei and cells expressing cyclin B1 compared with the wild type. These results suggest that TSK is required during the cell cycle and defects of TSK cause the arrest of cell cycle progression at G2/M phase.     

Developmental expression of fibrillarin and U3 snRNA in Xenopus laevis.

Fibrillarin is one of the protein components that together with U3 snRNA constitute the U3 snRNP, a small nuclear ribonucleoprotein particle involved in ribosomal RNA processing in eucaryotic cells. Using an antifibrillarin antiserum for protein detection and a fibrillarin cDNA and a synthetic oligonucleotide complementary to U3 snRNA as hybridization probes, the expression of these two components has been studied during Xenopus development. Fibrillarin mRNA is accumulated early in oogenesis, like many other messengers, and translated during oocyte growth. Fibrillarin protein is thus progressively accumulated throughout oogenesis to be assembled with U3 snRNA and used for ribosome production in the amplified nucleoli. After fertilization, the amount of U3 snRNA decreases while the maternally accumulated fibrillarin mRNA is maintained and utilized to produce more protein. After the mid-blastula transition, stored fibrillarin is assembled with newly synthesized U3 snRNA and becomes localized in the prenucleolar bodies and reforming nucleoli.     

Caffeine-induced uncoupling of mitosis from DNA replication in tobacco BY-2 cells

Caffeine induced a mitosis-like state in cultured tobacco (Nicotiana tabacum L.) BY-2 cells after DNA synthesis had been arrested by aphidicolin. Cells were synchronized upon removal of aphidicolin. When aphidicolin was readded, the cell cycle was again interrupted and caffeine, when added with aphidicolin, induced the mitosis-like state in 5–10% of cells.